清热解毒方连花汤提取物诱导子宫内膜癌HEC-1A及Ishikawa细胞自噬的作用及其机制研究

Effect and mechanism of Qingrejiedu Lianhua soup extracted on autophagy of HEC-1A and Ishikawa cells in endometrial carcinoma

目的探讨清热解毒方连花汤（黄连15 g、白花蛇舌草15 g、半枝莲15 g、生黄芪20 g等）正丁醇和水提取物对人子宫内膜癌细胞系HEC-1A及Ishikawa作用12 h、24 h后自噬相关蛋白微管相关蛋白1轻链3（light chain 3,LC3）表达及其相关通路的影响。方法采用Real-time PCR检测LC3mRNA;采用Westernbolt检测LC3蛋白;采用Phosflow（TM）检测雷帕霉素靶蛋白（MTOR）、蛋白激酶B（AKT）表达。结果与阴性对照组比较,正丁醇组12 h HEC-1A中LC3 mRNA升高但24 h下降,而LC3蛋白在12 h和24 h均下降,水提组则正好相反,雷帕霉素组LC3表达均下降,同时中药复方醇提和水提组AKT/mTOR通路均不同程度表达增加（P〈0.05）;而在Ishikawa中,与阴性对照组比较,三组处理后LC3 mRNA表达在12 h增加,除雷帕霉素组其余两组24 h均下降。醇提组12 h、水提组24 h、雷帕霉素组AKT/mTOR双表达与阴性对照组比较,差异均有统计学意义（P〈0.05）。结论在HEC-1A中,连花汤正丁醇提取物与雷帕霉素作用类似,可能激活AKT/mTOR通路抑制细胞自噬;而在Ishikawa细胞中连花汤正丁醇组与水提组均通过激活AKT/mTOR通路自噬先增加后抑制,与雷帕霉素作用后自噬均增加不同。

Objective To investigate the effects Lianhua soup which consists of Berberine 15 g, Hedyotisdiffusa 15 g, Scutellariabarbata 15 g and Health Astragalus 20 g extracted with N-butanol and water, and the expressions of autophagy-associated protein light chain 3（LC3） and the autophagy-related pathway proteins mTOR and AKT in HEC-1A and Ishikawa cells for 12 and 24 hours, respectively. Methods Real-time PCR was used to detect the expression of LC3 mRNA and Western-bolt was used to detect the expression of LC3 protein. The expressions of mTOR and AKT were detected by Phosflow^（TM）. Results Compared with the negative control group, the LC3 mRNA in n-butanol group increased at 12 h but decreased at 24 h in HEC-1A group, but LC3 protein expression decreased at 12 h and 24 h in HEC-1A group, while the expression of LC3 mRNA in rapamycin group decreased.While the expression of AKT/mTOR pathway in Chinese medicine extract group increased to some extent（P〈0.05）. In Ishikawa, compared with the negative control group, the expression of LC3 increased after 12 h in three groups, and decreased in the other two groups except rapamycin group. AKT/mTOR expression in alcohol extraction group 12 h, water extraction group 24 h, rapamycin group and negative control group were different. Conclusions In HEC-1A, L-butanol group and Lianhua Tang may activate AKT/mTOR pathway to inhibit autophagy. In Ishikawa cells, the n-butanol-treated group and the water-withdrawn group were increased by autophagy after activation of AKT/mTOR pathway, and then inhibited by autophagy. However, autophagy increased after treated with rapamycin.