TMEM16A Regulates Cell Cycle Of Pulmonary Artery Smooth Muscle Cell In High-flow Induced Pulmonary Arterial Hypertension

O?JECTIVE In order to investigate the regulation of TMEM16 A in rats pulmonary artery smooth muscle cells(PASMCs)with high pulmonary blood flow induced pulmonary arterial hypertension(PAH).METHEDS Thirty Sprague-Dawley(SD)rats were randomly divided into control,sham and shunt groups.In the shunt group,a rat model of high pulmonary blood flow pulmonary hypertension was established by abdominal aorta-inferior vena cava sputum shunt.The sham group was treated with abdominal aortic clipping for 3 minutes and no vascular puncture anastomosis and the remaining operation steps were treated with the shunt group method,and the control group did not do any treatment.The three groups of rats were kept in the same environment for 11 weeks.Right ventricular pressure(RVP),Right ventricular hypertrophy index(RVHI),HE staining of lung tissue sections and abdominal B ultrasoundwere measured 11 weeks after the operation.Three groups of SD rat pulmonary artery smooth muscle tissue(grade 2-4 blood vessels)were taken for primary pulmonary artery smooth muscle cell culture.The TMEM16 A gene was silenced by siRNA technology using lentivirus as a vector.The cell cycle of pulmonary artery smooth muscle was detected by flow cytometry.The expression of cyclin D,CDK2,p27 KIP and cyclin E in pulmonary artery smooth muscle cells of each group was detected by Westren Blot.RESULTS 1.The model established successfully: The right ventricular pressure(RVP)of SD rats in the shunt group was significantly higher than that in the normal control group and the sham group.The cardiac hypertrophy index(RVHI)of SD rats in the shunt group was significantly higher than that in the normal control group and the sham group.HE staining of lung tissue sections showed that the pulmonary artery of SD rats in the shunt group was significantly thicker than the normal control group and the sham group.And Abdominal B-ultrasound showed that there was obvious arteriovenous shunt in the blood of the SD rats in the shunt group.The above experimental results confirmed that the rat model of high pulmonary blood flow pulmonary hypertension was successfully established.2.The expression of TMEM16A: The expression of TMEM16 A in the shunt group SD rats was significantly higher than that in the normal control group and the sham operation group,but there was no significant difference in the expression of TMEM16 A between the sham group and the control group.3.Cyclin expression and cycle ratio of each experimental group:Compared with the control group,the proportion of G0-G1 phase in the smooth muscle cells of the shunt group was significantly decreased(P<0.05),but the proportion of S phase+M/G2 phase,the expression cyclin D1 and cyclin E were significantly increased(P<0.05).The proportion of G0/G1 phase in smooth muscle cells of rats in the lentivirus-interfering group was significantly higher than that in the empty virus group and the shunt group(P<0.05).The proportion of S phase+M/G2 phase,cyclin D1 and cyclin E protein expression of smooth muscle cells in the lentiviral interference group were significantly lower than those in the empty virus group and the shunt group(P<0.05).There was no significant difference in the expression of CDK2 protein and p27 KIP protein in each group(P>0.05).CONCLUSION In the rat model of pulmonary hypertension with high pulmonary blood flow,the expression of TMEM16 A protein in pulmonary artery smooth muscle cells was significantly increased,which led to an increase in the expression of cyclin D1 and cyclin E,which further promoted the transition of cell cycle from G1 to S phase.Thereby enhancing the proliferation of pulmonary artery smooth muscle cells,promoting pulmonary artery remodeling,may be one of the pathogenesis of high pulmonary blood flow pulmonary hypertension.