强精煎介导PI3K/Akt/mTOR通路调控少精子症大鼠精子发生的实验研究

Experimental Study of Qiangjing Decoction Regulating Spermatogenesis in Oligopermia Rats Based on PI3K/Akt/mTOR Pathway

目的:探索强精煎通过介导PI3K/Akt/mTOR通路及其下游靶因子4EBP1、p70S6K,调控环磷酰胺(CTX)诱导的少精子症模型大鼠精子发生的作用机制。方法:将75只SD雄性大鼠随机分为空白对照组、模型组、mTOR通路抑制剂组(雷帕霉素组)、强精煎+雷帕霉素组和强精煎组,每组15只。采用腹腔注射CTX法诱导少精子症大鼠模型。连续药物干预38 d后,剪开各组大鼠腹腔摘取睾丸,采用免疫组化法检测PI3K/Akt/mTOR通路蛋白及其下游通路蛋白(4EBP1、p70S6 K)、细胞增殖标记蛋白Ki-67表达;采用RT-PCT法检测睾丸组织PI3K/Akt/mTOR基因及其下游靶基因(4EBP1、p70S6K)表达。结果:与空白对照组比较,模型组、雷帕霉素组睾丸组织PI3K、Akt、mTOR、4EBP1、p70S6 K、Ki-67蛋白表达水平均显著降低(P<0.05),雷帕霉素组降低尤为明显。与模型组、雷帕霉素组相比,强精煎组睾丸组织PI3K、Akt、mTOR、4EBP1、p70S6K、Ki-67蛋白表达水平明显上调(P<0.05)。与空白对照组比较,模型组、雷帕霉素组睾丸组织PI3K、Akt、mTOR、4EBP1、p70S6K基因表达水平均显著降低(P<0.05)。强精煎组睾丸组织PI3K、Akt、mTOR、4EBP1、p70S6K基因表达水平明显上调(P<0.05)。结论:强精煎可能通过激活PI3K/Akt//mTOR/4EBP1/p70S6K蛋白/基因表达、激活Ki-67蛋白表达等多途径调控少精子症大鼠生精细胞的增殖,促进精子发生,提高精子数量。

Objective:To explore the mechanism of Qiangjing Decoction regulating spermatogenesis in cyclophosphamide(CTX)-induced oligospermia model rats by mediating PI3K/Akt/mTOR pathway and its downstream target factors of 4EBP1 and p70S6K.Methods:75 male SD rats were randomly divided into the blank control group,the model group,the Rapamycin group(mTOR pathway inhibitor group),the Qiangjing Decoction plus Rapamycin group and the Qiangjing Decoction group,with 15 rats in each group.The rat model of oligospermia was induced by intraperitoneal injection of CTX.After 38 days of continuous drug intervention,the abdominal cavity was cut and the testis of the rats in each group was removed.The expressions of PI3K/Akt/mTOR pathway protein and its downstream pathway protein of 4EBP1 and p70S6K,as well as cell proliferation marker protein of Ki-67 were detected by immunohistochemistry.The expressions of PI3K/Akt/mTOR gene and its downstream target genes of 4EBP1 and p70S6K were detected by RT-PCT.Results:Compared with those in the blank control group,the protein expressions of PI3K,Akt,mTOR,4EBP1,p70S6K and Ki-67 of the testicular tissues were significantly decreased in the model group and in the Rapamycin group(P<0.05);the decreases in the Rapamycin group were more significant.Compared with those in the model group and in the Rapamycin group,the protein expressions of PI3K,Akt,mTOR,4EBP1,p70S6K and Ki-67 of the testicular tissues were significantly up-regulated in the Qiangjing Decoction group(P<0.05).Compared with those in the blank control group,the gene expressions of PI3K,Akt,mTOR,4EBP1 and p70S6K of the testicular tissues were significantly reduced in the model group and in the Rapamycin group(P<0.05),which were significantly up-regulated in the Qiangjing Decoction group(P<0.05).Conclusion:Qiangjing Decoction may regulate the proliferation of spermatogenic cells,promote spermatogenesis and increase the number of sperm in oligospermia rats by activating the protein and gene expressions of PI3K/Akt/mTOR pathway and its downstream targets of 4EBP1 and P70S6K,as well as by activating the protein expression of Ki-67.