Panax ginseng C. A. Mey (PG) is an important herbal drug which has been used for benefiting Qi for thousand years. Panax ginseng C. A. Mey and its compounds (PGC) possess various pharmacological activities, including anti-fatigue. Here, we conducted a systematic review of both randomized clinical trials (RCTs) and preclinical animal studies to investigate the efficacy and safety of PGC for fatigue.Fatigue, as a complex, multidimensional symptom, is associated with many physical illnesses. Electronic searches were performed in 7 databases from the time of each database's inception to August 2019. The methodological quality of RCTs was assessed using 7-item checklist recommended by Cochrane Collaboration or by the CAMARADES 10-item quality checklist. All the data were analyzed using Rev-Man 5.3 and Stata SE software.Eight eligible RCTs and 30 animal studies were identified. The risk of bias scores in RCTs ranged from 4/7 to 7/7, and of animal studies varied from 4/10 to 7/10. Meta-analyses showed that PGC was superior to placebo according to their respective fatigue scales, heart rate recovery, and clinical effect (P < 0.05). There were a similar number of adverse effects between PGC and placebo group (P > 0.05). Meta-analyses showed that PGC can significantly decrease level of blood lactate, blood urea nitrogen, creatine kinase, malondialdehyde, and lactic dehydrogenase in serum, level of malondialdehyde in liver and level of gamma-aminobutyric acid, 5-hydroxytryptamine in brain tissue, and increase swimming time, level of glutathione peroxidase, glucose, superoxide dismutase in serum, level of glycogen and activity of superoxide dismutase, glutathione peroxidase, and catalase in skeletal muscle, level of hepatic glycogen in liver and level of dopamine, acetylcholine in brain tissue, compared with control (P < 0.05). Meta-analyses showed no significant difference in animal body weight between PGC and control (P > 0.05).The present findings supported, to a certain degree, that PGC can be recommended for routine use in fatigue. The possible mechanism of PGC resists fatigue, mainly through antioxidant stress, regulating carbohydrate metabolism, delaying the accumulation of metabolites, promoting mitochondrial function, neuroprotection, antiapoptosis, and regulating neurotransmitter disorder in central nervous system. Fatigue is a condition of lacking the energy and motivation in responding to physical activity, emotional stress, boredom or insufficient sleep . It is aPanax ginseng C.A. Mey (PG) has been a rising utilization in treating fatigue in Asia and elsewhere around world. Based on traditional Chinese medicine and herbal philosophy, PG is considered as an adaptation to help restore body balance , including energy and vitality, particularly during times of fatigue or stress , including cognition, sleep disorders, depression, pain, and the ability to regulate inflammatory cytokines  for fatigue  RCTs that evaluated the effectiveness and safety of PGC for fatigue; (2) the Cochrane risk of bias (ROB) tool met at least 4 out of the 7 domains; (3) Subjects had chronic fatigue syndrome (CFS) or healthy adults after exercise; Subjects were classified as CFS-like according to Evaluation and Classification of Unexplained Chronic Fatigue (ECUCF) ; (4) PGCThe inclusion criteria of animal studies were prespecified as follows: (1) PGC for fatigue animal models was established by forced movement; (2) The interventions of treatment group were PGC at any dose and control group were nonfunctional liquid  or no treatment; and (3) The primary outcome measures were forced movement time and/or serum biochemical parameters and/or skeletal muscle biochemical parameters and/or liver biochemical parameters and/or brain tissue biochemical parameters. The secondary outcome measures were body weight, organ index (organ weight/body weight) and possible mechanisms of PGC for anti-fatigue. The exclusion criteria were predefined as follows: (1) not fatigue model; (2) combined use of other drugs; and (3) no available data, duplicate publications, and lack of control group.Two independent researchers extracted the details from the included RCTs and animal studies according to two standardized data extraction forms, respectively. There are many manners for including outcomes, such as peak time point, last time point, and same time point. There is undeniable that any manner will lead to bias. In order to minimize bias, inclusion criteria were prespecified as follows: The result of the peak time point was included when the data were expressed at different times. If meta-analysis data were lost or expressed graphically, we would try to contact the author for more information. When no response was received, we used digital ruler software or exclusion software to measure data from charts. If the data in the primary RCT were missing or merely illustrated graphically, an effort was launched to obtain further information through contacting the authors. If failed, the digital ruler software was used for measuring data from the graphs or excluded.The methodological quality of the included RCTs was evaluated by using the Cochrane Collaboration's tool. The RoB of the included animal studies was assessed using 10-item quality checklist of the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) with minor modification. Divergences were well settled by correspondence author (GZ).2 - statistic test. An I2 value greater than 50% was considered as having substantial heterogeneity. When substantial heterogeneity was not observed, the fixed-effects model was reported. On the contrary, the random-effects model was reported. Simultaneously, considering the differences in subjects, interventions, and treatments, we used the Z-test for subgroup analysis. P <0.05 was considered to be statistically significant. If an outcome contained more than 10 RCTs, funnel plots, and Egger's test were used to examine publication bias.Analysis was conducted with RevMan 5.3 and Stata SE software. Continuous outcomes were presented as mean difference (MD) or standardized mean difference (SMD) with 95% confidence interval (CI). Dichotomous outcomes were presented risk ratio (RR) or odds ratio (OR) with 95% CI. Probability values P <0.05 were considered significant. In order to estimate heterogeneity across studies, we used IA total of 1331 relevant literatures were retrieved from the database, of which 667 were considered duplicates. Of the remaining 664 articles, qualified RCTs and animal experiments should be selected separately. For RCTs, 362 articles were eliminated because of reviews, case report, or animal studies. After scanning the remaining 302 full-text articles, 294 studies were excluded by reasons that they were (1) combined with other disease; (2) combined with other herbal treatment(s) in the intervention group; (3) no data available; (4) not real RCTs or quasi-RCTs; or (5) with the less than 4 domains “yes” according to the Cochrane RoB tool. Eventually, 8 RCTs were selected , I2 = 0%) , I2 = 0%) (Five studies  describeI2 = 0%)  showed tP = 0.001; heterogeneity χ2 = 3.80, df = 2 (P = 0.15), I2 = 47%) , I2 = 63%). Owing to obvious heterogeneity, we used sensitivity analyses and removed one study , I2 = 38%) , I2 = 90%). Two studies , I2 = 6%)   found th studies  may be c studies  utilized studies  utilized2 = 46.87, df = 2 (P < 0 00001), I2 = 96%). After sensitivity analyses, we removed 1 study , I2 = 0%) , I2 = 14%) , I2 = 49%) ; I2 = 42%) , I2 = 29%) , I2 = 38%) , I2 = 18%) , I2 = 20%)   showed t2 = 14%)  for redu2 = 49%)  for redu2 = 42%)  for redu2 = 29%)  for redu2 = 38%)  for incr2 = 18%)  for redu2 = 20%)  found th2 = 17.10, df = 5 (P =0.004), I2 = 71%). After removing 1 studies , I2 = 35%) , I2 = 66%). Sensitivity analyses were conducted to explore potential sources of heterogeneity. After removing 1 trial , I2 = 43%) , I2 = 5%) , I2 = 0%)   showed t 1 trial  that uti 1 trial  showed t2 = 43%)  showed tI2 = 5%)  for imprI2 = 0%) . There w2 = 58.77, df = 10 (P < 0 00001), I2 = 83%).Owing to obvious heterogeneity, we conducted subgroup analysis. These studies were divided into 4 subgroups according to different experiment interventions. Meta-analysis showed that there was significant difference in the level of glycogen between PGC and control in all subgroups, including ginseng , I2 = 0%) , I2 = 0%) (P < 0.05) for increasing activity of SOD compared PGC with control, whereas 2 studies (P > 0.05).Meta-analysis of 11 studies  showed t ginseng , Ginseno ginseng , ginseng ginseng , and gin ginseng  groups ( ginseng  showed tI2 = 0%)  showed nI2 = 0%) . Two stuI2 = 0%)  showed s studies  showed cP < 0.00001; heterogeneity: χ2 = 17.97, df = 4 (P = 0.001), I2 = 78%) and level of 5-HT , I2 = 74%) compared with control. One study , I2 = 3%) , I2 = 17%) , I2 = 59%). After removing one study , I2 = 6%) , I2 = 42%)   found sine study , meta-anne study  showed sI2 = 6%)  showed aP = 0.62; heterogeneity: χ2 = 8.87, df = 6 (P = 0.18), I2 = 32%) , which did not suggest an obvious publication bias. However, there was a significant bias in the BLA levels with Egger's test . Because the number of studies in the remaining outcomes was limited (n < 10), funnel plot and Egger's test were not appropriate.Funnel plots were conducted for two outcomes . PGC attenuated MPP+-induced MPTP-induced and apoptosis. PGC increased Ach and DA levels, and decreased GABA and 5-HT levels in brain tissue , reduced toxic oxidant peroxynitrite, and prevented mitochondrial dysfunction and lipid peroxidation. PGC may enhance fat mobilization and promote gluconeogenesis, increase the delivery of glucose, and maintain blood glucose level. PGC increased the LDH activity and the hepatic glycogen levels, and retarded the accumulation of BUN and BLA. PGC improved succinate dehydrogenase (SDH), NaThis is the clinical and preclinical systematic review to evaluate the efficacy and safety of PGC for fatigue. Eight RCTs with 678 participants and 30 studies with 2249 animals were selected. The quality of RCTs included was high, and animal studies were generally moderate. The findings of RCTs demonstrated that PGC was superior to placebo according to their respective fatigue scales, heart rate recovery, and clinical effect. There were a similar number of adverse effects between PGC and placebo group. The evidence available from animal studies showed that PGC could preserve physical function after exercise, mainly through antioxidant, regulating carbohydrate metabolism, delaying the accumulation of metabolites, promoting mitochondrial function, neuroprotection, antiapoptosis, and regulating