PUB) family genes encode the E3 ubiquitin ligase enzyme, which determines substrate specific recognition during protein ubiquitination. They are widespread in plants and are critical for plant growth, development, and response to external stresses. However, there are few studies on the functional characteristic of PUB gene family in the important staple crop, maize (Zea mays L.). In this study, the PUB gene in maize was aimed to identify and classify through whole-genome screening. Phylogenetic tree, gene structure, conserved motif, chromosome location, gene duplication (GD), synteny, and cis-acting regulatory element of PUB member were analyzed. The expression profiles of ZmPUB gene family in maize during development and under abiotic stress and hormones treatment were analyzed by the RNA-seq data. A total of 79 PUB genes were identified in maize genome, and they were stratified into seven categories. There were 25 pairs of segmental duplications (SD) and 1 pair of tandem duplication (TD) identified in the maize PUB gene family. A close relationship was observed between the monocot plant maize and rice in PUB gene family. There were 94 kinds of cis-acting elements identified in the maize PUB gene family, which included 46 biotic- and abiotic-responsive elements, 19 hormone-responsive elements, 13 metabolic and growth-related elements. The expression profiles of maize PUB gene family showed characteristics of tissue specificity and response to abiotic stress and hormones treatment. These results provided an extensive overview of the maize PUB gene family.The U-box E3 ( The ubiquitin proteasome system (UPS) is an energy dependent system that modulates protein activity and degeneration. It is involved in cellular growth, differentiation and apoptosis, secretion and endocytosis, gene transcription and expression, as well as signal transduction and immune response ,2. UPS iPUB gene family was detected in numerous species, namely Arabidopsis , rice , Chinese cabbage , soybean , barley , tomato , citrus , grape , cotton , and Medicago  . Un. UnZmPUBZmPUBs expression, we conducted RNA-seq under the hormone treatments . Based on our observations, 42 (53.16%) ZmPUB genes were detected in the leaf tissue (FPKM > 1)  and up-regulated at the late stage (12 h). Under IAA treatment, 15 ZmPUB genes were altered, among which 5 were reduced, and 10 were augmented. Under GA treatment, 17 ZmPUB genes were altered, 6 of which were significantly diminished at one stage, and 11 were significantly elevated at one or two stage. As a result, we identified eight ZmPUB genes that showed the highest degree of hormone-induced expression, and they were ZmPUB1, ZmPUB4, ZmPUB43, ZmPUB45, ZmPUB57, ZmPUB64, ZmPUB68, and ZmPUB78, respectively. These evidences suggested that these ZmPUB genes contributed to the hormone-responsive network.To further elucidate whether hormone affect PKM > 1) . Thirty-PUB genes are common among mammals, plants, and microorganisms, and they encode the PUB ligase enzyme, a key enzyme in ubiquitin proteasome-mediated degradation [PUB genes was performed in numerous species, and they were found to play essential roles in modulating plant development and response to biotic, abiotic, and hormonal stressors [ZmPUB genes in the maize genome and designated ZmPUB1 through ZmPUB79 based on their location on the chromosome.The radation . The gentressors ,11,22,23ZmPUB genes can be clustered into seven categories. Similar results were found in rice, soybean, barley, citrus, and banana (Musa paradisiaca), wherein the genes were classified into 6–8 groups using phylogenetic analysis [ZmPUB genes encoded proteins that harbor only the U-box domain, and 43 ZmPUB genes encoded proteins that contain the U-box domain, along with additional 10 domains, including the ARM repeat (25), coil coil region(10), pkinase-tyr (9), TPR (2), UFD2P_CORE (2), ZnF_TTF (1), WD40 repeat (1), KAP (1), RS4NT (1), and USP (1) domains , kinase (14), WD40 repeat (2), MIF4G (2), cyclophilin (1), UFD2 (1), and TPR domains (1) [PUB gene family, the U-box domain is evolutionarily conserved. Meanwhile, there are plenty of other domains observed in numerous species, which might introduce functional diversity to the PUB genes.Phylogenetic analysis revealed that these analysis ,16,18,46 domains . In Arabains (1) . In riceains (1) . In highPUB gene family, there was a wide variation in the number of exons; the mean number of exons per gene was 3.61, and it ranged from 1 to 16. Around 37.97% of the ZmPUB genes contained only one exon and no intron  at the 5′ and 3′ ends. The structural organization is closely related to gene function and family evolution . In the o intron . In prioUB genes ,21. The utations . In the  cabbage ,18,21,49UB genes . For insZmPUB genes) of SD and one pair of TD (ZmPUB43/ZmPUB44) in the maize PUB gene family , two genes to the coil coil region (ZmPUB62 and ZmPUB8), and one gene to the KAP domain (ZmPUB49) , mutation, and natural selection are the major sources of new genes and functions, and they provided a basis for biodiversity . Gene due family . GD is sZmPUB49) . The add–215 Mya . Based oonserved . In addibidopsis . This suPUB gene family. These were 19 hormone-response elements, 46 biotic- and abiotic-response elements, 13 metabolism- and development-related elements, and 13 unknown function elements  were highly expressed at certain stages of root development (FPKM > 10). Further analysis revealed that GA, IAA, and meristem expression-related elements were present in the promoter of these genes  were spotted during seed development, four of which harbored the gliadin metabolic regulatory element (O2-site) in their promoter regions  transporter 6 protein, which was highly expressed in the node, and it blocked the cadmium (Cd) upward transport [AtPUB18 and AtPUB19 were positively regulated by ABA and salt, and their double mutants could not respond well to ABA and the salt-based suppression of seed germination relative to wild-type plants [ZmPUB30 and ZmPUB57 were up-regulated by 0.5–12.5 or 3.67–84.82 times. The conserved motif analysis revealed that ZmPUB57 contained more of motifs 2, 5, and 8 than ZmPUB30. This may greatly enhance the stress response ability of ZmPUB57. ZmPUB31 and ZmPUB45 were a pair of duplicated genes. Under cold, heat, salt, drought, UV, and ABA stresses, ZmPUB31 and ZmPUB45 were up-regulated by 0.19–3.8 and 3.1–36.6, respectively ). The Hidden Markov Model (HMM) profile of the U-box domain (PF04564) was retrieved from the Pfam database (http://pfam.xfam.org/ (accessed on 5 March 2022)). The HMMER program was searched in the maize genome with default parameters and a significant e−3 value. Next, we confirmed the putative ZmPUB genes using the Pfam (http://pfam.xfam.org/ (accessed on 5 March 2022)), SMART (http://smart.embl-heidelberg.de/ (accessed on 5 March 2022)), and NCBI CDD (https://www.ncbi.nlm.nih.gov/cdd (accessed on 5 March 2022)) databases. The putative ZmPUB protein MW (molecular weight) and PI (isoelectric point) were analyzed using ExPASy (http://www.expasy.org (accessed on 5 March 2022)). WoLF PSORT (https://www.genscript.com/wolf-psort.html (accessed on 5 March 2022)) was employed to estimate the subcellular localization of ZmPUB proteins.The maize genome sequences (B73 RefGen_v4) were obtained from the Ensembl Plants database  using MEGA 7 [Oryza sativa and Arabidopsis thaliana PUB protein sequences were obtained from the NCBI website (https://www.ncbi.nlm.nih.gov/ (accessed on 6 May 2022)), as reported in previous studies [Multiple sequence alignments were performed using the U-box domain sequences of the 219 PUB proteins ), SMART (http://smart.embl-heidelberg.de/ (accessed on 6 May 2022)) and Pfam (http://pfam.xfam.org/ (accessed on 6 May 2022)) softwares [http://meme-suite.org (accessed on 6 May 2022)) [https://github.com/CJ-Chen/Tbtools (accessed on 6 May 2022)).The genetic structure analysis was conducted via comparison of the CDS and DNA sequences of the oftwares . The cony 2022)) , and visPUB genes from Arabidopsis, maize, and rice were aligned in BLASTp using an E-value < 1 × e−10 cut-off. MCScanX was employed to assess GD events and examine syntenic association between different species, and visualization was performed in Circos [−6, where λ = 6.5 × 10−9 [The gene lengths and position data were acquired from the maize genome (B73 RefGen_v4). Next, MapChart was employed for the construction of the chromosomal localization map . To assen Circos ,85. The 5 × 10−9 .ZmPUB gene. Then the cis-acting regulatory element analysis was carried out in PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 10 May 2022)) [We downloaded the 1500 bp upstream flanking sequences from the transcription start site of each y 2022)) .ZmPUB genes in numerous tissues, developmental stages, and stress conditions were acquired from NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 6 June 2022)) and MaizeGDB (https://www.maizegdb.org/ (accessed on 6 June 2022)) and were reported in previous studies [ZmPUB genes were drawn in the omicshare website (https://www.omicshare.com/ (accessed on 10 June 2022)).The RNA-seq information for  studies ,39,40,88Zea mays cv. B73 was employed for the examination of gene profile in response to multiple hormonal treatments. All seeds were cultivated in commercial soil at 28 °C in a photoperiod of 16 h light/8 h dark. Three-leaf stage seedlings were treated with 100 μmol/L ABA, 100 μmol/L IAA, and 100 μmol/L GA in 0.1‰ Tween-20, respectively. The controls were treated with water in 0.1‰ Tween-20. The leaves were harvested at 0, 3, 6, 12, and 24 h after hormonal treatments. Three replicates of three plants were used in each treatment. All samples were flash-frozen in liquid nitrogen prior to storage in −80 °C until subsequent analysis.Total RNA extraction employed Trizol . The quality of RNA was assessed via the Agilent 2100 Bioanalyzer . Transcripts were enrich-fragmented into small pieces, which were then converted to cDNA. The cDNA library was generated with the NEBNext Ultra RNA Library Prep Kit for Illumina , and sequencing was completed with Illumina Novaseq 6000 by Gene Denovo Biotechnology Co., . To quantify gene abundance, we filtered reads and mapped the clean reads to the reference genome with the fastp, HISAT2. 2.4, and StringTie v1.3.1 softwares ,90,91. FTo compare the variation of gene expression, SPSS 12.0  was employed to carry out the least significant difference (LSD) test. Data processing and visualization were done with GraphPad Prism 5. Individual data were presented as mean ± standard error (SE) of three experimental replicates.PUB genes in maize. We identified 79 ZmPUB genes, which were stratified into 7 categories. Each group exhibited similar exon-intron structures and motif compositions. Using gene duplication and synteny analysis of PUB genes, we obtained important clues regarding the evolutionary profiles of maize PUB genes. PUB genes strongly regulated plant development as well as response to abiotic stresses and hormones. Our phylogenetic and gene expression analyses provided useful information for enhancing our comprehension of the biological roles of the PUB genes in maize.Herein, we conducted an extensive analysis of


