五子衍宗方通过调节miR-let-7-Imp轴对老龄大鼠精原干细胞增殖的影响

Effect of Wuzi Yanzong Fang in Promoting Spermatogonial Stem Cells Proliferation in Aging Rats by Regulating miR-let-7-Imp Axis

目的:通过miR-let-7-Imp轴探讨五子衍宗方(WZ)对老龄大鼠精原干细胞增殖的作用及其机制。方法:将40只18月龄SPF级SD雄性大鼠随机分为老龄模型组,WZ低、中、高剂量组,每组10只,另以10只2月龄大鼠作为青年组。WZ低、中、高剂量组分别灌胃给药0. 4,0. 8,1. 6 g·kg^-1,青年组、老龄模型组分别灌胃给予生理盐水,每周停药2 d,持续给药4个月。末次给药后麻醉处死大鼠,快速取出睾丸组织。实时荧光定量聚合酶链式反应(Real-time PCR)检测miR-let-7,Imp mRNA表达水平,蛋白免疫印迹法(Western blot)和免疫荧光检测睾丸精原干细胞增殖相关信号通路磷酸化(p)-两面神激酶2(JAK2)/JAK2,p-信号传导及转录激活因子3(STAT3)/STAT3蛋白的表达与定位,免疫荧光检测精原干细胞数量及增殖相关蛋白增殖细胞核抗原(PCNA)的表达,苏木素-伊红(HE)染色观察睾丸组织形态学变化。结果:Real-time PCR结果显示,与青年组比较,老龄模型组miR-let-7 mRNA表达水平显著升高(P<0. 01),Imp mRNA表达水平显著降低(P<0. 01),而给予WZ干预后,miR-let-7 mRNA水平明显降低(P<0. 05,P<0. 01),Imp mRNA表达水平明显升高(P<0. 05);Western blot和免疫荧光结果显示,精原干细胞增殖相关信号通路蛋白p-JAK2,p-STAT3表达降低(P<0. 01),WZ处理后,p-JAK2,p-STAT3表达明显增多(P<0. 05,P<0. 01);免疫荧光结果显示,与青年组比较,老龄模型组中精原干细胞数量减少,PCNA表达下降,WZ处理后,精原干细胞数量增多,PCNA表达上调;HE结果也显示,WZ能够明显改善自然衰老所致的睾丸组织形态结构变化。结论:WZ能够促进老龄大鼠精原干细胞的增殖,其作用机制可能与调节睾丸内miR-let-7-Imp轴有关。

Objective: To explore the effect and mechanism of Wuzi Yanzong Fang( WZ) on proliferation of spermatogonial stem cells in aging rats by regulating miR-let-7-Imp axis. Method: A total of 40 18-month-old male SD rats were randomly divided into aging model group,and low,middle and high-dose WZ groups,with 10 rats in each group. Ten 2-month old rats were used as young control group. Low,middle and high-dose WZ groups were given by gastric WZ 0. 4,0. 8,1. 6 g·kg^-1 respectively. Young control group and aging model group were given normal saline for 4 months,which was suspended for 2 days every week. Rats were put to death after the final treatment with WZ,and then the testes were quickly removed from rats. The relative mRNA expression levels of miR-let-7 and Imp were detected by Real-time fluorescence quantitative polymerase chain reaction( Realtime PCR). The expressions and localization of p-JAK2/JAK2, phosphate( p)-signal transduction and transcriptional activators3( STAT3)/STAT3 signaling pathway proteins were detected by Western blot and immunofluorescence. The numbers of spermatogonial stem cells and the expression levels of proliferating cell nuclear antigen( PCNA) were detected by immunofluorescence. The testicular tissue morphology was observed using htoxylin eosin( HE) staining. Result: Compared with young control group,the expression levels of miR-let-7 mRNA were significantly increased,while the expression levels of Imp mRNA were decreased in the aging model group( P< 0. 01). Conversely,the expression levels of miR-let-7 mRNA were significantly decreased( P< 0. 05,P< 0. 01),while the mRNA expression levels of Imp mRNA were significantly increased after WZ treatment( P<0. 05). The results of Western blotting and immunofluorescence showed that the expression levels of p-JAK2,p-STAT3 in aging model group were decreased significantly( P< 0. 01),while WZ significantly increased the expression levels of p-JAK2,p-STAT3( P< 0. 05,P< 0. 01). Immunofluorescence results showed that compared with young control group,the number of spermatogonial stem cells and PCNA expression decreased in aging model group. After WZ treatment,the number of spermatogonial stem cells was increased,whereas PCNA expression was up-regulated. HE showed that WZ significantly improved the testicular tissue structure of aging rats. Conclusion:WZ effectively promote the proliferation of spermatogonial stem cells by regulating miR-let-7-imp axis in testis.