The Role And Mechanism Of Pannexin-1 In Pulmonary Hypertension

Objective:In this study,SD rats were used as experimental objects to establish a model of monocrotaline（MCT）-induced Pulmonary Artery Hypertension（PH）and primary cultured pulmonary artery smooth muscle cells（PASMCs）in vitro to explore whether Pannexin-1 can regulate the expression and function of P2X7 receptor,activate the MEK1/2-ERK1/2 pathway,promote the proliferation and migration of pulmonary artery smooth muscle cells,and thus affect pulmonary vascular remodeling in pulmonary hypertension.It aims to provide new insights into the pathogenesis of PH and provide new targets for the prevention and treatment of PH.Method:1)In vivo:PH model was established by intraperitoneal injection of MCT,which was divided into CON group,MCT group,MCT+probenecid（Prob,an inhibitor of Pannexin-1）group,and Prob group.The changes of pulmonary artery blood flow were detected by echocardiography.HE staining was used to detect the remodeling of right ventricle and pulmonary arterioles,right ventricular hypertrophy index,the percentage of vascular wall thickness（WT%）and the percentage of vascular wall area（WA%）were calculated.The expressions of Pannexin-1,P2X7,proliferating cell nuclear antigen（PCNA）,osteopontin（OPN）,MEK1/2-ERK1/2 in lung tissue and pulmonary vessels were detected by tissue immunofluorescence and Western blot.2)In vitro:Primary cultured PASMCs were divided into:1)CON group,DMSO group,PDGF-BB（Platelet-derived growth factor-BB）group,PDGF-BB+Prob group;2)CON group,DMSO group,PDGF-BB group,PDGF-BB+Prob group,PDGF-BB+U0126（MEK1/2-ERK1/2 pathway inhibitor）group;3)CON group,DMSO group,PDGF-BB group,PDGF-BB+A740003（P2X7 inhibitor）group.CCK-8,Ed U assay,scratch test and Transwell ^TM test were used to detect the effect of Pannexin-1 on the proliferation and migration of PASMCs;The changes of Pannexin-1,PCNA and OPN were detected by immunofluorescence assay;Western blot was used to detect the expression of Pannexin-1,PCNA,OPN,P2X7 and MEK1/2-ERK1/2 pathway proteins;ATP kit and calcium imaging were used to detect extracellular ATP content and the level of intracellular Ca²⁺.Result:1)In vivo:The expression of Pannexin-1 in the lung tissue of SD rats in MCT group was significantly increased,and Pannexin-1 inhibition significantly increased the pulmonary artery acceleration time（PAAT）（P<0.01,n=6）,decreased the mean pulmonary artery pressure（m PAP）（P<0.01,n=6）and the right heart hypertrophy index（P<0.05,n=6）,significantly improved the lung small vessel remodeling（P<0.01,P<0.05,n=6）and significantly down-regulated the expression of PCNA,OPN,P2X7,p-MEK1/2and p-ERK1/2 proteins in lung tissue and blood vessels induced by MCT（P<0.05,P<0.01,n=6）.2)In vitro:Pannexin-1 protein expression on PASMCs in PDGF-BB group was up-regulated,Pannexin-1 inhibition significantly inhibited the proliferation,migration and healing ability of PASMCs induced by PDGF-BB（P<0.01,n=6）;The protein expression of PCNA,OPN,p-MEK1/2 and p-ERK1/2 in PASMCs induced by PDGF-BB was significantly down-regulated（P<0.01,P<0.05,n=6）,while the extracellular ATP concentration of PASMCs（P<0.001,n=4）,the expression of P2X7（P<0.01,n=3）and Ca²⁺influx were inhibited.Conclusion:MCT induced an increase in the expression of Pannexin-1 in PASMCs in the middle and small vessels of the rat lung;Pannexin-1 up-regulates the expression of P2X7 receptor,and activates P2X7receptor by releasing ATP,thus increasing the influx of Ca²⁺,activating the MEK1/2-ERK1/2 pathway,promoting the proliferation and migration of PASMCs,and promoting pulmonary vascular remodeling.Inhibition of Pannexin-1 can down-regulate the expression of P2X7 receptor,inhibit P2X7 function by reducing the concentration of extracellular ATP,reduce the influx of Ca²⁺,inhibit the activation of MEK1/2-ERK1/2 pathway,inhibit the proliferation and migration of PASMCs,improve the remodeling of pulmonary small and medium-sized vessels,and play a role in the prevention and treatment of PH.