| 摘要: |
| 目的 探究表没食子儿茶素没食子酸酯（EGCG）在脓毒症后急性肝损伤中的保护作用及潜在机制。方法 8~10周龄雄性C57BL/6J小鼠随机分为盲肠结扎穿刺术（CLP）组（脓毒症后急性肝损伤模型小鼠）、CLP+EGCG低剂量（4 mg/kg，皮下注射）组、CLP+EGCG高剂量（8 mg/kg，皮下注射）组和假手术组（n＝6）。人正常肝细胞（L02细胞）根据干预方式不同分为脂多糖（400 ng/mL，脓毒症后急性肝损伤细胞模型）组、脂多糖（400 ng/mL）+高迁移率族蛋白B1（HMGB1）（100 ng/mL）组、脂多糖（400 ng/mL）+EGCG（100 μg/mL）组和对照组（加入PBS）组。术后24 h收集小鼠全血并分离肝组织，采用全自动生化分析仪检测小鼠血常规及肝功能，通过H-E染色观察肝组织病理学变化。采用ELISA检测小鼠血清及L02细胞培养上清液中炎症因子（HMGB1、TNF-α、IL-6）表达，采用蛋白质印迹法检测小鼠肝组织及L02细胞中HMGB1、Toll样受体4（TLR4）、NF-κB p65、核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）、焦亡相关蛋白[消皮素D（GSDMD）、caspase 1、caspase 11、IL-1β、IL-18]的表达，采用免疫组织化学染色分析小鼠肝组织中HMGB1、GSDMD的表达及定位情况。结果 CLP组小鼠白细胞计数、淋巴细胞计数、中性粒细胞计数、单核细胞计数均低于假手术组（P均＜0.05），血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）均高于假手术组（P均＜0.01），提示脓毒症后急性肝损伤模型小鼠构建成功。与CLP组相比，CLP+EGCG低剂量、高剂量组小鼠ALT、AST、HMGB1、TNF-α、IL-6表达水平均降低（P均＜0.05），且这些指标在CLP+EGCG高剂量组较CLP+EGCG低剂量组降低更显著（P均＜0.05）。组织病理学提示CLP+EGCG低剂量、高剂量组小鼠肝组织损伤较CLP组减轻，且以高剂量组较为显著。CLP+EGCG低剂量、高剂量组小鼠肝组织中HMGB1、TLR4、NF-κB p65、NLRP3和焦亡相关蛋白的表达水平均低于CLP组（P均＜0.05），且以高剂量组降低更为明显（P均＜0.05）。免疫组织化学染色显示GSDMD主要表达于肝细胞的细胞质内，而HMGB1在细胞质和细胞核均有表达；CLP+EGCG低剂量、高剂量组小鼠肝组织中HMGB1和GSDMD的表达均较CLP组减少（P均＜0.05），且CLP+EGCG高剂量组降低更为明显（P均＜0.05）。脂多糖组、脂多糖+EGCG组及对照组细胞培养上清液中HMGB1、TNF-α和IL-6的表达水平均低于脂多糖+HMGB1组（P均＜0.05），且该3个指标在脂多糖+EGCG组降低较脂多糖组更为显著（P均＜0.05）。脂多糖+HMGB1组中HMGB1、TLR4、NF-κB p65、NLRP3和焦亡相关蛋白的表达水平均高于脂多糖组、脂多糖+EGCG组和对照组（P均＜0.05），且脂多糖+EGCG组上述蛋白质的表达水平低于脂多糖组（P均＜0.05）。结论 EGCG对脓毒症后急性肝损伤有保护作用，其机制可能与通过减弱HMGB1/TLR4/NF-κB/NLRP3途径降低肝细胞焦亡有关。 |
| 关键词: 表没食子儿茶素没食子酸酯 脓毒症 急性肝损伤 高迁移率族蛋白B1 细胞焦亡 |
| DOI：10.16781/j.CN31-2187/R.20220048 |
| 投稿时间：2022-01-13修订日期：2022-04-29 |
| 基金项目:国家自然科学基金面上项目(81871608) |
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| Epigallocatechin gallate alleviates sepsis-induced acute liver injury by attenuating HMGB1/TLR4/NF-κB/NLRP3 pathway |
| LI Fei1△,WU Chuan-xin2△,HE Jia-hui2,ZHANG Jie3,LIU Hui-ling1,SUN Hang1* |
(1. Institute for Viral Hepatitis, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China;
2. Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China;
3. Department of Neonatology, Chengdu Shuangliu District Maternal and Child Health Hospital, Chengdu 610200, Sichuan, China
△Co-firsts author.
