The Experimental Study Of Polyphyllin ? Regulat Autophagy In Pulmonary Artery Smooth Muscle Cells And Inhibit Pulmonary Hypertension

Objective:Pulmonary hypertension(PH)is a hemodynamic condition in which pulmonary artery pressure increases beyond a certain threshold.It is characterized by pulmonary vascular remodeling and contraction,increased pulmonary vascular resistance,and progressive increase of pulmonary artery pressure,which eventually leads to right heart failure and death.The occurrence and development of PH is the result of the joint action and interaction of multiple factors and molecules,and its pathogenesis is particularly complex.There are various clinical types of PH,and its pathogenesis is also different.According to recent studies,targeted therapeutic drugs for PH,still have unsatisfactory therapeutic effects and long-term prognosis.In recent years,more and more attention has been paid to autophagy,which is an effective internal regulation mechanism for organisms to adapt to different environments Autophagy has become an important mechanism for the maintenance of normal physiological functions and the occurrence of pathological phenomena.The mechanism of autophagy in the occurrence and development of PH has also been gradually discovered.Polyphyllin ?(PP7)has been found to have many effects,such as anti-tumor effects by promoting apoptosis and autophagy death,anti-inflammatory,inhibit angiogenesis.We hypothesized that PP7 may prevent the occurrence and development of PH by regulating autophagy.In order to verify this scientific hypothesis and further reveal its mechanism,we had designed and completed the following experiments.Methods:Cell experiments:pulmonary artery smooth muscle cells(PASMCs)were primary culture.The optimal concentration of ET-1 induced the proliferation of PASMCs was selected.The cells were divided into 5 groups:control group,endothilin-1(ET-1)group and ET-1+PP7(concentration:0.25 mol·L-1,0.5 mol·L-1,0.75 mol·L-1)group.Cell counting kit-8(CCK8)and Transwell methods were used to detect the capacity of proliferation and migration of PASMCs in each group at 24h,48h and 72h,as well as the cycle changes and apoptosis rate detected by flow cytometry.The expressions of autophagy related proteins LC3B and p62/SQSTM1 of PASMCs in each group were detected by Western Blot at 24h.Animal model:adult male SD rats were randomly divided into 5 groups:control group(Con);Monocrotaline(MCT)-induced PH model group(MCT);MCT was combined with Sildenafil(10mg/kg/d)group(Sildenafil);both MCT and PP7(0.2mg/kg/d))treatment group(PP7 0.2);both MCT and PP7(1.0mg/kg/d)treatment group(PP7 1.0).After 21 days,we check each rat right ventricular systolic pressure(RVSP);right ventricular hypertrophy index(RVHI);pulmonary arteriolar wall remodeling indicators:the percentage of wall thickness of pulmonary arteriole(WT)in the arterial outer diameter(WT%)and the percentage of external diameter of pulmonary arterioles(WA%)in the total vascular area(WA%).The expressions of autophagy related proteins LC3B,Beclin 1,p62/SQSTM1 and autophagy related signaling molecules p-mTOR,p-ERK1/2 and ERK1/2 were detected by Western Blot.Results:In cell experiments,the optimal concentration of ET-1-induced the proliferation and migration of PASMCs was 10"7mol L-1.Compared with the group of ET-1-induced the PASMCs,different concentrations of PP7(0.25?mool·L-1,0.5?mol·L-1,0.75?mol·L-1)reduced the proliferation and migration of ET-1-induced PASMCs in a dose-dependent manner.The PP7 0.75?mol L-1 group much more reduced the proliferation and migration of ET-1-induced PASMCs.PP7 inhibited the cell cycle progression of PASMCs under the action of ET-1,induced the transformation from G0/G1 phase to S phase,and inhibited the transformation from S phase to G2/M phase.The results of apoptosis rate showed that the proportion of normal cells increased significantly and the proportion of late apoptotic/dead cells decreased significantly after the treatment of PASMCs with ET-1 for 48h compared with the control group.The addition of PP7 reversed this apoptosis rate,indicating that the apoptosis rate of PASMCs tended to be normal.In addition,the expression of LC3B?/? decreased in the ET-1-induced PASMCs group,but increased with the raised of PP7 concentration.The expression of p62/SQSTM1 increased in the ET-1-induced PASMCs group,and decreased in dose dependent manner.In animal models:RVSP,RVHI,WT%,WA%of MCT group was significantly higher than control group(P<0.001)and RVSP,RVHI,WT%,WA%of Sildenafil group was significantly reduced than MCT group(P<0.01).RVSP,RVH1,WT%of PP7 0.2 group was not significantly different from that in the MCT group.However,RVSP,RVHI,WT%of PP7 1.0 group was significantly reduced than MCT group Western Blot showed that,comparing with Con group,LC3B-?/?,Beclin 1 and p-mTOR,p-ERK1/2 protein expressions in rat lung tissues of MCT group significantly increased.Comparing with MCT group,following the increasing concentrations of PP7,LC3B-?/?,Beclin 1 and protein expressions in rat lung tissues significantly increased(P<0.05),and p-mTOR expression significantly decreased(P<0.001).With the increase of PP7 concentration,the expressions of p62/SQSTM1 and p-ERK 1/2 increased.Conclusion:1.PP7 inhibited the proliferation and migration of rats PASMCs induced byET-1,and induced the apoptosis of rats PASMCs.2.PP7 can effectively inhibit MCT-induced pulmonary vascular remodeling and PH in rats.3.The mechanism of PP7 inhibiting the proliferation and migration of ET-1-induced PASMCs and MCT-induced PH in rats may be related to the promotion of autophagy of PASMCs.4.PP7 may regulate autophagy in PASMCs through ERK and mTOR pathways.