Exercise may influence 25(OH)D levels in the circulation by regulating either the vitamin D metabolites stored in tissues or the utilization by target tissues. The effects of exercise on 25(OH)D levels in the circulation may be dependent on many factors, such as the vitamin D nutritional status, exercise type and intensity, and sex. Therefore, further research on the effects and mechanisms of exercise on vitamin D metabolites is required.Vitamin D deficiency has become a widespread public health problem owing to its potential adverse health effects. Generally, the nutritional status of vitamin D depends on sunlight exposure and dietary or supplementary intake. However, recent studies have found that exercise can influence circulating 25(OH)D levels; although, the results have been inconclusive. In this review, we focused on the effect of exercise on circulating vitamin D metabolites and their possible mechanisms. We found that endurance exercise can significantly increase serum 25(OH)D levels in vitamin D-deficient people but has no significant effect on vitamin D-sufficient people. This benefit has not been observed with resistance training. Only chronic endurance exercise training can significantly increase serum 1,25(OH) Due to its long half-life . In an analysis of both pre-pubertal and pubertal boys, the concentration of 25(OH)D3 increased 15 min after the VO2max test and dropped one hour after exercise, but these changes were not significantly different at specific time points   . Abboud  nmol/L] . In addi(FGF23)) ,71. PTH its mRNA . Moreovehe bones ,73. Theshe bones . Changes2D can bind to VDR and exert physiological functions, which may explain why exercise promotes health. Because 1,25(OH)2D utilization in the target tissue increases, so does conversion of 25(OH)D to 1,25(OH)2D3, resulting in reduced serum 25(OH)D levels [Endurance exercise can increase VDR mRNA levels ,51,55. ID levels .A summary of how endurance training may exert its effects on 25(OH)D in several ways can be seen in One human study and one animal study have investigated the effect of acute resistance exercise intervention on 25(OH)D . Barker Five human studies have investigated the effects of resistance exercise training on 25(OH)D levels . Resista2D3 D and 1,25(OH)2D3 . Buskermale mice .The predominant adaptation of resistance exercises is in the musculoskeletal system, including increases in muscle mass, muscle strength, and bone density . Muscle 3 in C2 myotubes and mouse muscle fibers and reduces its retention in myotubes [Moreover, 25(OH)D can be released from skeletal muscle ,82,83,84myotubes . In Barkmyotubes . In Zhanmyotubes . The incmyotubes ,75. Howemyotubes ,77. This2D3. Moreover, resistance training can increase target tissue VDR levels [2D3 utilization, which may explain why serum 25(OH)D levels are not altered in response to resistance training [2D; hence, the decrease in 1,25(OH)2D may be caused by increased degradation, while its synthesis remains unchanged [Resistance training can increase the level of CYP27B1 , which cR levels , increastraining . In additraining , which cnchanged . These fHow resistance training may exert its effect on 25(OH)D in various ways is briefly illustrated in The effect of endurance combined with resistance exercise training intervention on 25(OH)D was investigated in three human studies and one animal study, which did not yield consistent results . In the 2 levels are difficult to detect [Aside from the small number of relevant studies, there are many limitations. First, while mass spectrometry, enzyme-linked immunosorbent assays, and other methods can detect 25(OH)D levels, their accuracies vary greatly. Moreover, 25(OH)Do detect . Second,o detect  or outdoo detect , the vas2D levels may be influenced by whether it is performed under fed or fasted states. Third, vitamin D metabolites are primarily found in circulation, adipose tissue, and skeletal muscle [Because the nutritional status of vitamin D is influenced by various factors, we recommend incorporating the following considerations in future studies. First, due to their lipid solubility, vitamin D metabolites are sequestered in adipose tissue, leading to decreased bioavailability in obese subjects . Moreovel muscle . Thus, a2D levels, and this effect may be sex-dependent. Exercise may influence 25(OH)D levels in circulation by regulating either the release of vitamin D metabolites from storage tissues or the utilization of target tissue D levels in vitamin D-deficient subjects but has no significant effect on vitamin D-sufficient subjects. Moreover, resistance training did not significantly increase 25(OH)D concentrations. Only chronic endurance exercise intervention significantly increased serum 1,25(OH)t tissue . The eff


Arabidopsis thaliana.In flowering plants, pollen development is a key process that is essential for sexual reproduction and seed set. Molecular and genetic studies indicate that pollen development is coordinatedly regulated by both gametophytic and sporophytic factors. Tapetum, the somatic cell layer adjacent to the developing male meiocytes, plays an essential role during pollen development. In the early anther development stage, the tapetal cells secrete nutrients, proteins, lipids, and enzymes for microsporocytes and microspore development, while initiating programmed cell death to provide critical materials for pollen wall formation in the late stage. Therefore, disrupting tapetum specification, development, or function usually leads to serious defects in pollen development. In this review, we aim to summarize the current understanding of tapetum-mediated pollen development and illuminate the underlying molecular mechanism in  In higher plants, the alternation of the life cycle mostly depends on sexual reproduction, in which normal pollen development is necessary. In the past decades, extensive studies have shown that pollen development is a complex biological process, which includes anther primordial cell differentiation, pollen mother cell meiosis, microspore mitosis, tapetal cell differentiation and programmed cell death, pollen wall formation and pollen grain release ,2. AccorArabidopsis thaliana.Over the past two decades, extensive studies indicated that pollen development is coordinately controlled not only by gametophytic but also by sporophytic regulators, especially the tapetum for the latter case ,6,7. TheArabidopsis .DYT1 , ABCG26 (WBC27), and TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), which play an essential role in regulating tapetum development and pollen wall formation, are directly regulated by AMS , encoding a plasma membrane protein, is another potential sporopollenin precursor transporter .,97.25,97MYB2, is expressed in the tapetum from stage 5 to stage 11, and works as a key factor for the regulation of the tapetum PCD and pollen development [MYB2 will lead to delayed tapetal PCD, irregular and spare exine, and defective pollen. The vitality and gemmation of the survival pollen in the myb2 were greatly decreased [CEP1 and βVPE, both of which are required for tapetal cell degradation  . During efective . The SECelopment . A recenelopment ,107. SECn grains . The undn grains . Sar1b iArabidopsis: AP-1, AP-2, AP-3, AP-4, and AP-5 [β1 and β2 cannot be obtained, and in the β1-1β2-11/+ and β1-2β2-11/+, pollen shows defects in exine formation, gemination, and pollen tube growth [Adaptor protein complex (AP) plays an important role in clathrin-mediated vesicle formation, which has five types in and AP-5 . The AP and AP-5 . Two recand AP-5 ,109. β1 β1-2 β2-11/+ [ABCG9 and ABCG16, two plasma membrane transporters, are involved in pollen surface material transport from tapetal cells to the anther locules and primarily detected in the plasma membrane in wild-type plants, while are incorrectly localized to the TGN in  β2-11/+ . Taken t β2-11/+ . Differe β2-11/+ ,110,111. β2-11/+ .MON1 is highly expressed in the tapetum, and the mutation of MON1 results in delayed tapetum PCD, which lead to delayed degradation, impaired pollen coat deposition, and male fertility [mon1, the Cys protease RD21 exhibits abnormal accumulation in enlarged PVC. Collectively, these results suggest that MON1–Rab7 complex-mediated vacuolar trafficking is essential for tapetal PCD and pollen development [In a report, Cui et al. discovered that the proper timing of tapetum PCD is dependent on the function of MON1/CALCIUM CAFFEINE ZINC SENSITIVITY1 (CCZ1), which activates Rab7 and thus mediates vacuolar trafficking . In addiertility . Previouertility ,100. Theelopment .In higher plants, pollen development is an extremely complex process, which is regulated not only by gametophytic but also sporophytic factors, especially the tapetum for the latter case ,5,12. ThAlthough the underlying mechanisms of tapetum-mediated pollen development are well understood, many questions remain to be addressed, for example, how the signal communication between the microspores and tapetum takes place; what are the signals that induce tapetal PCD and how; and which transporters mediate the transports of the materials secreted by the tapetum to the pollen surface.


Retinal degenerative diseases are the major factors leading to severe visual impairment and even irreversible blindness worldwide. The therapeutic approach for retinal degenerative diseases is one extremely urgent and hot spot in science research. The sigma-1 receptor is a novel, multifunctional ligand-mediated molecular chaperone residing in endoplasmic reticulum (ER) membranes and the ER-associated mitochondrial membrane (ER-MAM); it is widely distributed in numerous organs and tissues of various species, providing protective effects on a variety of degenerative diseases. Over three decades, considerable research has manifested the neuroprotective function of sigma-1 receptor in the retina and has attempted to explore the molecular mechanism of action. In the present review, we will discuss neuroprotective effects of the sigma-1 receptor in retinal degenerative diseases, mainly in aspects of the following: the localization in different types of retinal neurons, the interactions of sigma-1 receptors with other molecules, the correlated signaling pathways, the influence of sigma-1 receptors to cellular functions, and the potential therapeutic effects on retinal degenerative diseases. The sigma receptor was once mistaken for one subtype of opioid receptor that consists of three subtypes: kappa, mu, and sigma . SubsequSigma receptors were classified as sigma-1 and sigma-2 subtypes, based on their distinct binding site and molecular mass ,7. The hAs the research moves forward, the sigma-1 receptor has been found to play an important role in both physiological and pathological conditions. The initial studies postulated that the protein contained only one transmembrane domain ,10,14,15The sigma-1 receptor is widely expressed in several organs , such asThe vertebrate retina is composed of three cell nuclear layers , respectPhotoreceptor cells are the initiators in visual signal transduction. Cell dysfunction and the death of photoreceptor cells drive visual impairment and even blindness. Studies have shown that the sigma-1 receptor is closely related to the function of photoreceptor cells. The expression of the sigma-1 receptor in the retina occurs in the early developmental stage. As showed from the IHC analysis, the sigma-1 receptor started to express as early as a day 16 embryo (E16) and increased progressively with the maturation of the retina from postnatal day1(p1) to p10 and kept stable at p30 in mice retina . The 661Akita/+Ins2 diabetes mice [Retinal ganglion cells integrate visual information from retinal interneurons and project to the visual cortex, although they are a very minor portion in whole retinal neurons. Retinal ganglion cells are tightly connected to energy