汉黄芩素通过调控miR-451/PI3K/AKT/RXRA/Bcl-2信号通路改善低氧诱导的肺动脉高压

Wogonin protects hypoxia-induced pulmonary hypertension via regulating miR-451/PI3K/AKT/RXRA/Bcl-2 signaling pathway

目的:评价汉黄芩素对低氧诱导的肺动脉高压(HPH)的保护作用及其相关分子机制。方法:在体内采用成年雄性野生型C57BL/6小鼠建立HPH模型,将小鼠随机分为3组:对照组(N)、低氧(H+Saline)组、低氧+汉黄芩素(H+w)组。造模3周后,使用压力传感器系统采集小鼠血流动力学指标;肺小动脉管壁直径/管总直径(WT/TT)、肺小动脉管壁面积/管总面积(WA/TA)来评估肺动脉结构重塑;基于网络药理学方法筛选汉黄芩素作用于HPH潜在靶点及分子信号通路。在体外采用小鼠肺动脉平滑肌细胞(MPASMCs),将细胞分为5组:常氧(N)组、低氧(H+PBS)组、低氧+低剂量汉黄芩素(H+40μmol/L w)组、低氧+高剂量汉黄芩素(H+80μmol/L w)组、低氧+高剂量汉黄芩素+740Y-P(H+80μmol/L w+740Y-P)组。N组在常氧(5%CO_(2),21%O_(2),74%N2,37℃)环境中培养48 h,H+PBS、H+40μmol/L w、H+80μmol/L w、H+80μmol/L w+740Y-P组在低氧(5%CO_(2),3%O_(2),92%N2,37℃)环境中培养48 h。通过CCK-8、Ed U法检测评估细胞增殖能力,Transwell和伤口愈合/划痕实验用于检测评估细胞迁移能力,Western blot检测PI3K/AKT/RXRA/Bcl-2信号通路的主要蛋白(P-PI3K、PI3K、P-AKT、AKT、RXRA、Bcl-2)的表达,并利用PI3K通路激活剂740Y-P进行功能回补实验;RT-q PCR检测汉黄芩素对miR-451表达水平的影响,Western blot检测汉黄芩素对PI3K/AKT/RXRA/Bcl-2通路上游调控因子CAB39和MIF表达的影响。结果:与对照组相比,低氧组MPASMCs的增殖、迁移能力以及小鼠右心室压力(RVSP)显著增加(P<0.05),肺动脉结构发生显著重构,经汉黄芩素干预后MPASMCs的增殖、迁移能力以及小鼠RVSP显著下降(P<0.05),肺动脉结构重塑得到显著改善。与对照组相比,低氧组中PI3K/AKT/RXRA/Bcl-2信号通路相关蛋白p-PI3K、p-AKT、RXRA、Bcl-2表达显著升高;与低氧组相比,汉黄芩素干预组中PI3K/AKT/RXRA/Bcl-2信号通路相关的蛋白表达水平显著下调(P<0.05);功能回补实验证实在缺氧条件下,激活PI3K通路能够显著削弱汉黄芩素对MPASMCs增殖抑制效应。与对照组相比,低氧组中CAB39和MIF表达水平显著上升,miR-451表达水平显著减少(P<0.05);与低氧组相比,汉黄芩素干预组中CAB39和MIF表达水平显著下降,miR-451表达水平显著上升(P<0.05);miR-451 inhibitor干预能够部分逆转汉黄芩素对MPASMCs增殖和迁移能力的抑制效应(P<0.05)。结论:汉黄芩素可能通过上调mi R-451的表达靶向调控CAB39和MIF进而抑制PI3K/AKT/RXRA/Bcl-2信号通路,抑制低氧诱导的MPASMCs的增殖和迁移、改善HPH小鼠右心室压力和肺动脉结构重塑,最终缓解肺动脉高压。