neurotransmitter disorder in central nervous system.Some methodological flaws existed in the primary RCTs. First of all, 3 studies used allocation concealment . Trials In addition, we cannot neglect the contribution of Korea and other countries worldwide in the study of ginseng in treatment of fatigue. Due to the limitation of language, the present study was not included studies which language was not English or Chinese. We can increase international cooperation to overcome the linguistic limitation.The evidence available from present study supported the routine use of PGC for fatigue, whereas the safety still needs more data because only five of eight studies reported. Given the gap between limitations of the primary RCTs and the quality of RCTs, we recommend that further design of the RCTs should consult the CONSORT statement , which oCurrently, there is no gold standard for measuring fatigue. Various scales were used to measure fatigue in different studies. Some scales have been calibrated, and some are homemade. Measurement of fatigue is challenging. Due to the wide range of conceptualizations of the problem and the concurrent development of questionnaires for many specific diseases, many questionnaires are used to measure fatigue. A comprehensive fatigue measurement, such as the Fatigue Severity Scale , Piper FDegree and scope of debilitating fatigue is a core component of health care where chronic diseases are receiving increasing attention. Current acute disease research models are not enough to solve chronic disorders affecting multiple regulatory systems and present complex constellations of symptoms. The identification of objective markers consistently associated with CFS is an important goal in relation to diagnosis and treatment, as the current case definitions are based entirely on physical signs and symptoms. Since the human body is an autonomous, fully integrated, and self-regulating system, it is not surprising that even localized muscle fatigue can present systemic biomarkers. There is a growing study devoted to understanding the biology of fatigue. Recognition of CFS biomarkers is an important part of this work. A complex construct of symptoms emerges from alterations and/or dysfunctions in the nervous, endocrine, and immune systems. Biomarkers, distributing across these systems, constituted complex biological networks. The acquisition of biomarkers required a comprehensive biological network-based analysis of fatigue biology . In addiPreclinical research is the key to convert preclinical data into clinical data . However+-K+-ATPase and Ca2+-Mg2+-ATPase are crucial enzymes to degrade ATP. NRF-1 and TFAM are positive regulators of transcription. PGC improved SDH, Na+-K+-ATPase, and Ca2+Mg2+-ATPase activities. PGC enhanced the mRNA expression of NRF-1 and TFAM and increased the mtDNA content, and thereby enhancing mitochondrial function and producing more ATP for energy supplementation  Antioxidant stress: PGC passed through the injured membrane, improved the activity of GSH-Px, CAT, and SOD, scavenged hydroxyl radical, and reduced the excessive ROS, and thus preventing lipid oxidation and protecting the corpuscular membrane to reduce the release of LDH, MDA, and CK into the serum . Anotherentation ; (5) Neuentation ; (6) Regentation .The findings of present study demonstrated that PGC exerted anti-fatigue function, mainly through antioxidant stress, regulation of carbohydrate metabolism, delaying the accumulation of metabolites, promotion of mitochondrial function, neuroprotection and antiapoptosis, and regulation of neurotransmitter disorder. And the findings supported, at least to an extent, the use of PGC for fatigue.Study conception and design: YL/G-QZ. Acquisition, analysis and/or interpretation of data: T-YJ/P-QR/H-YL/P-PZ/G-QZ/YL Final approval and overall responsibility for this published work: YL/G-QZ.This study was supported by a grant from the National Natural Science Foundation of China (81973657/H2902); priority speciality of integrative brain diseases, the second affiliated hospital of Wenzhou Medical University, Wenzhou China (2016).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.


SSTs) with dopamine receptors (DRs) has been discovered in NENs. This phenomenon results in hybrid receptors with enhanced functional activity. On these bases, chimeric molecules embracing somatostatin and dopamine features have been recently developed. The aim of this review is to provide a comprehensive overview of the available preclinical and clinical data regarding chimeric somatostatin-dopamine agonists as a new class of “magic bullet” in the therapy of NENs.Neuroendocrine neoplasms (NENs) are a widely heterogeneous family of neoplasms arising from neuroendocrine cells, which are interspersed throughout the body. Despite NENs are relatively rare, their incidence and prevalence are constantly increasing probably due to the improvement in earlier diagnosis and patients’ management. When surgery is not curative, particularly for patients with metastatic disease, several medical options are available. Somatostatin analogues (SSA) are the first-line medical therapy for well-differentiated NENs. Interestingly, the heterodimerization of somatostatin receptors ( SSTs) [SSTs and dopamine receptors (DRs) were performed in several NENs showing their co-expression. Interestingly, formation of heterodimers between SSTs and DRs was found to be associated with an enhanced activity bringing new insight for NENs treatment [Neuroendocrine neoplasms (NENs) are a rare family of tumors that arise from neuroendocrine cells. These cells are widely dispersed all over the human body and their main function is hormone secretion. NENs can seriously compromise organs homeostasis and quality of life of patients due to the excessive secretion of hormones able to generate disabling symptoms, specific syndromes and other complications ,2,3. SomSSTs) . Analysireatment . Speculareatment .In this review, we provide an overview concerning the complex interplay between somatostatin and dopamine signaling, analyzing the potential clinical application of chimeric somatostatin-dopamine molecules as “magic bullet” in the therapy of NENs.SSTs. Its physiological function is mainly related to inhibition of exocrine and endocrine secretion. Notably, in the central nervous system somatostatin acts as a neurotransmitter/neuromodulator playing an important role in learning and memory. In addition, the inhibitory effects of native somatostatin on hormone release and cell proliferation made this molecule a potential candidate as anticancer drug, particularly in NENs expressing SSTs. SSTs belong to the G-protein coupled receptor superfamily of which five human different receptors subtypes (SST1–SST5) have been identified [SST1, SST3, and SST4 genes are introneless, consequently they do not undergo to splicing events, while for SST2 and SST5 genes splicing variants are known. Truncated variants, resulting from splicing events of SST2 and SST5 genes, display an abnormal functional response to somatostatin ligands. In particular, the truncated form of SST5 (sst5TMD4), which is mainly expressed in medullary thyroid carcinoma cell lines, non-functioning pituitary adenomas (NFPAs), somatotropinomas, and pancreatic neuroendocrine tumors, is associated with a more aggressive tumor behavior and decreased response to SSAs [Somatostatin is a small regulatory peptide that can explicit a broad spectrum of effects including maintenance of homeostasis or involvement in pathological events, such as inflammation, through interactions with specific entified  and they to SSAs ,16,17,18SSTs are mainly bound to inhibitory G proteins (Gi/G0), which after ligand binding are able to decrease the activity of adenylyl cyclase, phopholipase-C stimulation and MAPK modulation through phosphotyrosine phosphatase (PTP) activation. SST2 has been reported to be coupled mainly with protein-Gα02/β2/γ3, which can activate inwardly rectifying K+ channels and/or inhibit voltage-dependent Ca2+ channel, providing an inhibition of secretion through hyperpolarization of membrane potential [SST3 is coupled with Gα12 or Gα14 activating pertussis toxin-independent mechanisms. All this evidence underlines that the activated signaling cascade depends on the SST subtype. Not only different subtypes of SSTs but also the nature of the ligand can affect tissue response [Somatostatin binds its receptor at transmembrane domain level and, based on the receptor subtype, it provides several down-streaming signals through activation of different G-proteins . Notablyotential . On the response . Indeed response ,22.SSTs are summarized in SST1, SST2, SST4, and STT5 have been shown to be associated with cell cycle arrest in G1/S phase, while SST3 induced a block in G2M phase [SST2 and SST3, which can also induce apoptosis through p-53/Bax, NFkB and PTPs/caspase8 signaling [SST1,SST3, and SST4 can directly mediate antiproliferative effects through inhibition of the Na+/H+ exchanger, especially in enteric endocrine cells.The main anti-tumor signaling pathways of 2M phase . Finally2M phase ,30. Thesignaling ,33,34,35SSTs signals act not only directly by dephosphorylation of growth factors receptors but also indirectly through down-regulation of their synthesis [SST2,SST3, and SST5 trigger inhibition of GH synthesis in pituitary somatotropinoma [SSTs signaling is the inhibition of angiogenesis, which is mainly mediated by SST5. On the other hand, SST3 was indicated as pivotal initiator for downregulation of vascular endothelial growth factor (VEGF), which is crucial for vessel development under tumor hypoxia conditions [ynthesis . After tynthesis ,38. Acturopinoma ,40,41 Moropinoma ,43. Anotnditions ,45.SSTs activity regulation is also provided through desensitization and internalization of receptors. Interestingly, SSTs show peculiar features for these mechanisms. Internalization process, especially for SST2, is activated by binding of SSAs, while there is no evidence on SSTs internalization induced by somatostatin antagonist. Moreover, time and magnitude of internalization can modify type and susceptibility of the downstream response to SSAs. In particular SST2, which is the most studied SST, is internalized and rapidly recycled back to the plasma membrane without going through degradation [SSTs status. Indeed, SSTs can form both homo- and hetero-dimers with members of the same or distantly related receptor subtypes [radation ,46,47,48subtypes ,36. AltoSST2 and less affinity for SST5. Over the years, octreotide and lanreotide compounds showed powerful effects, mostly as