metabolism, cellular stress, and axonal transporting. The death of retinal ganglion cells was associated with glaucoma . Substantes mice ,49. Howetes mice . The subtes mice . Similartes mice . Furthertes mice . One eartes mice .Müller cells are the major glial cell type spanning all retinal layers and normally contribute to retinal structure and homeostasis . An earlHorizontal cells, bipolar cells, and amacrine cells are three predominant interneurons in retina. They receive visual input from photoreceptor cells and convey excitatory or inhibitory outputs to retinal ganglion cells . StudiesApart from the expression in the retina, the sigma-1 receptor was also distributed in other ocular structures of various species and in other ocular cell lines associated with physiological and pathological functions. The earliest study concerning ocular tissue expressing sigma-1 receptors were the lacrimal glands . Since t2+ influx in RGC-5 cells by fura-2AM intracellular calcium imaging [2+ release [−/−rd10/sigR mice compared with rd10 mice. A (+)-PTZ intervention decreased miR-214-3p to about twofold. MiRNA targeted predictive program analysis revealed that the sigma-1sigma-1 receptor 3′UTR had a binding site for MMU-Mir-214-3p; it was the miR214-3p sequence GGACGAC, which can bind to the CCUGCUG sequence of the sigma-1 receptor [c−) to improve the level of antioxidant capacity [The sigma-1 receptor acts as a novel molecular chaperone that regulates several cellular functions by the interaction with ion channels, receptors, kinases, and even RNA . (+) SKF imaging . In anot imaging . In the  imaging ,63. (+)  release , suggest release . Sigma-1 release . An addi release . When cu release . MiFindereceptor . Under hcapacity .cis-acting elements, namely antioxidant response elements (ARE), and activates downstream transcription and the expression of antioxidation-related genes, such as NADPH, NQO1, HO-1, etc. [rd10Pde6b mice is a type of inherited retinitis pigmentosa mice model in which rod and cone photoreceptors were lost resulting from the missense mutation in the phosphodiesterase 6β subunit in rd10 mice. An in vivo investigation made it clear that sigma-1 receptor agonist administration was incapable of rescuing cone photoreceptor function in rd10 mice when Nrf2 was absent. The amplitude of cone responses was decreased in rd10/Nrf2−/− mice compared with that of rd10 mice by ERG recording. Meanwhile, there was a larger cone photoreceptor cell loss in Nrf2-deficient mice [−/−rd10/Sig1R retina compared with rd10 mice. In cultured primary cortical neuronal-glial cells, sigma-1 receptor deficiency promoted gliosis, and enhanced antioxidant capacity of cells under stressful conditions through Nrf2 signaling pathway [The transcription factor NF-E2 p45-related factor 2 (Nrf2) was regarded as one indispensable orchestrator to exert pleiotropic cytoprotective effects to maintain homeostasis, among which, antioxidant action was the primary function . Under p-1, etc. . Many st-1, etc. ,73,74. A-1, etc. . Meanwhi-1, etc. ,75. In t-1, etc. . Pde6brdent mice . Similarent mice . In the ent mice . In cultent mice . However pathway ,80. Alth pathway .Mitogen-activated protein kinases (MAPK) are three kinase signaling modules activated by multiple external and internal stimuli, and the pathways are composed of five subfamilies, the extracellular signal-regulated kinase (ERK1/2) pathway, the c-Jun kinase (JNK) pathway, the p38 pathway, the ERK3a/4 pathway, and the ERK5 pathway. Among them, ERK1/2 is the most thoroughly described and most extensively studied subset. ERK1/2 is widely expressed and activated by various inputs, leading to cellular proliferation, differentiation, survival, and cell cycle regulation ,83,84. S2+-dependent PLC-PKC pathway was involved [In addition to the two signaling pathways mentioned above, there were other related molecular mechanisms that might be also involved. The up-regulation of brain-derived neurotrophic factor (BDNF) was suggested to enhance the effect of antidepressants that blocked the reuptake of serotonin and norepinephrine to presynaptic terminals . The BDNinvolved . Researcinvolved . Anotherinvolved .rd10 mice, (+)-PTZ improved retinal structure, reduced cone death, and rescued the cone function by ERG recording. In the meanwhile, (+)-PTZ weakened the gliosis of Müller cells, and the activation of microglial cells and oxidative stress [Many studies have confirmed the protective role of the sigma-1 receptor in multiple retinopathy models and a variety of cell subtypes in the retina. (+)-PTZ, SA4503, and neuroactive steroids were the most widely used agonists, PRE-084, (+) SKF10047, and (−)-MR22 are also involved. BD-1047, BD-1063, and NE-100 were the most common antagonists used to inhibit the physiological and biochemical effects of agonists . Intravie stress ,95. In ae stress . In the e stress . Retinale stress . Hypotone stress . (+) SKFe stress . The sige stress  -PTZ decreased the expression of proapoptotic genes Cflar and STKS and increased the expression of the antiapoptotic gene Eef1a2d [Apoptosis is another fundamental common form of cell death that prevents the inflammatory response caused by the spillage of the intracellular components from dead cells . Under p Eef1a2d . In sigm Eef1a2d ,67,126.+/+σR1 Müller cells, (+)-PTZ reduced the translocation of NFκB, nevertheless, the transfer process of NFκB failed to be reversed in sigma-1 receptor knockout− mice [iba1 and GFAP, markers of microglial and Müller cells, respectively [rd10 mice [rd10 mice lacking the sigma-1 receptor [Retinal diseases usually showed increased inflammatory responses. Microglia are the important population of immune cells in retina, and they secret inflammatory cytokines aggravating the related retinal diseases . (+)-PTZut− mice . Hypoosmut− mice . Water aut− mice . The neuectively . By the d10 mice , whereasreceptor .With an increasing understanding of sigma-1 receptor, the more indispensable role it plays in physiological and pathological conditions. Defects of sigma-1 receptors cause a series of adverse consequences. All above studies implicate that the sigma-1 receptor may have the potential to be a therapeutic target in retinal diseases, such as glaucoma, retinitis pigmentosa, and diabetic retinopathy .Glaucoma is one retinal neurodegenerative disease leading to irreversible blindness. It is associated with an elevated intraocular pressure and accompanied with the death of retinal ganglion cells, mitochondrial dysfunction, and ER stress ,160. TheIns2Akita/+ diabetic retinopathy mice model, intraperitoneal injection of (+)-PTZ at the onset of diabetes for 22 weeks maintained retinal structural integrity, increased thickness of mouse retina, especially the IPL and INL, and rescued the cell number of the ganglion cell layer compared with the untreated groups. At the same time, (+)-PTZ treatment preserved the radial morphology of Müller cells, reduced the accumulation of reactive oxygen and reactive nitrogen species [Akita/+Ins2 mice indicated that (+)-PTZ injection reversed the expression of 23 genes that were associated with apoptosis, oxidative stress, axon outgrowth, calcium binding, and cell differentiation among 29992 genes [Akita/+Ins2 mice at 4 weeks or 8 weeks after the onset of diabetes decreased cell loss of GCL and gliosis of Müller cells and preserved the neatly nuclear layer and retinal architecture compared with no-treatment Akita/+Ins2 mice. Fundoscopy indicated vitreal opacities and retinal vessel leakage in sigma-1 receptor deficient Akita/+Ins2 mice at the age of 12 weeks or 16 weeks. Furthermore, retinal morphology was more fragmented when the sigma-1 receptor was absent in Akita/+Ins2 mice than that of Akita/+Ins2 mice, which suggested that a defect of the sigma-1 receptor accelerated the pathological features of diabetic mice [Diabetic retinopathy leads to severe visual impairment and even blindness as a complication of diabetes. It is often accompanied with retinal microvascular abnormalities, retina-vessel barrier impairment, the occurrence of inflammation, the thinning of retinal thickness, the loss of RGC reducing the reaction in electroretinography detection, and increased endoplasmic stress ,162. Ear species , and dec species . The res92 genes . Even thtic mice . Althougtic mice . The protic mice . Unlike tic mice .rd10 group at p42. Meanwhile, (+) PTZ treatment performed as a “protective molecule” in other processes, including the loss of GCL, the reactive gliosis of microglial and Müller cells, and the oxidative stress level of lipids and proteins. While in sigma-1 receptor-deficient rd10 mice, the above protective effects to cone cells were reversed, suggesting the essential role of the sigma-1 receptor in retinitis pigmentosa [rd10 mice presented a more profound protection to photoreceptors than treatment at p18, p21, and p24. Visual acuity by an optokinetic tracking system recoding, the amplitude of electroretinograms, and the retinal structure all performed better in the (+)-PTZ-rd10 group [rd10 mice [rd10 mice, the above results were suppressed. The subsequent exploration suggested that the antioxidant signaling pathway Nrf2 was involved in the neuroprotection of the sigma-1 receptor to photoreceptor cells [rd10 mice, the loss of rod photoreceptor cells occurred before 5 weeks of age, while the loss of cones was exacerbated after being 6 weeks old. Similarly, gliosis was accelerated in Müller cells with an increased expression of GFAP, oxidative and ER stress associated signal molecular increased. For example, the expression of the apoptotic protease caspase3, the autophagy marker LC3-II, and the ER stress marker CHOP, was markedly upregulated at the early stage in sigma-1 receptor knockout rd10 mice [Retinitis pigmentosa (RP) is a one class inherited retinal progressive degenerative disease characterized by abnormalities of the retinal pigment epithelium and impairment of photoreceptors, ultimately resulting in vison loss. Currently, one strategy for RP therapy is to acquire neuroprotection to maintain retinal architecture integrity . As mentgmentosa . Further10 group ,101. Whed10 mice . While ior cells . Sigma-1d10 mice ,131.Apart from the above retinal diseases laid out here, a series of studies indicated that the sigma-1 receptor was one potential therapeutic target for several other diseases including neurodegenerative diseases, chronic pain, cancer, drug addiction, and cardiovascular disease. The phenotype and progression of neurodegenerative disorders, consisting of Huntington disease ,163, AlzSince the original discovery of the sigma-1 receptor, multiple investigations have provided insight into the potential protective role of the sigma-1 receptor in the retina through oxidative stress, cell autophagy, cell apoptosis, and anti-inflammation. Synthetic and endogenous agonists and sigma-1 receptor-deficient mice were applied to identify the molecular mechanisms and signaling pathways in the retina. The reviewed pieces of research suggest the promising therapeutic role of the sigma-1 receptor in retinal degenerative diseases, especially glaucoma, diabetic retinopathy, and retinitis pigmentosa. Nevertheless, as one widespread distributed molecular chaperone, the more specific molecular mechanism of neuroprotective effects played by the sigma-1 receptor in retinal degenerative diseases remains to be deeply explored and further studied. For example, the conditional knockout of sigma-1 receptors in different retinal developing stages and distinct subtypes of retinal neurons.


