Experimental Studies On The Effects And The Mechanism Of Total Flavonoids Of Epimedium On Functional Decline In Aging Testicular Sertoli Cells

Objective To study the effects of total flavonoids of Epimedium(TFE)on the decline of secretory function in aging testicular Sertoli cells and explore its molecular mechanisms.Methods Experiment one: 36 SPF 18-month-old male SD rats were randomly divided into TFE low-and high-dose group and natural aging group.Another 10 SPF 2-month-old male SD rats were used as the young control group.TFE low-and high-dose groups were given 10 and 40 mg/kg dose by stomach lavaging,young control group and natural aging group were given the same concentration of 1% sodium carboxymethyl cellulose solution,lasting for 4 months.After weighing,anesthetized and executed,testicular tissue was taken and investigated:(1)Testicular weight and testicular index were calculated;(2)The testicular histomorphology was observed by HE staining;(3)The numbers of Sertoli cells in the testis of each group were detected by immunofluorescence;(4)Quantitative-real time-PCR and Western Blot were used to detect the levels of GDNF,BMP4 and SCF m RNA and protein expression in each group;(5)The IRE1α/XBP1 signal pathway-related proteins p-IRE1α and XBP1 in each group were detected by Western Blot;(6)The expression and localization of p-IRE1α in testis were detected by immunofluorescence.Experiment two: The mouse Sertoli cell line TM4 cells were stimulated by D-galactose to build Sertoli cell secretory dysfunction model,TFE were intervened.(1)PCR and Western Blot were used to detect the levels of GDNF,BMP4 and SCF m RNA and protein expression in each group;(2)The expression of p-IRE1α and XBP1 proteins in each group were detected by Western Blot.Results Experiment one:(1)TFE significantly increased testicular weight and testis index.(2)HE staining showed that the seminiferous tubules of rats in the young control group were intact,and the spermatogenic cells in the tubules were arranged closely and regularly.Compared with the young control group,the seminiferous tubules of the natural aging rats atrophy,the gap between spermatogenic cells increased.Furthermore,the numbers of spermatogenic cells were significantly reduced,Conversely,TFE significantly recovered testicular seminiferous tubules of morphological structure caused by aging.(3)There was no significant difference in the numbers of Sertoli cells in the testis tissue among these groups.(4)TFE significantly up-regulated m RNA and protein levels of Sertoli cell secretory factors,including GDNF,BMP4 and SCF.(5)Western Blot showed that TFE significantly promoted the expression levels of p-IRE1α and XBP1 proteins.(6)TFE increased the fluorescence intensity of p-IRE1α in the Sertoli cells.Experiment two:(1)TFE significantly up-regulated the expression levels of GDNF,BMP4 and SCF both at m RNA and protein levels,and improved the secretion dysfunction of TM4 cells induced by D-galactose stimulation.(2)TFE increased the expression levels of p-IRE1α and XBP1 protein.Conclusion TFE can improve the decline of sertoli cell function effectively,and its mechanism may be related to the regulation of IRE1α/XBP1 signaling pathway.