二甲双胍在慢性血栓栓塞性肺动脉高压大鼠肺动脉内皮间充质转化中的作用

The role of metformin in the endothelial-mesenchymal transition of pulmonary artery in chronic thromboembolic pulmonary hypertension rat

目的探索二甲双胍在慢性血栓栓塞性肺动脉高压(CTEPH)大鼠肺动脉内皮间质转化中的作用。方法60只SD(Sprague Dawley)大鼠随机分为正常对照组、二甲双胍对照组、CTEPH组、二甲双胍低剂量组[50 mg/(kg·d)]、二甲双胍高剂量组[100 mg/(kg·d)]。经颈静脉反复注入自体血栓柱制备CTEPH模型,二甲双胍低剂量组和二甲双胍高剂量组分别用二甲双胍50 mg/(kg·d)和100 mg/(kg·d)给大鼠灌胃。6周后,测量平均肺动脉压力(mPAP)、肺动脉管壁面积/血管总面积(WA%),检测肺动脉内皮波形蛋白(vimentin)、α平滑肌肌动蛋白(α-SMA)、CD31、上皮型钙黏蛋白(E-cadherin)、β-连环蛋白(β-catenin)和磷酸化腺苷酸活化蛋白激酶(p-AMPK)抗原和蛋白质表达情况,并进行比较和相关性分析。结果与正常对照组相比,CTEPH组mPAP、WA%明显升高,二甲双胍对照组mPAP、WA%差异无统计学意义;与CTEPH组相比,不同剂量二甲双胍组连续给药6周后均能降低mPAP、WA%,且高剂量组降低更明显[mPAP:正常对照组(12.82±1.80)比二甲双胍对照组(12.94±1.78)比CTEPH组(25.50±2.89)比二甲双胍低剂量组(21.69±2.59)比二甲双胍高剂量组(16.74±2.86)mm Hg,F=60.59,P<0.01;WA%:正常对照组(30.16±5.26)%比二甲双胍对照组(30.15±4.73)%比CTEPH组(63.81±6.25)%比二甲双胍低剂量组(49.96±6.85)%比二甲双胍高剂量组(41.47±5.68)%,F=72.48,P<0.01]。与正常对照组相比,CTEPH组波形蛋白、α-SMA、β-catenin抗原和蛋白表达增高,CD31、E-cadherin抗原和蛋白表达降低(均P<0.05);p-AMPK抗原和蛋白表达差异无统计学意义。与正常对照组相比,二甲双胍对照组波形蛋白、α-SMA、CD31、E-cadherin、β-catenin抗原和蛋白表达差异无统计学意义;p-AMPK抗原和蛋白表达增高(均P<0.05)。与CTEPH组相比,不同剂量二甲双胍组波形蛋白、α-SMA、β-catenin抗原和蛋白表达均不同程度下降,且高剂量组降低更明显(P<0.05);CD31、E-cadherin、p-AMPK抗原和蛋白表达均不同程度升高,且高剂量组升高更明显(均P<0.05)。结论二甲双胍可以改善CTEPH大鼠mPAP及肺血管结构重构,减弱CTEPH大鼠肺动脉内皮间充质转化,其机制可能与二甲双胍激活AMPK,下调β-catenin通路有关。二甲双胍对正常大鼠肺动脉mPAP及肺动脉内皮无影响。

Objective To investigate the role of metformin on endothelial mesenchymal transformation in rats with chronic thromboembolic pulmonary hypertension(CTEPH).Methods Sixty Sprague Dawley(SD)rats were randomly divided into 5 groups:normal control group,metformin control group,CTEPH group,metformin low-dose group[50 mg/(kg·d)],and metformin high-dose group[100 mg/(kg·d)].The autologous thrombus column was repeatedly injected through the jugular vein to establish the CTEPH model.The rats in low-dose metformin group and high-dose metformin group were given metformin 50 mg/(kg·d)and 100 mg/(kg·d)by gavage each day.After 6 weeks,the mean pulmonary artery pressure(mPAP),pulmonary artery wall area to total area of the tube ratio(WA%)and the antigens and protein expression of vimentin,α-smooth muscle actin(α-SMA),CD31,E-cadherin,β-catenin and p-adenosine 5’-monophosphate activated protein kinase(p-AMPK)in pulmonary artery were measured.Results Compared with the normal control group,mPAP,WA%were significantly increased in CTEPH group,while were not significant different in metformin control group;compared with the CTEPH group,mPAP,WA%reduced in metformin group,and the reduction in the high-dose group was statistically significant[mPAP:normal control group(12.82±1.80)vs metformin control group(12.94±1.78)vs CTEPH group(25.50±2.89)vs low-dose metformin group(21.69±2.59)vs metformin high-dose group(16.74±2.86)mm Hg,F=60.59,P<0.01;WA%:normal control group(30.16±5.26)%vs metformin control group(30.15±4.73)%vs CTEPH group(63.81±6.25)%vs low-dose metformin group(49.96±6.85)%vs metformin high-dose group(41.47±5.68)%,F=72.48,P<0.01].Compared with the normal control group,the antigen and protein expression of vimentin,α-SMA,β-catenin were significantly increased,while the antigen and protein expression of CD31,E-cadherin were decreased in the CTEPH group(all P<0.05).There was no significant difference in the antigen and protein expression of p-AMPK between the normal control group and CTEPH group.There was no significant difference of antigen and protein expression of vimentin,α-SMA,CD31,E-cadherin,and β-catenin between the normal control group and metformin control group,while the metformin control group had higher antigen and protein expression of p-AMPK(P<0.05).Compared with the CTEPH group,the antigen and protein expression of vimentin,α-SMA and β-catenin reduced in all rats treated with metformin,and the reduction in the high-dose group was statistically significant(P<0.05).Which was opposite to the antigen and protein expression of CD31,E-cadherin and p-AMPK,especially in the high-dose group(P<0.05).Conclusions Metformin can reduce mPAP and improve pulmonary vascular structure remodeling by attenuating pulmonary endothelial-mesenchymal transition in CTEPH rats,which may be related to the activation of AMPK and down-regulation of β-catenin pathway.Metformin has no effect on mPAP and pulmonary artery endothelium in normal rats.