PXG3, LOX2, CYP710A1, PAL and 4 down-regulated genes including FATA2, CYP86A1, LACS3, PLA2-ALPHA were selected. The expression of these eight genes in milk thistle was similar to Arabidopsis under drought stress. Thus, PXG3, PAL, LOX2 and CYP86A1 genes that increased expression were selected for protein analysis. Due to the lack of protein structure of these genes in the milk thistle, modeling homology was performed for them. The results of molecular docking showed that the four proteins CYP86A1, LOX2, PAL and PXG3 bind to ligands HEM, 11O, ACT and LIG, respectively. HEM ligand was involved in production of secondary metabolites and dehydration tolerance, and HEM binding site remained conserved in various plants. CA ligands were involved in synthesis of cuticles and waxes. Overall, this study confirmed the importance of lipids in drought stress tolerance in milk thistle.Milk thistle is an oil and medicinal crop known as an alternative oil crop with a high level of unsaturated fatty acids, which makes it a favorable edible oil for use in food production. To evaluate the importance of Milk thistle lipids in drought tolerance, an experiment was performed in field conditions under three different water deficit levels (Field capacity (FC), 70% FC and 40% FC). After harvesting seeds of the plant, their oily and methanolic extracts were isolated, and subsequently, types and amounts of lipids were measured using GC–MS. Genes and enzymes engaged in biosynthesizing of these lipids were identified and their expression in Arabidopsis was investigated under similar conditions. The results showed that content of almost all measured lipids of milk thistle decreased under severe drought stress, but genes (belonged to Arabidopsis), which were involved in their biosynthetic pathway showed different expression patterns. Genes biosynthesizing lipids, which had significant amounts were selected and their gene and metabolic network were established. Two networks were correlated, and for each pathway, their lipids and respective biosynthesizing genes were grouped together. Four up-regulated genes including  Recently, data acquired from genomics and metabolomics studies as well as application of tools to characterize biosynthetic pathways of enzymes have provided the understanding of processes engaged in production of plant metabolites2. Study of compounds derived from medicinal and aromatic plants, which have been used to make approximately 25% of drugs in use by humanity, improves our knowledge and introduces new compounds may be effective in curing diseases and ailments3. Milk thistle, Silybum marianum (L.) Gaertn, a well-known medicinal plant belongs to Asteraceae family, is recently in center of attentions because of having a wide range therapeutic applications. Milk thistle is one of the most studied plant for treatment of liver disease due to having a group of flavonolignans called silymarin in its fruit integument. The hepatoprotective function of silymarin is mainly attributed to its anti-free radical and anticarcinogenic roles4 although but it includes other biological activities such as antioxidant, anti-lipid peroxidative, antifibrotic, anti-inflammatory, immunomodulatory and liver regenerating activity5. Silymarin has clinical applications in alcoholic liver diseases, liver cirrhosis, Amanita mushroom poisoning, viral hepatitis,toxic anddrugdiseases ofthe liver,psoriasis and in neuroprotective and neurotropic activity5. Silymarin acts as a toxin blockade agent by inhibiting binding of toxins to hepatocyte cell membrane receptors6.Metabolomics, as a comprehensive metabolic profiling approach, enables a wide range of metabolite classes be analyzed simultaneously using bioinformatics method8. Lipid components, especially fatty acids, as high-value compounds used in food and pharmaceutical areas, play key roles in cell membranes structure and metabolic processes. Cell membranes maintenance, brain functions, transmission of nerve impulses, processes of transferring atmospheric oxygen to blood plasma, synthesis of hemoglobin, and cell division are the roles which are associated with Omega-series fatty acids in humans. Since human body is not able to synthesize these compounds, they have been known as curtail fatty acids9. Phytosterols, which mainly found in vegetables, fruits, nuts, and seeds, is another important dominant lipid component of this plant, which inhabits cholesterol absorption and is added to functional foods to increase their cholesterol-lowering ability10. Sitosterols, campesterols, and stigmasterols are the most extensive phytosterols in nature11. Beta-sitosterol (BS), one of phytosterols with a similar chemical structure to cholesterol, is synthesized in plants; whereas animals should acquire those through food diet12. BS possesses antimicrobial, antioxidant, immunomodulatory, angiogenic, anticancer, anti-inflammatory, antidiabetic, and antinociceptive activities without any major toxicity for human being13.Milk thistle contains 15–30% of triglycerides such as linoleic, oleic, and palmitic acids, and 30% of proteins, sugars , sterols, tocopherol, and flavonoids 14.Global milk thistle products market has been growing at a CAGR of 7% from 2018 to 2022. Milk thistle one of top-selling herb supplements in the U.S. mass market, has introduced new opportunities such as food and feed consumption, bioenergy production, cosmetic and cosmeceutical applications, phytoremediation, industrial applications through the medium-large scale cultivation of this plant16.Drought stress affects plant growth and development in many aspects, such as morphological, physiological, and phytochemical parameters, and reduces yield, dry biomass, and growth rate17. For example, drought conditions cause oxidative stress, which long chain fatty acids are transported from cytosol into mitochondrial membrane. Mitochondria break down sugar through citric acid cycle (CAC) and synthesize ATP to provide energy18. Oxygen is reduced to H2O at the end of CAC, leading to a reduction oxygen concentration and, subsequently, reducing reactive oxygen species (ROS) formation19. Therefore, plants ability to adapt to various environmental situations is affected by fatty acids and cell membrane lipids composition20.Furthermore, composition of fatty acids and lipid content of organisms are affected by environmental conditions. In plants, biotic and abiotic factors affect primary and secondary metabolisms1 to identify and measure bioactive compounds of milk thistle in field conditions under drought stress; (2) to identify genes involved in biosynthesis of these lipids and to determine interaction between genes and lipids; and finally, (3) to perform protein modeling and molecular docking of selected genes to confirm the role of these genes in stress tolerance.Considering the importance of lipids, this research was aimed Milk thistle seeds were obtained from the Pakanbazr, Isfahan, Iran. Plant study comply with relevant institutional, national, and international guidelines and legislation.2 (10 m wide and 15 m long). Seeds were sown every 0.5 m in the rows while 1 m distance was applied between the rows. Milk thistle seeds were purchased from the Pakanbazr Company, Isfahan, Iran. The seeds were sown on 16 March 2017, and irrigated every 2 days.The experiment was established on Shahid Beheshti University’s experimental field in Tehran, Iran (51.23°N and 35.48°E) in a moderate and mountainous climate. The experiment was designed in a randomized complete block design (RCBD) with four replicates in three different drought stress levels . The soil texture consisted of 1/3 sand, 1/3 clay and 1/3 leaf composts. Average annual rainfall and temperature were 145.2 mm and of 22 °C, respectively. Average of air temperature, precipitation, relative humidity and wind speed were recorded monthly in meteorological site , supplied with a DB-1 J&W capillary column . The chromatographic conditions were followed by an initial temperature at 70 °C for 5 min, a temperature rate: 5 °C/min, and a final temperature at 290 °C for 10 min. Injector temperature was 300 °C, transfer-line temperature was 300 °C with a split ratio of 20:1. The extracts were dissolved in n-hexane solvent separately and 1–2 µl of each extracts was injected into apparatus. GC–MS data and peaks were analyzed, components of each extract were identified and their amounts were measured.https://www.genome.jp/kegg/)23 and Metabolic Pathways From all Domains of Life (MetaCyc) (https://metacyc.org/)24 databases were used to identify biosynthesis pathways as well as enzymes and genes involved in synthesis of extracts components. For this purpose, the desired lipids name was searched through KEGG and MetaCyc databases and in the "Compounds" section, the desired lipid was selected and the required information was extracted. The genes involved in the synthesis of these lipids were selected from the Arabidopsis model plant because most of its genes are known. GENEVESTIGATOR-Visualizing the world's expression data software (https://genevestigator.com/)25 was applied to investigate expression of genes under drought stress. Then Biclustering tool was used to compare the expression level of selected genes in different Arabidopsis datasets. Functional protein association networks (STRING) (https://string-db.org/)26 and ShinyGO v0.61 programs (http://bioinformatics.sdstate.edu/go/)27 were used to determine proteins interaction and biology processes interactions, respectively. ANOVA analysis for all measured lipids were performed using R version 3.5.3 (https://www.r-project.org/)28 and RStudio version 1.1.463 (https://www.rstudio.com/)29. Data were analyzed in a completely randomized blocks design (CRBD) with treatments as fixed effects and replications as random effect. Mean comparison was calculated by Duncan test presented in the agricolae package at 5% significance level of probability30. Box plot to represent relative gene expression was also drawn by R software.Kyoto Encyclopedia of Genes and Genomes (KEGG)  and subsequently treated with DNase I  to eliminate probable genomic DNA contamination. The quality and quantity of the extracted RNA were assessed with 1% agarose gel and NanoDrop 1000 spectrophotometer , respectively. Purified RNA (Concentration 5 µg) was used to synthesize first-strand cDNA through cDNA Synthesis kit  following the manufacturer’s protocola, and stored at − 20 °C until use.Helianthus annuus and Carthamus tinctorius) were obtained from the NCBI database. These sequences were then blasted on the milk thistle assembly data (from our previous experiment) using the BlastStation tool and the most identity sequences were selected. To primer design for qPCR, areas near to end of poly adenine, with a length of 150–250 bp were chosen. Homodimer, heterodimer, stem-loop, GC percent, and TM temperature were evaluated using Oligo 7 software (https://www.oligo.net/)31 and Vector NTI® Express Designer Software (https://vector-nti.software.informer.com/11.0/)16. The 18SrRNA gene was used as the reference gene. Finally, primers were synthesized by Bioneer Company (South Korea). The sequence and other information of primers are listed in Table To design primers of the genes of interest, nucleotide sequences of same family plants . 