表没食子儿茶素没食子酸酯对H9N2猪流感病毒诱导小鼠肺损伤中Toll样受体-4表达的影响

Effect of Epigallocatechin Gallate on Toll-like Receptor-4 Expression in Mouse Model of Acute Lung Injury Induced by H9N2 Swine Influenza Virus

旨在探讨表没食子儿茶素没食子酸酯(EGCG)对猪源H9N2流感病毒感染诱导小鼠肺损伤及氧化应激相关信号通路Toll样受体(TLR)-4表达的影响。将6~8周龄、雌性SPF级BALB/c小鼠随机分为病毒感染致急性肺损伤(H9N2)组、H9N2+EGCG组、模拟感染(Mock)组、Mock+EGCG以及TLR-4抑制剂Eritoran5564(H9N2+E5564)对照组,观察各组小鼠肺组织病理学变化,测定肺湿/干重比;测定肺组织内MPO、T-SOD、抗HO·能力、MDA、IL-1β和TNF-α的含量;Western blot和RT-PCR方法检测肺内TLR-4 mRNA及蛋白质的表达。结果表明,H9N2组小鼠精神沉郁、呼吸困难、体重下降明显;肺组织学表现为肺泡壁水肿、炎性细胞浸润、出血为特征的弥漫性肺组织损伤。EGCG干预后,与H9N2组相比,EGCG治疗组小鼠临床症状较轻,一定程度上降低了死亡率,并明显延长小鼠存活时间(P<0.01);其肺组织损伤程度较轻,肺湿/干重比极显著下降(P<0.01);MPO和MDA的含量显著降低,与之相反T-SOD及抗HO·能力升高;同时,EGCG显著降低肺组织内IL-1β和TNF-α的含量。肺组织内TLR-4mRNA以及蛋白表达显著降低;E5564呈现与EGCG相似的干预效果,显著降低TLR-4mRNA以及蛋白表达,降低肺组织MPO、MDA、IL-1β和TNF-α的含量,减少T-SOD的消耗以及提高抗HO·能力。EGCG明显缓解小鼠肺损伤过程,其机制可能与其影响活性氧自由基的产生或清除进而显著降低TLR-4的表达有关,提示其在辅助预防和干预H9N2-SIV诱导的肺损伤方面具有潜在的应用前景。

The present research investigated effect of epigallocatechin gallate(EGCG)on toll-like receptor(TLR)-4expression in lung injury induced by H9N2 swine influenza virus infection in mice.A total of 400 female BALB/c(SPF)mice were randomized into five groups and treated as follow:(1)H9N2group,(2)H9N2+EGCG treatment group,(3)mock control,(4)mock+EGCG and(5)pharmacological inhibitor of TLR-4Eritoran5564(H9N2+E5564).The lung histopathology,lung water content,and MPO activity,OH · scavenging activity,the content of MDA and cytokines(IL-1βand TNF-α),and the mRNA and protein expression of TLR-4in the lung tissue were observed or investigated at 2,4,6,8,and 14 days after inoculation.The results showed:(1)H9N2virus-infected mice presented depression,dyspnea and weight loss dramatical-ly;Histopathologically,alveolar and interstital edema,hemorrhage and inflamatory cell infiltration were observed in H9N2-infected mice.(2)Compare to that of H9N2-inected mice,the EGCG treatment alleviated clinical signs and the histological lesion,prolonged survival time and decreased mortality(35% vs 65%,P<0.05).It also inhibited MPO activity,decreased MDA content,and increased T-SOD level and OH· scavenging activity.Moreover,the lung wet weight to dry weight ratio(P<0.05)and the content of IL-1βand TNF-αin lung tissue significantly decreased compare with that of H9N2-inected mice.(3)The EGCG treatment markedly down-regulated the levels of mRNA and protein of TLR-4in the lungs of H9N2-infected mice(P<0.05).Similarly,the E5564 inhibited the expression of TLR-4mRNA and protein in lung tissue of H9N2 virus infected mice;It also decreased dramatically the levels of MPO,MDA,IL-1βand TNF-α,but the levels of T-SOD and OH· scavenging activity were higher than H9N2 group.These data demonstrated that EGCG down-regulated remarkably the levels of mRNA and protein of TLR-4and effected the produce the levels of MPO,MDA,IL-1βand TNF-αduring H9N2 viral infection,thus supporting the use of EGCG for managing ALI induced by H9N2-SIV influenza in future.