萝卜硫素调控Nrf2预防肺动脉高压大鼠右心室损伤和肺血管重构的机制研究

Nrf2 Regulation by Sulforaphane to Prevent Right Ventricular Injury and Pulmonary Vascular Remodeling in Rats with Pulmonary Arterial Hypertension

目的:探究萝卜硫素通过调控转录因子核因子红细胞系2相关因子2(Nrf2)预防肺动脉高压(PAH)大鼠的右心室损伤和肺血管重构的机制。方法:30只用成年雄性SD大鼠随机分为对照组、模型组和萝卜硫素组,每组10只。模型组和萝卜硫素组大鼠构建PAH大鼠模型。经胸回声测量使用高频、高分辨率数字成像平台检测大鼠右心室心脏指数(CI)、肺动脉瓣速度时间积分(VTI)、肺动脉加速时间(PAT)、肺动脉射血时间(PET)、右心室舒张期内径(IDd)、右心室自由壁厚度(FW)、右心室等容松弛时间(IVRT)、三尖瓣舒张早期心肌收缩速度(RVE)、三尖瓣舒张早期心肌舒张速度(RVE′)、收缩压速度(S′)和心肌性能(Tei)指数。蛋白免疫印迹法(Western Blot)检测大鼠Nrf2和NQO1蛋白表达。实时荧光定量聚合酶链反应(RT-PCR)检测纤维连接蛋白(FN)、结缔组织生长因子(CTGF)、Ⅰ型胶原α1(COL1A1)、Ⅰ型胶原α2(COL1A2)mRNA表达以及肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β和IL-6 mRNA表达。检测氧化应激标志物丙二醛(MDA)、还原性谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和总抗氧化能力(TAC)。通过免疫组化分析大鼠全肺α-SMA蛋白表达。结果:模型组SOD、GSH-Px、TAC和GSH水平,CI、VTI、PAT、PET、RVE′、S′、Tei指数、Nrf2、α-SMA和NQO1表达较对照组降低(P<0.05),萝卜硫素组SOD、GSH-Px、TAC,GSH水平,CI、肺动脉瓣VTI、PAT/PET、RVE′、S′、Tei指数、Nrf2、α-SMA和NQO1表达较模型组升高(P<0.05)。模型组IDd、FW、IVRT、RVE/RVE′、FN、CTGF、COL1A1、COL1A2、TNF-α、IL-1β和IL-6 mRNA蛋白表达、MDA水平较对照组升高(P<0.05),萝卜硫素组IDd、FW、IVRT、RVE/RVE′、FN、CTGF、COL1A1、COL1A2、TNF-α、IL-1β和IL-6 mRNA蛋白表达、MDA水平较模型组降低(P<0.05)。结论:萝卜硫素通过上调Nrf2/NQO1减少缺氧暴露诱导的PAH中肺血管重构并改善右心室功能障碍,萝卜硫素作为一种预防PAH的新型辅助疗法可能具有重要意义。

Objective:To explore the mechanism of sulforaphane prevent right ventricular injury and reduce pulmonary vascular remodeling in rats with pulmonary arterial hypertension(PAH)by regulating transcription factor nuclear factor erythrocyte 2-associated factor 2(Nrf2).Methods:Thirty adult male SD rats were randomly divided into control group,model group and sulforaphane group,with 10 rats in each group.The rat model of pulmonary arterial hypertension was established in model group and sulforaphane group.Right ventricular heart index(CI),pulmonic velocity-time integral(VTI),pulmonic acceleration time(PAT),pulmonic ejection time(PET),right ventricular diastolic inner diameter(IDd),right ventricular free wall thickness(FW),and right ventricular isovolute relaxation time(IVRT)were measured,tricuspid valve early diastolic myocardial contraction(RVE),tricuspid valve early diastolic myocardial diastolic velocity(RVE′),systolic blood pressure velocity(S′)and myocardial performance(Tei)by using a high-frequency,high-resolution digital imaging platform.The expression of Nrf2 and NQO1 proteins in rats were detected by Western Blot.Real-time polymerase chain reaction(RT-PCR)was used to detect the mRNA expression of fibronectin(FN),connective tissue growth factor(CTGF),collagen-type Iα1(COL1A1),collagen-type Iα2(COL1A2),tumor necrosis factorα(TNF-α),interleukin-1β,and IL-6 mRNA expression.Oxidative stress markers malondialdehyde(MDA),glutathione(GSH),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and total antioxidant capacity(TAC)were tested.The whole lungα-SMA protein expression was analyzed by immunohistochemistry.Results:The levels of SOD,GSH-Px,TAC,and GSH,CI,VTI,PAT,PET,RVE′,S′,Tei index,Nrf2,α-SMA,and NQO1 in model group were lower than those in the control group(P<0.05).SOD,GSH-Px,TAC,GSH levels,CI,pulmonary valve VTI,PAT/PET,RVE′,S′,Tei index,Nrf2,α-SMA,and NQO1 expression in sulforaphane group were higher than those in the model group(P<0.05).IDd,FW,IVRT,RVE/RVE′,FN,CTGF,COL1A1,COL1A2,TNF-α,IL-1β,and IL-6 mRNA protein expression and MDA levels in model group were more than those in the control group(P<0.05).The expression of IDd,FW,IVRT,RVE/RVE′,FN,CTGF,COL1A1,COL1A2,TNF-α,IL-1β,and IL-6 mRNA,α-SMA protein,and MDA levels in sulforaphane group were mower than those in the model group(P<0.05).Conclusion:Sulforaphane reduces pulmonary vascular remodeling in hypoxic expose-induced PAH and improves right ventricular dysfunction by uspregulating Nrf2/NQO1.Sulforaphane may be useful as a novel adjuvant therapy for the prevention of PAH.