MYB transcription factors (TFs), as one of the largest gene families in plants, play important roles in multiple biological processes, such as plant growth and development, cell morphology and pattern building, physiological activity metabolism, primary and secondary metabolic reactions, and responses to environmental stresses. The function of MYB TFs in crops has been widely studied, but few studies have been done on medicinal plants. In this review, we summarized the MYB TFs that play important roles in secondary metabolism and emphasized the possible mechanisms underlying how MYB TFs are regulated at the protein, posttranscriptional, and transcriptional levels, as well as how they regulate the downstream target gene networks related to secondary metabolism in plants, especially in medicinal plants. These MYB TFs have important functions in the regulation of the biosynthesis of secondary metabolites in plants.Plant secondary metabolites include many types of compounds, such as flavonoids , stilbenes, various phenolic acids, and monolignols. The functions of these compounds have been determined and include mechanical strength , signaling molecules, pigments, ultraviolet (UV) light protectants, and phytoalexins. v-Myb is the first MYB TF identified in avian myeloblastosis virus (AMV) [Arabidopsis thaliana [A. thaliana, researchers have identified more than 1600 TFs, representing about 6% of the whole genome [Zea mays [A. thaliana, AtMYB2 shows considerable homology with C1 from Z. mays [The MYB TF is named by its conserved MYB domain at the N-terminus and is an evolutionarily conserved present in nearly all eukaryotes ,2. v-Mybus (AMV) . Subsequus (AMV) ,5,6. Comthaliana . In A. te genome ,8. The Me genome ,6. The MZea mays . In A. t Z. mays . The lar Z. mays . The MYB Z. mays ,12,13,14 Z. mays ,16,17. T Z. mays ,19,20, wOryza sativa, and 68 members in A. thaliana [Dendrobium candidum genome also encodes 42 1R-MYB genes. 1R-MYB TFs involve in flower and fruit development, response to phosphate starvation, circadian clock control, and phenylpropanoid-derived compounds [It is now clear that the common characteristic of MYB TFs is the conserved DNA-binding domain (MYB domain), which interacts with DNA ,2,15. Inompounds .O. sativa and 120 members in A. thaliana [Dendrobium candidum genome encodes 117 2R-MYB TFs, and the Jatropha curcas genome contains 123 2R-MYB TFs [2R-MYB, which contains two MYB domains, is the most widely existed protein, regulates the transcription of secondary metabolic processes in medicinal plants, and regulates cell differentiation. At the same time, recent studies have shown that 2R-MYB acts as a regulatory protein involved in the metabolic pathways of flavonoids and phenylpropanoids in medicinal plants . The 2R--MYB TFs . The exp-MYB TFs ,29,30,31-MYB TFs .O. sativa and five members in A. thaliana [D. candidum genome only encodes four 3R-MYB genes, and the Jatropha curcas genome only contains four 3R-MYB TFs. Their role, albeit divergent, is largely related to regulate cell differentiation and participate in cell cycle control [A. thaliana and one member in O. sativa [D. candidum genome only encodes two 4R-MYB TFs, and the J. curcas genome only contains one 4R-MYB TF. The function of the members of this 4R-MYB subfamily needs further study.The 3R-MYB subfamily, whose members contain three MYB domains, is an evolutionarily conserved group in plants. Genes encoding 3R-MYB proteins have been found amongst plant genomes. There are only five members in model plants thaliana . This ph control . The sma. sativa . In mediD. candidum genome encodes 117 2R-MYB genes, and only nine of them are regulated by low temperature. DoMYB28, DoMYB29, DoMYB54, DoMYB75, DoMYB78, DoMYB81, and DoMYB111 have been up-regulated under salinity stress [Nicotiana tabacum has confirmed the binding of Betula platyphylla BplMYB46 to the E-box, GT-box, and TC-box motifs in the promoters of the superoxide dismutase (SOD), peroxidase (POD), and phenylalanine ammonia-lyase (PAL) genes, which function in secondary wall biosynthesis and abiotic stress tolerance [Gossypium hirsutum GhMYB109, a homolog of AtMYBGL1 in Arabidopsis thaliana, is specifically expressed in cotton elongating fibers and fiber cell initials, indicating that this gene may be involved in the development of seed trichome in cotton [Epimedium sagittatum [Dendrobium officinale DoMYB75 in A. thaliana can significantly increase seed water-soluble polysaccharide content [Panax Ginseng PgMYB2, a nucleus localization protein, may play a key regulatory role in ginsenoside synthesis [The MYB TFs have a wide range of biological functions and participate in many life processes in plants, such as regulating plant growth and development, involved in cell morphogenesis, regulating primary and secondary metabolic reactions, and responding to abiotic and biotic stresses ,41,42,43y stress . Effectoolerance . Gossypin cotton ,46. Huangittatum . Heterol content . Panax Gynthesis .Chrysanthemum morifolium (CmMYB1), Leucaena leucocephala (LlMYB1), Panicum virgatum (PvMYB4a), Eucalyptus gunnii (EgMYB1), and Zea mays  are able to repress lignin biosynthesis [Malus domestica, MdMYB3, MdMYBA, and MdMYB1 can control the red-pigmented anthocyanins biosynthesis in the peel [MdMYB110a is associated with the red coloration of the fruit cortex during the later stages of fruit maturity, and MdMYB10 can participate in the biosynthesis of anthocyanins in foliage, flesh, and peel [Petunia hybrida, 2R-MYB TFs PHZ (purple haze), DPL (deep purple), and AN2 (anthocyanin2) are responsible for vegetative pigmentation, flower tube bud-blush/venation, and full petal color, respectively [A. thaliana, AtMYB111, AtMYB12, and AtMYB11  are connected in the middle by a heterocyclic pyran or pyran (with double bond) ring (C) ,88. The C1 gene in maize [P1 is also found to be very significant in maize [C1 gene mainly controls the color of the aleurone layer and the pigmentation embryo, while P1 mainly exists in vegetative tissues to regulate the anthocyanin synthesis and accumulation [C1 has a broader DNA-binding specificity than P1 in maize. From more similar studies, it can be concluded that MYB TFs play a similar role in the secondary metabolism of medicinal plants. For example, Yuan et al. (2015) found that overexpression of Scutellaria baicalensis SbMYB8 in tobacco could change the expression level of some flavonoid synthesis-related genes and ultimately regulate the biosynthesis of flavonoids [Camellia sinensis CsMYB4a negatively regulated the synthesis of lignin, phenolic acids, phenylalanine, and flavonoids [CsMYB4a could inhibit the promoter activity of five phenylpropanoid pathway genes  and two shikimate pathway genes (CsAROC and CsAROF) [CsMYB2 and CsMYB26 were involved in flavonoid biosynthesis by regulating the expression of CsF3′H and CsLAR in Camellia sinensis, respectively [Epimedium sagittatum’s two TFs (EsMYBA1 and EsMYBF1) and twelve structural genes. The transcriptional analysis suggested that EsMYBF1 and EsMYBA1, together with EsTTG1 (a WD40 TF) and EsGL3 (a bHLH TF), were probably involved in the coordinated regulation of synthesis of the flavonol-derived bioactive components and anthocyanins [SlCOS1 (costunolide synthase gene), which catalyzed the final key step of costunolide (sesquiterpene lactone) biosynthesis in Saussurea lappa [Antirrhinum majus, AmMYB305 activates the gene encoding phenylalanine ammonia-lyase (PAL) when it is co-expressed in tobacco protoplasts [Gentiana triflora TFs—GtMYBP4 and GtMYBP3—can activate the expression of flavonol biosynthesis genes  and, when heterologously expressed in A. thaliana, increase the content of flavonol [A. maju, VENOSA determines vein-associated anthocyanin patterning, whereas ROSEA1 and ROSEA2 are required for bud-blushed patterns, pale pink, bull’s-eye, and full red [The reaction and biosynthesis of flavonoids are shown in in maize , and thein maize . Studiesmulation . C1 has avonoids . Li et aavonoids . The CsM CsAROF) . Wang etectively . Huang eocyanins . Thakur ea lappa . In Antitoplasts . Both thflavonol . In A. mfull red ,96.Antirrhinum majus AmMYB308 and AmMYB330 repressed phenolic acid metabolism in transgenic tobaccos [Salvia miltiorrhiza, SmMYB39 negatively regulates enzyme activities and transcripts of tyrosine aminotransferase (TAT) and 4-hydroxylase (C4H) [SmMYB39 acts as a repressor and is participated in the regulation of the rosmarinic acid pathway [Saussurea involucrate, tomato, and tobacco of the potato genes PAP1, StMTF1, StAN1, as well as that of AtMYB12, increases the content of chlorogenic acid [A. thaliana AtPAP1 gene in Salvia miltiorrhiza can significantly increase the content of salvianolic acid B in transgenic plants. S. miltiorrhiza SmMYB4 gene can act as a transcriptional repressor to regulate C4H and ultimately affect the biosynthesis of rosmarinic acid [MYB TFs are also widely involved in the secondary metabolism of organic acids in medicinal plants. It has been found that the R2R3-MYB TFs of the fourth subfamily are involved in the metabolism of phenylpropanoid as a negative regulator in various medicinal plants. Tamagnone et al. 1998) found that overexpression of tobaccos . In Salvse (C4H) . SmMYB39 pathway . Constit found thnic acid  , and their induction triggers the expression of MYB58, MYB63, and MYB85, which, in turn, can interact with relevant promoter AC elements of the lignin synthesis genes to up-regulate these genes [A. thaliana [Eucalyptus gunnii [Z. mays [Pinus taeda [A. thaliana [Lignin is an aromatic heteropolymer mainly derived from the monolignols sinapyl alcohol, coniferyl, and p-coumaryl, which produce syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H) subunits, respectively. Lignin contributes to the rigidity and strength of stems, as well as providing most of the mechanical strength of the plant cell wall. MYB TFs are also widely involved in the secondary metabolism of lignin. For example, MYB83 and MYB46 from se genes ,101,102.thaliana . It has s gunnii ,105, ZmM[Z. mays , PtMYB1 us taeda ,108, andthaliana ,109.PttMYB21a in poplar would lead to the increase of CCoAOMT transcriptional abundance and lignin content, suggesting that this TF might be a transcriptional inhibitor [Chrysanthemum morifolium (CmMYB1 and CmMYB8) [Leucaena leucocephala (LlMYB1) [Panicum virgatum (PvMYB4a) [Eucalyptus gunnii (EgMYB1) [Musa nana (MusaMYB31) [Z. mays (ZmMYB31 and ZmMYB42) [Certain MYB TFs have been implicated as repressors of the monolignol pathway. Karpinska et al. (2004) found that reducing the expression of nhibitor . We also CmMYB8) ,111, Leu(LlMYB1) , PanicumPvMYB4a) , Eucalyp(EgMYB1) , Musa nasaMYB31) , and Z. ZmMYB42) ,52 can iA. thaliana NAC TFs—NST1/2/3 and VND6/7—are regulated by MYB TFs during secondary wall synthesis [AtMYB66, which directly regulates GL2 and CPC. In addition, other TFs can directly target MYB. For example, AGL15 (AGAMOUS-Like15) can bind to about 29 MYB genes and activate its transcriptional expression [The regulation network of gene expression is interrelated and mutually restrictive. MYB TF regulates the expression of downstream genes, and its expression is also regulated by upstream genes. For example, WD40 protein TTG1, as an important upstream regulator of MYB TFs, plays an important role in many regulatory pathways in MYB ,115. MYBynthesis . Recentlpression .MYB TFs can be divided into four different regulatory mechanisms: 1, The regulatory mechanism of protein interaction; 2, The regulatory mechanism of transcriptase; 3, The regulatory mechanism of a redox reaction. 2R-MYB is the most widely distributed MYB TF in plants ,49. The Functional, structural, and phylogenetic analyses have suggested that there are many homologs of MYB TFs with conserved MYB domains, which have similar activities and functions in divergent plant species. In a given subfamily, the biological function and primary protein structure of most MYB TFs are very conserved among angiosperm species, which means that they are evolved from a common ancestral gene. The MYB TFs play important roles in regulatory networks for flavonoid pathways in plants. As part of transcriptional networks, MYB TFs can be regulated by plant growth regulators, environmental signals, microRNAs, and other TFs. There have been more and more studies on the structure and function of MYB TFs, and it has been found that the functions of MYB TFs tend to be diversified.To date, little is known about the role of the secondary metabolism in plants, especially in medicinal plants. Further studies should focus on detecting the bHLH-binding and promoter- binding capacity of a greater variety of MYB TFs in medicinal plants. Identifying MYB-binding targets using ChIP-seq would provide additional data on targets on a whole-genome scale. Ultimately, detailed knowledge of MYB TFs will help us to further understand the fine transcriptional regulation of the secondary metabolism in plants, especially in medicinal plants.


