肝细胞NF-κB信号通路在雷公藤甲素诱发间接性肝损伤中的作用研究

doi:10.19803/j.1672-8629.20240855

摘要/Abstract

摘要： 目的探究肝细胞核转录因子κB（NF-κB）信号通路在雷公藤甲素（TP）增强肝脏对脂多糖（LPS）敏感性所致的间接性药物性肝损伤中的作用。方法将雌性C57BL/6N小鼠随机分为对照组（Con）、TP序贯LPS给药组（TP+LPS）、LPS序贯TP给药组（LPS+TP）,在TP与LPS共同作用6 h时,通过血液生化检测、肝重系数、苏木精伊红染色（HE）考察肝脏损伤情况,聚合酶链式反应（PCR）检测炎症因子、抗凋亡相关基因水平变化,Western Blot（WB）、免疫组织化学（IHC）考察肝脏凋亡/抗凋亡相关蛋白的表达并检测凋亡相关蛋白的酶活力变化;在LPS作用2 h（即TP+LPS共同作用2 h,LPS+TP共同作用1 h）时,WB检测NF-κB抑制性蛋白α（IκBα）、p65蛋白的表达,IHC检测p65入核情况,PCR检测抗凋亡相关基因水平变化。通过CCK8法、测定培养液上清乳酸脱氢酶（LDH）含量,分别确定TP与肿瘤坏死因子α（TNF-α）作用于AML12细胞的合适浓度,测定TP与TNF-α联合给药后,细胞培养液上清中LDH含量的变化,细胞内凋亡蛋白酶活力的变化,WB检测凋亡/抗凋亡蛋白的表达,以及TP作用后由TNF-α诱导的IκBα蛋白降解的变化,并通过免疫荧光（IF）观察p65的入核情况;通过给予AML12细胞佛波酯（PMA）预先刺激以激活NF-κB信号通路,考察凋亡蛋白酶活力变化,WB检测凋亡/抗凋亡蛋白的表达,并检测PMA预先给药对LDH释放量的影响。结果与Con组相比,（TP+LPS）组小鼠转氨酶水平显著增加,肝重系数显著上升,肝脏发生明显病理学变化,炎症因子水平显著上调,而（LPS+TP）组较Con组没有明显变化;（TP+LPS）组肝脏中Caspase-8酶活性显著上升,Cleaved Caspase-3、Cleaved PARP等凋亡相关蛋白表达显著增加,抗凋亡蛋白FLIP的表达显著降低,Cleaved Capsase-3 IHC阳性区域显著增加,而（LPS+TP）组上述指标较Con组均无显著变化;预先给予TP能显著抑制由LPS诱发的IκBα降解与p65入核,并下调抗凋亡相关基因的表达。在体外AML12细胞体系中,无毒浓度的TP与TNF-α联给后,LDH释放量显著增加,抗凋亡蛋白表达显著降低,凋亡相关蛋白表达显著增加,TNF-α 诱导的IκBα降解与p65入核被TP显著抑制;给予PMA预先激活NF-κB信号通路后,凋亡相关蛋白表达显著降低,抗凋亡蛋白表达显著增加,Caspase-8酶活性与LDH释放量均显著降低。结论 NF-κB信号通路在TP与LPS不同顺序序贯给药所致小鼠肝损伤差异中发挥了关键作用, TP通过抑制NF-κB信号通路的激活,导致肝细胞无法正常抵抗外源性病原体/凋亡信号刺激,引发肝细胞发生大量凋亡,最终诱导间接性药物性肝损伤的发生。