20 µl reactions included 10 μL 2 × SYBR, 1 μL of synthesized cDNA, 1 μL of each primer (20 nmol), and 7 μL RNase-free water. The thermal conditions consisted of an initial step for 5 min at 95 °C, followed by 35 cycles amplification . To investigate the specificity of each amplicon, post-amplification melting-curve ranging from 60 to 95 °C were assessed in every reaction. Cycle threshold values and PCR efficiency were computed by LinRegPCR program (https://www.gear-genomics.com/rdml-tools/)32, and relative expression levels were calculated by Relative Expression Software Tool (REST) (https://www.gene-quantification.de/rest.html)33 with the following formula. The E in the equation refers to the primer efficiency. All the qRT-PCR analysis were done in three biological and three technical replicates.RT-qPCR amplification was carried out by Rotor-Gene 2000  using SYBRhttps://www.expasy.org/)34. Homology modeling was implemented to obtain the 3D  structures of these proteins using trRosetta (https://yanglab.nankai.edu.cn/trRosetta/)35. HHsearch against the PDB70 database was used to detect template in trRosetta server. TM-score of the predicted models was estimated based on the probability of top predicted distance and convergence of top models. Next, 3D structures of proteins were used to create Ramachandran plot by ProFunc server (http://www.ebi.ac.uk/thornton-srv/databases/ProFunc/)36. Proteins functionality were predicted by Protein Structure Classification Database at UCL (CATH) (https://www.cathdb.info/)37. Molecular docking was performed by protein–ligand binding site prediction (COACH-D) (https://yanglab.nankai.edu.cn/COACH-D/)38 using 3D models as inputs. Atomistic interactions between proteins and predicted ligands were studied within 5A of ligand residues using VMD-Visual Molecular Dynamics software (https://www.ks.uiuc.edu/Research/vmd/)39. Hydrogen bonds between proteins and ligands were identified using PyMOL software (https://pymol.org/2/)40. All modeled structures also were visualized though PyMOL.Vector NTI software was used to identify ORFs of selected gene sequences, which were later translated into protein sequences using the SIB Swiss Institute of Bioinformatics (Expasy) . Cis-13-octadecenoic acid fatty acid was involved in cutin, oleate, sporopollenin precursors, suberin monomers biosynthesis and catalyzed by oleoyl-CoA thioesterase. The only identified ester was 1-oleoyl-glycerol, which was involved in triacylglycerol degradation by sn1-specific diacylglycerol lipase activity.Methanolic extract contained other fatty acids, esters, steroids and lipids. Fatty acids included Methyl linoleate, Methyl stearate, Methyl 9-octadecenoate, Methyl palmitate, Oleic acid and β-Monolinolein. The enzymes involved in these pathways were peroxygenase, lipoxygenase, monooxygenase, hydrolase and hydratase. Methylated fatty acids were synthesized by thioesterase acyl-CoA enzyme in acyl-CoA hydrolysis pathway. Oleic acid, the major fatty acid in methanolic extract, was active in biosynthesis pathways of fatty acids, cutin, suberine, wax, and plant secondary metabolites. β-Monolinolein fatty acid was responsible in linoleate biosynthesis pathway through acyl-lipid ω-6 desaturase (ferredoxin) activity.Esters comprised Methyl behenate, Linoleic acid ethyl ester, Glycerol β-stearate, Phthalic acid dioctyl ester and Hexadecanoic acid 2-(octadecyloxy) ethyl ester, which the engaged enzymes were carboxylesterase 3 and triacylglycerol lipase, which were active in triacylglycerol degradation pathway. Cholesterol belonged to steroids, which were active in steroid hormone biosynthesis, steroid biosynthesis and degradation, biosynthesis of alkaloids derived from terpenoid and polyketide, fat and vitamin digestion and absorption, cholesterol metabolism and metabolic pathways by involving 3beta-hydroxy-Delta5-steroid dehydrogenase, cholesterol oxidase, monooxygenase, hydroxylase and desaturase, 7-dehydrocholesterol and Delta24-sterol reductase, sterol acyltransferase and esterase, alcohol and bile-salt sulfotransferase, steryl sulfatase and glucosidase, steroid Delta-isomerase. The identified lipid was β-Sitosterol, which was synthesized by engaging sterol 22-desaturase enzymes in steroid, plant secondary metabolites and terpenoids biosynthesis pathways.Arabidopsis thaliana and Biclustering plot was drawn under drought stress , AT4G17470, AT5G47330, LOX3, LOX4, LOX2, CYP710A1 were the important up-regulated genes, while FATA2, CYP86A1, AT3G01690, LACS3, LACS1, PLA2-ALPHA, and CYP710A2 down-regulated under drought stress.Gene expression patterns of lipid synthesizing genes was extracted from Table ess Fig. . PXG3 (RLipids variations and respective involved genes at three different levels of irrigation (Field Capacity (FC), 70% FC, 40% FC) was studied  was performed at three different levels of drought stress with three replications. According to the results of Table FATA2 and FATA, respectively.The stearic acid content decreased with increasing stress intensity. Among genes involved in biosynthesis pathway of stearic acid, a significant decrease and a slight reduction was observed in expression levels of PXG3 (RD20) significantly increased in all pathways, whereas CYP86A1 and FATA2 expression significantly reduced.The oleic acid content in treatments FC and 40% FC was extremely high , but in treatment 70% FC (52% Area) was lower than two mentioned treatments. In oleic acid biosynthesis pathway, expression of AT5G47330 significantly raised when a drastic decrease of CYP86A1, LACS3, LACS1 expression were observed.The amount of palmitic acid has decreased over time with increasing water deficiency. In palmitic acid, expression of LOX3, LOX4, LOX2 and LOX1 and down-regulated genes were PLA2-ALPHA and LOX5.The linoleic acid content also decreased under drought stress. Up-regulated genes in linoleic acid biosynthesis pathway were DWF5 and DIM decreased. Expression of CYP710A1 and CYP710A2 significantly increased and declined, respectively, in β-Sitosterol biosynthesis pathway.The content of cholesterol and β-Sitosterol in treatment 70% FC had a relatively high value , but in two treatments 40% FC and FC was no significant. In cholesterol biosynthesis pathway, expression of The content of β-Monolinolein was relatively high (10.7% Area) in treatment 70% FC but in the other two treatments the amount was halved. The genes biosynthesizing this lipid did not show significant expression.The content of Linoleic acid ethyl ester and 1-Oleoyl-glycerol in treatment 70% FC had higher values  than the two treatments 40% FC and FC. Expression of linoleic acid ethyl ester and 1-oleoyl-glycerol biosynthesis pathway genes reduced , which include LACS1, LACS2, AT4G17470, AT5G47340, AT5G47330, AT4G17483 and AT3G60340 were categorized into group 3, yellow colors, which were active in palmitic acid and linoleic acid ethyl ester biosynthesis pathway. Group 4 with blue colors contained LACS6, LACS7 and LOX4, which engaged in biosynthesis pathway of palmitic acid and linoleic acid play role in the response to ozone.For genes and lipids, a network was drawn separately to examine the relationship between genes and lipids. Gene network Fig.  was diviCYP86A1, CYP710A1, FATA2, LACS3, LOX2, Palmitoyl-protein thioesterase (PAL), PLA2-ALPHA and PXG3 genes were not previously identified in the plant of our interest, so, the nucleotide sequence of these genes (based on our RNA-Seq data in previous research) was provided to submit in NCBI database. Accession numbers of selected genes are MW151571, MW151572, MW151573, MW151574, MW151575, MW151576, MW151577 and MW151578, respectively.After comparing protein and metabolite networks, 4 up- and 4 down-regulated genes with high interaction was chosen , and 4 down-regulated genes, FATA2, CYP86A1, LACS3 and PLA2-ALPHA, was compared at three irrigation levels  with three replications.Expression of 4 up-regulated genes, PXG3 gene expression increased 65.119 and 59.302 times in 40% FC and 70% FC treatments compared to FC, respectively. The expression of this gene was relatively similar in both treatments and had a significant increase compared to the control. Palmitoyl-protein thioesterase (PAL) expression significantly increased compared to control in treatment 70% FC (154.879) but no significant increase of expression was observed in treatment 40% FC (1.693). Expression of LOX2 was significantly increased in both 40% FC and 70% FC treatments while expression level of this gene in treatment 70% FC (1917.49) was significantly higher than treatment 40% FC (60.129) compared to control. A reduction of CYP86A1 gene expression was observed in both 40% FC and 70% FC treatments compared to the control . The expression of PLA2-ALPHA gene in two treatments 40% FC and 70% FC increased by 4.332 and 3.972, respectively, compared to the control although this increase was not significant. Expression of LACS3 was reduced in the 40% FC and 70% FC treatments at ratio of 0.044 and 0.068, respectively, compared to the control. CYP86A1 expression in both treatments 40% FC and 70% FC showed a significant increase compared to the control. Increased expression of this gene in treatment 70% FC (519.147) was significantly higher than treatment 40% FC (9.952) compared to control. FATA2 gene in treatment 40% FC showed an increase compared to FC (2.585), which was not significant while the expression of this gene decreased in treatment 70% FC (0.343)  with high expression level were selected and their structure were modeled. The model with the highest Confidence and TM-score was chosen for each protein among suggested five models by TrRosetta server Fig. . HomologApplication of Ramachandran plot with ProFunc server showed that 91.2, 87.7, 85.3 and 88.1% of amino acids in CYP86, LOX2, PAL and PXG3, in an order, were in the most favored regions, where the maximum points are observed , and four down-regulated, FATA2, CYP86A1, LACS3 and PLA2-ALPHA genes.Lipids as vital and major cellular constituents provide a structural basis for cell membranes and an energy resource for metabolism. In addition, lipids as signal mediators are involved in initiation of defense reactionsCYP710A1 and CYP86A1, as members of cytochrome P450 gene family, protect plants against multiple biotic and abiotic stresses through involving in plenty of detoxification activities and biosynthetic pathways45.LOXs act in signaling processes due to stressor effects and lipoxygenase activity characteristics, and may serve as molecular markers for plant stress tolerance studies46. Probable calcium-binding peroxygenases (PXG3) take part in storage lipid degradation of oil bodies in the abiotic stress signaling pathways as well as in drought tolerance by controlling stomata under water deficiency47. Palmitoyl-protein thioesterase (PAL) is responsible for removing palmitate group from its substrate proteins, which might contain cysteine string protein (CSP), presynaptic proteins like SNAP-25, dynamin, and synaptotagmin48.FATA2 plays an important function in chain termination within de novo synthesis of fatty acid and is also essential for plant viability49. Long-chain acyl-CoA synthetases (LACSs) synthesize long-chain acyl-CoAs from free fatty acids in plant cells. LACS2 is primarily involved in polyunsaturated linolenoyl-CoA production, which is vital to activate ethylene response transcription factors-mediated hypoxia signaling50. PLA2, as one of phospholipase A2, manages plenty of cellular processes, such as development, growth, defense, and stress responses51.CYP86A1, LOX2, Palmitoyl-protein thioesterase (PAL) and PXG3 increased while the other four genes did not change significantly.The results of gene expression in milk thistle were similar to the results of the Genevestigator program in Arabidopsis under different drought experiments. Under drought stress conditions, expression of four genes 52 were taken into account. Confidence of CYP86A1 and PAL was high, while for PXG3 and LOX2 were medium and low, respectively. The constructed models examined by Ramachandran analysis exhibited that these models are relatively close to reality. Protein interaction with the respective ligands was investigated to analyze their atomic interactions and functional preferences.To further investigate function mechanism of these genes, protein structure of the respected genes was examined. Due to lack of protein structure sequenced in milk thistle, both quality of homology modeling and accuracy to dock substrate to the substrate-binding site53, just seven P450 protein crystals of plants were found in Protein Data Bank (PDB) database because it is localized in the membrane and the structure is simply degraded and broken within purification and crystal growth54. Heme-containing P450s bonded to oxidoreductases catalyze stereo- and regio-selective oxidations, and also intensively challenging reactions, including decarboxylation, deamination, C–C cleavage, ring opening, coupling, expansion, migration, and dehydration55.Although approximately 1% of plant protein-coding genes are predicted to encode P450s56. Our results confirmed that the mentioned amino acids interacted with the HEM ligand. Residues interacting with HEM are mostly non-polar, particulalry aromatic amino acids, preparing a hydrophobic condition for HEM ring structure57. Our results demonstrated that nine amino acids interacting with HEM ligand were non-polar, which one of them had a hydrogen bond. Hydrogen bonds play important roles in protein folding58, protein–ligand interactions59, and catalysis60 and affect molecule physicochemical properties, such as distribution, partitioning, solubility, and permeability, which are essential to drug development60. A mutation in conserved glycine of heme-binding motif led to an inactive and unstable apoprotein, which supports a main function of glycine to bind the heme and probably regulating P450s activity61. Therefore, glycine mutant instability extremely restricts examination of its exact catalytic role62.For understanding the architecture of HEM binding site, one of biologically active motifs present in CYP, is crucial to establish the mechanism assembling basic metabolic machinery and facilitating secondary metabolites formation. It was previously reported that Phenylalanine, Glycine, Alanine, Arginine, Isoleucine, Cysteine and Proline amino acids were frequently repeated in HEM binding site of nine investigated plants by a proteome-wide identification of CYP enzymes analysis using