甘草酸二铵对百草枯诱导肺泡上皮Ⅱ型细胞高迁移率族蛋白1的影响

Effects of Diammonium Glycyrrhizinate on the Expression of HMGB1 in Alveolar Epithelial Cell with Paraquat

目的了解甘草酸二铵(DG)对百草枯(PQ)诱导肺泡上皮Ⅱ型细胞(alveolar epithelial cellⅡ,AECⅡ)中高迁移率族蛋白1(high mobility group box 1,HMGB1)的影响。方法 AECⅡ用含10%胎牛血清的RPMI 1640培养基,并加入青霉素100 U/ml、链霉素100μg/L,置于37℃含5%CO2的培养箱中培养。将培养后的细胞分为3组,空白对照组(NS组)不采用任何药物干预培养24 h;模型组(PQ组):以1000μmol/L PQ培养24 h;DG预治疗组(DG组):先以0.6 mg/ml DG培养2 h,再以0.6 mg/ml DG+1000μmol/L PQ培养24 h。采用MTT法测定DG对PQ诱导下AECⅡ的增殖作用;采用酶联免疫吸附法检测3组细胞中的HMGB1、Toll样受体4(TLR-4)、髓样分化因子88(My D88)、细胞核因子-κB(NF-κB)、肿瘤坏死因子(TNF)-α含量;采用实时荧光定量聚合酶链式反应测定3组细胞中HMGB1、TLR-4、My D88、NF-κB P65 mRNA表达。结果在1000μmol/L PQ诱导下,AECⅡ以0.6 mg/ml DG干预24 h时生存率最高,以0 mg/ml DG干预生存率最低。与NS组比较,PQ、DG组HMGB1、TLR-4、My D88、NF-κB P65、TNF-α水平均明显升高,且DG组升高程度低于PQ组,差异均有统计学意义(P<0.01);与NS组比较,PQ组、DG组HMGB1、TLR-4、My D88、NF-κB P65 mRNA表达均升高,且DG组升高程度低于PQ组,差异均有统计学意义(P<0.01)。结论 DG可降低HMGB1、TLR-4、My D88、NF-κB、TNF-α水平,减轻PQ诱导的AECⅡ损伤。

Objective To investigate the effects of Diammonium Glycyrrhizinate （DG） on the expression of HMGB1 in rat alveolar epithelial cell Ⅱ（AECⅡ）with paraquat （PQ）. Methods The AECⅡ was cultured in RPMI-1640 media containing 10% FBS, 100 U/ml penicillin and 100 μg/L streptomycin at 37＆#176;C in 5% CO2.The cells were divided into three groups： The NS group was induced 24 h without any drugs, the PQ group was induced 24 h by 1000 μmol/L PQ ,and the DG group was induced 2 h by 0.6 mg/ml DG, then, was induced 24 h by 0.6 mg/ml DG＋1000 μmol/L PQ.MTT assay was used to measure the proliferation effect of DG on AEC Ⅱ with PQ.The ELISA assay was applied to measuring the levels of HMGB1, TLR-4,MyD88,NF-κB, TNF-α in the three groups.The gene expressions of HMGB1,TLR-4, MyD88, NF-κB P65 mRNA in the three groups were detected by R T-PCR.Results The highest survival rate of AECⅡ induced 24 h by 1000 μmol/L PQ was in the group of 0.6 mg/ml DG, the lowest survival rate of AECⅡ induced 24 h by 1000 μmol/L PQ was in the group of 0 mg/ml DG.The levels of HMGB1, TLR4, MyD88, NF-κB P65, TNF-α in PQ and DG group were higher than those in NS group.While, the levels of HMGB1, TLR-4, MyD88, NF-κB P65, TNF-α in DG group were lower than those in PQ group （P〈0.01）.The expression of HMGB1, TLR-4, MyD88, NF-κB P65, TNF-α mRNA in PQ and DG group were higher than those in NS group.The expression of HMGB1, TLR4,Myd88,NF-κB P65,TNF-αmRNA in DG group were lower than those in PQ group（P〈0.01）. Conclusion DG can attenuate the injury in rat alveolar epithelial cells caused by paraquat poisoning.And it can decrease the expressions of HMGB1, TLR-4, MyD88, NF-κB and TNF-α.