Pharmacodynamics And Preliminary Mechanism Of Riluzole In The Treatment Of Oligospermia

Objective:With the aggravation of environmental pollution and social competition pressure,the incidence of oligospermia is increasing,which seriously affects the quality of life and family harmony.In view of the limitations of current gonadotropin replacement therapy and surgical treatment,a new drug with less side effects,quick curative effect and low cost is urgently needed to break through these limitations.Our previous study found that Riluzole could target Sertoli cells(SCs)and promote SCs to secrete cytokines required for the proliferation and differentiation of spermatogonial stem cells(SSCs),suggesting that Riluzole may have the function of regulating spermatogenesis.Therefore,in this study,we used Busulfan to establish a mouse model of oligospermia and studied the role of Riluzole in the treatment of oligospermia,which laid a theoretical and experimental foundation for the treatment of male oligospermia.Method:(1)The oligospermia model was established by single intraperitoneal injection of Busulfan(30mg/kg)to 4-week-old male Kunming mice.After the model was successfully established,model mice were intraperitoneally injected with Riluzole(3,6 mg/kg)or orally administered with Riluzole(5 mg/kg)for 7 days.After the administration,the mice continued to be fed normally for 8 weeks.At the 8th week after administration,The improvement of the pregnant ability of Riluzole on oligospermia mice was verified by co-cage mating experiment;the count,viability,motility and abnormal rate of sperm in the cauda epididymis of oligospermia mice were detected by CASA sperm analyzer;the histological changes of testis and epididymis in oligospermia mice were observed by HE staining;the effects of Riluzole on the expression of spermatogenesis related proteins(GFRα-1,PCNA,SYCP3 and TNP-1)in the testis of oligospermia mice were investigated by immunofluorescence.Based on the above experiments,the therapeutic effect of Riluzole on oligospermia model mice was clarified.(2)The effect of Riluzole on the integrity of the blood-testis barrier in oligospermia mice was investigated by fluorescent tracer method with streptavidin-conjugated biotin;the effects of Riluzole on the expression levels of blood-testis barrier related proteins(ZO-1 and Occludin)in the testis of oligospermia mice were detected by immunofluorescence.The in vitro SCs model was established,and the regulatory effect of Riluzole on the gene expression of related secretory factors in SCs was detected by q PCR;the effects of Riluzole on the expression levels of blood-testis barrier related proteins(ZO-1 and CX43)were detected by Western blot(WB)and immunofluorescence(IF);finally,the effect of the combined treatment of busulfan and Riluzole on the transmembrane resistance between SCs was detected by cell resistance meter,and the improvement effect of Riluzole on the damage of tight junction caused by busulfan was evaluated in vitro,and the mechanism of Riluzole in the treatment of oligospermia in mice was clarified from the perspective of improving the tight junction function of blood-testicular barrier.Results:(1)Intraperitoneal injection or oral administration of Riluzole promoted the recovery of testicular atrophy in oligospermia mice,thickened the wall of testicular seminiferous tubules,increased the diameter of seminiferous tubules,significantly increased the number of germ cells on the basement membrane of the tubules,and significantly reduced the empty tube rate of seminiferous tubules;in addition,Riluzole promoted the proliferation and differentiation of spermatogonia and sperm maturation,thus promoting sperm regeneration in oligospermia mice,increasing the count,viability and motility of sperm,significantly reducing the abnormal rate of sperm and restoring the pregnancy ability of oligospermia mice.(2)Busulfan could destroy the integrity of tight junctions in the blood-testis barrier of oligospermia mice.After Riluzole administration,the blood-testis barrier of oligospermia mice was more complete,and the expression levels of blood-testis barrier related proteins(ZO-1and Occludin)were significantly up-regulated.Further in vitro studies showed that Riluzole promoted the formation of tight junctions by up-regulating the expression levels of related secretory factors(Gdnf,Bmp4,Scf,Cxcl12,Inhibinb,Fgf2)and blood-testis barrier related proteins(ZO-1 and CX43)in SCs,improved the damage of blood-testicular barrier caused by busulfan,and restored the spermatogenesis ability of oligospermia mice.Conclusion:Intraperitoneal injection or oral administration of Riluzole could effectively improve the testicular injury of oligospermia mice,promote the proliferation and differentiation of spermatogonia,improve the sperm quality and reproductive ability of oligospermia model mice,and have a significant therapeutic effect on oligospermia model mice.This study is expected to provide reference for the treatment of male infertility and has potential application prospects in the field of medicine.