statistical weight matrix approach63. Lipoxygenases (LOXs) catalyze polyunsaturated fatty acid (PUFA), as a substrate, to synthesize hydroperoxides64. Therefore, iso-enzymes of LOX would be identified according to substrate’s peroxidation site65. LOXs generate HPOs, which act as reactions hubs because of having cytotoxic potential to cell membrane, therefore, are rapidly metabolized in chemical compounds engaged in plant defense, signaling and apoptosis66. Since the majority of studies have focused on compounds with lipoxygenases inhibitory activity, investigation of ligand 11O and its binding site has not undertaken yet and should it be taken into account for further studies.LOX2, a member of a large monomeric protein family with non-sulphur, non-heme, and iron cofactor containing dioxygenases, interacted with ligand 11O. Free fatty acids are considered to be the main substrates of LOX although phospholipids and glycerolipids were also identified as oxygenation substrates67, a hydrophobic domain and a proline knot motif probably to target protein to oil bodies (OBs)68. In Arabidopsis, Peroxygenase activity of AtCLO1 and AtCLO2, two members of caleosin family, required the presence of calcium and two conserved histidines, which subsequently proved by Aubert et al.69 that these two histidines were in the PXG3 sequence. Despite acting PXG3 as the putative OB-associated peroxygenase, it may participate in lipid modifications or signaling in stress responses of plants69. Biosynthesis of cuticular waxes and cuticle was increased under water and ABA deficiency and in Arabidopsis70, as a result of peroxygenase activity in wax and cuticle synthesis pathway71. It was exhibited that PXG3 takes part in drought tolerance mechanisms by regulating plant growth, stomatal aperture and water use efficiency69.PXG3, a member of caleosin family, interacted with CA ligand. PXG3 protein binding site includes two hydrogen bonds between two ASP and ILE amino acids with calcium ligand. Caleosins have a specific calcium-binding domain in N-terminal regionPalmitoyl-protein thioesterase interacted with ACT ligand. This protein was known only in Arabidopsis and no attributed function has been reported yet.72. Therefore, preservation of cellular membrane integrity, as a key factor to retain metabolic homeostasis73, is a prerequisite to survive during severe stress conditions74. In addition to identifying important lipids and genes involved in their synthesis, the method of identifying lipids is also of particular importance. Characterization and identification of main compounds in biological processes, such as metabolites, lipids and proteins have been facilitated by implementing high sensitivity and resolution in mass spectrometry (MS)75, highlighting modern lipidomic tools can be applied to study of plant lipid76 by relying on tailored and optimized MS-based strategies77.Lipid composition indicated a main impact on cellular membrane integrity and activities of intrinsic-membrane proteins under stress conditionsPXG3, LOX2, CYP710A1, PAL, FATA2, CYP86A1, LACS3, and PLA2-ALPHA) that had differential expression and examined their expression in milk thistle. Out of 8 selected genes, 4 that had high expression  were selected, and protein structures were obtained by homology modeling. Next, the ligands attached to these proteins were examined. Examination of ligands, binding site amino acids, and atomistic interactions confirmed that the interactions between proteins and ligands are powerful and useful. The product of these proteins, which contains lipids, also plays the final role in confirming the role of these genes in stress tolerance.Our results highlighted the importance of lipids as main components of plant cells, which play a major role in stress conditions. Regulating gene expression is one of mechanisms in plants to reduce the stress influences. Lipid components and respected biosynthesizing genes of milk thistle were identified. Determined the genes of these lipids and examined their expression in various plants under drought stress. Next, we selected 8 genes (


The giant cell arteritis (GCA) pathophysiology is complex and multifactorial, involving a predisposing genetic background, the role of immune aging and the activation of vascular dendritic cells by an unknown trigger. Once activated, dendritic cells recruit CD4 T cells and induce their activation, proliferation and polarization into Th1 and Th17, which produce interferon-gamma (IFN-γ) and interleukin-17 (IL-17), respectively. IFN-γ triggers the production of chemokines by vascular smooth muscle cells, which leads to the recruitment of additional CD4 and CD8 T cells and also monocytes that differentiate into macrophages. Recent data have shown that IL-17, IFN-γ and GM-CSF induce the differentiation of macrophage subpopulations, which play a role in the destruction of the arterial wall, in neoangiogenesis or intimal hyperplasia. Under the influence of different mediators, mainly endothelin-1 and PDGF, vascular smooth muscle cells migrate to the intima, proliferate and change their phenotype to become myofibroblasts that further proliferate and produce extracellular matrix proteins, increasing the vascular stenosis. In addition, several defects in the immune regulatory mechanisms probably contribute to chronic vascular inflammation in GCA: a defect in the PD-1/PD-L1 pathway, a quantitative and qualitative Treg deficiency, the implication of resident cells, the role of GM-CSF and IL-6, the implication of the NOTCH pathway and the role of mucosal-associated invariant T cells and tissue-resident memory T cells. Giant cell arteritis (GCA) is a granulomatous vasculitis affecting large vessels, especially the aorta and its major branches, mainly subclavian arteries and cranial arteries of the external carotid system. This vasculitis affects adults after 50 years (peak incidence between 70 and 80 years) and women are affected two to three times more frequently than men ,2,3,4,5.The incidence of GCA follows a north to south gradient. The highest prevalence is observed in Scandinavian countries and in Olmsted County, Minnesota , where the population has a similar ethnicity, suggesting that the occurrence of GCA is favored by one genetic background ,12,13,14HLA-DRB1*04, particularly the HLA-DRB1*0401, DRB1*0404 or DRB1*0408 haplotypes, have been shown to be associated with the occurrence of GCA in independent cohorts [+ T cells. Importantly, a genome-wide association study (GWAS) assessing 2134 GCA patients from 10 independent populations of European ancestry and 9125 controls confirmed that class-II MHC is the genomic region with the strongest association with GCA [HLA-B*15:01 allele and GCA, regardless of the clinical phenotype of the disease, and that there was an increased risk of developing GCA, whatever its phenotype, if HLA-B*15:01 and HLA-DRB1*04:01 were expressed together [PLG (gene of plasminogen) and P4HA2 . Interestingly, these genes are involved in a wide spectrum of physiological processes, some of which are relevant to the GCA pathogenesis, such as wound healing, angiogenesis, lymphocyte recruitment and inflammation for PLG and vascular remodeling for P4HA2 [In the last 20 years, several studies have reported genetic polymorphisms affecting genes involved in the immune response and inflammation that are associated with an increased risk of having GCA . The mos cohorts ,25,26,27 cohorts ,24,28. Twith GCA . More retogether . This laor P4HA2 ,31.IFNG, IL21, IL23R, IL17RA, TNF, IL6, IL1B, IL2, IL18, LTA, LTB, CCR7, CD6, NLRP1), as well as RUNX3, which is involved in the production of IFN-γ and CD40LG, which encodes for CD40L, a protein involved in the cross-talk between T and B cells [CD28) and proteins implicated in T-cell activation pathways , were also hypomethylated in GCA arteries when compared to non-GCA arteries [In addition to genetic factors, research has shown that a dynamic relationship exists between genetic predispositions and environmental factors via epigenetics, which is defined as the changes in gene expression that occur without altering the underlying DNA sequence but through DNA methylation, histone modifications and microRNAs . Differe B cells ,34. The  B cells ,36,37. AAge is an essential factor in the onset of GCA, which occurs after 50 years with an increasing incidence. This could, therefore, naturally be a major etiological factor in the GCA pathogenesis . The agi+CD28− and CD8+CD28− senescent T cells [+ Treg [Furthermore, the aging process has been associated with modifications to multiple cells implicated in the immune response and vascular remodeling, such as dendritic cells (DC), T cells, endothelial cells and vascular smooth muscle cells (VSMC) . In part T cells ,43,44,45 [+ Treg , thus le [+ Treg ,47. Duri [+ Treg ,49. This [+ Treg ,51,52,53 [+ Treg . Similar [+ Treg . Thus, aChlamydia pneumonia, but neither Epstein-Barr virus nor human herpesvirus 6 [Several studies have investigated the potential roles of a large number of viruses and/or bacteria in the pathogenesis of GCA. The condition appears to have a seasonal variation, suggesting the involvement of an infectious trigger in the induction of this vasculitis ,55. Manysvirus 6 . Howeversvirus 6 . Recentlsvirus 6 . VZV is svirus 6 . This vasvirus 6 ,59. VZV svirus 6 . In contsvirus 6 ,64,65,66svirus 6 . Neverthsvirus 6 . Thus, tsvirus 6 , even whsvirus 6 ,77,78,79svirus 6 . Rather The role of DC in the pathogenesis of GCA suggests a relationship between a potential infection and the initiation of GCA. It is generally assumed that an infectious agent can activate adventitial DC and trigger immunologic processes leading to the development of vasculitis. However, this hypothesis has never been fully resolved, mainly because no specific pathogen triggering GCA has been identified. Indeed, very recent work analyzing the microbiome of temporal arteries or thoracic aortic aneurysms due to GCA provided contradictory and inconclusive results ,82. It iThe immunopathological model of GCA can be divided into four main phases .+CD11c+CCR6+CD83−MHC-IIlow) are physiologically localized in the adventitia where they are involved in immune surveillance [+CD11c+CCR7+CD83+CD80/86+MHC-IIhigh) and the production of cytokines and chemokines responsible for the recruitment of CD4+ T cells in the arterial wall. Once they are activated, DCs express high levels of class-II major histocompatibility complex (MHC-II) and co-stimulatory molecules (CD80 and CD86), making them capable of activating CD4+ T cells. In GCA, DCs produce CCL19 and CCL21 chemokines and express their receptor (CCR7) so that they are trapped in the arterial wall instead of migrating into satellite lymph nodes, as is the case in classical immune responses [Immature myeloid DCs  in this model resulted in a significant decrease in lesions of vasculitis and IFN-γ mRNA expression . The tro+ T cells, which are physiologically absent from the walls of healthy arteries, play a major role in the pathogenesis of GCA, as highlighted by the fact that their depletion in immunocompromised mice engrafted with GCA arteries strongly decreases vasculitis lesions [+ T cells [+ T cells are recruited through the chemokines  produced by activated DCs [+ T cells expressing CCR6, the ligand of CCL20. Interestingly, the nature of the initial danger signal determines the architecture of the vasculitis. TLR4 ligands trigger the recruitment of CD4+CCR6+ T lymphocytes, a phenotype matching Th17 cells [Vasa vasorum [CD4 lesions . Studies T cells ,89,90. T T cells . In GCA,ated DCs . Among t17 cells ,91,92,93 vasorum .Vasa vasorum of the adventitia, in which endothelial cells express adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) [T cells are thought to be recruited via (VCAM-1) ,56. Endo(VCAM-1) . Along t(VCAM-1) .+ T cells are activated by DCs that present still unidentified antigen(s). The presence of proinflammatory cytokines  in the microenvironment polarizes CD4+ T lymphocytes toward Th1 and Th17 cells [+CD161+ T cells, which express CCR6 and are prone to polarize into Th17 and Th1 cells, with a high level of plasticity between these two subsets that is highlighted by the presence of a high number of double-positive cells (IFN-γ+IL-17+) [Once recruited in the arterial wall, CD417 cells ,37,97. T17 cells . This po+IL-17+) .IL-17 production by Th17 is rapidly decreased by corticosteroid treatment ,36,37,98Therefore, therapeutic strategies targeting Th1 cells more specifically seem to be required to improve the treatment of GCA. This could be achieved by therapeutic strategies inhibiting intracellular cytokine signaling such as Janus kinase (JAK) inhibitors. Environment-cell communications rely on cytokine signals that trigger the JAK and signal transducer and activator of transcription (STAT) pathway. Th1 lineage commitment is strictly linked to STAT1- and STAT4-mediated gene induction, and STAT3 is considered the master regulator for Th17 cell differentiation. The effect of tofacitinib, a JAK3 and JAK1 inhibitor, has been studied in immunocompromised mice engrafted with temporal arteries and reconstituted with T cells and monocytes from GCA patients. The authors showed that, compared to healthy arteries, GCA arteries were characterized by an increased expression of STAT1- and STAT2-targeted genes, thus highlighting the activation of the IFN-γ pathway. Furthermore, pharmacologic inhibition of JAK3 and JAK1, the latter being involved in the IFN-γ pathway, effectively decreased vascular inflammation, the Th1 immune response, neoangiogenesis and intimal hyperplasia .