Objective:To evaluate the potential protective effect of wogonin on hypoxic pulmonary hypertension(HPH)and to elucidate the molecular mechanism underlying these advantageous outcomes.Methods:In vivo,18 C57BL/6 male wild-type mice were randomly divided as normoxia group(N),hypoxia group(H+Saline)and hypoxia+wogonin group(H+w).The mouse hemodynamics were detected using a pressure sensor system 21 days after modeling.The degree of pulmonary artery remodeling was calculated by wall area/total area and wall thickness/total thickness.Prediction and screening of potential targets and molecular signaling pathways of wogonin in hypoxic pulmonary hypertension were performed based on network pharmacology.In vitro,mouse pulmonary artery smooth muscle cells(MPASMCs)were cultured under the respective condition of normoxia(N),hypoxia(H+PBS),hypoxia+low dose wogonin(H+40μmol/L w),hypoxia+high dose wogonin(H+80μmol/L w),hypoxia+high dose wogonin(H+80μmol/L w)+740Y-P(20μg/mL).The N group was exposed to normoxic(5%CO_(2),21%O_(2),74%N2,37℃)environment for 48 h.The H+PBS,H+40μmol/L w,H+80μmol/L w and H+80μmol/L w+740Y-P groups were exposed to hypoxic(5%CO_(2),3%O_(2),92%N2,37℃)environment for 48 h.Cell proliferation was assessed with the CCK-8 and EdU assay,respectively.The migration capacity of MPASMCs was determined by transwell and scratch wound healing assays.Protein expression profiles of the PI3K/AKT/RXRA/Bcl-2 signaling pathway(P-PI3K,PI3K,P-AKT,AKT,RXRA,Bcl-2)were analyzed using the Western blot method and the PI3K pathway activator 740Y-P was used for functional complementation experiments.The expression level of miR-451 was detected using RT-qPCR to assess the impact of wogonin.The expression of CAB39 and MIF,upstream regulatory factors of the PI3K/AKT/RXRA/Bcl-2 pathway,was examined by Western blot to detect the effect of wogonin on their regulation.Results:Comparative to the controls,hypoxic MPASMCs showed significantly increased proliferation and migration abilities(P<0.05).Moreover,the hypoxia group exhibited a significant increase in right ventricular systolic pressure(RVSP)and pulmonary vascular remodeling in comparison to the control group(P<0.05).After intervention with wogonin,the proliferation and migration activity of MPASMCs,RVSP and pulmonary vascular remodeling were rescued(P<0.05).The hypoxia group exhibited a significant increase in the expressions of p-PI3K,p-AKT,RXRA,and Bcl-2,which are proteins related to the PI3K/AKT/RXRA/Bcl-2 signaling pathway,when compared to the normoxia group.However,after wogonin intervention,the PI3K/AKT/RXRA/Bcl-2 signaling pathway was significantly inhibited(P<0.05).Functional restoration experiments confirmed that under hypoxic conditions,activation of the PI3K pathway can significantly weaken the inhibitory effect of wogonin on the proliferation of MPASMCs.In the context of the PI3K/AKT/RXRA/Bcl-2 pathway,it has been observed that wogonin exhibited a notable inhibitory effect on the upstream regulators CAB39 and MIF expression.Compared with to the normoxia group,the expression levels of CAB39 and MIF exhibited a significant up-regulation,while the expression of miR-451 demonstrated a significant downregulation in the hypoxia group(P<0.05).After wogonin intervention,the expression levels of CAB39,MIF and miR-451 were reversed.Further experimentation suggested that the intervention of a miR-451 inhibitor demonstrated a partial reversal of the inhibitory impact exerted by wogonin on the proliferation and migration of MPASMCs(P<0.05).Conclusion:Wogonin has the ability to hinder the PI3K/AKT/RXRA/Bcl-2 signaling pathway through the up-regulation of miR-451 expression.This intervention leads to the amelioration of abnormal proliferation and migration in hypoxic MPASMCs,as well as the remodeling of pulmonary vascular structure in mice afflicted with HPH.Consequently,the administration of wogonin results in a reduction of pulmonary hypertension.