palliative therapeutic options for treating symptoms related to NENs  [SSTs: SST5 > SST2 > SST3 > SST1 [SST2, SST4, and SST5 [Although endogenous somatostatin triggers powerful route for the negative control of hormone secretion and tumor progression, its clinical administration was limited by its short half-life ~2.5 min) . Therefoa, etc.) ,55,56,57a, etc.) . Over tha, etc.) ,5. Notab min . Th3 > SST1 ,60. Thes3 > SST1 ,62. More3 > SST1 ,64, pasi3 > SST1 ,66,67. A3 > SST1 . A phase3 > SST1 , while a3 > SST1 . In addi3 > SST1 . In 2001and SST5 ,73,74. Aand SST5 .SSTs, belong to the G-protein coupled receptor family and five subtypes of DRs were isolated, named D1R to D5R. The third intercellular loop of DRs interacts with G-proteins leading to cAMP regulations, while the hydrophobic transmembrane domain is crucial for DAs and antagonists ligand binding [D1-like group (D1R and D5R) and D2-like group  [2R, which differ for the third cytoplasmic domain, giving them different functional characteristics. The longer form, named D2RL, interacts with Gi-proteins, whereas the shorter one, D2RS, is able to activate phospholipase D providing important anti-proliferative effects [2RL and D2RS, is related to inhibition of MAPK signaling together with activation of phospholipase C, phospholipase D and different membrane channels such as calcium and potassium channels, Na+/K+ exchangers and Na+-K+-ATPase [Dopamine is one of the most relevant neurotransmitter belonging to catecholamine family. Its action triggers a broad profile of functions, not only in the central nervous system but also in the periphery. Indeed, it is associated with regulation of cardiovascular and renal function, and it is mainly involved in regulation of hormone secretion . Dopamin binding . DRs canand D4R) . D1-like effects ,79. D2-l+-ATPase ,77.2R signaling [+ voltage activated channels, while several signaling pathways contribute synergistically to anti-proliferative effects of DAs. Indeed, it has been demonstrated that prolactinomas treated with DAs, able to decrease cAMP intracellular concentration, show not only hormone secretion inhibition but also tumor shrinkage [2R activated pathway leads to cell death and apoptosis through the well-known p38 and JNK MAPK pathways [2RS induces stimulation of phospholipase D leading to inhibition of proliferation [2R and D5R has been observed to inhibit pituitary tumor growth by autophagic cell death both in vitro and in vitro [2R knockout mice show overexpression of VEGF, which is one of the main factors promoting neoangiogenesis and tumor growth, it was demonstrated that this growth factor is under dopaminergic control indicating a novel potential role of this class of receptors in oncology [Increasing evidence shows that DRs are associated with the regulation of tumor behavior and proliferation, with relevant clinical applications in the therapy of pituitary NENs, particularly prolactinoma. The main anti-secretory and anti-tumor functions of dopamine appear to be modulated through Dignaling . These phrinkage . D2R actpathways . D2RS inferation ,83. Morein vitro . Furtheroncology .2R is extensively expressed in all pituitary tumor types [2R, is highly used in the therapy of these tumors and shows less side effects and more convenient dosing schedule than the older bromocriptine. Several in vivo experiments were performed to better understand the role of this receptor in cellular development and tumor progression. Indeed, it has been showed that D2R knockout mice develop prolactinomas with age [Over the years, DAs administration becomes largely used for the treatment of different pituitary NENs. It was demonstrated through immunohistochemistry and in situ hybridization that Dor types . DAs repor types ,86. Cabewith age . In hypewith age . In a lawith age . After 1with age . On theswith age . In acrowith age ,92. In awith age . In Cushwith age . Cabergowith age ,96,97. Iwith age ,99.There are also few evidences about the potential antitumor activity of DA in non-pituitary origin NENs. In 1992 Farrell observed promising effect of bromocriptine on regulation of ACTH precursor secretion in small cell lung cancer cell line . SubsequSSTs and DRs in NENs. Altogether, in vitro autoradiography, real-time PCR and immunohistochemistry analyses have detected a large expression of SSTs and DRs in several types of human NENs. The most prevalent SST in NENs is SST2, expressed in almost all pituitary NENs [SST3 as the most expressed SSTs [SST5 [SST2 is found in 80–100% of GEP NENs, whereas it is expressed in 50–70% of insulinomas [2R expression has been demonstrated by immunohistochemistry in several pituitary adenomas [SSTs in NENs. SSTs and D2R co-expression has been demonstrated in BON-1, a pancreatic neuroendocrine cell line, and in several GEP NENs [SST2, SST5 and D2R specifically in a broad variety of NENs including bronchial, GEP NENs and ovarian carcinoid, in particular with higher expression of D2R in low-grade rather than high-grade [The well-studied signaling cascade of both somatostatin and dopamine in regulating hormone secretion inhibition has built-up one of the main cornerstones in the treatment of NENs. Several studies evaluated the expression profile of both ary NENs . NFPAs ssed SSTs . ActuallTs [SST5 ,106. Morulinomas . On the adenomas ,109,110 adenomas ,112. FolGEP NENs ,114. Recgh-grade ,117,118.SSTs and DRs have been showed to interact via hetero- or homo- oligomerization. It has been demonstrated that heterodimerization of D2R and SST5 resulted in an enhanced functional activity and, more recently, also heterodimerization of D2R with SST2 has been reported [2R and SSTs (SST2 and SST5) heterodimers display a reciprocal influence on their effectors and an increased ability to their response [It is worthy of note that reported ,119. Actresponse . These hresponse . Specularesponse ,123,124.SSTs and DRs due to the functional interactions between these receptors. On these assumptions, novel chimeric compounds . Savenau et al. [SST2) or BIM-53097 (full agonist for D2R), either alone or in combination, in suppressing both GH- and PRL secretion in primary pituitary adenoma cells from patients previously treated with octreotide and/or quinagolide (a selective D2R agonist). The reason for the enhanced potency seems to be related to the heterodimerization of SSTs and DRs creating a novel receptor with distinct functionality. In 2003, another study analyzed mechanisms of action of BIM-23A387 in both human and rat pituitary adenoma primary cells. Ren et al. [2R antagonist, but not SST2 antagonist, abolished the SST2-active component of BIM-23A387 to inhibit GH secretion. In addition, the structure of the D2R/SST2 heterodimer might be different from the individual receptor forms, since it showed peculiar features: the heterodimer binding domain had a higher affinity to DA-selective ligand and the intracellular signaling occurred mainly via SST2 pathways [2R signaling in primary cultures of DA-resistant prolactinomas, since its action did not provide an enhanced response when SST2 was successfully overexpressed [The first chimeric compound was synthetized by Ipsen Biomeasure, Inc. . This molecule, named BIM-23A387, combines structural elements of both SSAs and DAs. Based on above mentioned studies on receptor expression profile, BIM-23A387 retains affinity for u et al.  demonstrn et al. , showed pathways . Interespathways  tested spathways . These rpathways . In thispathways . Florio pathways . Moreovepathways ,131. In pathways . Neverthpathways . BIM-23Axpressed . In primxpressed , while Zxpressed . Neverthxpressed ,137. Subxpressed . BIM-23Axpressed . Very rexpressed .Unfortunately, this evidence about BIM-23A760 failed to bring positive results in clinical human trials. Although the compound showed a clean safety profile in phase I and a significant therapeutic efficacy in a phase IIa single-dose study in patients with acromegaly, after repeated administration, BIM-23A760 produced a long-lasting and highly potent dopaminergic metabolite. Circulating accumulation of this metabolite gradually reduced the effects of the parent compound BIM-23A760, compromising its GH suppression activity ,141.2R and SST2 and a partial agonist at SST5  [SST-D2R multi-receptor targeting drugs were able to inhibit CgA and serotonin secretion, but not cell growth [SSTs and no interference with the chimera (TBR-065) activity [In order to overcome clinical limitations, recently, a novel chimeric compound named TBR-065 (formerly BIM-23B065), was synthetized by IPSEN Pharma . It is a full agonist at D at SST5 . Testingd NFPAs)   . In thisl growth . Recentll growth . Newly, l growth . In anotactivity . Due to activity . Furtheractivity ,149.In the view of these findings, further research is needed for TBR-065, since it appears to be the most promising somatostatin-dopamine chimera with potential clinical applications in NENs.SSTs and DRs displayed a large co-expression in several types of NENs. The most abundant receptors subtypes were SST2, SST5, and D2R. Interestingly, the formation of heterodimers between these receptors is associated with an enhanced antitumor activity. This evidence promoted the research for the development of new chimeric multi-target molecules. TBR-065 targeting SST2, SST5 and D2R appear to be a new molecule with relevant perspectives in the therapy of well-differentiated NENs, as shown by several preclinical and preliminary clinical studies. The great capability of TBR-065 to decrease in vivo GH, IGF-1 and PRL serum levels is encouraging.After almost twenty years from the development of the first somatostatin-dopamine chimeric molecule, prominent positive conclusions in the neuroendocrinology field can be drawn. First, it became clear the central role of SSAs and DAs in the therapy of well-differentiated NENs. Second, it has been observed that both 2R of available somatostatin-dopamine chimeric molecules is lower compared to that reported for the SST2. The development of new molecules with higher affinity for D2R could enhance the antitumor activity of these compounds.Nevertheless, the knowledge of these chimeric molecules is leaving behind some outstanding questions which may be worth elucidating in further studies. For instance, both the hypothetical signal transduction pathway, that we have described in In conclusion, despite the encouraging read-out about TBR-065, additional clinical studies are required to clearly define the safety, antitumor activity and the place of this molecule in the therapeutic algorithm of patients with NFPA, acromegaly, and advanced well-differentiated NENs.


