基于蛋白质组学探讨双补丸佐制雷公藤多苷片生殖毒性的机制研究

Study on the mechanism of Shuangbu Pills in reducing reproductive toxicity induced by Tripterygium Glycoside Tablets based on proteomics

目的:应用标记定量(TMT)蛋白质组学技术,探究双补丸减轻雷公藤多苷片致雄性生殖损伤的作用机制。方法:将21只4周龄雄性幼鼠随机分为对照组、雷公藤组(12.5 mg·kg^(-1)·d^(-1))和双补丸组(3.125 g·kg^(-1)·d^(-1)),每组7只,连续灌胃12周。采用苏木精-伊红(HE)染色观察大鼠睾丸、附睾组织病理情况;采用TMT标记联合液相色谱-串联质谱技术(LC-MS/MS)进行蛋白质组学分析,并应用Western Blot验证富集通路的蛋白表达情况。结果:HE病理结果表明,双补丸能显著减轻雷公藤多苷引起的睾丸、附睾病理损伤,增加生精细胞及线性精子数量。蛋白组学共鉴定出蛋白质7381个。与对照组比较,雷公藤组有34个差异表达蛋白,经双补丸干预后有21个差异表达蛋白显著回调,其主要涉及与生殖相关的磷脂酰肌醇3激酶(PI3K)-蛋白激酶B(Akt)信号通路、过氧化物酶体增殖物激活受体(PPAR)信号通路。Western Blot结果表明,双补丸可显著上调与生殖密切相关的PI3K-Akt通路上磷酸化磷酸肌醇3激酶(p-PI3K)、磷酸化蛋白激酶B(p-Akt)、B细胞淋巴瘤/白血病-2(Bcl-2)蛋白表达(P<0.05)。结论:双补丸能够佐制雷公藤多苷片生殖毒性,其作用机制可能与调节PI3K-Akt介导的凋亡通路有关。

Objective:To explore the mechanism of Shuangbu Pills in alleviating the male reproductive damage induced by Tripterygium Glycoside Tablets(TGT)by using quantitative labeling(TMT)proteomics.Methods:Twenty-one 4-week-old male rats were randomly divided into Control group,TGT group(12.5 mg·kg^(-1)·d^(-1))and Shuangbu Pills group(3.125 g·kg^(-1)·d^(-1)),7 rats in each group,and gavaged continuously for 12 weeks.HE staining was used to observe the pathological changes of rat testis and epididymis;TMT labeling combined with liquid chromatography tandem mass spectrometry(LC-MS/MS)was used for proteomic analysis,and Western Blot was used to verify the protein expression of enrichment pathway.Results:The pathological results showed that Shuangbu Pills could significantly reduce the pathological damage of the testis and epididymis caused by TGT and increase the number of spermatogenic cells and linear sperm.A total of 7381 proteins were identified by proteomics.Compared with the control group,there were 34 differentially expressed proteins in the TGT group,and 21 differentially expressed proteins were significantly increased after the intervention of Shuangbu Pills,which are mainly involved in the PI3K-Akt signaling pathway and the PPAR signaling pathway related to reproduction.Western Blot results showed that Shuangbu Pills could significantly up-regulate the expression of p-PI3K,p-Akt,and Bcl-2 proteins on the PI3K-Akt pathway closely related to reproduction(P<0.05).Conclusion:Shuangbu Pills may play a role in assisting the reproductive toxicity of TGT by regulating PI3K-Akt mediated apoptosis pathway.