关键词: 雷公藤, 雷公藤甲素, 间接性药物性肝损伤, 脂多糖, 凋亡, 抗凋亡, 肝细胞核转录因子, 小鼠

Abstract: Objective To investigate the role of hepatocellular nuclear factor-κB (NF-κB) signaling pathway in increasing hepatic sensitivity to lipopolysaccharide (LPS) mediated and drug-induced indirect liver injury caused by triptolide (TP). Methods In the in vivo experiment, female C57BL / 6N mice were randomly divided into the control group (Con), TP sequential LPS group (TP+LPS), and LPS sequential TP group (LPS+TP). Six hours after coadministration of TP and LPS, blood biochemistry, liver weight coefficient and hematoxylin eosin staining (HE) were detected to assess liver injury. Polymerase chain reaction (PCR) was used to detect the levels of inflammatory factors and genes related to anti-apoptosis. The expressions of apoptosis / anti-apoptosis related proteins were detected by Western Blot (WB) and immunohistochemistry (IHC), and the enzyme activity of apoptosis related proteins was also measured. Two hours after administration of LPS, the protein expressions of NF-κB inhibitory protein α (IκBα) and p65 were detected by WB. The translocation of p65 from the cytoplasm to the nucleus was observed by IHC. Changes in the levels of anti-apoptosis related genes were determined by PCR. In the in vitro experiment, the LDH content, enzyme activity of apoptosis related proteins, and levels of apoptosis / anti-apoptosis related proteins were detected after coadministration of TP and TNF-α. After administration of TP, changes in degradation of IκBα protein induced by TNF-α were determined by WB, and translocation of p65 was observed via immunofluorescence (IF). Phorbol 12-myristate 13-acetate (PMA) was selected to activate NF-κB signaling pathway in AML12 cells. The enzyme activity of apoptosis related proteins, protein levels of apoptosis / anti-apoptosis related proteins, and the content of LDH were measured to evaluate the changes caused by PMA pre-administration. Results Compared with the Con group, mice in the (TP+LPS) group showed a significant increase in aminotransferase levels, liver weight coefficient, and inflammatory factors, with significant pathological changes in the liver. However, there were no significant changes in the (LPS+TP) group compared with the Con group. The enzyme activity of caspase-8, expressions of apoptosis-related proteins, and IHC positive regions of cleaved caspase-3 increased significantly in the liver tissue of the (TP+LPS) group, and the expression of the anti-apoptotic protein FLIP showed a significant reduction, while the above-mentioned indexes were not significantly changed in the (LPS+TP) group. Pre-administration of TP significantly inhibited LPS-induced degradation of IκBα and p65 entry into the nucleus, and downregulated the expressions of anti-apoptosis related genes. In AML12 cells in in vitro, after coadministration of TP and TNF-α at a non-toxic concentration, the release of LDH and the expressions of apoptosis related proteins were significantly increased while the expressions of anti-apoptotic proteins were significantly decreased. TNF-α induced degradation of IκBα and the entry of p65 into the nucleus were significantly inhibited by TP. After pre-activation of the NF-κB signaling pathway in AML12 cells by administration of PMA, the expressions of apoptosis-related proteins, the enzyme activity of caspase-8 and the release of LDH were significantly decreased, while the expression of anti-apoptotic proteins was significantly increased. Conclusion NF-κB signaling pathway can make a big difference to the liver injury resulting from different sequential administrations of TP and LPS. TP can impair hepatocytes' ability to withstand the stimulation of exogenous pathogens and apoptosis signaling by inhibiting NF-κB activation, triggering a significant increase in apoptosis among hepatocytes, ultimately leading to the development of indirect drug-induced liver injury.

Key words: Tripterygium wilfordii Hook.f., Triptolide, Indirect Drug-Induced Liver Injury, Lipopolysaccharide (LPS), Apoptosis, Anti-Apoptosis, Hepatocellular Nuclear Factor-κB(NF-κB), Mice

中图分类号:

唐千茴, 朱英, 张昊然, 张陆勇, 江振洲. 肝细胞NF-κB信号通路在雷公藤甲素诱发间接性肝损伤中的作用研究[J]. 中国药物警戒, 2025, 22(2): 128-138.

TANG Qianhui, ZHU Ying, ZHANG Haoran, ZHANG Luyong, JIANG Zhenzhou. Roles of Hepatocellular NF-κB Signaling Pathway in Triptolide-Induced Indirect Liver Injury[J]. Chinese Journal of Pharmacovigilance, 2025, 22(2): 128-138.

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