Kidney function decline is one of the complications of diabetes mellitus and may be indicated as diabetic nephropathy (DN). DN is a chronic inflammatory disease featuring proteinuria and a decreasing glomerular filtration rate. Despite several therapeutic options being currently available, DN is still the major cause of end-stage renal disease. Accordingly, widespread innovation is needed to improve outcomes in patients with DN. Chemokines and their receptors are critically involved in the inflammatory progression in the development of DN. Although recent studies have shown multiple pathways related to the chemokine system, the specific and direct effects of chemokines and their receptors remain unclear. In this review, we provide an overview of the potential role and mechanism of chemokine systems in DN proposed in recent years. Chemokine system-related mechanisms may provide potential therapeutic targets in DN. Kidney failure is one of the major serious complications from diabetes mellitus (DM). About 30% to 40% of patients with diabetes develop diabetic nephropathy (DN), and DM accounts for 30% to 50% of the incident cases of end-stage renal disease (ESRD) in the United States . DN deveTreatment strategies currently target glycemia, blood pressure, the renin–angiotensin system (RAS), blood cholesterol and so on, which can be initiated once abnormal albuminuria and renal function are detected . The inhInflammation has been suggested to be a novel mechanism linked to the development of DN . MacrophChemokines, small molecular weight chemotactic cytokines, and their receptors play key roles in the recruiting and physiological direction of cell migration. They can be broadly divided into four subfamilies—the CC, C, CXC, and CX3C families—according to their N-terminal cysteine-motifs . The CXCChemokines are shown to be closely related to DN in clinical settings. Mesenchymal stem cells can orchestrate local and systemic innate and adaptive immune responses through the release of chemokines when exposed to an inflammatory environment in DN . In DN aThe most well-known CC chemotactic chemokine family is CCL2, also called macrophage chemokine-1 (MCP-1), which is suggested to reflect the level of tubular injury and renal inflammation in DN ,19,20. Tγ and activated NF-κB signaling, accompanied by up-regulated CCL2 and IL-6 [Several studies indicate that DN could be attenuated or improved in combination with decreased levels of CCL2, suggesting the potential role of CCL2 in the progression of DN ,17,23,24and IL-6 . Furtherand IL-6 . In humaand IL-6 . Fetuin-and IL-6 . The lacand IL-6 . High glucose or TGF-β1-induced IL-20 leads to apoptosis by activating caspase-8. Meanwhile, IL-20 can up-regulate MMP-9, CCL2, TGF-β1 and vascular endothelial growth factor (VEGF) expression in podocytes . TGF-β1 The inhibition of TLR4 prevents the release of CCL2 and keratinocyte chemoattractant and decreases the podocyte death induced by palmitic acid alone or in combination treatment with Fetuin-A . MoreoveDianthus superbus-EtOAc soluble fraction has a renoprotective effect with decreasing albumin excretion, plasma creatinine, kidney injury molecules-1 (KIM-1), C-reactive protein, TGF-β, and CCL2 levels. It can also directly reduce inflammation and fibrosis in human renal mesangial cells [In STZ-induced DN mice, a glucagon-like peptide-1 (GLP-1) analog attenuates the levels of ROS, proinflammatory cytokine and chemokine including TNF-α, IL-1β, CCL2, ICAM-1, and fibrosis-related molecules including TGF-β1 and fibronectin with reduced tubular injury and macrophage infiltration . Furtheral cells . More inal cells .The NADPH oxidase-derived production of ROS in the kidney plays a role in modulating DN. The vascular smooth muscle cell and mesangial cell-specific overexpression of Nox5 isoform of NADPH oxidase leads to increased glomerular ROS production, glomerulosclerosis, mesangial expansion, and ECM accumulation with increased macrophage infiltration and CCL2 levels in a mouse model of DN . AdditioIn a clinical setting, the CCL2-2518 GG genotype and G allele increase the risk of progression to ESRD in type 2 DM . ElevateIn clinical trials, the CCL2 inhibitor emapticap pegol and the Janus kinase (JAK)1/JAK2 inhibitor baricitinib seem to be promising drugs for DN . BaricitIn summary, CCL2 has direct signaling effects on cell migration, proliferation, and differentiation . The inhPrevious studies also provided the explanation for the role of other chemokines in the CC chemotactic family in DN. For example, plasma levels of plasminogen activator inhibitor-1, syndecan-1, VEGF, IL-1β, interleukin-1 receptor antagonist, and CCL4 are increased in type 1 DM patients with kidney failure . The renSeveral members of the chemokine CXC family, including CXCL1, 7, 8, 9, 10, 11, 12, and 16, may be involved in diabetic nephropathy. The expression of CXCL1 is increased in podocytes of OVE, db/db and STZ diabetic mouse models . The glomerular endothelium dysfunction plays an important role in the pathogenesis of early DN. In primary rat glomerular endothelial cells, platelet microparticle-derived CXCL7 results in glomerular endothelial injury, which can be reversed by CXCL7 inhibition with a CXCL7 neutralizing antibody .CXCL8 inhibition improves the urine volume, urine albumin/creatinine ratio, blood urea nitrogen, and creatinine clearance rate with decreased mesangial expansion, glomerulosclerosis, and extracellular matrix deposition in DN mice. Furthermore, CXCL8 inhibition attenuates high glucose-induced inflammatory and fibrotic-related proteins in human renal mesangial cells . Serum and urine levels of CXCL9 are enhanced in patients with DN compared with healthy controls. Urinary CXCL9 and CXCL11 mRNA levels correlate with baseline renal function. The rate of renal function decline is associated with urinary CXCL9 mRNA level in DN . AGEs dePlasma CXCL10 levels are decreased in diabetic db/db mice compared with non-diabetic mice; nevertheless, the concentrations of CXCL9 and CXCL11 are similar. Furthermore, recombinant murine CXCL10 can reduce mesangial and peritubular matrix expansion, albuminuria, and glomerular hypertrophy in vivo. In the in vitro part, CXCL10 can inhibit kidney fibroblast collagen and TGF-β production under high glucose conditions . In contDPP-4 inhibitor increases GLP-1 and CXCL12 in type 2 diabetes patients with or without DN compared with the placebo group, suggesting its pro-vasculoregenerative ability in type 2 DM . The DPPCompared with untreated db/db mice, db/db mice, which received casein, have accelerated tubulointerstitial injury with increased secretion of ECM and cholesterol accumulation in tubulointerstitium, which is accompanied by the induction of the CXCL16. In HK-2 cells under high glucose conditions, CXCL16 knocked down by siRNA can reverse IL-1β-induced cholesterol accumulation, ROS production, and ECM protein expression . IncreasIn summary, anti-chemokines may be potential therapeutic targets due to the majority of chemokines in the CXC family seeming to be pro-inflammation and related to disease progression in DN. Nevertheless, the roles of CXCL10 and CXCL12 are multifaceted.In the chemokine CX3C family, CX3CL1, also known as fractalkine, is mainly produced by glomerular endothelial cells and the tubular epithelium, which can be also detected in podocytes, mesangial cells, renal tumor cells and stromal cells . CX3CL1 Inflammatory cell recruitment, infiltration, and activation play a critical role in the development of DN. CCL2 can activate C-C chemokine receptor type 2 (CCR2) to stimulate the release of monocytes from the bone marrow and lead to the recruitment of monocytes to inflammation area . The majIn an STZ-induced diabetic mouse model with hypertension, the combination treatment of CCR2 antagonist and ACEi exhibits better renal protection than ACEi alone . In diabIn DN patients, CCR2 expression is up-regulated by glomerular podocytes, suggesting the potential role of CCR2 beyond the recruitment of monocytes . A previC-C chemokine receptor type 5 (CCR5) is a receptor for CCL5. Increased CCR5 expressions are observed in inflammatory kidney diseases and transplant rejection . HoweverChemokine CXC motif receptor (CXCR) 1 and CXCR2 inhibitors can reverse CXCL7-induced injury in primary rat glomerular endothelial cells . CXCL8 bChemokine (CX3C motif) receptor 1 (CX3CR1) is expressed on mononuclear cells, lymphatic leucocytes and especially in inflammatory renal tissues ,110. A hMoreover, the atypical chemokine receptor ACKR2 is a chemokine scavenger receptor for several CC chemokines, which does not induce typical downstream G-protein mediated signaling pathways. ACKR2 can bind CC-chemokines, such as CCL2, CCL5, CCL3, CCL4, CCL7, CCL8, CCL11, CCL13, CCL17, CCL22, CCL23, and CCL24, and then leads to their degradation and reduces local inflammatory chemokine expressions as a result ,115. LevIn this review article, we have cited most studies related to chemokines and chemokine receptors in DN over the past five years . Recent


TBX1 gene haploinsufficiency. Other genes, implicated in thymic organogenesis include FOXN1, associated with Nude SCID syndrome, PAX1, associated with Otofaciocervical Syndrome type 2, and CHD7, one of the genes implicated in CHARGE syndrome. More recently, chromosome 2p11.2 microdeletion, causing FOXI3 haploinsufficiency, has been identified in 5 families with impaired thymus development. In this review, we will summarize the main genetic, clinical, and immunological features related to the abovementioned gene mutations. We will also focus on different therapeutic approaches to treat SCID in these patients.Combined Immunodeficiencies (CID) are rare congenital disorders characterized by defective T-cell development that may be associated with B- and NK-cell deficiency. They are usually due to alterations in genes expressed in hematopoietic precursors but in few cases, they are caused by impaired thymic development. Athymia was classically associated with DiGeorge Syndrome due to  Foxn1) expression (Pax1), Eyes absent homolog 1 (Eya1), sine oculis homeobox (Six), homeobox A3 (Hoxa3), and T-box 1 (Tbx1) drive the outgrowth of the thymic epithelial anlage from the 3rd PP  might be implicated in the development of both neural crest cell-derived mesenchyme and pharyngeal endoderm-derived thymic epithelial cells (TECs). Pax3 and Hoxa3 expression in mesenchymal cells allows the detachment of the thymic lobes from the pharynx (CCL25), delta like canonical Notch ligand 4 (Dll4), and Hoxa3, allowing thymocyte recruitment and TECs differentiation in cTECs and mTECs. Mesenchymal cells are implicated in the recruitment of hematopoietic thymic seeding progenitors, as well  . The epie 3rd PP . Hoxa3 aal cells –8. Studi pharynx , 10. Thycells Chd might be pharynx –14. In t as well , 16. Thy as well –19. In s as well , 21, tha as well .FOXN1, PAX1, TBX1, CDH7 impair T-cell development in humans (Bone marrow derived hematopoietic stem cells (HSCs) enter the thymus through cortico-medullary junction, where they proliferate . The VD, 25. Memn humans . AlteratFOXN1 is located on chromosome 17q11.2 and it is composed of eight exons (30 kb) , implicated in the recruitment of hematopoietic progenitors and of molecules, as DLL4 notch ligand, implicated in maturation of the progenitors toward the T-cell line  , 38. Thi (30 kb) . During and TECs , 41. FOXand TECs  42–44).–44.FOXN1ell line  45–47).