TLR4 promotes liver inflammation by activating the JNK pathway
TLR4 promotes liver inflammation by activating the JNK pathway
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DOI：
10.26355/eurrev_201909_18889
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发表时间：
2019-01-01
影响因子：
3.3
通讯作者：
Wang, Y.
中科院分区：
文献类型：
--
作者：
Li, W.;Yang, G-L;Wang, Y.
目的：药物性肝损伤已成为一个不容忽视的严重公共卫生问题。尽管对乙酰氨基酚(APAP)致肝损伤的机制已被研究了几十年，但仍有许多不足之处。然而，只有深入研究其机制，才能为APAP所致肝损伤的防治提供更有效的措施。本研究旨在探讨Toll样受体4是否参与和调节APAP所致的肝损伤，为临床药物性肝炎的防治提供新的方向。材料和方法：WT小鼠给予APAP(300 mg/kg)或相当于PBS的治疗。分别于给药后1h、3h、6h、12h取小鼠肝脏。采用实时荧光定量聚合酶链式反应(RT-qPCR)检测肝组织TLR4基因表达水平。在证实TLR4参与APAP诱导的肝损伤后，我们研究了TLR4的表达与肝脏炎症的关系。WT和TLR4-/-小鼠禁食16h后腹腔注射APAP(3000 mg/kg)，分别于禁食后8h和24 h采血，测定血清丙氨酸氨基转移酶(ALT)和还原型谷胱甘肽(GSH)活性。苏木精-伊红(H&E)染色观察大鼠肝组织的组织学变化。逆转录-定量聚合酶链式反应(RT-qPCR)和酶联免疫吸附试验(ELISA)检测促炎症细胞因子(如肿瘤坏死因子α、白介素1β、单核细胞趋化蛋白1、白介素6)的表达。分离小鼠肝脏单个核细胞后，用流式细胞仪检测细胞活化水平及巨噬细胞和中性粒细胞的浸润情况。Western blotting分析WT和TLR4-/-小鼠肝脏中磷酸化JNK和p38信号通路的激活情况。此外，经APAP刺激后，RAW264.7细胞中TLR4的沉默可激活磷酸化的JNK和p38信号通路。结果：APAP刺激后，WT小鼠比TLR4-/-小鼠表现出更严重的肝损伤，ALT水平高于TLR4-/-小鼠，GSM水平低于TLR4-/-小鼠，坏死或凋亡细胞增多。TLR4-/-小鼠肝组织中MCP-1、IL-6等炎性细胞因子水平明显低于WT小鼠，同时TLR4-/-小鼠肝组织中的巨噬细胞和中性粒细胞数明显低于WT小鼠。WT小鼠经APAP刺激后，JNK信号通路的激活明显增强，而TLR4-/-小鼠经相同剂量的APAP刺激后，JNK信号通路的激活无明显差异。同样，在RAW264.7细胞中，TLR4-siRNA显著抑制了磷酸化JNK和p38的激活，而对照组则被激活，这在活体上是一致的。结论：APAP处理的TLR4-/-小鼠与WT小鼠相比，肝损伤较轻。已证实TLR4可激活JNK信号通路，诱导炎性因子的分泌和巨噬细胞的浸润，从而促进APAP诱导的肝损伤。这一发现可能为临床药物性肝炎的防治提供新的思路。
OBJECTIVE: Drug-induced liver injury has become a serious public health problem that cannot be ignored. Although the mechanism of acetaminophen (APAP)-induced liver injury has been investigated for several decades, there are still many deficiencies. However, only a deeper study of its mechanism can provide more effective measures of prevention and treatment for APAP-induced liver injury. The aim of this study was to investigate whether toll-like receptor 4 (TLR4) participates in and regulates APAP-induced liver injury, which may provide a new direction for the prevention and treatment of clinical drug-induced hepatitis.MATERIALS AND METHODS: WT mice were treated with APAP (300 mg/kg) or equivalent PBS. The livers of mice were taken at 1 h, 3 h, 6 h and 12 h after treatment. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect TLR4 mRNA expression level in the liver. After TLR4 involving in APAP-induced liver injury was confirmed, we investigated the relationship between TLR4 expression and hepatic inflammation. WT and TLR4-/- mice received APAP (3000 mg/kg) intraperitoneal injection after 16 h of fasting; serum was collected after 8 h and 24 h, and serum alanine aminotransferase (ALT) and reduced glutathione (GSH) activity were measured. Rat liver tissue was observed for histological changes by hematoxylin and eosin (H&E) staining. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) assay were performed to analyze proinflammatory cytokines expression (such as TNF-alpha, IL-1 beta, MCP-1, IL-6). After isolating mononuclear cells (MNCs) in the liver of mice, flow cytometry was used to detect cell activation level and infiltration of macrophages and neutrophils. Western blotting was used to analyze the activation of phosphorylated JNK and p38 signaling pathways in livers of WT and TLR4-/- mice. In addition, after stimulated with APAP, the silence of TLR4 in RAW264.7 cells could activate phosphorylated JNK and p38 signaling pathways.RESULTS: After APAP stimulation, WT mice exhibited more severe liver injury than TLR4-/- mice, with higher ALT levels, lower GSM levels, and more necrotic or apoptotic cells. TLR4-/- mice have lower levels of inflammatory cytokines including MCP-1 and IL-6; at the same time, the number of infiltrating macrophages and neutrophils in liver tissue of TLR4-/- mice was significantly lower than that of WT mice. The activation of JNK signaling pathway was strikingly enhanced in WT mice treated with APAP, but no significant difference was observed in the activation of JNK phosphorylation in TLR4-/- mice after the same dose of APAP stimulation. Similarly, in RAW264.7 cells, the activation of phosphorylated JNK and p38 was remarkably inhibited by TLR4-siRNA, but was activated in the control group, which was consistent in vivo.CONCLUSIONS: APAP-treated TLR4-/- mice showed milder liver injury compared to WT mice. It was confirmed that TLR4 could activate the JNK signaling pathway to induce the secretion of inflammatory factors and the infiltration of macrophages to promote APAP-induced liver injury. This finding might provide a new prevention and treatment idea for clinical drug-induced hepatitis.