| 1.Disruption of Anchoring Junctions in the Testis of Experimental Varicocele RatsVaricocele is a common disease of the male reproductive system,which is the main inductor of male infertility;however,its pathomechanism is still debatable.The anchoring junctions(AJ)in the testis are located between Sertoli cells or between Sertoli cells and germ cells.Intact and functional AJ is crucial for spermatogenesis.In this study,the histomorphology,ultrastructure of AJ,cell cycle,expression of AJ structural proteins,and the level of AJ-associated signaling molecules were investigated in the left testis of experimental varicocele rats at 8 and 12 weeks after the operation.The results showed that varicocele induced the loss of premature germ cells from the seminiferous epithelium,damage of the AJ ultrastructure,disorientation of the spermatid head,deregulation of the cell cycle,down-regulation of the AJ structural proteins and cytoskeletal proteins,up-regulation of p-FAK-Tyr397 and its important down-stream adapter p-Src-Tyr416,and activation of the ERK signaling pathway.In summary,varicocele results in the disruption of AJ in the rat left testis,while the up-regulation of p-FAK-Tyr397 may contribute to the damage of AJ,probably by activating the ERK signaling pathway and disturbing actin-based filament network,which is crucial for understanding the varicocele pathomechanism that contributes to male infertility and to identify new targets for the development of new treatments for varicocele.2.Effects of Marinda officinalis Polysaccharide on Experimental Varicocele RatsMorinda officinalis is a kind of traditional Chinese herbal medicine,and has been used in clinical for a long time in China to strengthen yang and tonify the kidney.Varicocele(VC)is considered as the main inducement of male infertility,but the effective therapy and pathophysiology contribute to male infertility are yet-to-be discovered.In the research,the repair effects of Morinda officinalis Polysaccharide(MOP)on damaged reproductive system of rats induced by experimental varicocele was investigated.The result showed that VC resulted in the disruption of TJ structure and seminiferous epithelium,down-regulated sperm count in left epididymis,the expression of TJ protein,testosterone(T)level both in serum and left testis,the concentration of inhibin B(INHB)in serum;while increased the levels of TGF-?3 and TNF-? in the left testis,the levels of up-stream sexual hormones(GnRH,FSH,and LH),and the concentration of antisperm antibody(AsAb)in serum.MOP can repair the damaged structure of TJ and seminiferous epithelium,reduced the apoptosis of germ cells,the left testicular cytokines level,the levels of GnRH,FSH,LH,and AsAb in serum;meanwhile increase the sperm count,the expression TJ protein,serum and left testicular T level,and the concentration of INHB in serum.We conclude that MOP can repair the damaged male reproductive system induced by varicocele,such repair effects of MOP may be realized via down-regulating left testicular cytokines level and accommodating the abnormal sex hormones to the normal level.MOP is a worthy component of Morinda officinalis,which can be used in clinical to treat male infertility induced by varicocele,or combined with varicocelectomy to promote spermatogenesis and provide the patients more effective treatment.3.MicroRNA profiles in the experimental varicocele rat left testis and microRNA-181d-5p may induce the disruption of TJ via PTEN in TM4 cell lineMicroRNAs(miRNA)are recognized as a class of small non-coding and single-stranded RNAs(22-25 nucleotides),which can down-regulate the expression of target gene post-transcriptionally via combining with the 3'-untranslated region(3'-UTR)of mRNAs.According to the related research recent years,miRNAs are confirmed to take part in a wide variety of biologic activities(e.g.,cell migration,proliferation,differentiation,spermatogenesis)and be involved in multiple pathological processes.In this study,we perform the sequencing analysis of miRNAs in experimental varicocele rat left testis,and investigate the target mRNA of miRNA-181d-5p and its effect on TJ between Sertoli cells(TM4 line),as well as the underlying mechanism.The result show that miRNA-181d-5p can combine with the 3'-UTR region of PTEN mRNA and down-regulate the expression of PTEN in 293T and TM4 cell line.In addition,the over-expression of miRNA-181d-5p in TM4 cell line result in the decrease of Trans-epithelium electrical resistant(TER)and the up-regulation of p-FAK-Tyr397.In summary,miRNA-181 d-5p can interact with the 3'-UTR region of PTEN mRNA and down-regulate the expression of PTEN.The up-regulation of p-FAK-Tyr397 and activation of PI3K/Akt signaling pathway induced by low-level of PTEN may contribute to the decrease of TER between TM4 cells after over-expression of miRNA-181d-5p.4.TMT-based Quantitative Proteomic Analyses of Left Testis in Experimental Varicocele Rats Treated with 300mg kg-1 Morinda officinalis PolysaccharideThe proteome is a new branch of science and has been a hot area of research for several years,which is defined as revealing the law of biopsies from the whole protein aspect.To elucidate the pathophysiology of varococele and the mechanism of repair effect of Morinda officinalis polysaccharide(MOP)on male reproductive system injury,in this study,we performed the TMT-label based quantitative proteomic analyses of left testis in experimental varicocele rats treated with 300mg kg-1 MOP to detect the differentially expressed proteins,as well as do the bioinformatics analysis.We also performed the chemical fingerprint analyses of MOP,including the Fourier Transform Infrared(FT-IR)analysis,molecular mass distribution of MOP analysis using Gel-permeation Chromatography(GPC),and 1H-Nuclear Magnetic Resonance(1H-NMR).The number of total detected polypeptide,protein,quantifiable protein and differentially expressed proteins are 14742,2510,2412 and 57 respectively.The number of differentially expressed proteins between the VC model group and sham group,the VC+300 mg kg-1 MOP treatment group and VC model group,the VC+300 mg kg-1 MOP treatment group and sham group is 46,21 and 5.Genes and Genomes(KEGG)and protein-protein interaction prediction analyses showed that the revealed that differentially expressed proteins predominantly enrich on regulation of apoptotic signaling pathway,ribosome biogenesis,regulation of hormone levels,humoral immune response,oxidoreductase activity,positive regulation of hormone secretion,steroid hormone biosynthesis temperature homeostasis,positive regulation of blood circulation biological process,and so on.We concluded that MOP can decrease the number of differentially expressed proteins induced by VC in rat left testis,make the expression of protein be similar to the sham group and decrease the discrepancy according to the result of hierarchical cluster analysis of the total differentially expressed proteins.The enriched biological process may contribute to the VC pathophysiology and the MOP repair efficacy.Our study provide new insights into molecular mechanisms that may be involved in VC and repairment of traditional Chinese herbal medicine. |
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