–47.FOXN1ell line , 49 by iell line . FOXN1 aelection . Knockoufunction . Foxn1 e and Wnt , 54.in vitro , 72PAX1  balance . In miceficiency . On the  to SCID . The dif lacking .PAX1 mutations. Different biallelic deleterious PAX1 variants cause OTFCS2 and SCID, characterized by absent thymic shadow, chronic diarrhea, recurrent respiratory infections, pneumonia, and also Omenn Syndrome  is an autosomal dominant disorder characterized by short stature, facial dysmorphism , shoulder girdle abnormalities, hearing loss, and mild intellectual disability . Two difSyndrome . DiffereSyndrome , 71, 77.The immunological phenotype of patients with OTFC2 was described very recently in six patients who showed T-cell lymphopenia, impaired proliferative response to mitogens, and normal levels of B and NK cells. In one of them, 98% of CD4 and CD8 T cells were CD45RO+ cells, while CD45RA+CD31+ cells and TRECs were undetectable. TCRVβ analysis showed an oligoclonal spectrum. Lymph node biopsy of another patient showed absence of germinal centers and almost total absence of CD3+ T cells. Patients with Omenn Syndrome also showed eosinophilia and increased IgE levels .de novo microdeletion of 22q11.2  (TBX1 mutations may be rarely reported in patients with DGS (TBX1 gain-of-function mutations can result in the same phenotypic spectrum of loss-of-function mutations (DiGeorge syndrome (DGS) is usually associated with 3 or a 1.5 Mb  22q11.2 , 79. In  9 exons  and enco 9 exons , 82. It  9 exons , 84, in  9 exons . It is i 9 exons . However 9 exons . On the  9 exons . Reduced 9 exons . TBX1 re 9 exons . Ffg recde (Whn)  89). Mo. Mode node (Whn) . Isolatewith DGS  and TBX1utations .Tbx1 and other molecules implicated in thymus organogenesis including Pax1, Pax9, Hoxa3, Fgf8, and Bmp4 (ZNF717), MicroRNA-1243 (MIR-1243), and MIR-4273 genes was identified (DGCR8) haploinsufficiency, is implicated in the pathogenesis of immunological, cardiac, endocrinological, and neurological phenotype (CRKL), a gene encoding a 39-kDa adapter protein belonging to the Crk family, implicated in many cellular functions, including cell migration and adhesion, are associated with impaired T-cell proliferation in response to TCR triggering , recombinase activating gene 1 (Rag1)  syndrome is associated to haploinsufficiency in ome 8q12 . CHD7 isome 8q12 . Impairehe heart , 123. CHpression  124). C. CCHD7 gpression , 125.CHARGE syndrome is characterized by different degrees of thymic alterations and even by complete thymic aplasia, resulting in combined immune deficiency . A wide Most of the patients have increased risk of recurrent infections, including recurrent otitis media, sinusitis, conjunctivitis, dermatitis, respiratory tract infections, pneumonia, and sepsis . Severe FOXI3, a member of FOX family transcription factors, implicated in development of brachial arch-derived structures was considered the candidate gene for this phenotype  represents the cornerstone for the treatment of SCID. However, in SCID due to genetic defects that impair development and function of the thymic epithelium, theoretically thymus transplantation would represent the most appropriate therapy. Thymic tissue is obtained from infants undergoing to median sternotomy for open heart surgery. Cultured postnatal human thymic tissue is then transplanted in thin slices into the quadriceps muscle . In casesurvived , 144 whisurvived , 140.FOXN1 deficient patients underwent HSCT and two survived . In other cases, HSCT may not be curative since the defect involves thymus development. However, genome-wide association studies have shown that a large proportion of variants likely to cause human disease are located outside of the protein-coding domains, so whole genome wide approaches might lead to proper identification and thus correct treatment plans for immunodeficiency disorders resulting from aberrant expression of some of the genes discussed in the present review.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. GG, CB, MD, EC, and RR reviewed the literature, organized and wrote the manuscript. CP wrote the manuscript and supervised all the work. RP did the picture. All authors reviewed and approved 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.


Circular RNAs (circRNAs) identification, expression profiles, and construction of circRNA-parental gene relationships and circRNA-miRNA-mRNA ceRNA networks indicate that circRNAs are involved in flag leaf senescence of rice.Circular RNAs (circRNAs) are a class of 3′-5′ head-to-tail covalently closed non-coding RNAs which have been proved to play important roles in various biological processes. However, no systematic identification of circRNAs associated with leaf senescence in rice has been studied. In this study, a genome-wide high-throughput sequencing analysis was performed using rice flag leaves developing from normal to senescence. Here, a total of 6612 circRNAs were identified, among which, 113 circRNAs were differentially expressed (DE) during the leaf senescence process. Moreover, 4601 (69.59%) circRNAs were derived from the exons or introns of their parental genes, while 2110 (71%) of the parental genes produced only one circRNA. The sequence alignment analysis showed that hundreds of rice circRNAs were conserved among different plant species. Gene Ontology (GO) enrichment analysis revealed that parental genes of DE circRNAs were enriched in many biological processes closely related to leaf senescence. Through weighted gene co-expression network analysis (WGCNA), six continuously down-expressed circRNAs, 18 continuously up-expressed circRNAs and 15 turn-point high-expressed circRNAs were considered to be highly associated with leaf senescence. Additionally, a total of 17 senescence-associated circRNAs were predicted to have parental genes, in which, regulations of three circRNAs to their parental genes were validated by qRT-PCR. The competing endogenous RNA (ceRNA) networks were also constructed. And a total of 11 senescence-associated circRNAs were predicted to act as miRNA sponges to regulate mRNAs, in which, regulation of two circRNAs to eight mRNAs was validated by qRT-PCR. It is discussed that senescence-associated circRNAs were involved in flag leaf senescence probably through mediating their parental genes and ceRNA networks, to participate in several well-studied senescence-associated processes, mainly including the processes of transcription, translation, and posttranslational modification , oxidation–reduction process, involvement of senescence-associated genes, hormone signaling pathway, proteolysis, and DNA damage repair. This study not only showed the systematic identification of circRNAs involved in leaf senescence of rice, but also laid a foundation for functional research on candidate circRNAs.The online version contains supplementary material available at 10.1007/s00425-020-03544-6. With rapid development of high-throughput sequencing technology, a large number of noncoding RNAs, such as long noncoding RNAs and microRNAs, were identified and confirmed as the crucial regulator for gene expression and biological function in different biological processes  resulted in a decreased abundance of the parental gene phytoene synthase 1, and overexpression of a circRNA derived from phytoene desaturase showed similar results  could increase the abundance of the cognate exon 6-skipped alternative splicing variant (SEP3.3) by forming R-rings with its own genomic locus. The overexpression of this circRNA led to the formation of flowers with increased petals but fewer stamens , a monocotyledonous model organism, is one of the major food crops. Thus, deciphering leaf senescence in rice is of great importance for understanding its molecular regulatory mechanism. In the past years, enough efforts to reveal the molecular mechanisms underlying leaf senescence have been mainly made by transcriptomic (mRNA), proteomic and metabolomic researches  circRNAs were identified and functional enrichment analysis of their parental genes was performed. Subsequently, 39 DE circRNAs were considered to be highly associated with leaf senescence through WGCNA. Furthermore, circRNA-parental gene regulatory relationships and circRNA-miRNA-mRNA ceRNA networks of these senescence-associated DE circRNAs were constructed to decipher their functions on leaf senescence of rice. This study not only showed the systematic identification of circRNAs involving in leaf senescence of rice, but also laid a foundation for functional research on candidate circRNAs.Oryza sativa L. subsp. indica) was used as the experimental material in this study. The 93-11 seeds were maintained in our laboratory by strict selfing.Rice cultivar 93-11 , flowering stage (FL2), filling stage (FL3), milking maturity stage (FL4) and dough stage (FL5) were collected, respectively. Three biological replicates were used for samples in each stage, with each sample pooled with three flag leaves from three independent plants, flash-frozen in liquid nitrogen, and stored at − 80 °C for subsequent analysis.Total RNAs from each sample at five stages were extracted with TRIzol reagent  following the manufacturer’s instructions, respectively. Subsequently, the purity and concentration of total RNAs were detected using NanoDrop 1000 . Then, the integrity of total RNAs was evaluated using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 . To ensure the application of qualified samples for sequencing, samples with RNA integrity number ≥ 7 were used for subsequent analysis.® UltraTM Directional RNA Library Prep Kit for Illumina®  according to manufacturer’s instructions and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was conducted on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS  following the manufacturer’s instructions.After quality confirmation of the total RNAs, the library construction for circRNAs was performed according to the protocol as previously described .http://rice.plantbiology.msu.edu/) using the HISAT2 software (version 2.0.5). The reads that could not be mapped to the genomes were obtained. For these unmapped reads, the 20-nt anchors were first extracted from both ends and aligned independently to the rice reference genomes to identify the unique anchor positions by a widely using find_circ software  from our transcriptome, the clean reads were mapped to the rice reference genome using HISAT2 software  as an indicator of their expression levels. To calculate the expression of protein-coding transcripts, the fragments per kilobase of transcript per million mapped reads (FPKMs) value were calculated by StringTie software (1.3.1). And a pairwise differential expression analysis between any two stages  was performed using the DESeq R package (1.10.1). In this study, circRNAs with a http://ibi.zju.edu.cn/plantcircbase/). Then, we analyzed the genomic sequences and back-splicing sequences of circRNAs in the PlantcircBase database to seek for the sequence regulation. Results showed that the genomic sequences linked head with tail to form the complete circRNAs. The back-splicing sequences were produced by the combination of 100 nt at the tail end of the genomics sequences following with 100 nt at the front end of the genomics sequences once the genomics sequences were over 200 nt, thus the junction sites were located in the middle of back-splicing sequences. When the length of genomic sequences was less than 200 nt, the second half of the genomic sequence was moved to the front of the first half to assemble into back-splicing sequences, making the junction sites be located in the middle of back-splicing sequences and the length of back-splicing sequences be equal to those of genomic sequences. Accordingly, we reassembled all identified circRNAs in our study into back-splicing sequences following this strategy. The BLAST analysis  was performed to find the conserved circRNAs using the reassembled back-splicing sequences as the query , Pfam , KOG/COG , Nr , Swiss-Prot , and KEGG  were used to perform function annotation of parental genes. GO