Phosphatidylinositol and its phosphorylated derivatives, the phosphoinositides, play many important roles in all eukaryotic cells. These include modulation of physical properties of membranes, activation or inhibition of membrane-associated proteins, recruitment of peripheral membrane proteins that act as effectors, and control of membrane trafficking. They also serve as precursors for important second messengers, inositol  trisphosphate and diacylglycerol. Animal models and human diseases involving defects in phosphoinositide regulatory pathways have revealed their importance for function in the mammalian retina and retinal pigmented epithelium. New technologies for localizing, measuring and genetically manipulating them are revealing new information about their importance for the function and health of the vertebrate retina. Including PtdIns, a total of eight different PI head groups are commonly found in eukaryotic cells (Phosphoinositides (abbreviated here as “PI”) include phosphatidylinositol (PtdIns), in which the 1′-position of the inositol ring is attached via a phosphodiester bond to the ic cells  4,5].,5.sn-3 pPtdIns is a relatively substantial component of the membranes of most cells in metazoans, with mole fractions ranging from ~4%–20% of total phospholipid ,8,9. Int3 in preparations of rod cells that contain both outer segments and fragments of the inner segments and demonstrated levels of PI(3)P at 0.0035 mol% of total phospholipid under illumination conditions that yielded the highest levels of that lipid, and at least 10-fold lower  levels of PIP3. PI(4)P and PIP2 are, in general, found at much higher levels than the 3-phosphorylated PI, but even those appear to be present in rods at very low levels, which are only about 10-fold higher than the levels of PI(3)P, i.e., on the order of 0.04 mol% .As a negatively charged lipid, which typically contains one polyunsaturated side chain fatty acid, such as arachidonic acid , PtdIns 2 and PI(3)P, the change is generally less than 30% inositol, reported that light led to decreases in PIP2 levels without generation of InsP3, suggesting light-dependent activity of a phosphatase rather than of phospholipase C [2 in membranes could affect the activity of components of the phototransduction cascade [2 on phototransduction or of effects of light on PIP2 metabolism remains untested, and it seems to be possible to explain the entire time-course of rod light responses without invoking any participation by phosphoinositide metabolism [αq/11 PLC-coupled subunits revealed the presence of PLCβ4 and Gαq/11 in rod outer segments, and Gαq and other PKC isoforms elsewhere in the retina [In contrast, another group  reportedlipase C . In vitr cascade ,87, incltabolism . Antibode retina . The fune retina .3, with reports of effects of light on the activity of this enzyme in bovine rod outer segments [Interest has turned toward Type I PI-3 kinase and PIPsegments ,92, whicsegments ,94,95. Dsegments . Likewissegments .3, seem to be much less important for rod function. Rod-specific ablation of the p85α gene using two different rod-specific Cre transgenes yielded no obvious defects in retinal morphology or rod cell survival [3 levels in rods isolated from dark-adapted or light-adapted retinas revealed levels of this phosphoinositide of more than one order magnitude lower than those found for PI(3)P in the light, or at least two orders of magnitude lower than light-stimulated levels of PIP2 [In contrast, Type I PI-3 kinase and its product, PIPsurvival ,97, althIP2 .Opn4 gene, a visual pigment which is more closely related to invertebrate opsins than to vertebrate opsin [αq/11/14 class G-protein, which activates the phosphoinositide-specific phospholipase C isoform, PLCβ4; the phospholipase presumably acts on PIP2 as in other cell types, including Drosophila photoreceptors, which contain a homologous phospholipase, to produce diacylglycerol and InsP3. PIP2 hydrolysis, in turn, leads to the activation of the cation channels TRPC6 and TRPC7 [In addition to image-forming light detection mediated by rods and cones, the vertebrate retina also contains intrinsically photosensitive retinal ganglion cells. These contain phototransduction cascades reminiscent of that found in invertebrate rhabdomeric photoreceptors ,103,104.te opsin . In M1-tnd TRPC7 .2, on a timescale of tens of minutes; effective stimuli included fetal bovine serum, agonists for muscarinic, histamine, and serotonin, peptides, including bradykinin, arginine vasopressin, bombesin and oxytocin, [3, following stimulation by light [3 in response to induction of phagocytosis by addition of isolated rod outer segments [3 release was not observed in cells from Royal College of Surgeon (RCS) rats, which have a defect in OS phagocytosis due to a deficiency in the receptor tyrosine kinase, MERTK [3 [A variety of extracellular stimuli acting on tyrosine kinase-associated receptors or G protein-coupled-receptors have been reported to stimulate release of inositol phosphates in cultured RPE cells, presumably derived from PLC action of PIPxytocin, ,108,109.by light . Acutelysegments ; this Ine, MERTK . As in mMERTK [3 ,112,113.MERTK [3 ,115 and MERTK [3 ,117 are 2 and lysosomal fusion may involve other phosphoinositides such as PIP2 and PI(5)P [A number of important processes in RPE are known to rely on phosphoinositides, but how they are regulated in these cells is not well understood. Phagocytosis, autophagy, endocytosis and endosome processing, establishment of epithelial cell polarity and extension of microvilli membranes are all known to critically depend on phosphoinositides. For example, both autophagy and phagocytosis involve the recruitment of the ubiquitin-like protein, LC3 ,119,120,d PI(5)P ,124,125.In mammals, there are 47 genes encoding 19 PI-kinase and PIP-kinases and 28 PIP phosphatases . 2 as their substrate to produce PIP3, and there is little evidence for a substantial portion of cellular PI(3)P being formed by these enzymes. When the Type III PI-3 kinase, Vps34, was knocked out in mouse rod cells and phosphoinositide levels measured, the results suggested complete ablation of PI(3)P despite the presence of the Type I enzyme [2 in most cells [The kinase isoforms are divided into three major families: PI 4-kinases (PI4Ks), the PI 3-kinases (PI3Ks), and PIP (PIP) kinases (PIPKs) . The nomI enzyme . SeveralI enzyme ,57,128. st cells . Of thesst cells  and in ost cells ,131 and st cells . Type IIst cells . The prost cells .Pip4kc2 gene was found to lead to hyperactivation of the immune system, but the retinal phenotype was not examined [The phosphoinositide phosphatases encoded by Inpp1, Inpp4a, Inpp4b, Inpp5e, Mtmr2 and Synj1 (synaptojanin-1) have also been detected in retina by proteomics . As noteexamined .Rods and cones have a very high rate of metabolism and biosynthesis of membrane components, due to the energetic demands of the phototransduction cascade and the daily shedding of ~10% of the disk membranes P3, and its critical role in the cilia suggests that regulating levels of PIP2 may be important in ciliogenesis and cilium stability.Inherited defects in the phosphoinositide phosphatase, INPP5E, are associated with the multi-syndromic ciliopathies, Joubert syndrome and Bardet–Biedl syndrome ,149,150,Synaptojanins are phosphoinositide phosphatases associated with synaptic function ,152,153,Defects in another phosphoinositide phosphatase, OCRL, are associated with a severe ciliopathy, oculocerebrorenal syndrome of Lowe . SymptomDrosophila retinal degeneration gene, rdgB, and defects in it are associated with autosomal dominant cone-rod dystrophy CORD5 [The phosphatidyl inositol transfer protein PIT3NM3 (aka CCL118) is the mammalian homologue of the hy CORD5 ,158,159.hy CORD5 . In zebrhy CORD5 .tub/TUB gene [tubby mice causes obesity, deafness and blindness. The widely expressed members of the tubby family, including TULP1-TULP4, have a characteristic carboxyl-terminal tubby domain consisting of an alpha helix surrounded by a beta barrel which has been shown to bind to specific phosphoinositides [Proteins with phosphoinositide-binding domains have also been associated with retinal disease. These include the product of the TUB gene ,163 and TUB gene ,165,166.ositides . Both Tuositides ,168.While the overall field of phosphoinositide regulation and signaling has made dramatic advances in recent years, our understanding of specific regulatory pathways in specific cell types of the retina remains limited. The emergence of new technologies for cell-type-specific gene manipulation, along with imaging techniques for subcellular localization of phosphoinositides, their regulatory enzymes and their effector proteins, and the development of more sensitive methods for measuring PI levels, bodes well for rapid progress in understanding the roles of retinal phosphoinositides in the coming years.


