Toll样受体4调控的程序性坏死和铁死亡对对乙酰氨基酚肝损伤的影响

doi:10.6040/j.issn.1671-7554.0.2023.1016

摘要/Abstract

摘要： 目的探究Toll样受体4(toll-like receptor 4, TLR4)是否通过调控程序性坏死及铁死亡进一步影响对乙酰氨基酚(acetaminophen, APAP)肝损伤过程及其发生机制。方法体外培养人正常肝细胞L-02,采用CCK-8法检测细胞活力,筛选出最佳APAP和TAK-242浓度。将实验分为对照组、APAP组(1、4、12 h)和APAP+TAK-242组(1、4、12 h),比较各组细胞TLR4 mRNA表达;检测各组细胞匀浆液的丙氨酸氨基转移酶(alanine aminotransferase, ALT)和天门冬氨酸氨基转移酶(aspartate aminotransferase, AST)水平;检测各组细胞核因子-κB(nuclear factor-κB, NF-κB)、白细胞介素-6(interleukin-6, IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)水平;检测各组细胞高迁移率族蛋白1(high mobility group box 1, HMGB1)、受体相互作用蛋白激酶1(receptor interacting protein kinase 1, RIP1)、受体相互作用蛋白激酶3(receptor interacting protein kinase 3, RIP3);检测各组细胞内Fe²⁺含量以及NF-κB、 P53、重组溶质载体家族7成员11(solute carrier family 7 member 11, SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4, GPX4)水平。结果通过CCK-8法,确定5 mmol/L APAP和100 nmol/L TAK-242作为后续实验浓度。与对照组比较,各时间点APAP组TLR4 mRNA水平上调(P<0.05);与APAP组比较,同一时间点APAP+TAK-242组TLR4 mRNA水平下调(P<0.05/3=0.016 7)。与对照组比较,各时间点APAP组ALT、AST水平升高(P<0.05);与APAP组比较,同一时间点APAP+TAK-242组ALT、AST水平下降(P<0.05/3=0.016 7)。与对照组比较,各时间点APAP组NF-κB、IL-6、TNF-α mRNA表达均上调(P<0.05);与APAP组比较,同一时间点APAP+TAK-242组NF-κB、 IL-6、TNF-α mRNA表达均下调(P<0.05/3=0.016 7)。与对照组比较,各时间点APAP组HMGB1、RIP1、RIP3蛋白水平均升高(P<0.05);与APAP组比较,同一时间点APAP+TAK-242组HMGB1、RIP1、RIP3蛋白水平均降低(P<0.05/3=0.0167)。与对照组比较,各时间点APAP组Fe²⁺含量、NF-κB和P53蛋白水平均上升(P<0.05),而SLC7A11和GPX4蛋白水平和mRNA表达均降低(P<0.05);与APAP组比较,同一时间点APAP+TAK-242组Fe²⁺含量、NF-κB和P53蛋白水平均降低(P<0.05/3=0.016 7),而SLC7A11和GPX4蛋白水平和mRNA表达均升高(P<0.05/3=0.016 7)。结论抑制TLR4可通过调节TLR4/HMGB1信号通路下调程序性坏死,以及可能通过调节TLR4/NF-κB信号通路下调炎症反应和铁死亡来减轻APAP肝损伤。

关键词: 对乙酰氨基酚, Toll样受体4, 程序性坏死, 铁死亡, 炎症

Abstract: Objective To explore whether toll-like receptor 4(TLR4)further affects the process of acetaminophen(APAP)induced liver injury and its mechanism by regulating programmed necrosis and ferroptosis. Methods Human normal hepatocytes L-02 were cultured in vitro and cell viability was detected by the CCK-8 method, and the concentrations of APAP and TAK-242 were evaluated. The experiment was divided into control group, APAP groups(1, 4, 12 h)and APAP+TAK-242 groups(1, 4, 12 h), and the TLR4 mRNA levels were compared in each group. The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in cell homogenates of different groups were detected; The levels of nuclear factor-κB(NF-κB), interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were detected in each group; The levels of high mobility group box 1(HMGB1), receptor interacting protein kinase 1(RIP1)and receptor interacting protein kinase 3(RIP3)were detected in each group; The intracellular Fe²⁺ content and the level of NF-κB, P53, recombinant solute carrier family 7 member 11(SLC7A11), glutathione peroxidase 4(GPX4)were detected in each group. Results The concentration of 5 mmol/L APAP and 100 nmol/L TAK-242 was determined by the CCK-8 method. Compared to the control group, the TLR4 mRNA levels of the APAP groups were positively regulated at each time point(P<0.05); Compared to the APAP groups, the levels of TLR4 mRNA in the APAP+TAK-242 groups were negatively regulated at the corresponding time points(P<0.05/3=0.016 7). Compared to the control group, the levels of ALT and AST in the APAP groups increased at each time point(P<0.05); Compared to the APAP groups, the levels of ALT and AST in the APAP+TAK-242 groups decreased at the corresponding time points(P<0.05/3=0.016 7). Compared to the control group, the mRNA expressions of NF-κB, IL-6 and TNF-α were up-regulated in the APAP groups at each time point(P<0.05); Compared to the APAP groups, the mRNA expressions of NF-κB, IL-6 and TNF-α were all down-regulated in the APAP+TAK-242 groups at the corresponding time points(P<0.05/3=0.016 7). Compared to the control group, the levels of HMGB1, RIP1, and RIP3 increased in the APAP groups at all time points(P<0.05); Compared to the APAP groups, the levels of HMGB1, RIP1, and RIP3 decreased in the APAP+TAK-242 groups at the corresponding time points(P<0.05/3=0.016 7). Compared to the control group, the content of Fe²⁺, NF-κB and P53 was increased(P<0.05), but the levels of SLC7A11 and GPX4 decreased in the APAP groups at all time points(P<0.05); Compared to the APAP groups, the content of Fe²⁺, NF-κB and P53 were decreased(P<0.05/3=0.016 7), but the levels of SLC7A11 and GPX4 increased in the APAP+TAK-242 groups at the corresponding time points(P<0.05/3=0.016 7). Conclusion Inhibition of TLR4 can negatively regulate programmed necrosis by regulating the TLR4 / HMGB1 signaling pathway and can negatively regulate the inflammatory response and ferroptosis by regulating TLR4/NF-κB signaling pathway to alleviate APAP-induced liver injury.

Key words: Acetaminophen, Toll-like receptor 4, Programmed necrosis, Ferroptosis, Inflammation

中图分类号:

R575

沈海涛,乔亚琴,董萍,路燕. Toll样受体4调控的程序性坏死和铁死亡对对乙酰氨基酚肝损伤的影响[J]. 山东大学学报 (医学版), 2024, 62(4): 1-8.

SHEN Haitao, QIAO Yaqin, DONG Ping, LU Yan. Effects of programmed necrosis and ferroptosis regulated by toll-like receptor 4 on acetaminophen-induced liver injury[J]. Journal of Shandong University (Health Sciences), 2024, 62(4): 1-8.

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