The Role Of Metformin In The Endothelial-mesenchymal Transition Of Pulmonary Artery In CTEPH Rat

Objective:To investigate the role of metformin in endothelial mesenchymal transformation in rats with chronic thromboembolic pulmonary hypertension(CTEPH).Methods:Sixty SD rats were randomly divided into the control group and the experimental group,which were divided into the normal control group and the metformin control group,and the experimental group was divided into the CTEPH group,the low dose of metformin group(50mg/kg/d),and the high dose of metformin group(100 mg/kg/d)(see ref.13 for dosage).The left common jugular vein of the experimental group was isolated after anesthesia.Each rat was injected with autologous thrombus column and normal saline containing tranexamic acid through the left jugular vein to prepare the chronic thromboembolic pulmonary hypertension model.The low-dose group and the high-dose group were given metformin 50mg/kg/d and 100 mg/kg/d,respectively,every day.After 4 days and 7 days,three embolizations were performed by the same method.After each embolization,penicillin was intramuscularly injected to prevent infection.Tranexamic acid was intraperitoneally injected throughout the procedure.Normal control group and metformin control group were injected with normal saline instead of thrombus column into the left common jugular vein.The remaining operations were performed in the CTEPH group and the high-dose metformin group,respectively.After 6 weeks of feeding,mean pulmonary artery pressure(mPAP)and pulmonary artery wall area/total tube area(WA/TA)were measured.Rat pulmonary artery was isolated and immunohistochemical method was used to ob-serve the expression changes of pulmonary artery endothelial antigen vimentin,α-SMA,CD31,E-cadherin and p-AMPK.Protein expression of pulmonary artery vimentin,α-SMA,CD31,E-cadherin,β-catenin and p-AMPK was determined by western blot.Real-time fluorescence quantitative PCR(QPCR)method was used to determine the mRNA levels of pulmonary artery vimentin,α-SMA,CD31,E-cadherin,β-catenin and p-AMPK,and to conduct comparison and correlation analysis.Results:The measurement results of mPAP,WA% and RVHI showed that compared withthe normal control group,mPAP,WA% and RVHI in the CTEPH group were significantly increased,and the difference was statistically significant(P<0.05).There was no significant difference in mPAP,WA% and RVHI measurements in the metformin control groupcompared with the normal control group.Compared with the CTEPH group,mPAP,WA% and RVHI in the lung were decreased in the metformin group after 6 weeks of continuous administration,and the difference was statistically significant as the dose increased(P<0.05).The results of vimentin,α-SMA,CD31,E-cadherin,β-catenin and p-ampk showed that compared with the normal control group,the mRNA and protein expressionsof vimentin,α-SMA,β-catenin in the CTEPH group increased,and the difference was statistically significant(P<0.05);CD31,E-cadherin mRNA and protein expression decreased,the difference was statistically significant(P<0.05);There was no significant difference between p-AMPK mRNA and protein expression.There was no significant difference in mRNA and protein expression of vimentin,α-SMA,CD31,E-cadherin,β-catenin in metformin control group compared with normal control group.P-AMPK mRNA and protein expression increased,and the difference was statistically significant(P<0.05).Compared with the CTEPH group,the mRNA and protein expressions of Vimentin,α-SMA,and β-catenin in the metformin group all decreased to different degrees after 6 weeks of administration,and decreased as the dose increased,with statistically significant differences(P<0.05);CD31,E-cadherin,p-AMPK mRNA and protein expression increased to varying degrees and increased with dose,with statistically significant differences(P<0.05).In the experimental group,mPAP was positively correlated with the expression of vimentin,α-SMA,and β-catenin(r=0.801,P<0.05;r=0.816,P<0.05;r=0.843,P<0.05),negatively correlated with the expression of CD31,E-cadherin and p-AMPK(r=-0.909,P<0.05;r=-0.897,P<0.05;r=-0.857,P<0.05).p-AMPK was positively correlated with the expression of CD31 and E-cadherin(r=0.841,P<0.05;r=0.797,P<0.05),negatively correlated with vimentin,α-SMA and β-catenin protein expression(r=-0.703,P<0.05;r=-0.747,P<0.05;r=-0.874,P<0.05).Conclusion : The endothelium of the pulmonary artery of CTEPH rats underwent obvious mesenchymal transformation.Metformin can improve the mPAP and pulmonary vascular structure in CTEPH rats,and weaken the endothelial mesenchymal transformation in the pulmonary artery of CTEPH rats,which may be related to the activation of AMPK by metformin and the down-regulation of the β-catenin pathway.Metformin had no effect on pulmonary mPAP and Pulmonary endothelium in normal rats.