In middle-aged women, the decline of ovarian follicle reserve below a critical threshold marks menopause, leading to hormonal, inflammatory, and metabolic changes linked to disease. The highest incidence and mortality of sporadic ovarian cancer (OC) occur at post-menopause, while OC risk is reduced by full-term pregnancies during former fertile life. Herein, we investigate how parity history modulates the ovarian transcriptome related to such declining follicle pool and systemic inflammation in reproductively-aged mice. Female C57BL/6 mice were housed under multiparous and virgin (nulliparous) breeding regimens from adulthood until estropause. The ovaries were then subjected to follicle count and transcriptional profiling, while a cytokine panel was determined in the sera. As expected, the follicle number was markedly decreased just by aging. Importantly, a significantly higher count of primordial and total follicles was observed in aged multiparous relative to aged virgin ovaries. Consistently, among the 65 genes of higher expression in aged multiparous ovaries, 27 showed a follicle count-like pattern, 21 had traceable evidence of roles in follicular/oocyte homeostasis, and 7 were transforming-growth factor beta (TGF-β)/bone morphogenetic protein (BMP) superfamily members. The remaining genes were enriched in cell chemotaxis and innate-immunity, and resembled the profiles of circulating CXCL1, CXCL2, CXCL5, CSF3, and CCL3, chemokines detected at higher levels in aged multiparous mice. We conclude that multiparity during reproductive life promotes the retention of follicle remnants while improving local (ovarian) and systemic immune-innate surveillance in aged female mice. These findings could underlie the mechanisms by which pregnancy promotes the long-term reduced OC risk observed at post-menopause. Aging is characterized by a wide range of cumulative damage that affects tissue homeostasis, thus predisposing to disease initiation. Cell signaling and metabolism, antioxidant defense, epigenetic status, intercellular communication, and genome stability, among other functions, become significantly impaired . In the Increased rates of chronic diseases, including several types of cancer, are observed in menopause. The declining follicle number has been recently proposed to play a role in ovarian cancer (OC) etiology by promoting a pre-neoplastic phenotype of the ovarian surface epithelium (OSE) ,4,5, a cEqually important, the observed OC risk at menopause is significantly decreased by reproductive history during the previous fertile life. Conditions characterized by a reduced number of ovulatory events, namely parity and the use of oral contraceptives, have been recognized to reduce OC risk in various populations . The undMouse models have been used to study OC risk factors, including obesity , inflammn = 6 per group) around 16-months old was euthanized to collect ovaries for RNA extraction and subsequent gene expression profiling with microarrays (see below). At 20 months old, another subset of 34 mice  was euthanized to dissect ovaries for follicle count. Sera were obtained from a third subset (n = 8 per group) from the blood samples collected twice per month from 17–20 months old. Both ovarian tissue and serum samples from four-month-old mice (n = 6 per group) were used as a reference.This study with female C57BL/6 mice was approved by the Bioethics Committee, Faculty of Medicine, University of Chile (CBA # 0536 FMUCH). The care and monitoring of the two experimental groups, virgin and multiparous, have recently been described in detail . Figure p-formaldehyde and embedded in paraffin. The largest longitudinal sections, 5-μm thick, were stained with haematoxylin-eosin (HE) and mounted in Entellan® . Primordial, primary, preantral, secondary, and Graafian follicles were identified according to the morphological criteria of Griffin et al. [The dissected ovary pairs were fixed in 1% n et al. , with onn = 6), aged virgin (n = 5), and aged multiparous (n = 6) ovary samples. The total ovarian RNA was isolated with an All-Prep kit , treated with Turbo-DNAse , precipitated with sodium acetate plus absolute ethanol, and stored at −80 °C until use. The RNA was quantified by UV spectrophotometry (A260/A280 nm) and verified for integrity with an Agilent 2100 Bioanalyzer . All of the RNA samples had RNA integrity (RIN) values > 7.5. Transcriptional profiling was performed by the service provider Macrogen Inc. . Further experimental details are given in One-channel bead-DNA microarrays  composed of 25,697 probes (80-mer) covering 17,640 unique coding genes were used. Each probe was represented 25–45 instances in a single microarray for a total of over 900,000 probes per microarray. A set of 17 microarrays comprised young (http://nciarray.nci.nih.gov). After quantile normalization, the differentially expressed genes (DEGs) were determined by limma tests with FDR adj p-values <0.05 and log2 fold-change (FC) >±1.0 for the young-aged comparison. The cut-offs for the aged virgin-aged multiparous comparison were FDR adj p-values <0.10 and log2 FC >±0.8. Quality threshold clustering (QTC), heatmaps, and centroid plots were done with MeV (http://mev.tm4.org/). Gene ontology (GO) enrichment was done with Visual Annotation Display(VLAD) v1.5.1 (http://proto.informatics.jax.org/prototypes/vlad/) and WebGestalt . Gene Set Enrichment Analysis (GSEA) was done at (http://software.broadinstitute.org/gsea/index.jsp). Protein–protein interaction networks were analyzed with STRING-v11.0 (https://string-db.org/). Mouse immune cell gene expression data were extracted from the Ref-DIC database (http://refdic.rcai.riken.jp/profile.cgi).The scanned images were analyzed with the Illumina Genome Studio v2011.1, gene expression module v1.9.0. The raw data was deposited at the NIH’s-National Cancer Institute microarray database , following instructions by the manufacturer, as previously described [Mouse cytokines CXCL1, CXCL2, CXCL5, CCL3, and CSF3 were quantified in serum using the multiplex, magnetic bead-based assay MCYTMAG-70K-PX32 panel from Milliplexescribed . Before One µg of DNAse-treated RNA was reverse-transcribed with 20 U of M-MLV , oligo-dT plus random hexamer primers for 1 h at 42 °C, and then inactivated for 10 min at 70 °C. Quantitative PCR was performed by combining an aliquot of the resultant cDNA with 1 µM final of each forward and reverse primers and Sensimix Sybr HI-ROX reagent . Primer pairs were designed with the Primer3-Plus software . Three sp < 0.05. The analysis was performed with the GraphPad Prism 5.0 software.The follicle counts and cytokine levels were plotted as mean ± standard error of the mean. The data did not follow a normal distribution, and were compared as non-paired data between the aged groups by using Mann–Whitney tests (non-parametric). Significance was set at 2 ratio range with a related previous study [We recently described the reproductive records, circulating gonadotropin levels, systemic inflammatory status, and intraperitoneal tumor spread in estropausal C57BL6 female mice, as well as the differential accumulation of lipofuscin and hemosiderin in the ovaries of these animals according to their divergent parity history ,22. In eus study , in whicThe follicles at various stages were identified and counted in the ovaries of the young and the two aged conditions. Based on the mean follicle counts, the ovaries of both aged groups contained significantly lower total follicle counts compared with the young ovaries, as expected, because of the age-dependent ovarian decline  versus aged ovaries irrespective of parity status (n = 5 + 6 = 11) resulted in 905 DEGs (adj p < 0.05 and ±1.0 log2 FC). This comparison allowed us to interpret parity differences below (n = 3–4 per group) sample sizes , CD68, CD86, class II H2 complex , Ly6 complex , CD3 complex , CD44, and proteases (Cpa3 and Cma1). Furthermore, all of the GO terms describing the activation of the leukocyte, lymphocyte, macrophage, neutrophil, and mast cells were significantly enriched in the aged relative to young ovaries. The same trend was observed for the terms of “phagocytosis”, “lysosome”, “cell surface receptors”, and “cytokine-mediated signaling”, which are closely related to the SASP. Consistent with this local ovarian inflammation associated with the reproductive decline, we previously showed an age-dependent increase of systemic levels of several cytokines in this mouse cohort, including TNF-alpha, IL1-beta, CCL2, IL-10, IL-5, and IL-4, among others [The gene ontology (GO) analysis indicated that, regardless of parity history, aged ovaries expressed a minimal repertoire of mitotic, cell cycle, and DNA repair genes, but a high content of genes implicated in inflammatory/immune responses, cell adhesion, TNF production, peptidase activity, and wound healing A. These g others .n = 5) versus multiparous (n = 6). This resulted in 177 DEGs , which were decomposed in 65 up-regulated and 112 down-regulated genes in aged multiparous respective to aged virgin ovaries.The second comparison was done between aged ovaries only, and was based on their parity history, that is, virgin  signaling”, while Fst and Grem1 are classified as TGF-β antagonists. The GSEA analysis analogous to that of Bmp2 knock-out mice [Fst and Grem1 antagonists. An additional follicle-related pathway enriched in the aged multiparous ovary was the “steroid metabolic process” comprising the genes Hsd17b1, Apoa1, Apoa4, and Nr5a2. Including the Hedgehog signaling members Ihh and Dhh, known to modulate steroid biosynthesis in various organs, including the ovaries [out mice , and thi ovaries , there wAs shown in Apoa1, Nlrp14, Nlrp5, S100a8, and Zp3), “positive regulation of immune system process” , “chemotaxis” , and “cell migration” . Among these 12 genes, Fpr2 and S100a8 overlap in three of the four GO terms mentioned. Fpr2 codes for a member of the G-protein coupled formyl peptide receptor family, whereas S100a8 codes for calgranulin-A, which is Ca/Zn binding protein that heterodimerizes with the calgranulin-B isoform. Both proteins confer a chemoattractant capacity to neutrophils and monocytes (see discussion).Included here were the GO terms “regulation of inflammatory response” . Six of these 14 genes  are annotated under the term “transcription factor activity, sequence-specific DNA binding”.This function comprised the GO term “positive regulation of transcription, DNA-templated” .The relevant GO terms containing the genes of the three themes above described were “cell death” B and theInhba, Gdnf, Grem1, Hsd17b1, and Kcne2. Finally, the genes Apoa1, Gdnf, Grem1, Ihh, Inhba, and Zp3, were common between the follicle/oocyte and immune/inflammatory themes based on GO. Similarly, Inhba, Gdnf, and