enrichment analysis on the parental genes of DE circRNAs was carried out using the topGO R packages (version 2.7). Statistical enrichment on the parental genes of DE circRNAs in KEGG biological pathways was performed using KOBAS software . Subsequently, the identified DE circRNAs and DE mRNAs were used as target prediction library. Then, psRNATarget was used with default parameters to identify miRNA target binding sites  with divergent primers. Second, total RNA was isolated using Trizol reagent (Ambion). Then, total RNA solutions without DNA contamination were subjected to reverse transcriptase reactions with the PrimeScript RT Reagent Kit  according to manufacturer’s protocol. To validate circRNAs of flag leaf in rice, PCR was performed using divergent and convergent primers. Convergent primers were used as positive controls for linear transcripts, and divergent primers were used to detect the candidate circular template. For each PCR amplification, cDNA or genomic DNA was used with T3 DNA polymerase , and 36 cycles of PCR were performed to amplify back-spliced junction sites of circRNAs. The PCR products were separated using 1% agarose gel and Sanger sequencing were carried out to further confirm the junction reads by Sangon Biotech Company .UBC (LOC_Os02g42314), ARF (LOC_Os05g41060) and Profilin-2 (LOC_Os06g05880) were used as internal reference genes to normalize the qRT-PCR data  was performed using SYBR Green PCR kit (TaKaRa) on a CFX 96 Real-Time PCR system (Bio-Rad) to validate the expression levels of circRNAs and protein-coding genes according to the manufacturer’s instructions. Three genes, Oryza sativa reference genome ranged 98.61–99.79% , flowering stage , filling stage , milking maturity stage  and dough stage  were individually constructed, with three biological replicates for each stage . A genome-wide high-throughput RNA sequencing on these 15 libraries was performed using Illumina Hiseq 2500 platform. More than 110 million raw reads were generated from each library, respectively . Among these identified 6612 circRNAs, 1234, 1320, 1993, 1669, 2151 were, respectively, detected in FL1, FL2, FL3, FL4 and FL5 stage, which had the largest number of identified circRNAs in dough stage (FL5) than others Fig. . BesidesThe features of these 6612 identified circRNAs were further analyzed. Genomic origin analysis showed that 4151 (62.78%) of the identified circRNAs were exonic circRNAs, 450 (6.81%) were intronic circRNAs, and that the remaining 2011 (30.41%) were intergenic circRNAs , 35 (FL1 vs. FL3), 15 (FL1 vs. FL4), 41 (FL1 vs. FL5), 29 (FL2 vs. FL3), 15 (FL2 vs. FL4), 42 (FL2 vs. FL5), 27 (FL3 vs. FL4), 29 (FL3 vs. FL5) and 33 (FL4 vs. FL5) DE circRNAs were identified in these ten groups, respectively , among which 7, 23 and 33 GO terms were classified under cellular component, molecular function and biological process, respectively . To further investigate the potential functions of the identified DE circRNAs involving in flag leaf senescence, the functional enrichment analysis on these 50 predicted parental genes of 51 DE circRNAs was performed. The Gene Ontology (GO) enrichment analysis showed that these parental genes were assigned to 63 GO terms ’, ‘ABC transporters (ko02010)’ and ‘Nicotinate and nicotinamide metabolism (ko00760)’ (Suppl. Table S6). The result may imply that parent genes of circRNAs in flag leaf of rice were involved in oxidation–reduction process and signal transduction.To systematically explore the potential regulation functions of circRNAs associated with flag leaf senescence, WGCNA was performed to identify gene sets associated with a specific biological process. First, the protein-coding transcripts were identified by rRNA-depleted library RNA-seq similar to the identification of circRNAs. Then, the DE mRNA transcripts were obtained by pairwise comparison (Suppl. Table S7). Next, the TPM matrix of DE circRNA transcripts and FPKM matrix of DE mRNA transcripts were combined. After bioinformatics processing, a total of seven modules were obtained, in which major tree branches define the modules (labeled with different colors), as shown in the dendrogram Fig. a. The moIn addition, the heatmaps of ‘MEbrown’ module, ‘MEblue’ module and ‘MEturquoise’ module showed the comprised transcripts which were the most highest expressed in flag leaf at booting stage , dough stage  and filling stage , respectively  of the circRNA 3:34,220,472|34,220,857, was identified to be a TALE transcription factor (Table Os03g0348200) of the circRNA 3:14,657,773|14,658,742 was annotated to possess terpene synthase activity (Table Os01g0830700) of circRNA 1:38,955,712|38,956,176 was identified to be a leaf senescence-related protein (Table Os11g0671000) of circRNA 8:29,762,968|29,763,202 was annotated to encode auxin-responsive protein (Table Os01g0559600) of circRNA 1:23,496,730|23,497,935 was annotated to possess cysteine-type endopeptidase activity (Table Os04g0429850) of circRNA 4:19,220,719|19,225,326 was annotated to be involved in NAD biosynthesis (Table Os10g0536450) of circRNA 10:19,878,758|19,879,082 was predicted to participate in the oxidation–reduction process (Table Os10g0521700) of circRNA 10:19,167,135|19,167,568 was annotated to be involved in signal transduction (Table Os03g0732100), and that of up-expressed circRNA 1:23,496,730|23,497,935 and its parental gene BGIOSGA003766 (Os01g0559600), and that of turn-point circRNA 4:19,220,719|19,225,326 and its parental gene BGIOSGA016430 (Os04g0429850) were verified by qRT-PCR  . Seventeen target genes were annotated to be related with posttranslational modification and another eight were with protein glycosylation. Twenty-two target genes were related with oxidation–reduction process. Eighteen hormone-related genes were involved in the signaling pathways of jasmonic acid, salicylic acid, abscisic acid, ethylene, cytokinin and auxin. Six target genes were associated with proteolysis and three with DNA damage repair. Thus, the senescence-associated circRNAs might play important roles in flag leaf senescence by regulating a large number of mRNAs related to senescence processes through the ceRNA regulatory network.A GO functional enrichment analysis was performed using all these protein-coding genes in ceRNA networks. The results suggested that these mRNAs were involved in many biological processes associated with leaf senescence, including ‘protein phosphorylation ’, ‘oxidation–reduction process ’, ‘response to abscisic acid ’, ‘fatty acid biosynthetic process ’, ‘signal transduction ’, ‘membrane lipid metabolic process ’, ‘cytokinin-activated signaling pathway ’, ‘lipid catabolic process ’, and ‘negative regulation of programmed cell death ’ (Suppl. Table S8). In addition, since many biological processes are reported to be associated with leaf senescence, according to the annotation information for mRNAs, these protein-coding genes in ceRNA networks were further classified into different categories referring to those well-known senescence-related biological processes, including transcription regulation, posttranslational modification , oxidation–reduction process, hormone signaling pathway, proteolysis and DNA damage repair (Suppl. Table S9). Eventually, a total of 13 target genes were various transcription factors, belonging to NAC, bHLH, HD-ZIP, SBP, ZF-HD, WRKY, MYB and ERF transcription factor families identified by searching on Plant Transcription Factor Database (ONAC022), BGIOSGA002195 (cytokinin oxidase 2), BGIOSGA026629 (auxin-responsive gene) and BGIOSGA000214 (probable glucuronosyltransferase), all showing higher expression levels at the turn-point stage (FL3) of the leaf senescence process , BGIOSGA029541 (SBP family transcription factor), BGIOSGA004410 (glycosyltransferase), and BGIOSGA017903 (glycosyltransferase) decrease with the flag leaf developing to senescence    Fig. . HoweverOryza sativa and Arabidopsis thaliana, more than 300 orthologous parental genes generating circRNAs from a similar position were identified, which may imply that circRNAs in plants have the conservation feature as in animals . Generally, the sequence conservation of circRNAs demonstrated that these circRNAs may have similar potential biological functions, and the functions of these conserved circRNAs in the plants need further investigation and verification.An increasing number of studies have reported that the sequence conservation of circRNAs was widespread in various plants. For example, in an conservation analysis for two model plants, Functional studies on circRNAs are very limited, particularly in various plants. Here, a WGCNA method was employed to investigate the co-expression network of 113 DE circRNAs and 9412 DE protein-coding mRNAs and further inferred the potential biological functions of circRNAs. Interestingly, three out of seven modules were considered to be highly associated with flag leaf senescence Fig. . The DE Os03g0732100) of a down-regulated circRNA 3:34, 220, 472|34, 220, 857 was identified to be a TALE transcription factor  of a down-regulated circRNA 3:14, 657, 773|14, 658, 742 was annotated to possess terpene synthase activity  of a turn-point circRNA 4:19,220,719|19,225,326 was annotated to be a nicotinate phosphoribosyltransferase (Table Os10g0536450) of turn-point circRNA 10:19, 878, 758|19, 879, 082 was also found to involve in the oxidation–reduction process, which may imply its corresponding role in leaf senescence  of an up-regulated circRNA 1:38,955,712|38,956,176 in this study was annotated to be a leaf senescence-associated protein with O-acetyltransferase activity on the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/), which may indicate its roles in leaf senescence (Table Os05g0136900) of a turn-point high-expressed circRNA 5:2, 405, 419|2, 405, 859 was annotated with N-acyltransferase activity (Table Os01g0559600) of up-regulated circRNA 1:23, 496, 730|23, 497, 935 was annotated to possess cysteine-type endopeptidase activity  of up-regulated circRNA 5:23, 221, 948|23, 223, 370 was annotated to be an arginyl-tRNA-protein transferase  of up-regulated circRNA 6:30,705,916|30,706,427 was annotated to encode DNA repair protein REV1, which may play role in leaf senescence through involving in DNA repair pathway , encoding a probable GTP-binding protein OBGC1, was regulated by up-regulated circRNA 7:26, 274, 523|26, 274, 796  was identified to be transcription factor ONAC022 and possibly regulated by circRNA 4:15, 277, 633|15, 363, 855 (Suppl. Table S9). In addition, the HD-ZIP gene family plays important roles in plant growth and development. It has been reported that overexpression of the HD-ZIP gene OsHox32 could result in pleiotropic effects on plant-type architecture and leaf development in rice  in our ceRNA network was identified to be a HD-ZIP member OsHOX27, which may possess the similar functions in leaf senescence. This transcription factor gene is predicted to be regulated by three circRNAs 1:31, 309, 262|31, 309, 883, 4:15, 277, 633|15, 363, 855, and 8:17, 248, 521|17, 266, 134 (Suppl. Table S9).It is well known that transcription factors play vital roles in leaf senescence. Accordingly, in this study, a total of 13 target genes in the ceRNA network were identified to be various transcription factors (Suppl. Table S9). In rice, overexpression of a stress-responsive NAC-type transcription factor premature leaf senescence 2 (PLS2), encoding a glycosyltransferase, is involved in leaf senescence  was successfully identified to catalyze the biosynthesis of UDP-N-acetylglucosamine, which is one of key substrates for N-glycan, and function inactivation of UAP1 induced early leaf senescence and defense responses in rice . Most importantly, increasing evidences have suggested that