+CD25highFoxP3+) and the low expression of FoxP3 in the arteries of GCA patients [The implication of Th17 cells, the proportion of which is increased in the blood of GCA patients and that infiltrate GCA arteries ,36,37, cpatients . These dpatients  and are patients  and the patients .+IL-21+ T cells is increased in the blood of GCA patients and correlates with the levels of Th1 and Th17 cells. Moreover, IL-21 increases Th1 and Th17 polarization, whereas it decreases Treg differentiation in vitro [In addition to IL-6, IL-21, a pro-inflammatory cytokine mainly produced by follicular helper T cells (TFH) , is alsoin vitro .Interestingly, the predominant cytokine profile could play a role in the emergence of the various histological patterns of GCA ,105. It + T cells expressing CXCR3 [+ T cells, we showed that the repertoire of circulating CD8+ T cells is also oligoclonal, thus supporting their antigen-driven activation. Furthermore, CD8+ T cells infiltrate the arterial wall and produce cytokines (IL-17 and IFN-γ) and cytotoxic molecules (granzymes and perforin) [+ T cells could be of particular importance for initiating vascular remodeling pathways, as supported by the fact that strong CD8+ T cell infiltration into temporal arteries is associated with more severe disease [The strong infiltration of Th1 and Th17 cells into the arterial wall is responsible for the production of large amounts of IFN-γ and IL-17, respectively. IFN-γ induces the production of several chemokines  by VSMCs . CXCL9, ng CXCR3 . As was erforin) . Through disease .brightCD16neg) mainly depends on the CCR2-CCL2 axis, while that of non-classical monocytes (CD14dimCD16+) depends on the CX3CR1-CX3CL1 axis [Monocytes are precursors of tissue macrophages. There are different subsets of monocytes, which differ in their phenotype, function and chemotaxis pathway. Recruitment of classical monocytes  and reactive oxygen species (ROS). In contrast, M2 macrophages, which express CD206 and FRβ, have anti-inflammatory and tissue-repairing functions. Like T cells, macrophages are highly plastic, and their heterogeneous phenotype has been reported in GCA ,113. It nd M-CSF .+MMP-9+ macrophages are located at the site of tissue destruction, whereas FRβ+ macrophages are located in the inner intima of arteries with degrees of intimal hyperplasia, and that this pattern was specific to GCA lesions and not seen in atherosclerotic lesions. Further experiments showed that GM-CSF upregulated CD206 expression, whereas FRβ was increased in M-CSF-skewed macrophages [+ macrophages that produce MMP9 involved in the destruction of the arterial wall. These CD206+ macrophages can also produce M-CSF, which induces the expression of FRβ that produce mediators, mainly PDGF-AA, promoting myofibroblast migration and proliferation and thus intimal hyperplasia, leading to ischemic signs of GCA [Macrophages play a critical role in vascular remodeling by promoting angiogenesis, intimal hyperplasia and tissue destruction. A recent study investigated the special distribution of different macrophage phenotypes in GCA arteries and showed that CD206rophages . This les of GCA .+MMP9+ macrophage subset in GCA inflamed temporal arteries [In addition, the same group demonstrated an increase in YKL-40 produced by the CD206arteries . Consistarteries .+YKL-40+MMP-9+ macrophages localized in the media and media borders where elastic lamina degradation takes place, and that these cells support tissue destruction and neoangiogenesis [MMPs are also key players in vascular remodeling. MMP-2 and MMP-9 produced by VSMCs and macrophages degrade elastin and destroy cellular matrix proteins, causing the destruction of the media and digestion of the internal elastic lamina ,116. MMPogenesis ,115. MMPogenesis .Current knowledge also suggests that IFN-γ, which is produced by Th1 cells, is the main lymphocytic cytokine inducing vascular remodeling. IFN-γ-activated macrophages, giant cells or injured VSMCs produce growth factors, essentially platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) . PDGF isvasa vasorum, which are physiologically restricted to the adventitia, are observed in the media and intima of arteries affected by GCA and correlate with internal elastic lamina digestion and infiltration by giant cells. IL-33, which is an alarmin belonging to the IL-1 family, is overexpressed in GCA arteries and also involved in angiogenesis [VEGF is responsible for neoangiogenesis, which increases the recruitment of other immune cells in the arterial wall . Illustrogenesis .+ cells in structures defined as artery tertiary lymphoid organs (ATLOs) [+ and CXCR5+ switched memory B cells are significantly reduced in GCA and PMR compared to healthy controls and inversely correlate with the serum levels of their complementary chemokines CXCL9 and CXCL13. Suggesting the implication of these chemokines in the recruitment of B cells in the arterial wall, CXCR3+ and CXCR5+ B cells were observed in areas with high expression levels of CXCL9 and CXCL13 [In GCA, B cells are much rarer than T cells in the arterial wall. They are localized in the adventitia and the media next to CD3 (ATLOs) . Studies (ATLOs) . Furtherd CXCL13 .Nevertheless, the humoral immune response does not appear to play as important a role in GCA as in Takayasu arteritis, which is the other primary large-vessel vasculitis. Indeed, a recent study found an increased TFH signature in both circulating and aortic CD4 T cells in Takayasu arteritis but not in healthy controls or GCA patients. This highlights that the cooperation between T and B cells is probably more critical in Takayasu arteritis than in GCA . AnotherAs discussed above, the mechanisms involved in inflammation and vascular remodeling are becoming better understood and are promising therapeutic targets. In addition, some mechanisms allow inflammation to become chronic, which could also represent interesting therapeutic targets to replace glucocorticoids.−/− mice, in which the PD-1/PD-L1 pathway was lacking, displayed elevated Th1 and Th17 levels [+ T cells and the emergence of typical lesions of GCA, such as intimal hyperplasia and neoangiogenesis [+ and VISTA+ (a negative immune checkpoint V-domain immunoglobulin-containing suppressor of T cell activation) T cells were decreased in the blood of GCA patients because they were recruited in GCA lesions. However, contrasting with PD-1/PDL-1, VISTA-Ig engagement failed to suppress Th1, Th17 and TFH lineage development in GCA [Programmed death-1 (PD-1) is a surface protein expressed by activated T cells, and its ligation to PD-L1 or PD-L2, which are expressed by antigen-presenting cells, induces T-cell apoptosis, T-cell anergy and the production of IL-10 by T cells or their polarization into Treg. By contrast, PD-17 levels . In GCA,ogenesis . Along tt in GCA .1. Due to the competitive binding of CTLA4 to CD80/CD86, abatacept dampens T-cell activation by impeding the interaction between CD28 and CD80/86. In contrast, the occurrence of PMR/GCA has recently been reported in patients treated with antagonists of CTLA-4 or PD-1 used in metastatic melanoma, ipilimumab and nivolumab [The implication of immune checkpoints in the GCA pathogenesis was also highlighted by the recently demonstrated efficacy of abatacept for the treatment of GCA . Abataceivolumab ,139.+CD25highFoxP3+) together with their absence from arterial lesions of GCA [Tregs are major actors in immune tolerance and protect patients from inflammation and autoimmunity . Our teas of GCA . The Tres of GCA . Confirms of GCA ,142. Thes of GCA . In addis of GCA . Anothers of GCA .+ T cells, a defect in CD8+ regulatory T cells was also reported in GCA [+CCR7+FoxP3+ phenotype, control the proliferation and activation of CD4+ T cells through the production of exosomes containing NADPH oxidase 2 (NOX2). These exosomes are captured by neighboring CD4+ T cells, in which NOX2 disrupts activation, survival and proliferation pathways [As for CD4d in GCA . CD8 Trepathways . Interespathways .Our team recently investigated the implication of mucosal-associated invariant T cells (MAIT) in GCA. MAIT cells are innate-like lymphocytes characterized by the expression of a semi-invariant T-cell receptor (TCR) composed of a constant α chain (TCRVα7.2-Jα33) and a β chain . Interaction between the two is restricted to major histocompatibility complex (MHC)-related protein 1 (MR1). MAITs display an immediate effector function on stimulation, and they have a high clonal volume . It has +CD103+ memory T cells that are barely detectable in the peripheral blood. This TRM population is sustained by IL-7, IL-9 and IL-15, which are present in the tissue microenvironment of GCA lesions. TRMs require JAK3/1-dependent signaling through γc chain-containing cytokine receptors to survive. Along this line, researchers demonstrated that the inhibition of JAK1/3 activity with tofacitinib minimized the in situ proliferation of CD4+CD103+ TRM. The persistence of vascular wall inflammation in GCA arteries may, therefore, also depend on a small, highly specialized population of CD4+CD103+ TRMs characterized by their ability to survive in the tissue microenvironment [Another study also suggested a role of tissue-resident memory T cells (TRMs) in chronic inflammation. Specialized to remain resident in the tissue microenvironment, TRMs are thought to provide rapid and effective immune responses when they re-encounter antigens, and they are considered pathogenic in chronic inflammatory diseases. They have a pro-inflammatory effector function and secrete pro-inflammatory cytokines such as IFN-γ, IL-17, IL-9 and TNF-α. In GCA arteries, there is a population of CD4ironment .VSMCs are major components of the vessel wall. They are characterized by contractile functions, a synthetic function that produces components of the extracellular matrix and they are also involvement in tissue repair through their ability to migrate and proliferate . In GCA,+ T cells that express Notch 1 and Jagged 2 [+Treg cells lies in aberrant Notch 4 signaling that deviates endosomal trafficking and minimizes exosome production. By transcriptionally controlling the profile of RAB GTPases, Notch 4 signaling restricts the vesicular secretion of the enzyme NADPH oxidase 2 (NOX2) [The interplay between T cells and resident cells of the arterial wall also involves the NOTCH pathway, a highly conserved cell-signaling system that is important for cell-cell communication, gene regulation mechanisms controlling cell differentiation processes during embryonic and adult life, and the development of blood vessels. NOTCH receptors and their ligands (Jagged and Delta) are transmembranous proteins. The ligation of NOTCH to Jagged or Delta ligands triggers proteolytic events leading to the translocation of the intracellular domain of NOTCH into the nucleus, where it interacts with transcription factors regulating the destiny of cells ,152. SevJagged 2 . In GCA,Jagged 2 . FurtherJagged 2 . The bloJagged 2 . Further2 (NOX2) .Emerging as a key cytokine in inflammation, granulocyte-macrophage colony-stimulating factor (GM-CSF) may play a role in promoting inflammation in GCA. GM-CSF contributes to the pathophysiology of GCA by regulating inflammatory macrophages, DCs and Th1 and Th17 cells and is involved in angiogenesis and vascular remodeling ,160,161.IL-6 is produced by many cells, especially monocytes and macrophages, and has pleiotropic effects. IL-6 signaling depends on gp130, a transmembranous glycoprotein that triggers the phosphorylation of STAT3. Gp130 is activated through its ligation to a complex composed of IL-6 and its receptor, either membranous (mIL-6R) or soluble (sIL-6R). While the expression of gp130 is ubiquitous, the expression of mIL-6R is restricted to hepatocytes, monocytes, macrophages, a few B and T cells, megakaryocytes and endothelial cells . ClassicIn GCA, the concentration of serum IL-6 is very high and correlates positively with disease activity ,36. The Knowledge of the mechanisms involved in the pathogenesis of GCA has improved considerably in recent years, leading to the identification of new therapeutic targets to improve patient treatment and reduce the use of glucocorticoids. The most recent work also showed the involvement of processes leading to the amplification and/or maintenance of inflammation and vascular remodeling, such as regulatory T response defects (regulated by IL-6), GM-CSF and the roles of macrophages and NOTCH, along with probably a major role of arterial wall resident cells. These mechanisms identify targets for the development of new therapeutics such as tocilizumab , and mor