Grem1, besides linking the follicle/oocyte and immune/inflammatory themes, exhibit a transcriptional modulation activity and take part in the cytokine-like gene regulatory network . The quality threshold clustering (QTC) algorithm was applied to the normalized expression levels of the 65 DEGs between the aged ovaries, and included the expression levels observed in young ovaries. This resulted in the following three clusters: 27 B, 32, an network B.Previously, we reported circulating levels of seven cytokines that increased with reproductive aging irrespective of parity records, and then decreased in response to tumor induction exclusively in multiparous compared with virgin mice . Here, wFpr2 and S100a8 were among the six-genes cluster showing the highest expression levels in the three conditions  macrophages show an increased Fpr2 expression in response to a pro-inflammatory stimulus (LPS) at various times and doses, while the Fpr2 expression by neutrophils is constitutively high. The Serpina3g expression was enhanced only in LPS-stimulated peritoneal macrophages and in the liver neutrophils from Tcra knockout mice. On the other hand, S100a8 was weakly induced by LPS in macrophages, but highly expressed by the three neutrophil types shown without any stimulus. Regarding the cluster of 32 genes, Ref-DIC demonstrated high  and moderate  expression levels in the J7774.1 macrophages. Interestingly, Ihnba showed a high expression exclusively in peritoneal macrophages (data not shown). A recent report found that LPS treatment improved the innate immunity in a xenograft model of SKOV3 cells in CD-1 mice, resulting in extended survival and increased CXCL1 levels [As shown in 1 levels , one of Fst, Fshr, Zp3, Bmp15, Oas1d, and Padi6 as major interconnected hubs among the descriptors at the bottom.The genes comprising the follicle and cytokine clusters display some degree of overlap in enriched GO terms shown in S100a8 is indexed in three of the four GO terms at the bottom, and showed links with Fpr2 and the three CXC cytokines only. In turn, all of the cytokines, except CSF3, were connected to Fpr2  measured as antral follicle counts and serum anti-müllerian hormone (AMH) . Then, oInhba codes for the β-A subunit of inhibin-A, activin-A, and activin-AB dimers expressed by granulosa cells. Circulating inhibins repress, while activins stimulate pituitary FSH synthesis and release. Female reproductive aging is characterized by increased levels of circulating activin and FSH [Inhba expression in the aged multiparous ovary suggests elevated levels of the activin-A homodimer. Intraovarian activins promote the autocrine expression of Fshr and Cyp19a1 (aromatase) in pre-antral follicles, while depressing steroidogenesis in advanced-stage follicles and lutein-granulosa cells [Fshr expression in aged multiparous with respect to aged virgin ovaries. The coexpression of Cyp19a1 showed the same trend, although not reaching statistical significance. In agreement with our findings, the FSH receptor (FSHR) and aromatase have been localized in the human postmenopausal ovary  and Grem1 (Gremlin-1) [Fst is expressed as three isoforms that regulate the balance between germ-cell nest breakdown and the subsequent assembly of primordial follicles. Mouse Fst overexpression blocks follicle maturation before the antral stage, possibly by inhibiting granulosa cell proliferation [Other TGF-β related genes highly expressed by the multiparous ovary were the ligand antagonists emlin-1) . The proemlin-1) . Althougemlin-1) , it coulemlin-1) . Ovarianferation , a role Bmp15 was another TGF-β/BMP transcript showing a parity-dependent differential expression. This BMP is detectable at the primordial stage and regulates the ovulatory rate by preventing the premature activation of primordial follicles [Fshr, also restricts activation of primordial follicles as observed in fertility studies [Bmp15, Fshr, and Fst are interconnected in the protein network  essential for luteinization [Foxo1, Fshr, and Nr5a2 co-expression under persistent FSH stimulation in the presence of activin-A (Inhba), suggest phosphorylated Foxo1 in the aged multiparous ovary. This conclusion is reflected in the follicular-like protein network shown in Foxo1 with membrane-localized Fshr to form an atypical signaling complex would preclude the nuclear translocation of Foxo1 in aged multiparous ovarian cells, as observed in HEK 293 cells [Six genes up-regulated in aged multiparous ovaries display an activity of transcription factor. In general, age-dependent up-regulation involves stress-responsive transcription factors. The forkhead box (FOXO) family of transcription factors is implicated in longevity and aging-related processes, including cell cycle arrest, oxidative stress, DNA repair, apoptosis, and autophagy . Foxo1 isa cells . It is hsa cells . During  in mice ,51. In tg others . In turnnization . Thus, o93 cells .Hey2 codes for a myc-type basic helix–loop–helix transcription factor highly expressed in the adult mouse ovary (https://www.ncbi.nlm.nih.gov/gene/15214#gene-expression). Hey2 connects TGF-β and Notch pathways during proliferation of pre-antral granulosa cells [Also, the Notch target sa cells  and modusa cells . Importasa cells .Ovarian processes, including ovulation, luteinization, and atresia, depend on a delicate pro-/anti-inflammatory balance relying on leukocyte activities ,28,29,58Cxcr1 gene was upregulated almost six-fold (adj-p 2.96 × 10−5) in aged with respect to young ovaries, regardless of parity status (not shown).Higher circulating levels of CXCL1, CXCL2, CXCL5, CSF3, and CCL3 were detected in aged multiparous mice. This cytokine set participates in the innate immune response to endogenous tissue damage and infection mainly in initial phases . A role Fpr2, S100a8, Sema5a, Fgf13, Gdnf, and Grem1 . This Ca-/Zn-binding protein forms homo- and hetero-dimers (with S100a9), which stimulate the migration of neutrophils to inflammatory sites [Cd68 expression in aged relative to young ovaries, irrespective of parity status . Furthermore, the S100A8/9 heterodimer  is abundant in neutrophils, increases with age in the majority of mouse and human tissues [p = 8.4 × 10−3). Neutrophil recruitment by S100A9 requires interaction with TLR2 and CXCL2 [Tlr2 overexpression in aged relative to young ovaries .Neutrophils, monocytes, and activated macrophages express high levels of S100-calgranulin A -associated molecular patterns (DAMPs), allowing for neutrophil activation and the subsequent resolution of inflammation [igenesis . Fpr2 chnd CXCL8 . Consistammation .In aged tissues, apoptosis and senescence may play complementary roles as tumor-suppressor mechanisms . Thus, tApoptosis might be also related to the roles of the resident leukocytes of the aged ovary see . The SASTgf/Bmp antagonists and the modulation of Fshr-Foxo1 signaling. Coincident with this residual follicular reserve, past parity would enhance the immune-surveillance capacity of resident ovarian macrophages and neutrophils by improving their phagocytic ability on apoptotic atretic cells, thereby minimizing the deleterious effects of SASP. In summary, the observed protection of pregnancy (or progestin-induced anovulation) against ovarian neoplasia may be regarded as a long-term “imprint”, leading to persistent remnant follicles with concomitant robust innate immunity in the aged multiparous ovary. How this imprint is generated during a woman’s fertile life and overcomes the effects of ovarian aging it remains to be investigated.Here, we applied an integrative analysis  to the ovarian transcriptome and systemic chemokine profiles observed in the naturally aged C57BL/6 mouse ovary so as to gain insight into the molecular basis of OC risk reduction by parity history. The mice were maintained in multiparous versus virgin regimens until estropausal age, equivalent to early post-menopause in women. Regardless of parity status, the aged ovary expressed a high content of genes involved in immune/inflammatory response, cell adhesion, TNF synthesis, proteolysis, and wound healing, thus reflecting a low-grade chronic inflammation due to endogenous tissue damage, which is characteristic of SASP. Leukocyte markers, including macrophage, neutrophil, lymphocyte, and mast cell transcripts, were also detected in the aged ovary. More importantly, aged multiparous ovaries showed a higher residual content of primordial and total follicles, relative to aged virgin ovaries. This finding agreed with the higher transcript levels of the 26 DEGs—out of the 65 total—with reported evidence of the roles in the homeostasis of oocytes, follicles, and the ovary, particularly genes preventing the exhaustion of the primordial follicle pool. The remaining DEGs were enriched in innate immunity mainly related to neutrophil and macrophage chemotaxis, while their expression profile resembled the circulating levels of the CXCL1, CXCL2, CXCL5, CSF3, and CCL3 chemokines implicated in the repair of endogenous tissue damage. We conclude that the aged multiparous ovary retains a remnant follicle reserve that evades either maturation or atresia, probably due to the combined action of paracrine


