淫羊藿苷通过调钙质改善运动性疲劳模型小鼠精子发生的作用及机制OA

Objective:This paper aims to investigate the effects of icariin on spermatogenesis in mice with exercise-induced fatigue and explore the underlying mechanisms.Methods:ICR male mice were screened by swimming and randomly divided into normal group,model group,vitamin C group,icariin groups with low,medium,and high doses,and medium-dose icariin+N-nitro-L-arginine methyl ester(L-NAME)group,with 10 mice per group.Except for the normal group,all the other groups underwent weighted swimming training to establish an exercise-induced fatigue model.No gavage was administered during the first two weeks of the weighted training.From week three to four,the icariin groups with low,medium,and high doses received 0.03,0.06,and 0.12 g·kg-1 icariin via gavage,respectively.The vitamin C group received 0.2 g·kg-1 vitamin C.The L-NAME group received 0.06 g·kg-1 icariin and 0.01 g·kg-1 L-NAME via intraperitoneal injection.The normal and model groups received equivalent physiological saline.After the experiment,body weight and the last exhaustive swimming time were recorded.Blood urea nitrogen(BUN),lactate(LA),lactate dehydrogenase(LDH),malondialdehyde(MDA),testicular testosterone(T),testicular Ca2+/Mg2+-adenosine triphosphatase(ATPase)(micro-assay),and the levels of testicular cyclic guanosine monophosphate(cGMP)were measured by using kits.Sperm CD46 levels were detected by flow cytometry.Testicular seminiferous tubules were observed via hematoxylin-eosin(HE)staining,and the testicular morphometric score(TMS)was used to evaluate the spermatogenic function.Protein expression of regucalcin(RGN,SMP30),cGMP-dependent protein kinase 1(PKG),and cGMP-dependent protein kinase anchoring protein(GKAP1)was detected by Western blot.Testicular regucalcin expression was examined by immunofluorescence(IF).The epididymal sperm quality of mice was observed under a microscope.Fluorescence-stained sections of stimulated by retinoic acid gene 8(STRA8),synaptonemal complex protein 3(SCP3),and transition protein 1(TNP1)in testicular seminiferous tubules were assessed by immunohistochemistry(IHC).Results:Compared with the normal group,the model group showed decreased body weight and exhaustive swimming time(P＜0.01),significantly increased fatigue markers(LA,LDH,and BUN)and lipid peroxidation product MDA(P＜0.01),reduced testicular RGN,PKG,GKAP1,testosterone,Ca2+/Mg2+-ATPase,and cGMP levels(P＜0.01),decreased sperm motility,sperm count,and TMS scores,and downregulated the expression of STRA8,SCP3,and TNP1.Compared with the model group,the icariin group with high dose exhibited increased exhaustive swimming time(P＜0.01),reduced LA,LDH,BUN,and MDA levels(P＜0.01),elevated superoxide dismutase(SOD)(P＜0.01),upregulated testicular RGN,PKG,GKAP1,testosterone,Ca2+/Mg2+-ATPase,and cGMP levels(P＜0.01),improved sperm motility,sperm count,and TMS scores,and enhanced STRA8,SCP3,and TNP1 expression.Compared with the L-NAME group,the icariin group with medium dose showed increased expression of STRA8,SCP3,and TNP1 in the testicular tissue(P＜0.01)and elevated cGMP and GKAP1 levels(P＜0.01).Conclusion:Exercise-induced fatigue reduces the expression of RGN and cGMP/PKG/GKAP1 in mice,thereby causing abnormal spermatogenesis and impairing reproductive function in mice.Icariin ameliorates spermatogenic dysfunction in exercise-induced fatigue mice by promoting the expression of RGN and cGMP/PKG/GKAP1,thereby mitigating the damage of exercise-induced fatigue to the reproductive system.