Chronic itch is one of the most prominent clinical characteristics of diverse systematic diseases. It is a devastating sensation in pathological diseases. Despite its importance, there are no FDA-labelled drugs specifically geared toward chronic itch. The associated complex pathogenesis and diverse causes escalate chronic itch to being one of the top challenges in healthcare. Humanized antibodies against IL-13, IL-4, and IL-31 proved effective in treatment of itch-associated atopic dermatitis but remain to be validated in chronic itch. There are still no satisfactory anti-itch therapeutics available toward itch-related neuropeptides including GRP, BNP, SST, CGRP, and SP. The newly identified potential itch targets including OSM, NMB, glutamate, periostin, and Serpin E1 have opened new avenues for therapeutic development. Proof-of-principle studies have been successfully performed on antagonists against these proteins and their receptors in itch treatment in animal models. Their translational interventions in humans need to be evaluated. It is of great importance to summarize and compare the newly emerging knowledge on chronic itch and its pathways to promote the development of novel anti-itch therapeutics. The goal of this review is to analyze the different physiologies and pathophysiologies of itch mediators, whilst assessing their suitability as new targets and discussing future therapeutic development. Chronic pruritus (CP) or chronic itch is a devastating clinical condition that entails continuous itch for more than 6 weeks, but there are limited medications to help with this condition. The complexity and heterogeneity of this clinical condition pose a major challenge for clinical care. Genetic studies and single-cell sequencing data have provided in-depth neural–immune networks encoding itch information in different mechanisms of itch. The detailed signature of itch-sensing neurons has been characterized but is still largely unknown. There are no therapeutics that are generally effective. Dysregulated communications between sensory and immune cells, skin resident cells, as well as central systems trigger chronic itch and chronification. When the skin is irritated, itch signals are sent to the peripheral nerve of the cutaneous nerve endings, which originated from dorsal root ganglion (DRG), then are ascended to the spinothalamic tract and the parabrachial nucleus at the other side before reaching the somatosensory thalamus, and then are projected on the cortex ,2,3,4. DNeuropeptides are critical in the transmission of itch sensation from the peripheral nervous system to the spinal nervous system and to higher levels. Diverse neuropeptides including calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP) can promote skin inflammation ,31,32. BTRPV1+ sensory neurons release CGRP to act on diverse immunocytes, including T cells, B cells, dendritic cells (DCs), mast cells, macrophages, and LCs, causing neuro-inflammation, neurogenic vasodilation, and immune response [CLA)+ T cells) may exaggerate neurogenic inflammation response and induce the release of itch-related cytokines, i.e., interleukin-4 (IL-4) or interleukin-13 (IL-13) from immune cells [CGRP+ interneurons mediate spinal itch transmission but not pain signals [CGRPα+ sensory neurons in mice showed a reduced sensitivity to histamine and chloroquine-induced itch [CGRP is an extensively studied vasodilator that is expressed in the sensory neurons, motor neurons, and many other cell types, including monocytes/macrophages , Langerhresponse . CGRP rene cells . CGRP wane cells . The pla signals . Althougced itch , intradeced itch . An antaced itch , and alsced itch . The proSP is an important transmitter of the afferent neurons in the PNS and CNS. SP acts on diverse immunocytes, including eosinophils, mast cells, and T cells, and promotes skin inflammation ,48. AlthBNP is a central itch mediator . ReleaseMrgprA3+ pruritoceptors [MrgprA3+ neurons, how the itch signal is coded with GRP is not clear. These data confirm a critical role of dorsal horn GRP neurons in spinal itch transmission [GRP is a spinal itch-selective transmitter known to be highly expressed in a population of spinal cord dorsal horn (DH) interneurons ,65,66,67oceptors . Since gsmission . GRP levsmission . Despitesmission . It remaMrgprA3+ neurons, which form monosynaptic, glutamatergic connections with NMBR+ and NMBR− DH neurons and function in a context-dependent manner  to enhance the activity of NMBR+ DH neurons [NMBR+ DH neurons is enhanced by NMB from MrgprA3+ afferents [MrgprA3+ and NPPB+-neurons are in distinct neuronal subsets, and each are labelled by different itch biomarkers and receptors , Mrgpr, IL-31RA, IL-13RA, IL-4R, CysLT receptors, TSLP, and endothelin receptors, etc. ,92,93. TH2 cells [Histamine is a common inflammatory mediator and the most known itch inducer. It is released mainly by the mast cells and basophils and occasionally by keratinocytes. It activates eosinophils, mast cells, basophils, and TH2 cells ,99,100. H2 cells , resultiH2 cells ,95,96. SH2 cells ,102. HowH2 cells .Specific serine and cysteine proteases, including tryptase, KLK 5 and 14, and cathepsin activate PAR2, which plays a critical role in itch sensation and cutaneous inflammation in mice and humans . IntradeMrgprA1+ sensory fibers respond to bilirubin and SP to induce itch [MrgprA3+ and its human ortholog MRGPRX1 respond to CQ, BAM8-22, and mucunain [MrgprD+ neurons innervating the epidermis sense itch, and nonpeptidergic MrgprD+ neurons are implicated in immunoregulatory function and cutaneous immune homeostasis [+ account for 95.2% of MrgprC11+ neurons, and MrgprD+ account for 18.5% MrgprC11+ neurons; however, these have distinct roles, particularly in glabrous skin itch, which is sensed mainly by MrgprC11 [TRPV1+ sub-population in DRG. In addition, like mouse MrgprA1, sensory neuronal MRGPRX4 in humans is activated by bilirubin to sense cholestatic itch [ClinicalTrials.gov Identifier: NCT04510090).Among 50 mouse and 10 human Mrgpr subjects, mouse MrgprA1, A3, B2, C11, and D, and human X1, X2, and X4, are characterized as itch receptors. Mouse uce itch . MrgprA3mucunain . Mouse Mmucunain ,112,113.mucunain . Mouse Meostasis . Mouse MMrgprC11 . SP alsoMrgprC11 . Mouse MMrgprC11 . In humatic itch ; howevertic itch ,119. MRGtic itch , howeverThe neurotransmitter 5-HT is important in the CNS and is an inflammatory mediator released by the mast cells, melanocytes, and platelets . In addiThe leukotrienes are eicosanoids which are synthesized from arachidonic acid under the catalyst of lipoxygenase (LO). Leukotriene C4 (LTC4) and LTB4, but not LTD4 and LTE4, induce itch. LTC4 activates peripheral neuronal CysLT2R to induce acute itch, and LTC4 is implicated in the MC903-induced AD-like itch, but not dry skin itch . LTB4 inH2 cells is increased, the inflammation related to specific cytokines and the generation of eosinophilia and immunoglobulin E (IgE) are promoted, whilst the generation of epidermal barrier proteins and antibacterial peptides is inhibited. IL-4 and IL-13 are typical type 2 cytokines and have been proven to directly stimulate the sensory neurons via the Janus Kinase 1 (JAK1) signals and promote itch sensation, and IL-5 is considered as a critical link in the growth, differentiation, and migration of the eosinophils and the recruitment of the eosinophils.The cytokines build “a bridge of communication” between the immune system and the nervous system. AD-related skin lesions and itch are aggravated under the mutual interaction of neural–epidermal immune signal pathways. Pruritus is caused by a variety of pruritus-derived cytokines, including TSLP, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-13 (IL-13), IL-31, interleukin-33 (IL-33), etc. ,143,144,IL-13Rα2, suggestive that IL-13Rα2 is a downstream target and contributes to the relief of AD itch [IL-13 levels are increased in skin and serum from AD patients, and IL-13 participates in the initiation of AD and itching. Together with IL-4, it aggravates epidermal barrier dysfunction by downregulation of the filaggrin (FLG) and involucrin (IVL) expression in the keratinocytes ,148. The AD itch . STAT6 h AD itch .Nppb transcription from both murine and human sensory neurons [IL-31 plays an important role in the induction of itch and inflammation in AD and chronic contact dermatitis in mice and humans ,152,153. neurons ,144, furIL-33 is an effective amplifier of type 2 immune reaction and is an important target for dry skin pruritus and chronic pruritus of unknown origin (CPUO). IL-33 receptor ST2  is expressed in DRGs , keratin+ T cells and their differentiation to TH2 cells [2+ signals in the astrocytes are necessary for the continuous signal transducer and activator of transcription (STAT3) activation, LCN2 expression, and chronic itch [IL-6 is predominantly expressed in dendritic cells, keratinocytes, macrophages, and neurons. The dendritic-cell-derived IL-6 level is linked to AD. IL-6 facilitates production of IL-4 expression by CD4H2 cells ,163. It H2 cells ,165,166.H2 cells . IL-6-innic itch ,165,166.nic itch ,169.IL-2 is an itch inducer as well as an autocrine cytokine, and its single intradermal injection induces a long-time low-intensity local skin itch that lasts 48–72 h, as well as erythema in human AD patients and healthy subjects . Serum lH2-mediated inflammation and enhances periostin release from keratinocytes, thereby promoting itch signaling, and this effect is susceptible to JAK2 inhibitor SD1008 and the STAT3 inhibitor niclosamide [H2 inflammatory response [TSLP is a pro-allergic cytokine that is mainly released from keratinocytes, and is the prime target in AD. TSLP drives Tlosamide . Upon relosamide , and senlosamide . Biologilosamide . Intraderesponse . TSLPR aresponse . Howeverresponse ,178. Aparesponse , which mH2 cytokines IL-13 and IL-4, then activates integrin aVβ3 on a fraction of SST+/NPPB+ sensory itch fibers [H2 cytokines such as IL-31 [H2 cytokine–periostin feedback loop  . A CCL2 Although substantial research has been conducted to fill the gaps pertaining to the mystery of itching, this debilitating clinical condition is still faced with a highly inapt medical need for efficient treatment. No neuropeptide-targeting therapeutic or agonist/antagonist achieved has successful chronic itch treatment in humans. Many humanized antibodies approved for itch-related diseases have great potential in chronic itch treatment. However, this closely depends on the disease phenotypes and dominance of inflammatory pathways. The combination of different biological agents has resulted in significant advances in the efficacy in some diseases, opening new avenues for future treatment on diseases which are refectory to single treatment. Here, a detailed summary of current development of anti-itch therapeutics is listed in Although pruritus is not the main target in the approval of dupilumab for the treatment of AD , the recH2 to TH17, and thus an IL-17 antagonist must be followed. Likewise, treatment with a psoriatic drug can improve psoriasis but cause atopic dermatitis, depending on the individual. Dose optimization and combination of different drugs are required. Therefore, it is important that a modern systematic assessment of the existing evidence be conducted to summarize the effects of current studies. Research into the enhanced understanding of the mechanisms of itch are ongoing and studies identifying novel mechanisms of pruritis in animal and human models are continuing, with the goal to improve the current development of potential therapeutics for chronic itch cases.Chronic itch is modulated by a complex network involving diverse cell types and systems, and is classified into dermatologic, systemic, neurologic, psychogenic, mixed, and other pruritus. It ranks at the top position of all dermatological diseases. The quality of life of affected individuals is dramatically reduced, as symptoms such as the itching–scratch cycle are combined with a social stigmatization. All chronic itch diseases can show a life quality that is diminished to a degree comparable to diabetes, cancer, or serious cardiovascular events. The most important contribution to this high disease burden comes from inflammatory skin diseases such as psoriasis and AD, two distinct diseases differing in the characteristics of immune response, inflammation, and pathogenesis . However