二甲双胍诱导子宫内膜异位症在位内膜细胞凋亡的研究

Induction of Eutopic Endometrial Apoptosis by Metformin in Endometriosis

目的:研究二甲双胍对大鼠子宫内膜异位症模型在位内膜细胞凋亡的影响。方法:采用手术自体移植法建立大鼠子宫内膜异位症模型,随机将成模鼠分为A组(二甲双胍50mg/kg·d),B组(二甲双胍100mg/kg·d)和C组(二甲双胍200mg/kg·d)各10只,D组(0.9%氯化钠溶液对照)8只。用药4周后采用TUNEL法检测细胞凋亡指数,逆转录PCR(RT-PCR)技术分别检测在位子宫内膜组织中Bcl-2、Bax和P53基因转录水平;Westernblotting检测磷酸化AMPK和磷酸化mTOR蛋白的表达;流式细胞仪分析细胞周期的分布情况。结果:A、B、C组凋亡率均高于D组(P<0.05),其中B组效果更显著;A、B、C组Bcl-2mRNA、BaxmRNA、P53mR-NA、Bcl-2mRNA/BaxmRNA比值、P-AMPK、P-mTOR与D组比较,差异均有统计学意义(P<0.05)。A、B、C组在位内膜细胞G0/G1期比例明显高于D组(P<0.05),且B组高于A组(P<0.05)、A组高于C组(P<0.05)。结论:二甲双胍调节Bcl-2、Bax和P53基因表达,从而诱导在位内膜细胞凋亡,以100mg/kg·d效果最明显。

Objective：To investigate the effects of metformin on eutopic endometrial apoptosis in experi- mentally induced endometriosis in a rat model and to identify the pathways involved in the effects. Methods： The model of endometriosis in rats was made by surgically autotransplanting endometrium to the peritoneum, and the 38 SD rat models were randomly divided into four groups. They were given 50,100 and 200 mg/kg/ day of oral metformin in group A （n = 10）, B （n = 10） and C （n = 10）, respectively, for 28 days. GroupD （n=8） was given saline as placebo. Then Bcl-2, Bax and P53 expressions were determined by RT-PCR. Modulation of protein expression of phospho-AMPK and phospho-mTOR were determined by western blot- ting. Apoptosis and cell cycle analysis were assessed by TUNEL and flow cytometry. These measurements and evaluations were made by the operators who blinded to the study. Results： Metformin induced apoptosis （15.29±0. 035, 29. 65±0. 259, 25.96±0. 178, 7. 14±0. 052, P 〈 0. 05） in eutopic endometrium by TUNEL, the apoptosis index was highest after 100 mg/kg/day metformin; Western blotting showed an in- crease of phospho-AMPK activated form, and an decrease of phospho-mTOR activated form （P 〈 0.05） ; RT-PCR demonstrated that the metformin could induce apoptosis in eutopic endometrium by down-regulating Bcl-2 protein expression, and up-regulating Bax and P53 protein expression （ P 〈 0. 001） ; Metformin pro- voked a cell cycle arrest in the G0/G1 phase （P〈O. 05） ; Moreover, protein expression of phospho-AMPK was not linear correlated with the number of cells in the G0/G1 phase （r = 0. 834, P = 0. 166）. Conclu- sions： We established that metformin could induce apoptosis by down-regulating Bcl-2 and up-regulating Bax and P53 expression. Bcl-2, Bax and P53 targeted strategies were suggested to constitute an effective therapeutic tool for the treatment of endometriosis.