protein glycosylation play important roles in cell death or senescence. For example, rice Os05g0528000) is identified as the OsRBOHc gene, and two circRNAs 4:15, 277, 633|15, 363, 855 and 8:17, 248, 521|17, 266, 134 possibly participated in flag leaf senescence by regulating this gene (Suppl. Table S9). In addition, a novel gene, OZONE-RESPONSIVE APOPLASTIC PROTEIN1 (OsORAP1), enhances cell death in ozone stress in rice by influencing the production of reactive oxygen species  is the OsORAP1 gene, and it might be regulated by circRNA 4:15, 277, 633|15, 363, 855 in the ceRNA network (Suppl. Table S9). At last, a total of 22 protein-coding genes regulated by seven circRNAs in the ceRNA network were predicted to be involved in oxidation–reduction process (Suppl. Table S9).The generation of reactive oxygen species is one of the earliest responses of plant cells under abiotic stresses and senescence  was identified to be the cytokinin oxidase 2, and it was predicted to be regulated by three circRNAs 1:31, 309, 262|31, 309, 883, 4:15, 277,633|15, 363, 855, and 8:17, 248, 521|17, 266, 134 (Suppl. Table S9). Moreover, early studies indicate that auxins have a role in the suppression of leaf senescence  is a leaf senescence-associated gene encoding an auxin-responsive protein in rice , oxidation—reduction process, hormone signaling pathway, proteolysis and DNA damage repair Fig. b.XPH drafted the manuscript; HYZ provided the bioinformatics analysis; QW, RG and XZL performed the paddy field management and carried out the qRT-PCR experiments; WGK, HYS and JLL provided assistance in experiments and data analysis. YJH participated in the experimental design; ZHW provided the overall guidance and the manuscript revision. All authors read and approved the final manuscript.Supplementary file1 (JPG 1259 KB)Supplementary file2 (XLSX 17 KB)Supplementary file3 (XLSX 966 KB)Supplementary file4 (XLSX 2837 KB)Supplementary file5 (XLSX 850 KB)Supplementary file6 (XLSX 41 KB)Supplementary file7 (XLSX 18 KB)Supplementary file8 (XLSX 3048 KB)Supplementary file9 (XLSX 22 KB)Supplementary file10 (XLSX 64 KB)Below is the link to the electronic supplementary material.


Allergic asthma has been considered as a respiratory disorder with pathological features of airway inflammation and remodeling, which involves oxidative stress. Formononetin (FMT) is a bioactive isoflavone obtained from Chinese herb Radix Astragali, and has been reported to have notable anti-inflammatory and antioxidant effects in several diseases. The purpose of our study was to elaborate the effects of FMT on asthma and the underlying mechanisms. To establish allergic asthma model, BALB/c mice were given ovalbumin (OVA) sensitization and challenge, treated with FMT  or dexamethasone (2 mg/kg). The effects of FMT on lung inflammation and oxidative stress were assessed. In OVA-induced asthmatic mice, FMT treatments significantly ameliorated lung function, alleviated lung inflammation including infiltration of inflammatory cells, the elevated levels of interleukin (IL)-4, IL-5, and IL-13, immunoglobulin (Ig) E, C-C motif chemokine ligand 5 , CCL11 , and IL-17A. In addition, FMT treatments eminently blunted goblet cell hyperplasia and collagen deposition, and remarkably reduced oxidative stress as displayed by decreased reactive oxygen species (ROS), and increased superoxide diamutase (SOD) activity. Furthermore, to clarify the potential mechanisms responsible for the effects, we determined the inflammation and oxidation-related signaling pathway including nuclear factor kappa β (NF-κB), c-Jun N-terminal kinase (JNK), and the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). FMT treatments appeared to dramatically inhibit the activation of NF-κB and JNK, significantly elevated the expression of heme oxygenase 1 (HO-1) but failed to activate expression of Nrf2. In conclusion, our study suggested that FMT had the therapeutic effects in attenuating airway inflammation and oxidative stress in asthma. Asthma is a chronic respiratory disorder of the conducting airways where the epithelial barrier working together with the adaptive and innate immune cells respond to a diverse range of exogenous inhaled stimuli such as allergens and air pollutants . Asthma Oxidative stress is involved in the pathogenesis of asthma . AllergeA growing number of evidences implicate that nuclear factor kappa β (NF-κB) signaling is involved in airway inflammation. In response to allergen challenge, cytokines, chemokines, and bacterial and viral infections, NF-κB acting as a proinflammatory transcription factor binds to the proinflammatory genes promoter region, and thereby upregulates the expression of many mediators and growth factors important in inflammatory cascade of asthma . In addiAstragalus membranaceus var. mongholicus or A. membranaceus, described as Huangqi in Chinese herb), which has been common applied for treatment of airway inflammation and allergic disease  is a bioactive isoflavone isolated from Radix Astragali , bought from Shanghai Jiesijie Laboratory Animal Co. Ltd (license number: SCXK (Hu) 2013-0006), were accommodated in pathogen-free cage with constant temperature and humidity, food, and water available. All animal experimental protocols in our study were in accordance with the Guide for the Care and Use of Laboratory Animals, and ratified by the Animal Care and Use Committee of the Fudan University (authorization number: 2018-10-HSYY-DJC-01).We established OVA-induced murine model of allergic asthma and set doses of formononetin and dexamethasone based on previous studies with minor modification . Sixty mL) were recorded when mice were exposed to Methacholine  at increasing dose of 0, 3.125, 6.25, and 12.5 mg/ml.24 h after the last OVA challenge, mice were anesthetized with an intraperitoneal injection of 2% phenobarbital sodium (50 mg/kg) to maintain a spontaneous breath. After that, mice were tracheostomized, intubated, and placed in a whole-body plethysmograph connected to the ventilator  to detect lung function. To evaluate AHR, lung dynamic compliance (Cdyn) and airway resistance .Mice were anesthetized as above, and then intubated with a cannula inside the trachea. Bronchoalveolar lavage ﬂuid (BALF) was collected by intratracheal instillation with 300 µl aliquots of cold PBS twice, centrifuged at 500The left lung was resected, fixed with 4% phosphate-buffered formalin, embedded in paraffin, and cut into 4-µm sections. The tissues were stained with H&E, Masson’s trichrome and Periodic Acid Schiff (PAS). The images were viewed and captured under a light microscopy connecting to a digital camera with a magnification of 100. Inflammation was scored in the light of previous protocols . In brieIL-4, IL-5, IL-13, IL-17A, eotaxin, CCL-5, IgE levels in BALF were determined by ELISA Kits  in accordance with the manufacturer’s protocol.Inflammatory cells in lung tissue were analyzed to identify eosinophils (CD45+CD11b+Ly6C-SiglecF+), Th17 cells (CD45+CD4+IL-17A+), regulatory T (Treg) cells (CD45+CD4+CD25+Foxp3+) by flow cytometry as previously described . BrieflyThe same weight of the lung tissue homogenates was prepared with 0.05 M Tris–HCl buffer (pH 7.4). The supernatant of homogenates was applied to determine the concentration of SOD and nitric oxide (NO) using commercial kits  as manufacturer’s protocol. In addition, frozen lung sections were stained with dihydroethidium  to detect ROS, analyzed by Image-Pro Plus 6.0. At least three 200× fields of image were randomly selected from each lung sections for photographing. The level of ROS was expressed as area density which is the ratio of the integrated optical density value (IOD) to the pixel area of the tissue (AREA).g for 10 min, 4°C. Before adding sample loading buffer, a small part of the supernatant was retained for protein quantitation. The concentration of total protein was detected by Pierce BCA Protein Assay Kit (Thermo Scientiﬁc). Samples were separated by 10% SDS-PAGE and transferred to 0.25 um PVDF membranes. Then, the membranes were blocked with 5% milk at room temperature, incubated with primary antibodies (1:1000) overnight at 4°C and then incubated with HRP-conjugated secondary antibody (1:15000) 1.5 h, and ultimately exposed by LAS-4000 mini . Primary antibodies used in this experiment were purchased from Cell Signaling Technology Inc, including anti-HO-1 Abs, anti-SOD1 Abs, anti-NF-κB Abs, anti-phospho NF-κB Abs, anti-JNK1/2 Abs, and anti-phospho JNK1/2 Abs, except for anti-Nrf2 Abs bought from Proteintech.Total protein was extracted from lung tissue. In brief, lung tissues were minced and homogenized in cold RIPA Lysis Buffer containing phosphatase inhibitors and a protease inhibitor, followed by centrifugation at 14,000GraphPad Prism 8  was used for all data, which were expressed as the mean ± SD and analyzed by one-way or two-way analysis of variance (ANOVA).When P value < 0.05, it is statistically significant and expressed as: * p < 0.05, ** p < 0.01, and *** p < 0.001.L at Mch dose of 6.25 (p = 0.068) and 12.5 mg/ml (p < 0.001) . In addition, FMT exhibited a significant increment in Cydn especially in the asthma +FMT 40 mg/kg group, which was able to gradually normalize Cydn responding to Mch of 6.25 and 12.5 mg/ml (p < 0.05) , CCL11 . The results demonstrated the significant elevation of total Ig E, CCL5, and CCL11 level in asthma group compared with control group (p < 0.05). However, this increment was abolished by FMT and Dex treatment , manganese superoxide dismutase (SOD2) and extracellular superoxide dismutase (SOD3)  . Meanwhie (SOD3) . SOD1 wae (SOD3) . Previoue (SOD3) . In accoTo gain more insight into the potential mechanisms of the anti-inflammatory and antioxidant effects of FMT on asthma, we detected inflammation and oxidative stress signaling. NF-κB is known to acts as one of major contributors to inflammatory pathways. The phosphorylation of NF-κB p65 and IκB kinase is highly activated in airway inflammation . Consistin situ ROS production, increasing SOD activity; (f) upregulating the antioxidative signaling pathway of Nrf2/HO-1. Thus, these results provided new information about the protective effects of FMT against asthma.Our study demonstrated the potential therapeutic effect of FMT in the asthmatic murine model, which might be mediated through its anti-inflammatory and antioxidant activities. In summary, our data suggested that FMT exerted anti-inflammatory effects as evidenced by (a) alleviating AHR, inflammatory cell infiltration, goblet cell hyperplasia, and collagen deposition in airways; (b) diminishing elevated expression of IL-4, IL-5, IL-13, IL-17A, Ig E, CCL5, CCL11; (c) reducing Th17 cells and eosinophil recruitment in lung tissue; (d) inhibiting the inflammatory signaling mediated through NF-κB and JNK. Our finding also indicated that FMT restored OVA-induced imbalance of the oxidation and antioxidation as demonstrated by (e) decreasing The original contributions presented in the study are included in the article further inquiries can be directed to the corresponding authors.The animal study was reviewed and approved by the Animal Care and Use Committee of the Fudan University.LY was responsible for the original ideas and designed the experiments. JC, WW, WT, FT, XZ, JQ, TW, and JS assisted LY to conduct experiments. LY analyzed the data and wrote the draft manuscript. YW and JD were responsible to supervise manuscript writing and revised it. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China , Shanghai science and technology commission (grants 17401930300 and 18401971300), and Young Elite Scientists Sponsorship Program by China Association for Science and Technology (grant 2018QNRC001).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.