Background: Microcystic stromal tumor is a recently described subtype of ovarian tumor characterized by microcystic pattern and diffuse immunoreactivity for CD10, vimentin, and β-catenin and negative for EMA. However, its diagnostic criterion and standard treatment remain unclear.Case presentation: We report a rare case of a left side microcystic stromal tumor with diameter about 7 cm in a 25-year-old female and summarize all cases of MCST reported in this study. The present patient underwent left ovarian tumor resection. Generally, the tumor was solid and cystic mixed. Immunohistochemically, the tumor was expressed CD10, WT1, cyclin D1 and vimentin, and nuclear immunoreactivity for β-catenin but negative for α-inhibin, calretinin, CK AE1/AE3, PLAP, SALL-4, CK7, P53, EMA, CD99, AFP, desmin, CgA, E-cadherin, and melanA.Conclusion: Unilateral ovary, solid-cystic, and a larger than 4–8 cm pelvic mass without serious abdominal pain are its clinical features. The immunophenotype of vimentin+/CD10+/WT-1+/β-catenin+(nuclei)/cyclin D1+ is supportive of diagnosis. For these patients, unilateral oophorectomy dissection could be selected. Ovarian microcystic stromal tumor (MCST) is a rare ovarian tumor that was first characterized by Irving and Young in 2009 . They re*5.7 cm*3.9 cm increased left ovary with various cystic masses. The largest of them is 2.5 cm*1.8 cm. The serum levels of tumor markers, such as carcinoembryonic antigen (CEA), CA199, CA153, CA125, and a-fetoprotein (AFP), were within the normal range. The serum levels of total cholesterol were 5.50 mmol/L, triglyceride 2.21 mmol/L, apolipoprotein 1.01 g/L, specific beta hcG 0.10 mIU/ml, E2 144 pg/ml LH 7.74 mIU/ml, FSH 2.14 mIU/ml, PROG 11.54 ng/ml, PROL 75.4 ng/ml, and TESTO 0.48 ng/ml. Based on the chief complaint and imaging results, the diagnosis was considered as an ovarian chocolate cyst at first. Laparoscopic left ovarian tumor resection was subsequently performed at the local hospital. During laparoscopy, the surgeons discovered a 7 cm*6 cm cystic-solid mass and a 2-cm cystic mass present on the left increased ovary, and the uterus and right ovary were unremarkable. Frozen section of the left ovarian mass demonstrated an ovarian sex-stromal tumor, mostly like an ovarian granulosa cell tumor. After discussing treatment options with her family to balance curative treatment and fertility preservation and receiving her consent, the surgeons in the local hospital finally chose left ovarian tumor resection. No further oncologic therapy was administered.A 25-year-old woman, gravida 2 and para 2, visited her local hospital due to a pelvic mass discovered via health examination with no relevant past medical story, no abdominal discomfort, no complaints of menstrual disorder and no urination or defecation disturbance. She also denied a family history of cancer. Upon gynecological examination, a 7-cm mainly cystic component was discovered in the left adnexal area. B ultrasonography revealed a 7 cmThirty days after surgery, the patient visited our hospital and Peking Union Medical College Hospital for pathology consultation and was diagnosed with ovarian microcystic stromal tumors. And till now, 4 months after surgery, the B ultrasonography and serum levels of tumor markers are all within the normal range.The resection specimens were processed on 4-μm slides and stained with haematoxylin and eosin in the local hospital. Immunochemical staining of inhibin-α, calretinin, cytokeratin AE1/AE3 (CK AE1/AE3), PLAP, SALL-4, CK7, WT1, P53, epithelial membrane antigen (EMA), CD99, AFP, vimentin, CD10, desmin, synaptophysin (Syn), CD56, chromogranin (CgA), Ki67, and LCA was performed in the local hospital. We reviewed the haematoxylin and eosin-stained and immunohistochemistry sections and performed β-catenin (Zsbio Store) and melanA (Zsbio Store) staining for diagnosis with the Ventana Benchmark XT automated staining system  according to standard techniques. Peking Union Medical College Hospital subsequently added CD10, E-cadherin, cyclin D1 and Ki-67 staining, and reviewed all of the slides.The tumor was confined to the left ovary and had a smooth outer surface. It measured 7 cm in maximum diameter and revealed a solid-cystic mixed appearance. Microscopically, the tumor consists of diffuse cells, with large, darkly variable sizes, round, oval, and fusiform nuclei . Tumor gAnother small cyst was also fixed in the left ovary, measuring 2 cm at the greatest dimensions. The cyst wall consisted of corpus luteum tissues with blood and infrequent tumor tissues.The tumor cells commonly showed diffuse and strong cytoplasmic membranous expression for CD10, WT1, cyclin D1, and vimentin and nuclear immunoreactivity for β-catenin . Cells wThis patient was initially diagnosed with a sex cord stroma tumor at her local hospital. Subsequently, the histopathological diagnosis was revised and confirmed as MCST of the ovary by the authors and two experienced pathologists .This case is a very rare tumor of ovarian MCST, which is a newly categorized ovarian tumor first described by Irving and Young in 2009 . To dateHowever, given the rarity of this tumor and the limited investigation of the clinical and immunohistochemical profile, we compared our results with others reported and summarized their similarities. The clinical features of 45 cases of ovarian MCST have been summarized  1–13). –13. 1–13Immunochemical features of all 50 cases are summarized  1–13). –13. 1–13We presented a rare case of MCST and summarized the distinct clinical, histological, and immunohistochemical features of all the cases reported. Pathologists and clinicians should be aware of the existence of this unique neoplasm, unilateral ovary, solid-cystic, larger than 4–8 cm pelvic mass without serious abdominal pain. Serum levels of tumor markers are always within the limits; sometimes, CA125 may be elevated. The immunophenotype of vimentin+/CD10+/WT-1+/β-catenin+(nuclei)/cyclinD1+/EMA-/α-inhibin-/calretinin-/ E-cadherin- is supportive of diagnosis as MCST. For these patients, unilateral oophorectomy with or without salpingo dissection is a good choice. The findings presented here provide insights into this tumor and will facilitate future studies. In addition, further research is needed regarding the hormone levels, prognosis and tumourigenic mechanisms of these tumors.The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.Written informed consent was obtained from the participant for the publication of this case report. Ethical approval was given by the Medical Ethics Committee of the China Japan Friendship Hospital.LD, BL, and JL diagnosed the patient. LD and JL carried out experiments. LD analyzed the experimental results. All authors wrote and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila neuroblasts (NBs), as well as the underlying mechanisms by which those TSGs act in this process. Finally, we also describe what we know about the consequences of mutating these genes in impairing the process of asymmetric NB division and promoting tumor-like overgrowth.The Scribble polarity module is composed by Scribble (Scrib), Discs large 1 (Dlg1) and Lethal (2) giant larvae (L(2)gl), a group of highly conserved neoplastic tumor suppressor genes (TSGs) from flies to humans. Even though the Scribble module has been profusely studied in epithelial cell polarity, the number of tissues and processes in which it is involved is increasingly growing. Here we discuss the role of the Scribble module in the asymmetric division of  The third component of the module, scrib, was identified twenty years ago in a screen for mutations that altered the embryonic epithelial architecture in Drosophila [scrib phenotype was identical to that shown by l(2)gl and dlg1 mutants in embryonic, follicular and imaginal disc epithelia, strongly suggesting that all three genes acted in the same pathway to regulate cell growth and cell polarity [scrib, dlg1 and l(2)gl was confirmed by epistatic and strong genetic interactions between them, as well as by their cell localization and activity interdependence [scrib, dlg1 and l(2)gl, do not overgrow, have poor survival, and are finally eliminated by cell competition, a process in which the surrounding wild-type cells activate a Jun N-terminal kinase (JNK)-mediated apoptosis in the mutant cells [V1Ras2, can prevent the cell death in these TSG single mutant clones by transforming the pro-apoptotic function of JNK into a pro-growth effect, inducing the formation of big neoplastic tumoral masses [l(2)gl null mutant alleles, that l(2)gl single mutant clones in eye-antennal discs behave differently from scrib/dlg1 clones, in the sense that they are not eliminated by cell competition. In fact, l(2)gl clones overproliferate without affecting cell polarity by repressing the Hippo signaling pathway [l(2)gl but also dlg1 single mutant clones, in antennal discs, overgrow by activating the Ras-MAPK signaling pathway, while scrib clones do not [The Scribble polarity module is composed by Scribble (Scrib), Discs large 1 (Dlg1) and lethal 2) giant larvae (L(2)gl), all of which represent highly conserved neoplastic tumor suppressor genes (TSGs) from flies to humans  giant la,4. l(2)gin 1930s , but thel(2)gl4) . Thus, lon stage ,8,9, alton stage ,11. dlg1osophila . scrib ppolarity . A collapendence . This, apendence ,14,15,16nt cells ,21,22. Al masses ,19,21,23 pathway ,25. Intrs do not . Thus, iScrib, Dlg1, and L(2)gl are all cytoplasmic, membrane-associated proteins that co-localize to the baso-lateral domain of epithelial cells. Scrib and Dlg1 are multi PDZ (PSD-95/Dlg1/ZO-1) domain-containing proteins that act as scaffolds, whereas L(2)gl is a non-muscle myosin II binding protein with WD-40 repeats and, unlike Scrib and Dlg1, is cortically uniform  1,3,12,,27,28.Drosophila the adaptor protein GUK-holder (Gukh) links Scrib and Dlg1 [Some physical interactions between the Scribble module proteins have been described in mammals ,30, and and Dlg1 . In addiand Dlg1 . Among tand Dlg1 . Some ofDrosophila central nervous system (CNS), divide asymmetrically to give rise to another NB that keeps on dividing and a daughter cell called ganglion mother cell (GMC) that will start a differentiation program [NBs, the neural stem cells of the  program ,33,34. T program ,41,42,43Drosophila) and the atypical protein kinase C (aPKC)  motor protein that interacts with astral microtubule plus-ends, anchoring the spindle to the apical cortex [TPR domain and, additionally, with the Dynein molecular motor, which binds the astral microtubule minus-ends promoting pulling forces on them and reinforcing the apical-basal orientation of the spindle [A group of proteins located at the apical cortex of metaphase NBs control, in turn, the basal sorting of cell-fate determinants, as well as the orientation of the mitotic spindle along the NB apico-basal axis of polarity, two key processes to ensure the asymmetry of the division. This apical complex is an intricate protein network that includes the conserved partitioning defective proteins Par-6 and Par-3   [44,45,4C (aPKC)  [49,50,5bound to ,57,58,59l cortex ,61,62. M spindle  gl was not necessary for the localization of Dlg1. Hence, it was proposed that, at least for its localization, although not necessarily for its function, Dlg1 would act upstream of L(2)gl [Drosophila, at interphase. At metaphase, when L(2)gl is inactivated by aPKC, it was proposed that myosin II becomes active and, in turn, promotes the cortical exclusion of the cell-fate determinant adaptor protein Mira from the apical NB cortex gl ,68. Dlg1f L(2)gl ,68. In ff L(2)gl . Both prf L(2)gl . This isf L(2)gl ,71. Baz,f L(2)gl  gl   both in Drosophila and in mammals [Linker domain must be phosphorylated by the mitotic kinase AurA to physically interact with the Dlg1 GK domain [MBS) with the GK motif of Dlg1 at the cortex, and then with astral microtubule plus-ends through its motor domain (Khc-73motor)  a. This P73motor)  corticallignment . The meclignment  gl, dlg1 and scrib embryonic mutant NBs, symmetric divisions  and even inverted divisions  were detected [As mentioned above, Scrib was identified later than L(2)gl and Dlg1  and consdetected . Scrib, detected . More redetected . Thus, odetected . HoweverDrosophila larval brain as a model system [cno/AFDN or for each of the Scribble module (l(2)gl, dlg1 and scrib) do show ectopic NBs within the clone but they do not overgrow [scrib mutant clones are smaller than control NB clones and they do not appear very frequently. As it occurs in epithelial scrib mutant clones, in scrib NB clones a JNK activity-dependent apoptosis is also triggered [scrib and cno/AFDN in these larval NB clones overcomes the scrib/JNK-induced apoptosis and causes massive tumor-like overgrowths [V12 scribRas double mutant clones [cno l(2)gl nor cno dlg1 double mutant clones show the strong synergistic cooperation displayed in cno scrib mutant clones. In fact, the former double mutant clones are very similar to cno single mutant clones [cno l(2)gl and cno dlg1 mutant clones is that JNK is not activated in l(2)gl nor in dlg1 NB single mutant clones, even though in epithelia JNK is activated in each of those single mutant clones [l(2)gl or dlg1 single NB mutant clones is probably not the only explanation, as V12 scribRas NB mutant clones do not show the tumor-like overgrowth shown by cno scrib NB mutant clones [cno scrib double mutant clones. Hence, in asymmetric NB division, the Scribble module does not seem to be so functionally interdependent as in epithelia.ACD is a fundamental process during development to generate cell diversity. In addition, as we have learned over the past years, ACD is also a relevant process to take into account in the context of cancer and stem cell biology. A connection between failures in the process of ACD and tumorigenesis was first shown in the lab of C. González using the neural stem cells or NBs of the l system . In thesl system . Howeverl system . For exaovergrow . In factriggered . Howeverrgrowths . This efrgrowths ,90. Actit clones ,19,23,26t clones  and thatt clones . HoweverDrosophila neural stem cells. In this process, L(2)gl regulates cell-fate determinant localization by counteracting aPKC activity, and Dlg1 is a key component of the spindle orientation machinery by interacting with different microtubule-binding proteins, such as Khc-73 and Gukh. Nevertheless, while the mechanisms by which L(2)gl and Dlg1 modulate asymmetric NB division have been elucidated in more detail, the mode of Scrib action remains to be unveiled. Future work will be necessary to enlighten this point, as well as to further clarify the interdependence of these TSGs during asymmetric NB division.The Scribble module has been thoroughly analyzed in epithelial cell polarity. However, over the past decades this group of TSGs has been involved in additional processes and in different contexts, including the asymmetric division of


Andrographis paniculata, is a medicinal compound effective against infections, inflammatory disorders, and various cancers. In the present study, the effects of ANDR on NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation, caspase-8-mediated apoptosis and pyroptosis, and extra cellular matrix (ECM) degradation were analyzed in A431, MDA-MB231, and SKOV-3 cell lines. Results showed that ANDR inhibited the growth and proliferation of cancer cell lines by inhibiting NFkB signaling. A significant decrease in phospho-p65 level was observed upon increasing ANDR concentration in epidermoid carcinoma and breast cancer cell lines, A431 and MDA-MB231, respectively. Accordingly, upon ANDR treatment, the expression of caspase-8 was increased, whereas no significant induction of caspase-1 expression was observed. Moreover, we observed a significant increase in the expression of tissue inhibitor of metallopeptidase-1 (TIMP1) upon increasing ANDR concentration. Such induction of TIMP1 inhibited the activity of matrix metallopeptidase-7 (MMP-7), thus preventing the degradation of ECM. Therefore, as ANDR shows cytotoxicity towards cancer cells via the NFkB signal transduction pathway without inducing pyroptosis and blocks breast and ovarian cancer invasion by inhibiting MMP-7 expression through TIMP1 up-regulation, it has the potential to be developed as a drug targeting ovarian and breast cancers.Andrographolide (ANDR), isolated from  TAndrographis paniculata, is a bioactive herbal medicine with several applications. It has been reported to have antioxidant, anticancer, antibacterial, antifungal, antiviral, including dengue and human immunodeficiency (HIV) viruses, antihyperglycemic, anti-platelet aggregation, antidiabetic properties [In vitro and in vivo studies showed its cytotoxic effect on cell cycle progression, apoptosis, cell invasion, migration, and angiogenesis [Various natural products have been successfully employed to treat ovarian and breast cancers , 10, 11.operties , 17, 18.operties . ANDR isoperties , 21. In ogenesis , 23, 24.As cancer cells develop multi-drug resistance , new dru22.1ANDR  was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (10 mM) and stored at room temperature. Fetal bovine serum (FBS), phosphate buffer solution (PBS), penicillin, Dulbecco’s modified eagle medium (DMEM), and trypsin-ethylenediaminetetraacetic acid (EDTA) were procured from HiMedia Laboratory (P) Ltd., India. A protease and phosphatase inhibitor cocktail was obtained from Pierce  and the antibodies against phospho-p-65, p-65, and vinculin were obtained from Cell Signaling Technology, USA. Caspase-8, caspase-1, MMP-7, and TIMP-1 antibodies were obtained from Cistron Biolabs, India. Secondary mouse and rabbit antibodies conjugated with horseradish peroxidase were obtained from Santa Cruz . Enhanced chemiluminescence solution was obtained from GE Healthcare .2.22.2.12 and 95% air. The cells were harvested from the logarithmic phase of cultures and the media was replaced once every two days.MDA-MB-231 human breast cancer cell line, SKOV-3 human ovarian cancer cell line, and A431 epidermoid carcinoma cell line were obtained from National Centre for Cell Sciences . The cell lines were cultured in high glucose (4.5 g/L) DMEM supplemented with 10% FBS, 100 mg/mL streptomycin, and 100 units/mL penicillin at 37 °C under 5% CO2.2.2A stock solution (10 mM) of ANDR was prepared in DMSO, and further dilutions were made in the cell culture media. The inhibitory concentration of ANDR on cancer cell lines was determined from available literature (Supplementary Table 1), and the cells were treated with 10 μM, 20 μM or 50 μM of ANDR for 4 h .2.2.3Cancer cells were seeded in 100 mm cell culture dishes and left overnight for the cells to attach. After 24 hours, the cells were treated with 10 μM, 20 μM, and 50 μM of0 ANDR for 4 h. For the isolation of whole cell protein, radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and the protease/phosphatase inhibitor cocktail was used. The concentration of proteins was measured spectrophotometrically using a Bio-Rad protein estimation kit . Equal amounts of protein lysates were separated by 8% SDS polyacrylamide gel electrophoresis and transferred to the nitrocellulose membrane. The membranes were blocked with 5% BSA (Bovine Serum Albumin) for 1 h, and then incubated overnight at 4°C with the desired primary antibodies, followed by incubation with the respective secondary antibody for 1 h. The proteins were visualized using an Enhanced ChemiLuminiscence kit .2.2.4MDA-MB 231 and SKOV-3 cell lysates were collected after ANDR treatment. Standards and analytes were diluted with coating buffer and added to the wells. After 2 h of incubation, the wells were washed with wash buffer and incubated for 1 h with blocking solution at room temperature. After three washes, the analytes were incubated with primary antibodies against MMP-7/TIMP-1/caspase-8/caspase-1 for 2 h, followed by incubation with the respective secondary antibody for 1 h with intermittent washing. TMB (Tetramethylbenzidine) substrate (100 μL/well)  was added and the colorimetric reaction was stopped after 10 min by adding the stop solution (100 μL/well). The readings were recorded at an absorbance of 450 nm using spectrophotometer.2.2.5P-values. Differences at P <0.05 were considered statistically significant.Each experiment was performed three times. GraphPad PRISM was used for the statistical analysis. Statistical significance was evaluated by calculating the 33.1As ANDR was observed to affect tumor development, we examined the levels of total p65 and its phosphorylation status upon ANDR treatment by western blot analysis. Results showed a decrease in phospho-p65 levels with increasing concentrations of ANDR in epidermoid carcinoma and breast cancer cell lines, A431 and MDA-MB231, respectively  and b. TANDR has multiple targets at the membrane, cytoplasm, and nuclear levels, which in turn affected the targets in our study directly or indirectly . The tra3.2As ANDR inhibits the expression and activation of NFkB, which is essential for the inhibition of caspase activity, we explored the expression levels of caspase-8, an initiator caspase after ANDR treatment in breast and ovarian cancer cell lines MDA-MB231 and SKOV-3 by ELISA. We observed caspase-8 induction with increasing concentrations of ANDR in both breast and ovarian cancer cell lines  and b. AWe found that ANDR inhibited NFkB signaling in the different cancer cell lines used in the present study, and as NFkB inhibits apoptosis by inducing the expression of FLIP (FLICE-like inhibitory protein) and XIAP (X-linked inhibitor of apoptosis protein), inhibitors of caspase-8 and apoptosis, respectively , we expe3.3Using ELISA, we determined the effect of ANDR on the expression of MMP-7 in epidermoid carcinoma and breast cancer cell lines, A431 and MDA-MB231, respectively. We observed that MMP-7 levels were decreased in both the cancer cell lines upon increasing the concentration of ANDR  and b. DIt is known that cancer cells undergo metastasis once they are detached from the primary tumor site.Angiogenesis is one such mechanism exhibited by cancer cells that leads to tumor development and progression . AngiogeCurrently, a large number of drugs are under investigation, because they have the ability to inhibit the migration and invasion of cancer cells through the down-regulation of MMP-7; however, their use in clinical practice has been limited because of their side effects . Among t4We found that ANDR treatment could be used to prevent tumor invasion and migration . This ef


