连朴饮含药血清对肺泡Ⅱ型上皮细胞炎症反应的干预机制

Intervention Mechanism of Serum Containing Lianpuyin on Inflammatory Response of Alveolar Type II Epithelial Cells

目的:基于GR基因沉默探讨连朴饮改善急性肺损伤(ALI)炎症反应的机制。方法:构建沉默GR基因的慢病毒载体,制备含药血清,将培养的原代肺泡Ⅱ型上皮细胞(AECⅡ)随机分为9组:正常对照组、模型对照组、地塞米松0.2 mg/kg组、连朴饮9.3 g/kg组、干扰对照组、GR干扰组、GR干扰模型组、GR干扰地塞米松0.2 mg/kg组、GR干扰连朴饮9.3 g/kg组;运用QRT-PCR法检测AECⅡ的GR基因沉默效率,运用QRT-PCR、Western blot法检测各组TNF-αmRNA及蛋白的表达,运用ELISA检测各组细胞上清液IL-8和IL-1β的含量,FITC-Dextran法检测细胞通透性。结果:与干扰对照组比较,GR干扰组的GR mRNA表达水平显著下调,GR mRNA沉默效率达67%(P<0.05或P<0.01);与连朴饮9.3 g/kg 5%含药血清组比较,GR干扰连朴饮组TNF-αmRNA及蛋白的表达显著上调,IL-8和IL-1β的含量、细胞通透性显著增高(P<0.05或P<0.01)。结论:9.3 g/kg连朴饮5%含药血清能抑制TNF-α、IL-8和IL-1β分泌,降低细胞通透性,改善AECⅡ炎症反应,从而防治ALI,其部分通过GR为介导是改善AECⅡ炎症反应的作用途径之一。

Objective:To explore the mechanism of Lianpuyin on improving inflammation of ALI based on GR gene silencing.Methods:Lentiviral vector containing GR gene was constructed and serum containing drug was prepared.Primary cultured AEC II was randomly divided into 9 groups,the control group,the model group,0.2 mg/kg dexamethasone group,9.3 g/kg Lianpuyin group,interference control group,GR interference group,GR interference+model group,GR interference+0.2 mg/kg dexamethasone group and GR interference+9.3 g/kg Lianpuyin group.The silencing efficiency of GR gene of AEC II was detected by QRT-PCR,the expressions of TNF-αgene and protein in each group were detected by QRT-PCR and Western blot,the contents of IL-8 and IL-1βin cell supernatant were detected by ELISA,and the cell permeability was detected by FITC-Dextran.Results:Compared with the interference control group,the expression level of GR gene in the GR interference group was significantly decreased,the silent efficiency of GR gene was 67%.Compared with 5%serum containing 9.3 g/kg Lianpuyin group,the expressions of TNF-αmRNA and protein,the contents of IL-8 and IL-1β,the cell permeability in GR interference+9.3 g/kg Lianpuyin group were increased.Conclusion:The serum containing Lianpuyin can inhibit the expression of TNF-α,IL-8 and IL-1β,it may also reduce cell permeability and improve the inflammation reaction of AEC II,thus prevent and treat ALI.Part of its mechanism may be mediated by GR to improve the inflammation reaction of AEC II.