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-1β（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Bad mRNA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 kinase β，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-1β、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; mRNA 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 mRNA expressions and Bcl-2/Bax, Bcl-xl/Bad (P < 0.01). Tormentic acid significantly down-regulated TLR4, MyD88, phosphorylated IκB kinase β (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.

R285.5

湖北省生物酵素工程研究技术研究中心项目（JS2018-06）；宜昌市科学技术局资助项目（A18-302-a2，A21-2-044）；三峡大学硕士学位论文培优基金资助项目（2020SSPY114，2019SSPY159，2018SSPY140，2017YPY086）