Exercise training is one of the most effective interventional strategies for sarcopenia in aged people. Nevertheless, the underlying mechanisms are not well recognized. Increasing studies have reported abnormal regulation of autophagy in aged skeletal muscle. Our current study aims to explore the efficiency of exercise interventions, including treadmill exercise, resistance exercise, alternating exercise with treadmill running and resistance exercise, and voluntary wheel running, on 21-month-old rats with sarcopenia and to detect the underlying mechanisms. Results showed the declined mass of gastrocnemius muscle with deficient autophagy and excessive apoptosis as a result of up-regulated Atrogin-1 and MuRF1, declined Beclin1 level and LC3-II/LC3-I ratio, accumulated p62, increased Bax, and reduced Bcl-2 levels, and also exhibited a defective mitochondrial quality control due to declined PGC-1α, Mfn2, Drp1, and PINK1 levels. However, 12-week exercise interventions suppressed the decline in mass loss of skeletal muscle, accompanied by down-regulated Atrogin-1 and MuRF1, increased Beclin1 level, improved LC3-II/LC3-I ratio, declined p62 level, and reduced Bax and increased Bcl-2 level, as well as enhanced mitochondrial function due to the increased PGC-1α, Mfn2, Drp1, and PINK1 levels. Moreover, exercise interventions also down-regulated the phosphorylation of Akt, mTOR, and FoxO3a, and up-regulated phosphorylated AMPK to regulate the functional status of autophagy and mitochondrial quality control. Therefore, exercise-induced autophagy is beneficial for remedying sarcopenia by modulating Akt/mTOR and Akt/FoxO3a signal pathways and AMPK-mediated mitochondrial quality control, and resistance exercise exhibits the best interventional efficiency. As the population undergoes rapid aging, the number of elderly patients with aging-related diseases is also increasing. By 2050, the number of individuals over 65 years old will increase to over 1.5 billion . Aging aFor the experiment, 60 6-month-old male Wistar rats  were ordered from the Experimental Animal Center for Hubei Provincial Center for Disease Control and Prevention . All animals were raised with standard food and water with free accessibility under a specific pathogen-free (SPF)-grade environment with a room temperature of 22–24°C and a 12 h dark-light cycle. The experimental protocols were approved by the Institutional Animal Care and Use Committee at Wuhan Sports University. Upon reaching 21 months of age, rats were randomly divided into five groups consisting of the normally aged model group (OC), treadmill exercise group (OT), resistance exercise group (OR), alternating exercise with treadmill and resistance exercise group (OM), and voluntary wheel running group (OV), with 10 mice in each group. Another 10 6-month-old Wistar rats were utilized as the young control group (YC). After exercise interventions for 12 successive weeks, all rats fasted for 24 h and were sacrificed by decapitation to gather experimental samples. Subsequently, the harvested gastrocnemius muscle samples were quickly frozen in liquid nitrogen and afterwards stored at −80°C freezer.The rats went through treadmill running with weight bearing at 6:00–8:00 pm 3 days per week (once every other day). In order to adapt to the apparatus and avoid subsequent exercise-induced stress, the rats were placed on the treadmill for 30 min without running on the first day. After adaptive training on the first day, the rats began running at a speed of 4.2 m/min, with a progressive increase to a final speed of 12 m/min at a speed increment of 1 m/min every 30 s, and then maintained this speed for exercise training for 12 weeks with 60 min during each training time.As mentioned previously, resistance exercise was carried out through ladder-climbing training . The ladTreadmill running and ladder-climbing training were alternated at 6:00–8:00 pm 3 days per week (once every other day) based on the above-mentioned protocols.The rats were provided with a free lifestyle for living in the cage with a rotating running wheel (diameter in 33 cm and width in 10 cm) and rats were able to climb the running wheel freely for voluntary exercise.3 of gastrocnemius muscle sample was fixed, rinsed, cut (70 nm), and stained. The sections were subsequently examined and analyzed under a transmission electron microscope  at the Research Center for Medicine and Structural Biology of Wuhan University.The transmission electron microscopic assay was executed as illustrated previously ; in brieAfter euthanizing the rats, gastrocnemius muscle samples were harvested and embedded in Tissue-Tek OCT (optimum cutting temperature) compound and stored at −80°C after being snap-frozen in liquid nitrogen. After being cut at 5 μm, the sections were dewaxed, rehydrated, and stained with hematoxylin-eosin (HE). The sample sections from each group were observed under a light microscope  with an imaging system  at 400x zoom. The cross-sectional areas (CSA) of 150 skeletal muscle fibers were gauged using ImageJ software.Thr172, Akt, p-AktSer473, mTOR, p-mTORSer2448, FoxO3a, p-FoxO3aSer253 , and GAPDH . After being washed three times for 15 min in 0.1% TBS-T buffer, the membrane was incubated with goat horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG  at room temperature for 1 h, and the target protein was detected by enhanced chemiluminescence (ECL) reagent imaged by ultra-sensitive fluorescence/chemiluminescence imaging system ChemiScope6300 .As detailed formerly , total pp < 0.05.All data were expressed as mean ± standard deviation (M ± SD). The difference among all experimental groups was analyzed using one-way ANOVA through IBM SPSS Statistics 20 . The significant difference was considered at It is well recognized that aging is related to a slowly progressive decline of mass and function in skeletal muscle. Consequently, the reduction of skeletal muscle mass is dependable evidence for the aging process. To detect the mass loss of skeletal muscle in aged rats, we examined gastrocnemius muscle weight-body weight ratio (GMW/BW) as the sarcopenia index (SI) for indicating an indirect indicator of skeletal muscle atrophy . As showUnder normal circumstances, myofilaments showed a regular arrangement, following the nucleus situated at the side of skeletal muscle fibers; however, irregular shapes and nucleus aggregation were observed in the skeletal muscle of the rats from the OC group. After exercise interventions, the irregular structure of skeletal muscle fibers was clearly improved . MeanwhiThe aging of skeletal muscle is characterized by damaged or swollen mitochondria, disordered arrangement of myofilaments, or irregular fiber structure. Compared to the YC group, skeletal muscle from the OC group revealed a disordered arrangement of myofilaments and swollen and vacuous mitochondria. However, the ordered arrangement of myofilaments, normal mitochondria, and autophagosomes were observed in the skeletal muscle of rats from the OT, OR, OM, and OV groups , suggestE3 ubiquitin ligases including Atrogin-1 (atrophy gene-1) and MuRF1 (muscle ring finger protein 1) play a crucial role in the atrophy of skeletal muscle . As showBoth autophagy and apoptosis are highly correlated with biological procedures, and play an essential function in differentiation, development, and homeostasis in an interdependent or interrelated manner. In the present study, lower Beclin1 expression and LC3-II/LC3-I ratio and higher p62 expression in the skeletal muscle of the rats from the OC group were measured. In contrast, exercise interventions, especially resistance exercise, significantly up-regulated Beclin1 expression, increased LC3-II/LC3-I ratio, and down-regulated p62 expression ,B, suggeSer473/Akt, p-mTORSer2448/mTOR, and p-FoxO3aSer253/FoxO3a ratios in the skeletal muscle of the OC group were detected to reveal a significant increase, whereas exercise interventions, especially resistance exercise, could decrease the ratios as above , thus resulting in the activation of DNA damage response and cell aging. Mitochondrial quality control ensures the relative stability of the quantity and quality of mitochondria, maintains mitochondrial homeostasis through regulating mitochondrial biosynthesis, mitochondrial dynamic balance and mitophagy, and alleviates cell aging; in contrast, dysregulated mitochondrial quality control can contribute to sarcopenia .Thr172 and the expression of PGC-1α are increased after exercise interventions in aged skeletal muscle, thus affirming that exercise interventions, especially resistance exercise, can activate the AMPK/PGC-1α signal pathway and promote mitochondrial synthesis in the skeletal muscle of aged rats (Mitochondrial biogenesis is a complicated procedure that promotes a development in mitochondrial content to fulfill cellular energy as needed. PGC-1 is the master regulator of mitochondrial biogenesis. Various factors leading to the increase of AMP/ATP ratio, such as ischemia, hypoxia, exercise, and hunger, can activate AMPK or stimulate its expression , which tged rats ,B.Thr172 and the expression of Drp1 and PINK1 proteins are increased after exercise interventions in aged skeletal muscle, suggesting that exercise interventions, especially resistance exercise, can promote AMPK-mediated mitophagy (Mitochondrial dynamics, including fusion and fission, have been acknowledged as essential events in mitochondrial metabolism. Several studies on the molecular mechanism of exercise-mediated mitochondrial fusion report variant results, ranging from a significant decrease, to a little bit of change, to a noticeable increase , 2016. Titophagy ,C.in vivo, including substrate protein ubiquitination and degradation of ubiquitinated proteins. The E3 ubiquitin ligases, such as Atrogin-1 and MuRF1, are essential in protein ubiquitination. They selectively identify substrate proteins and promote ubiquitin transfer to substrate proteins; through this, then ubiquitinated protein substrate is finally recognized and degraded by 26S proteasome in skeletal muscle. Previous studies have found that E3 ubiquitin ligase, including Atrogin-1 and MuRF1, are closely related to skeletal muscle atrophy, and are significantly up-regulated in a variety of skeletal muscle atrophy models (The ubiquitin proteasome pathway applies to the whole cell and involves numerous substrates and reaction processes y models . In conty models . In addiy models , and exey models . In thisy models ,B, whichy models , of whicy models . Furthery models . TherefoIn summary, exercise interventions, especially resistance exercise, can induce autophagy, maintain mitochondrial quality control, and suppress E3 ubiquitin ligase in aged skeletal muscle, which may provide the exercise-induced advantageous effect on alleviating sarcopenia. Mechanistically, the modulation of the Akt/mTOR, Akt/FOXO3a, and AMPK signal pathways are implicated in the mitigation of sarcopenia upon exercise interventions .All datasets presented in this study are included in the article/supplementary material.The animal study was reviewed and approved by Institutional Animal Care and Use Committee at Wuhan Sports University.ZZ and NC conceived and designed the project. ZZ, JiL, LW, HZ, and JuL performed experiments. ZZ, JiL, HZ, and NC analyzed data and prepared