*Corresponding author) |
| Abstract: |
| ObjectiveTo explore the protective effect and underlying mechanism of epigallocatechin gallate (EGCG) in sepsis-induced acute liver injury.MethodsMale C57BL/6J mice aged 8-10 weeks old were randomly divided into cecal ligation and puncture (CLP) group (sepsis-induced acute liver injury model mice), CLP+EGCG low-dose (4 mg/kg, injected subcutaneously) group, CLP+EGCG high-dose (8 mg/kg, injected subcutaneously) group, and sham group (n＝6). According to the intervention methods, human liver cells (L02 cells) were divided into lipopolysaccharide (LPS) (400 ng/mL, cell model of sepsis-induced acute liver injury) group, LPS (400 ng/mL)+high mobility group protein B1 (HMGB1) (100 ng/mL) group, LPS (400 ng/mL)+EGCG (100 μg/mL) group, and control (PBS treatment) group. The whole blood of mice was collected and the liver tissues were isolated 24 h after operation. The routine blood test and liver function of mice were detected by automatic biochemical analyzer, and the pathological changes of liver tissues were observed by hematoxylin-eosin (H-E) staining. The expression of inflammatory factors (HMGB1, tumor necrosis factor α[TNF-α], and interleukin[IL]-6) in mouse serum and L02 cell supernatant was detected by enzyme-linked immunosorbent assay. The expression of HMGB1, Toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB) p65, nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) and pyroptosis-related proteins (gasdermin D [GSDMD], cysteine aspartic acid specific protease [caspase] 1, caspase 11, IL-1β, and IL-18) was detected by Western blotting. The expression and localization of HMGB1 and GSDMD in mouse liver tissues were analyzed by immunohistochemical staining.ResultsThe white blood cell count, lymphocyte count, neutrophil count and monocyte count in the CLP group were significantly lower than those in the sham group (all P < 0.05), and the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly higher than those in the sham group (both P < 0.01), indicating that the sepsis-induced acute liver injury mouse model was successfully constructed. Compared with the CLP group, the expression levels of ALT, AST, HMGB1, TNF-α and IL-6 in the CLP+EGCG low-dose and high-dose groups were significantly decreased (all P < 0.05), and these indexes were significantly lower in the CLP+EGCG high-dose group than in the CLP+EGCG low-dose group (all P < 0.05). The results of H-E staining showed that the liver tissue injury in the CLP+EGCG low-dose and high-dose groups was less than that in the CLP group, especially in the high-dose group. The expression levels of HMGB1, TLR4, NF-κB p65, NLRP3 and pyroptosis-related proteins in the liver tissues of the CLP+EGCG low-dose and high-dose groups were significantly lower than those in the CLP group (all P < 0.05), and the decrease was more significant in the high-dose group (all P < 0.05). Immunohistochemical staining showed that GSDMD was localized in the cytoplasm of liver cells, while HMGB1 was localized in both cytoplasm and nucleus; the expression levels of HMGB1 and GSDMD in the CLP+EGCG low-dose and high-dose groups were significantly lower than those in the CLP group (all P < 0.05), and the decrease was more significant in the high-dose group (both P < 0.05). Compared with the LPS+HMGB1 group, the expression levels of HMGB1, TNF-α and IL-6 in the cell supernatants of the LPS group, LPS+EGCG group and control group were significantly decreased (all P < 0.05), and the decrease of these 3 indexes in the LPS+EGCG group was more significant than that in the LPS group (all P < 0.05). The expression levels of HMGB1, TLR4, NF-κB p65, NLRP3 and pyroptosis-related proteins in the LPS+HMGB1 group were significantly higher than those in the LPS, LPS+EGCG and control groups (all P < 0.05), and the expression levels of the above-mentioned proteins in the LPS+EGCG group were significantly lower than those in the LPS group (all P < 0.05).ConclusionEGCG has some protective effect on sepsis-induced acute liver injury, and the underlying mechanism may be related to alleviating hepatocellular pyroptosis by attenuating the HMGB1/TLR4/NF-κB/NLRP3 pathway. |
| Key words: epigallocatechin gallate sepsis acute liver injury high mobility group protein B1 pyroptosis |