MicroRNAs (miRNAs) are a group of small noncoding RNA molecules with significant capacity to regulate the gene expression at the post-transcriptional level in a sequence-specific manner either through translation repression or mRNA degradation triggering a fine-tuning biological impact. They have been implicated in several processes, including cell growth and development, signal transduction, cell proliferation and differentiation, metabolism, apoptosis, inflammation, and immune response modulation. However, over the last few years, extensive studies have shown the relevance of miRNAs in human pathophysiology. Common human parasitic diseases, such as Malaria, Leishmaniasis, Amoebiasis, Chagas disease, Schistosomiasis, Toxoplasmosis, Cryptosporidiosis, Clonorchiasis, and Echinococcosis are the leading cause of death worldwide. Thus, identifying and characterizing parasite-specific miRNAs and their host targets, as well as host-related miRNAs, are important for a deeper understanding of the pathophysiology of parasite-specific diseases at the molecular level. In this review, we have demonstrated the impact of human microRNAs during host−parasite interaction as well as their potential to be used for diagnosis and prognosis purposes. Caenorhabditis elegans by Lee et al. , endogenous, evolutionarily conserved regulatory ncRNAs that are implicated in the post-transcriptional regulation of cellular signaling pathways in both animals and plants  so far supports the idea that miRNAs are involved in complex regulatory networks. Their role has been established in several pathophysiologies including cancer, autoimmune, and metabolic disorders  and quickly invades liver cells undergoing rapid multiplication causing malaria  , has been associated with increased susceptibility to P. falciparum infection in placental samples of pregnant women , Myeloid differentiation primary response 88 (Myd88), TIR domain-containing adaptor protein-inducing Interferon β (TRIF), TRAF6, and IRAK1 in macrophages, suggesting that these miRNAs are negative regulators of fine-tuned inflammatory reactions. Likewise, an upregulation of let-7a, miR-25, miR-26a, miR-132, and miR-140 in L. major-infected human macrophages led to a negative correlation on the expression of their specific chemokine targets CCL2, CCL5, CXCL10, CXCL11, and CXCL12 inhibiting macrophage stimulation , Filaggrin-2 (FLG2) and Natural Killer cell Granule protein 7 (NKG7) , regulatory factor X1 (RFX1) and B-cell receptor-associated protein 31 (BCAP31) involved in both immune response regulation and cell survival under endoplasmic reticulum stress during infection; thus, miR-346 could be considered as an enticing target for anti-Leishmania approaches. Ghosh et al. (L. donovani infection and Huh7 cells miR-122 levels (downregulation) by targeting DICER1. Moreover, Geraci and colleagues  in THP-1 and human monocyte-derived macrophages has been reported to be triggered by the upregulation of miR-30a-3p during infection  signaling pathway, ubiquitin-mediated proteolysis, mRNA surveillance pathways, and apoptosis. Interestingly, the amoeba can induce apoptosis in host cells by partially altering miRNAs that regulate genes involved in lipid metabolism, the PI3K/AKT signaling pathway, and apoptosis. Of particular note, the aforementioned six modulated microRNAs potentially target apoptosis-related genes. Thus, the expression of B Cell Lymphoma 2 (BCL-2) protein family, BCL2 Antagonist/Killer 1 (BAK1), BCL2 Interacting Protein 3 Like (BNIP3L), X-linked Inhibitor of Apoptosis Protein (XIAP), Apoptosis Inducing Factor Mitochondria associated 2 and 3(AIFM 2 and AIFM3), Apoptosis Inhibitor 5 (API5), BCL2 Associated transcription Factor 1 (BCLAF1), Apoptosis-Associated Tyrosine Kinase (AATK), BAG1, BAG3, BMF, Nuclear Apoptosis Inducing Factor 1 (NAIF1), CFLAR, and Caspase 7 and 8 (CASP7 and CASP8) can potentially be regulated by the aforesaid miRNAs suggesting their possible usage in the clinical diagnosis of amoebiasis  is an anthropozoonosis caused by the protozoan parasite uzi Fig. c which iuzi Fig. . The infuzi Fig. . Among cuzi Fig. . Over thuzi Fig. . Linhareuzi Fig.  detecteduzi Fig.  reportedSchistosoma and it is one of the most prevalent zoonotic diseases, affecting over 258 million individuals in 54 countries , S. haematobium (Africa and the Middle East), and S. japonicum (China and Southeast Asia) are the main species that cause this human disease , Metallothionein-1H (MT1H), PL1N1, Vanin 1 (VNN1), Catenin Alpha-3 (CTNNA3), SLC39A8, and Guanylate-Binding Protein 5 (GBP5) are involved in crucial processes implicated in hepatic fibrosis progressions, such as cellular proliferation and differentiation, reorganization of the extracellular matrix, lipolysis, and cellular detoxification. To date, four schistosomal miRNAs have been isolated from extracellular vesicles in sera from Schistosoma infected individuals, such as Bantam, miR-2c-3p, miR-3488, and miR-2a-5p suggesting that can be used both as a diagnostic tool for infection and to monitor treatment effectiveness  is a parasitic infection caused by several species of blood-flukes of the genus Toxoplasma gondii, one of the most common human parasites in the world, is a ubiquitous pathogen that is the causative agent of toxoplasmosis and can infect a large range of hosts including humans. The transmission begins with the ingestion of contaminated raw meat and then the parasite starts to infect as many cells as possible. Manifestations are highly variable, ranging from asymptomatic to severe, especially in cases of brain and eye infection   and reported an upregulation of miR-146a in HFF infected with Me49 and a downregulation in the RH infected cells, targeting IRAK and TRAF which are fine modulator of TLR-receptor signaling pathway. Furthermore, it is also known that T. gondii affects the human brain in many pathways related to epilepsy, neurodegeneration, and cancer. A positive regulation of miR-132 has been reported in the human neuroepithelial cell line infected with RH-2F, PRU, and CTG strains which have been related to a modulation in the expression of genes involved in the metabolism of dopamine, such as apoptotic protease-activating F-factor 1 (APAF1), Kirsten Rat Sarcoma (KRAS), MAPK3 and PPP2R5E  expression, coordinating the subsequent release of exosomes carrying antimicrobial peptides  proteins that are negative regulators of cytokine signaling , and MAPK3.The genus um Table . For exaum Table . SpecifiClonorchis sinensis through the consumption of raw or undercooked freshwater fish, containing metacercariae of C. sinensis. Clonorchiasis generally appears as bile duct obstruction, biliary inflammation, liver cirrhosis, hepatic carcinoma, and cholangiocarcinoma  revealed that the expressions of nine miRNAs  were similarly regulated, showing that the cell proliferation and inhibition of tumor suppression mediated by these miRNAs are common to both cancerous and noncancerous cells  and Echinococcus multilocularis, respectively . Likewise, Alizadeh and colleagues (Cystic echinococcosis (CE) and alveolar echinococcosis (AE), two severe zoonotic tapeworm diseases, are triggered by lleagues  reportedlleagues .Identifying and characterizing parasite-specific miRNAs and their targets in hosts, as well as miRNAs interfering with host pathology, are crucial for a better understanding of the pathophysiology of parasitic diseases at the molecular level. The biological knowledge acquired about miRNAs, especially in biomedical research, is expected to be widely translated in the field of parasitology in the coming years since miRNAs have great potential to lead to a transition to a novel class of theranostic tool. Thus, we can expect the discovery of more and more specific miRNAs with highly specialized functions linked to cellular processes in parasites that can provide novel guidelines for the management of parasitic diseases.