白藜芦醇通过Nrf2/ARE通路减轻柯萨奇病毒B3感染的乳鼠心肌细胞炎症和氧化应激反应

Resveratrol Alleviates Inflammation and Oxidative Stress in Coxsackievirus B3 Infected Cardiomyocyte of Neonatal Rat Through Nrf2/ARE Pathway

柯萨奇病毒B3(Coxsackievirus B3,CVB3)是引起病毒性心肌炎的主要病毒株,CVB3感染能够通过激活炎症反应及氧化应激反应来造成心肌细胞损伤。白藜芦醇(Resveratrol,RES)是具有抗炎、抗氧化作用的多酚化合物,已经被证实能够减轻缺血缺氧、缺氧复氧、脂多糖、过氧化氢等病理因素引起的心肌细胞损伤,但RES对CVB3感染引起心肌细胞损伤的保护作用尚未明确。为了研究RES通过核因子-E2相关因子2(Nuclear factor E2-related factor 2,Nrf2)/抗氧化反应元件(Antioxidant response element,ARE)通路减轻CVB3感染乳鼠心肌细胞的炎症和氧化应激反应,本研究采用原代培养大鼠乳鼠心肌细胞,分为DMEM处理的对照组、CVB3感染的CVB3组、CVB3感染及RES处理的RES组、CVB3感染及RES、Nrf2抑制剂鸦胆苦醇处理的RES+鸦胆苦醇组、CVB3感染及RES、血红素加氧酶-1(Heme oxygenase-1,HO-1)抑制剂锌原卟啉-9(Zinc protoporphyrin-9,ZnPP9)处理的RES+ZnPP9组,比较5组间心肌酶、CVB3拷贝数、炎症因子、氧化应激指标、Nrf2/ARE通路分子的差异。结果显示:与对照组比较,CVB3组培养基中乳酸脱氢酶(Lactate dehydrogenase,LDH)、肌酸激酶同工酶-MB(Creatine kinase isoenzyme-MB,CK-MB)、白介素(Interleukin,IL)-1β、IL-6、IL-8、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、活性氧簇(Reactive oxygen species,ROS)、丙二醛(Malondialdehyde,MDA)的含量及细胞中CVB3拷贝数、Nrf2、NADH依赖的醌氧化还原酶1(NAD(P)H:quinone oxidoreductase-1,NQO-1)、HO-1的表达量明显增多(P<0.05),培养基中超氧化物歧化酶(Superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(Glutathione peroxidase,GPx)的含量明显减少(P<0.05);与CVB3组比较,RES组培养基中LDH、CK-MB、IL-1β、IL-6、IL-8、TNF-α、ROS、MDA的含量明显减少(P<0.05),培养基中SOD、GPx的含量及Nrf2、NQO-1、HO-1的表达量明显增多(P<0.05),CVB3拷贝数无明显变化(P>0.05);与RES组比较,RES+鸦胆苦醇组、RES+ZnPP9组培养基中LDH、CK-MB、IL-1β、IL-6、IL-8、TNF-α、ROS、MDA的含量明显增多(P<0.05),培养基中SOD、GPx的含量明显减少(P<0.05),CVB3拷贝数无明显变化(P>0.05)。本研究揭示RES能够减轻CVB3感染引起的乳鼠心肌细胞炎症和氧化应激反应,激活Nrf2/ARE通路可能是其分子机制。

Coxsackievirus B3(CVB3)is a major viral strain causing viral myocarditis. CVB3 infection can cause cardiomyocyte damage by activating inflammatory and oxidative stress response. Resveratrol(RES) is a polyphenol compound with anti-inflammatory and antioxidant effects. It has been proved that RES can alleviate cardiomyocyte damage caused by ischemia-hypoxia,hypoxia-reoxygenation,lipopolysaccharide,hydrogen peroxide and other pathological factors. However,the protective effect of RES on cardiomyocyte injury induced by CVB3 infection has not been clarified. This research is to study the effect of RES on reducing inflammation and oxidative stress in neonatal rat cardiomyocytes infected with CVB3 through Nrf2/ARE pathway. Neonatal rat cardiomyocytes were primaryly cultured and divided into DMEM-treated control group,CVB3-infected CVB3 group,CVB3-infected and RES-treated RES group,CVB3-infected and Nrf2 inhibitor brucepin-treated RES+brucepin group,CVB3-infected and RES,HO-1 inhibitor ZnPP9-treated RES+ZnPP9 group. The differences of myocardial enzymes,copies of CVB3,inflammatory factors,oxidative stress indicators and Nrf2/ARE pathway molecules were compared among the five groups. Results showed that compared with the control group,the contents of LDH,CK-MB,IL-1β,IL-6,IL-8,TNF-α,ROS and MDA in culture medium and the copies of CVB3,the expression of Nrf2,NQO-1 and HO-1 in cells in CVB3 group were significantly increased(P<0.05),while the contents of SOD and GPx in culture medium were significantly decreased(P<0.05).Compared with CVB3 group,the contents of LDH,CK-MB,IL-1β,IL-6,IL-8,TNF-α,ROS and MDA in culture medium were significantly decreased(P<0.05),the contents of SOD,GPxin culture medium and the expression of Nrf2,NQO-1 and HO-1 in cells were significantly increased(P<0.05),while the copies of CVB3 did not change significantly(P>0.05). Compared with RES group,the contents of LDH,CK-MB,IL-1β,IL-6,IL-8,TNF-α,ROS and MDA in culture medium were significantly increased in RES+brucepin group and RES+ZnPP9 group(P<0.05),the contents of SOD,GPx in culture medium were significantly decreased(P<0.05),while the copies of CVB3 did not change significantly(P>0.05). The present study suggested that RES could alleviate inflammation and oxidative stress induced by CVB3 infection in neonatal rat cardiomyocytes and activating Nrf2/ARE pathway was a possible molecular mechanism.