基于PI3K/Akt通路观察五子衍宗方对老龄大鼠精原干细胞增殖的影响

目的:探讨五子衍宗方(Wuzi Yanzong Fang,WYF)基于磷酯酰肌醇3-激酶(PI3K)/丝氨酸苏氨酸蛋白激酶(Akt)信号通路对老龄大鼠精原干细胞增殖的作用及其机制研究。方法:以自然衰老大鼠为研究对象,将40只18月龄SPF级SD雄性大鼠随机分为老龄模型组,WYF低、中、高剂量组,每组10只,另以10只2月龄大鼠作为青年组。WYF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。免疫荧光法检测Sertoli细胞数量,实时荧光定量聚合酶链式反应(Real-time PCR)检测胶质细胞源性神经营养因子(GDNF),前髓细胞锌指蛋白(PLZF),干细胞因子(SCF)和骨形态蛋白4(BMP4)mRNA表达水平,免疫荧光法检测精原干细胞数量及增殖细胞核抗原(PCNA)的表达,蛋白免疫印迹法(Western blot)和免疫荧光法检测磷酯酰肌醇3-激酶(PI3K)和磷酸化丝氨酸苏氨酸蛋白激酶(p-Akt)的表达与定位。结果:与青年组比较,老龄模型组Sertoli细胞数量未见明显差异,而GDNF,PLZF,SCF和BMP4 mRNA表达水平显著降低,精原干细胞数量减少,PCNA表达下降,PI3K,p-Akt蛋白表达降低(P0.01);与老龄模型组比较,WYF各剂量组GDNF,PLZF,SCF和BMP4 mRNA表达水平显著升高,精原干细胞数量增多,PCNA表达上调,PI3K,p-Akt表达明显增多(P0.05,P0.01)。结论:WYF能够明显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PI3K/Akt信号通路有关。

Effect of Wuzi Yanzong Fang on Proliferation of Spermatogonial Stem Cells in Aging Rats Through PI3K/Akt Signaling Pathway

Objective:To investigate effect of Wuzi Yanzong Fang (WYF) on proliferation of spermatogonial stem cells in aging rats through phosphoinositol 3-kinase(PI3K)/protein kinase(Akt) signaling pathway. Method:The 18-month-old male SD rats were randomly divided into aging model group, and low, middle and high-dose WYF groups, with 10 rats in each group. Ten 2-month-old rats were used as young control group. low, middle and high-dose WYF groups were given WYF 0.4, 0.8, 1.6 g·kg^-1 by gavage for 4 months, respectively. Young control group and aging model group were given normal saline, and the administration was suspended for 2 days a week. The administration lasted for 4 months. The rats were put to death after the final administration of WYF, and then the testes were quickly collected. The numbers of Sertoli cells were detected by immunofluorescence. The relative mRNA expression levels of glial cell line-derived neurotrophicfactor (GDNF), promyelocyte Zinc finger protein (PLZF), bone morphogenetic protein4 (BMP4) and stem cell factor (SCF) in testicular tissues were detected by Real-time quantitative PCR (Real-time PCR). The numbers of spermatogonial stem cells and the expression levels of PCNA were detected by immunofluorescence. The expressions and localizations of PI3K, Akt, p-Akt were detected by Western blot and immunofluorescence. Result:Compared with young control group, there was no significant difference in the numbers of Sertoli cells in each group, the mRNA expression levels of GDNF, PLZF, BMP4 and SCF in testis of aging model group were significantly decreased. The number of spermatogonial stem cells and levels of PI3K, p-Akt, PCNA expression decreased(P<0.01). After WYF treatment, the levels of GDNF, PLZF, BMP4 and SCF were significantly increased, the numbers of spermatogonial stem cells were increased, and PCNA, PI3K, p-Akt expressions were up-regulated(P<0.05,P<0.01). Conclusion:WYF can effectively promote the proliferation of spermatogonial stem cells in aging rats through PI3K/Akt signaling pathway.