基于Toll样信号通路探讨强精片改善少弱精子症大鼠的作用机制

Qiangjing Tablets Improves Reproductive Function in Oligospermia Rats through Toll-like Signaling Pathway

目的探讨中药强精片(QJP)通过调控睾丸Toll样信号通路改善解脲支原体感染及环磷酰胺诱导两种少弱精子症大鼠模型生精功能的作用机制。方法将40只雄性SD大鼠随机分为5组,正常对照组(NC组)、解脲支原体感染少弱精子症组(UU组)、环磷酰胺少弱精子症组(CTX组)、强精片干预解脲支原体感染少弱精子症组(UU+QJP组)、强精片干预环磷酰胺少弱精子症组(CTX+QJP组),每组8只。造模后NC、UU、CTX组给予生理盐水灌胃,UU+QJP组、CTX+QJP组给予强精片0.45 g/(kg·d)灌胃,均给药4周。记录大鼠一般状态、睾丸附睾湿重,测定精子数量;HE染色观察睾丸及附睾结构变化;测量血清性激素水平;免疫组织化学染色分析睾丸Toll样受体4(TLR4)、髓样分化因子(MYD88)、肿瘤坏死因子受体相关蛋白6(TRAF6)蛋白表达;RT-PCR测定TLR4、MYD88、TRAF6 mRNA表达。结果与NC组比较,CTX、UU组的大鼠一般状态较差,HE染色显示生精细胞不同程度减少,免疫组化显示TLR4、MYD88表达增多,TRAF6表达减少,CTX+QJP组及UU+QJP组有所改善。与NC组比较,UU组、CTX组精子总数、A+B级精子、A+B+C级精子、促黄体激素(LH)、睾酮(T)水平、TRAF6 mRNA表达下降(P<0.05),卵泡刺激素(FSH)、雌二醇(E_(2))水平、TLR4、MyD88 mRNA表达增加(P<0.05)。与UU组和CTX组比较,UU+QJP组和CTX+QJP组精子密度、A+B级精子、A+B+C级精子、LH、T水平、TRAF6 mRNA表达增加(P<0.05),FSH、E_(2)水平、TLR4、MyD88mRNA表达下降(P<0.05)。结论强精片可改善解脲支原体感染以及环磷酰胺所致少弱精子症大鼠的精子水平,其机制可能与调控睾丸中Toll样信号通路的关键靶点TLR4、MYD88、TRAF6有关。

Objective To explore the mechanism of Qiangjing Tablet(QJT)on improving the spermatogenesis function by regulating the Toll-like signal pathway of the testis in two oligoasthenospermia rat models induced by ureaplasma urealyticum infection or cyclophosphamide.Methods Forty male SD rats were randomly divided into 5 groups,normal control group(NC group),ureaplasma urealyticum infection oligoasthenospermia group(UU group),cyclophosphamide oligoasthenospermia group(CTX group),QJT intervention for ureaplasma urealyticum infection oligoasthenozoospermia group(UU+QJT group),QJT intervention for cyclophosphamide oligoasthenospermia group(CTX+QJT group),8 in each group.After modeling,the NC,UU,and CTX groups were given saline intragastrically,and the UU+QJT and CTX+QJT groups were given QJT 0.45 g·kg^(-1)·d^(-1)for 4 weeks.The general state of the rat,the wet weight of the testis and epididymis were recorded,and the number of sperm was measured;HE staining was used to observe the structural changes of the testis and epididymis;the serum sex hormone level was measured;immunohistochemical staining was used to to analyze the testis toll-like receptor 4(TLR4),myeloid differentiation factor 88(MYD88),TNF receptor associated factor 6(TRAF6)protein expression;RT-PCR was used to to determine TLR4,MYD88,TRAF6 mRNA expression.Results Compared with the NC group,the rats in the CTX and UU groups were in poor condition.HE staining showed that the spermatogenic cells decreased.Immunohistochemistry showed that the expression of TLR4 and MYD88 increased,and the expression of TRAF6 decreased,the CTX+QJT group and UU+QJT group recovered.Compared with the NC group,sperm density,A+B grade sperm,A+B+C grade sperm,luteinizing hormone(LH),testosterone(T)level,TRAF6 mRNA expression decreased in UU group and CTX group(P<0.05),fol icle stimulating hormone(FSH),estradiol(E_(2))level,TLR4,MYD88 mRNA expression increased(P<0.05).Compared with UU group or CTX group,sperm density,A+B grade sperm,A+B+C grade sperm,LH,T level,TRAF6 mRNA expression increased(P<0.05),FSH,E_(2) level,TLR4,UU+QJP group or CTX+QJP group MYD88 mRNA expression decreased(P<0.05).Conclusions QJT can improve the sperm level of Ureaplasma urealyticum infection and cyclophosphamide-induced oligoasthenospermia rats.The mechanism may be related to the regulation of the key targets TLR4,MYD88,and TRAF6 of the Toll-like signaling pathway in the testis.