伞形酮通过抑制RhoA/ROCK信号通路和自噬改善慢性低氧性肺动脉高压

Umbelliferone improves chronic hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy

本研究考察低氧对肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)中RhoA/Rho激酶(Rho-kinase,ROCK)信号通路和自噬的影响,探讨伞形酮改善低氧性肺动脉高血压的作用。体外培养PASMCs,并将其分为4组:对照组、低氧组、低氧+伞形酮组、常氧+伞形酮组,免疫荧光染色评估平滑肌肌动蛋白α和LC3,Western blot检测RhoA、ROCK2、p-MYPT1、LC3-II、LC3-I、Beclin-1、p62、C-Caspase3、Bax和Bcl-2蛋白表达水平。在体研究中,将Sprague-Dawley(SD)大鼠分为3组:对照组、低氧组、低氧+伞形酮组,评估右心室重量和左心室加室内隔重量之比[RV/(LV+S)],HE染色评估肺动脉形态特征。结果显示,与对照组相比,低氧组PASMCs中LC3-II/LC3-I比例和Beclin-1表达显著增加,p62表达显著降低;RhoA、ROCK2和p-MYPT1蛋白表达显著增加(P<0.05)。而相比低氧组,低氧+伞形酮组PASMCs中的LC3-II/LC3-I比例和Beclin-1表达降低,p62表达显著增加;RhoA、ROCK2和p-MYPT1蛋白表达显著降低(P<0.05)。低氧大鼠的肺动脉壁增厚,RV/(LV+S)比值增高,而低氧+伞形酮组有明显改善(P<0.05)。这些结果表明伞形酮通过抑制PASMCs的RhoA/ROCK信号通路和自噬改善低氧性肺动脉高血压。

This study aimed to investigate the effects of hypoxia on RhoA/Rho-kinase(ROCK)signaling pathway and autophagy in pulmonary artery smooth muscle cells(PASMCs),and to explore the underlying mechanism of Umbelliferone(Umb)in ameliorating chronic hypoxic pulmonary hypertension.PASMCs were cultured from Sprague-Dawley(SD)rats and randomly divided into control group,hypoxia group,hypoxia+Umb intervention group and normoxia+Umb intervention group.Alpha smooth muscle actin(α-SMA)and LC3 were assessed by immunofluorescence staining.Protein expression of RhoA,ROCK2,p-MYPT1,LC3-II,Beclin-1,p62,C-Caspase 3,Bax and Bcl-2 was analyzed by Western blotting.In in vivo study,SD rats were divided into control group,hypoxia group and hypoxia+Umb intervention group.Weight ratio of the right ventricle(RV)/left ventricle plus septum(LV+S)was detected,and pulmonary arterial morphological features were examined by HE staining.The results indicated that compared with the control group,the LC3-II/LC3-I ratio and expression of Beclin-1 were significantly increased,while p62 expression was significantly decreased,and the expressions of RhoA,ROCK2 and p-MYPT1 were significantly increased in PASMCs of hypoxia group(P<0.05).The changes of LC3-II/LC3-I ratio,the expressions of Beclin-1,p62,RhoA,ROCK2 and p-MYPT1 in PASMCs were reversed by Umb treatment(P<0.05).Consistently,the pulmonary arterial wall was thickened and the RV/(LV+S)ratio was increased in hypoxic rats,which were significantly improved by Umb treatment(P<0.05).These results suggest that Umb can improve hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy in PASMCs.