Effects And Mechanism Of TMEM16A On Pulmonary Artery Smooth Muscle Cells Proliferation In Pulmonary Arterial Hypertension Induced By High Pulmonary Blood Flow In Rats

Objectives: We investigated the proliferation change and its potential mechanism of TMEM16 A on pulmonary artery smooth muscle cells(PASMCs)in pulmonary arterial hypertension induced by high pulmonary blood flow in rats.Methods: Thirty Sprague-Dawley(SD)rats weighing 180-220 g were randomly divided into control group,sham group and shunt group.The rats in the shunt group were created the shunt fistula from abdominal aorta to inferior vena cava to create the PAH model induced by high pulmonary blood flow,the rats in the sham group received blocking-up for blood flow in abdominal aorta for ten minutes,and the rats in the control group were without any treatment.After 11 weeks from creation of the shunt fistula,right ventricular hypertrophy index(RVHI),pulmonary arterial pressure and pulmonary arteriole HE staining were used to verify the success of the PAH model.The PASMCs were primary cultured in each group rats.The m RNA and protein expression of TMEM16 A were measured by q RT-PCR and Western Blot respectively.The protein expression of PCNA,AKT,p-AKT,ERK,p-ERK were measured by Western Blot.The proliferation of PASMCs in each group was measured by cell proliferative assay(CCK8).Then,cells of the shunt group were transfected by lentivirus or blocked by AKT pathway inhibitor(LY294002)and ERK pathway inhibitor(U0126),respectively,and divided into shunt group,shunt + lentivirus negative group,shunt + lentiviral interference group,shunt + LY294002 group,and shunt + U0126 group.Protein expression of TMEM16 A,PCNA,AKT,p-AKT,ERK and p-ERK were detected by western blot,and cell proliferation was detected by CCK8 assay.Results: The PAH rat model of pulmonary arterial hypertension induced by high pulmonary blood flow was successfully established measured by right ventricular pressure,right ventricular hypertrophy index and pulmonary arteriole HE staining.The m RNA expression of TMEM16 A in the PASMCs of shunt group was significantly increased compared with the control group(P<0.05),while there was no statistically significant difference between the sham group and the control group(P>0.05).The protein expressions of TMEM16 A,PCNA,p-AKT and p-ERK in the PASMCs of shunt group were significantly increased compared with the control group(P<0.05),while there was no difference between the control and sham group(P>0.05).The protein expressions of TMEM16 A,PCNA,p-AKT and p-ERK in the PASMCs of the shunt + lentiviral interference group were significantly decreased compared with the shunt group(P<0.05),while there were no statistically significant differences between the shunt + lentivirus negative group and the shunt group(P>0.05).The protein expressions of PCNA,p-AKT and p-ERK in the PASMCs of the shunt+LY294002 group were decreased compared with the shunt group(P<0.05),the protein expressions of PCNA and p-ERK in the PASMCs of the shunt+U0126 group were decreased compared with the shunt group(P<0.05),and the expressions of p-AKT was no difference compared with the shunt group(P>0.05).The optical density(OD)of PAMSCs in shunt group in 48 hours and after were significantly higher than that in the control group(P<0.05),the OD of PAMSCs in shunt + lentiviral interference group in 48 hours and after were significantly decreased than that in the shunt group(P<0.05),which was no significant difference between shunt group and shunt + lentivirus negative group at each time nodes,as well as between sham group and control group(P>0.05).The OD of PASMCs in shunt+LY294002 group and shunt +U0126 group in 48 hours and after were significantly decreased compared with the shunt group(P<0.05).Conclusion:(1)TMEM16A is highly expression in PAH PASMCs and promote PASMCs proliferation in the PAH rats induced by high pulmonary blood flow.(2)TMEM16A may be involved in the pathogenesis of PAH induced by high pulmonary blood flow by promoting the phosphorylation of EKR and AKT to promote the proliferation of PASMCs.