基于NLRP3炎性小体活化探讨地龙提取物对肺动脉高压的保护机制

Protective effect of Pheretima extract on pulmonary arterial hypertension based on NLRP3 inflammasome activation

目的研究地龙提取物对肺血管内皮细胞损伤及肺动脉高压的保护作用及机制。方法雄性SD大鼠随机分成对照组、模型组、模型+地龙提取物组及地龙提取物组,给予地龙提取物干预后,采用颈静脉插管检测各组大鼠右心室收缩压,计算右心室肥大指数;分离肺组织,采用苏木素-伊红(HE)染色观察肺血管形态学变化;采用免疫荧光及Western blotting法检测肺组织Nod样受体家族的吡啶结构域3 (Nod-like receptor family pyrin domain-containing protein 3,NLRP3)蛋白表达;采用流式细胞术检测肺组织活性氧(reactiveoxygenspecies,ROS)水平;采用ELISA法检测肺组织白细胞介素-1β(interleukin-1β,IL-1β)、IL-6和IL-18水平。体外培养人肺动脉内皮细胞(human pulmonary arterial endothelial cell,HPAEC),给予NLRP3激动剂尼日利亚菌素诱导细胞损伤,考察地龙提取物对HPAEC凋亡、线粒体功能、ROS水平及IL-1β、半胱氨酸蛋白酶-1 (Caspase-1)和IL-18蛋白表达的影响。结果与对照组比较,模型组大鼠右心室收缩压和右心肥大指数显著升高,肺血管壁增厚(P<0.01),肺组织NLRP3蛋白表达水平显著升高,ROS、IL-1β、IL-6和IL-18水平升高(P<0.01);地龙提取物可显著降低模型大鼠右心室收缩压和右心肥大指数(P<0.01),改善肺血管重构,同时下调肺组织NLRP3蛋白表达、ROS、IL-1β、IL-6和IL-18的水平(P<0.05、0.01)。尼日利亚菌素诱导HPAEC线粒体结构损伤及ROS增强,导致细胞凋亡(P<0.01);地龙提取物对尼日利亚菌素诱导的HPAEC凋亡、ROS水平、线粒体功能损伤及IL-1β、IL-6和IL-18蛋白表达有显著抑制作用(P<0.05、0.01)。结论地龙提取物通过降低肺血管内皮NLRP3诱导的线粒体ROS水平升高和炎症反应,从而减轻肺动脉高压。

Objective To elucidate protective effect and mechanism of Dilong(Pheretima) extract on pulmonary vascular endothelial cell damage and pulmonary arterial hypertension. Methods Male SD rats were randomly divided into control group, model group,model + Pheretima extract group and Pheretima extract group. After intervention with Pheretima extract, jugular vein cannula was used to detect right ventricular systolic pressure(RVSP), and right ventricular hypertrophy index(RVHI) was calculated. Lung tissue was isolated, and pulmonary vascular morphology changes were observed by HE staining. Nod-like receptor family pyrin domaincontaining protein 3(NLRP3) expression was detected by immunofluorescence and Western blotting. Flow cytometry was used to detect reactive oxygen species(ROS) level;Interleukin-1β(IL-1β), IL-6, and IL-18 levels in supernatants were detected by ELISA.Human pulmonary artery endothelial cells(HPAEC) were cultured in vitro, and NLRP3 agonist nigericin was applied to stimulate cell damage. Effects of Pheretima extract on HPAEC apoptosis, mitochondrial function, ROS level and IL-1β, Caspase-1 and IL-18 protein expressions were investigated. Results Compared with control group, RVSP and RVHI of rats in model group were significantly increased, pulmonary vessel wall was thickened(P < 0.01);NLRP3 protein expression in lung tissue was increased, ROS, IL-1β, IL-6 and IL-18 levels were significantly increased(P < 0.01). RVSP and RVHI of model rats were significantly reduced(P < 0.01),pulmonary vascular remodeling was improved, NLRP3 protein expression, ROS, IL-1β, IL-6 and IL-18 levels in lung tissue were down-regulated by Pheretima extract(P < 0.05, 0.01). HPAEC mitochondrial structure damage and ROS enhancement which leading to cell apoptosis were induced by nigerian(P < 0.01);Nigericin-induced HPAEC apoptosis, ROS levels, mitochondrial function damage and IL-1β, IL-6 and IL-18 protein expressions were significantly inhibited by Pheretima extract(P < 0.05, 0.01). Conclusion Pheretima extract reduces pulmonary arterial hypertension by ameliorating increases in mitochondrial ROS level and inflammatory factors induced by NLRP3 in pulmonary vascular endothelial cells.