figures. ZZ, JiL, and NC drafted the manuscript. NC edited and revised the manuscript. ZZ, JiL, LW, HZ, JuL, and NC approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The number of patients with central nervous system disorders is increasing. Despite diligent laboratory and clinical research over the past 30 years, most pharmacologic options for the prevention and long-term treatment of central nervous system disorders and neurodegenerative disorders have been unsuccessful. Therefore, the development of drugs and/or functional foods to prevent the onset of neurodegenerative disorders is highly expected. Several reports have shown that polymethoxylated flavones (PMFs) derived from citrus fruit, such as nobiletin, tangeretin, and 3,3′,4′,5,6,7,8-heptamethoxyflavone, are promising molecules for the prevention of neurodegenerative and neurological disorders. In various animal models, PMFs have been shown to have a neuroprotective effect and improve cognitive dysfunction with regard to neurological disorders by exerting favorable effects against their pathological features, including oxidative stress, neuroinflammation, neurodegeneration, and synaptic dysfunction as well as its related mechanisms. In this review, we describe the profitable and ameliorating effects of citrus-derived PMFs on cognitive impairment and neural dysfunction in various rat and murine models or in several models of central nervous system disorders and identify their mechanisms of action. The number of patients with neurodegenerative diseases and neurological disorders associated with dementia, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and cerebrovascular dementia, is increasing ,2. Curre21H22O8), tangeretin , and 3,3′,4′,5,6,7,8-heptamethoxyflavone  [Numerous natural resources contain bioactive substances, which allows them to function as treatments and means of preventing geriatric and neurodegenerative diseases ,17,18,19O9, HMF) ,24,25,26O9, HMF) ,28, obesO9, HMF) ,30, diabO9, HMF) ,32, cardO9, HMF) ,34, and O9, HMF) ,36,37. ICitrus reticulata and Citrus depressa [Nobiletin is abundant in citrus fruits, such as depressa ,20. NobiAD is one of the most commonly diagnosed neurodegenerative diseases ,5. The cThe effect of nobiletin on Aβ1-42–induced spatial learning and memory impairment in mice has also been evaluated . The admTwo main enzymes are involved in generating Aβ from amyloid precursor protein (APP): β-secretase (BACE1) and γ-secretase ,43. AnotThe triple-transgenic mouse model of AD (3xTg-AD) have mutations in the genes for presenilin 1 (PS1), APP, and tau . These mAging is the most strongly-associated additive factor in neurodegenerative disease pathogenesis . The detIn AD patients, learning and memory are impaired by the degeneration and loss of cholinergic nerves in the cerebral cortex and hippocampus . The OBXN-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission is linked with AD-induced behavioral changes and cognitive deficits in addition to depressive disorders and suicidal behavior [Dysfunctional behavior . Adminisbehavior ,59. Sevebehavior . Furtherbehavior . Nobilet2+/calmodulin–dependent protein kinase II (CaMKII) phosphorylation were confirmed. However, nobiletin significantly improved these functional declines [PD is characterized by motor dysfunction due to the degeneration of dopaminergic neurons in the substantia nigra pars compacta . Approxideclines . Studiesdeclines . NobiletCerebral infarction is a cerebrovascular disorder that contributes to the development of vascular dementia . During Although blood flow is restored to ischemic brain tissue (ischemia/reperfusion), superoxide and hydroxy radicals are generated, which promotes cerebral edema and neuronal apoptosis . In a moNeurodegeneration due to excessive inflammation is involved in the development of AD and PD ,8. NumerMultiple sclerosis is a complex chronic inflammatory and degenerative disorder of the central nervous system . DemyeliDepression is a common among patients with AD, especially during the early and middle stages ,85. NobiDDIT3, TRIB, and ASNS in the SK-N-SH human neuroblastoma-derived cell line. Meanwhile, the expression of CCNA2, a cell cycle control gene, and TXNIP, which encodes a thioredoxin-binding protein, decreased [Central nervous system disorders have numerous pathological similarities at the sub-cellular and molecular levels, including oxidative stress, neuroinflammation, and memory impairment ,87,88,89ecreased . Furtherecreased . These rLearning and memory demand the formation of new neural networks and synthesis of new mRNA and proteins in the brain . IntraceThe AMPA glutamate receptor contains four subunits (GluR1-R4) and is closely involved in synaptic plasticity, learning, and memory ,103. IncThe NMDA-type glutamate receptor is a heterotetramer composed of the essential subunit NR1 and a regulatory subunit (NR2A-D) that provides diversity in function . The disIn the brains of AD patients, there are degeneration and loss of cholinergic nerves . Further2 (PGE2) production [N-terminal kinase (JNK) and p38 levels without affecting the ERK1/2 level [The Aβ fragment 25–35 (Aβ25–35) is also involved in the pathogenesis of AD ,113. Resoduction . Moreoveoduction . Moreove/2 level . NobiletRecently, it has been reported that neprilysin can degrade Aβ ,116. HowThus, nobiletin might be a potential candidate for amelioration of neurological diseases, such as AD, via antioxidative, anti-inflammatory, anti-Aβ pathology and regulation of signal cascades related to neurotransmission and CRE-mediated transcription .Tangeretin is a citrus PMFs with a structure similar to that of nobiletin. Tangeretin is found in the peel  of tangerine and other citrus fluits . It appeThe effects of tangeretin in numerous PD animal models were investigated. Unilateral injection of 6-hydroxydopamine (6-OHDA), a synthetic catecholaminergic neurotoxin, into rats’ medial forebrain bundle significantly decreased the number of tyrosine hydroxylase-immunopositive (TH+) cells in the substantia nigra and declined the striatal dopamine content . TangereAnother study investigated that the neuroprotective effect of tangeretin in the prevention of neuroinflammation and improvement of dementia in MPTP-infused PD model rats . TreatmeER stress induces a signaling reaction known as the unfolded protein response (UPR), which aims to restore proteostasis via the induction of adaptive programs when stress is chronic and/or unrepaired . AbnormaDrosophila melanogaster) [In addition, Fatima et al. studied the effect of tangeretin on the symptoms of PD exhibited by PD model transgenic flies (ogaster) ,127. Tanogaster) ,127.2, iNOS, COX-2, IL-1β, toll-like receptor 4, interferon-γ, TNF-α, and IL-6 and the oxidative stress parameters SOD, GSH, GPx, catalase, glutathione reductase and MDA, in the serum and brain tissue of experimental rats. Thus, tangeretin could have neuroprotective and anti-inflammatory effects against ischemia–reperfusion injury in rats through suppression of inflammatory reaction and oxidative stress [Researchers have investigated the protective effect of tangeretin against ischemia–reperfusion injury in the rat . Ischemie stress .Epilepsy is a common neuronal disorder characterized by recurrent seizures ,130. TheCKD is a progressive loss of kidney function, and a significant health problem with limited therapeutic options . Both oxPTSD is a stress-related psychiatric/mental disease ,137. ResAntioxidative and neuroprotective activity of tangeretin against oxygen–glucose deprivation (OGD)-induced injury on human brain microvascular endothelial cells (HBMECs) was studied . The resOn the other hand, the Nrf2 signaling cascade is one of the major pathways involved in the protection of cells against oxidative stress through upregulation of the expression of neuroprotective genes such as SOD . Tangere2;, TNF-α, IL-1β, and IL-6 in a dose-dependent manner. In addition, it inhibited the LPS-induced expression of iNOS and COX-2 in microglial cells [2 and NF-κB caused by IL-1 [To suppress microglial activation-mediated neuroinflammation has become a promising target for the development of medicates or functional foods to treat neurodegenerative diseases . Shu et al cells . Tangerial cells . The antal cells . Tangere by IL-1 . These rHMF is another PMF found in citrus fruits . AlthougThe ERK/CREB cascade is one of the most important pathways in the regulation of synaptic plasticity as well as development of long-term memory ,146. AftAnother study using a cerebral ischemia mouse model investigated the effects of HMF on protection against cognitive decline and neural cell death . The autHMF significantly induced the activation of ERK/CREB cascade in cultured cortical neurons . ResearcThe effect of HMF on inflammation in the hippocampus was investigated using mice injected intrahippocampally with LPS . HMF preOne study investigated the antidepressive effects of HMF in mice injected subcutaneously with corticosterone at a dose of 20 mg/kg/day for 25 days . HMF treThe effect of HMF on the CUMS model was also investigated . ResearcNeurotrophic effect of HMF in vitro using rat C6 rat glioma cells was investigated . HMF enhMoreover, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HMF), an analog of HMF, was shown to efficaciously prompt neurite outgrowth of PC12 through the upregulation of growth-associated protein 43, a neural differentiation marker . 5-OH-HMThe three types of PMFs exerted neuroprotective and neurotrophic effects in various experimental models. These natural compounds share common mechanisms such as antioxidant and anti-inflammatory effects. The antioxidant and anti-inflammatory effects of PMFs may be an important mechanism of neuroprotective effect in various neurological models. On the other hand, PMFs seem to activate neural function through activation of several intracellular signal cascade and gene expression. Therefore, structure-activity relationship studies may be needed in order to understand the precise mechanism of action of the neuroprotective effects of PMFs.We further mention herein the facts that there is one exciting discovery of PMFs’ function which deserve additional discussion. Circadian rhythms are biological activity rhythm driven by internal circadian clocks and are a fundamental mechanism to regulate various pathways and pathophysiology . CircadiCitrus reticulata Blanco, Citrus reticulata or Citrus sinensis, which contain high concentrations of PMFs, have no harmful effects on animals [In this paper, we reviewed the neuroprotective effect of citrus PMFs, nobiletin , tangere animals ,169, and animals ,171. Citrus peels and/or extract have been reported to have various beneficial effects on humans, e.g., body weight control , promote