委陵菜酸对幽门螺杆菌诱导GES-1细胞损伤的保护作用

Protective effect of tormentic acid on Helicobacter pylori induced GES-1 cells injury

目的探索委陵菜酸对幽门螺杆菌(Helicobacter pylori,Hp)诱导人胃黏膜上皮细胞GES-1损伤的影响。方法GES-1细胞与Hp共培养,给予委陵菜酸和NOD样受体家族3(NOD-like receptor family 3,NLRP3)抑制剂MCC950,考察各组细胞存活率、乳酸脱氢酶(lactate dehydrogenase,LDH)释放率、集落形成、凋亡率、线粒体膜电位和活性氧自由基(reactive oxygen species,ROS)变化;采用ELISA法检测各组细胞上清液中单核细胞趋化蛋白-1(monocyte chemotactic protein-1,MCP-1)、角质细胞趋化因子(keratinocyte chemokines,KC)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-Iβ(interleukin-1β,IL-1β)、IL-6、IL-18、IL-4和IL-10水平;采用试剂盒检测各组细胞谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)活性和丙二醛(malondialdehyde,MDA)含量;采用qRT-PCR检测各组细胞Toll样受体4(toll-like receptor 4,TLR4)、髓样分化因子88(myeloid differentiation factor 88,MyD88)、Bcl-2、Bcl-xl、Bax和BadmRNA表达情况;采用Western blotting法检测各组细胞TLR4/NLRP3/核因子-κB(nuclear factor-κB,NF-κB)和线粒体凋亡信号通路相关蛋白表达情况。结果委陵菜酸显著抑制Hp诱导的GES-1细胞LDH释放率(P<0.05、0.01),促进细胞集落形成(P<0.05、0.01),抑制细胞凋亡(P<0.05、0.01);升高细胞线粒体膜电位(P<0.01),降低ROS水平(P<0.01);降低上清液中MCP-1、KC、TNF-α、IL-1β、IL-6和IL-18水平(P<0.01),升高IL-4和IL-10水平(P<0.01);升高细胞GSH-Px、SOD和CAT活性(P<0.01),降低MDA含量(P<0.01);降低细胞TLR4、MyD88、Bax、Bad mRNA表达水平(P<0.01),升高Bcl-2、Bcl-xl mRNA表达水平和Bcl-2/Bax、Bcl-xl/Bad(P<0.01);下调细胞TLR4、MyD88、磷酸化IκB激酶β(phosphorylated inhibitor kappa B kinaseβ,p-IKKβ)、p-IκBα、NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein,ASC)、半胱氨酸蛋白酶-1前体(pro-Caspase-1)、Caspase-1、硫氧还蛋白互作蛋白(thioredoxin-interacting protein,TXNIP)、pro-IL-lβ、pro-IL-18、Bax、Bad、细胞色素C、凋亡酶激活因子-1(apoptotic protease activating factor-1,Apaf-1)、剪切型Caspase-9(cleaved Caspase-9)、cleaved Caspase-3和胞核p65蛋白表达水平(P<0.01),上调Bcl-2、Bcl-xl、pro-Caspase-9、pro-Caspase-3和胞质p65蛋白表达水平(P<0.01)。结论委陵菜酸对Hp诱导的GES-1细胞损伤具有明显保护作用,其作用机制可能与增强内源性抗氧化系统功能、抑制氧化应激、炎性反应及TLR4/NF-κB/NLRP3炎症小体信号通路激活,从而减少线粒体介导的凋亡密切相关。

Objective To explore the effect of tormentic acid on Helicobacter pylori(Hp)-induced human gastric mucosal epithelial cells GES-1 injury.Methods GES-1 cells were co-cultured with Hp,and then given tormentic acid and NOD-like receptor family 3(NLRP3)inhibitor MCC950.Cell survival rate,lactate dehydrogenase(LDH)release rate,colony formation,apoptosis rate,mitochondrial membrane potential and reactive oxygen species(ROS)were investigated;Levels of monocyte chemotactic protein-1(MCP-1),keratinocyte chemokines(KC),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,IL-18,IL-4 and IL-10 in supernatant of GES-1 cells were detected by ELISA;Glutathione peroxidase(GSH-Px),superoxide dismutase(SOD),catalase(CAT)activities and malondialdehyde(MDA)levels were detected by kits;m RNA expressions of toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),Bcl-2,Bcl-xl,Bax and Bad were detected by qRT-PCR;The related protein expressions of TLR4/NLRP3/nuclear factor-κB(NF-κB)and mitochondrial apoptosis signal pathways were detected by Western blotting.Results Tormentic acid significantly inhibited the release rate of LDH induced by Hp(P<0.05,0.01),promoted cells colony formation(P<0.05,0.01),inhibited cell apoptosis(P<0.05,0.01),increased mitochondrial membrane potential(P<0.01),decreased ROS level(P<0.01),reduced levels of MCP-1,KC,TNF-α,IL-1β,IL-6 and IL-18 in supernatant(P<0.01),increased levels of IL-4 and IL-10 in supernatant(P<0.01).Tormentic acid significantly increased activities of GSH-Px,SOD and CAT(P<0.01),reduced MDA level(P<0.01).Tormentic acid significantly reduced TLR4,MyD88,Bax and Bad mRNA expressions(P<0.01),increased Bcl-2,Bcl-xl m RNA expressions and Bcl-2/Bax,Bcl-xl/Bad(P<0.01).Tormentic acid significantly down-regulated TLR4,MyD88,phosphorylated IκB kinaseβ(p-IKKβ),p-IκBα,NLRP3,apoptosis-associated speck-like protein(ASC),pro-Caspase-1,Caspase-1,thioredoxin-interacting protein(TXNIP),pro-IL-1β,pro-IL-18,Bax,Bad,cytochrome C,apoptotic protein activating factor-1(Apaf-1),cleaved Caspase-9,cleaved Caspase-3 and nuclear p65 protein expressions(P<0.01),and up-regulated Bcl-2,Bcl-xl,pro-Caspase-9,pro-Caspase-3 and cytoplasmic p65 protein expressions(P<0.01).Conclusion Tormentic acid has a notable protective effect on Hp-injured GES-1 cells,and its mechanism might be closely related to enhancing the function of endogenous antioxidant system,inhibiting the oxidative stress,inflammatory response,and inflammatory activation of TLR4/NF-κB/NLRP3 inflammasome signaling pathway,thereby reducing mitochondrial mediated apoptosis.