TLR4特异性抑制剂对大鼠心脏死亡器官捐献供肝缺血再灌注损伤的作用

Effect of selective TLR4 inhibitor on ischemia reperfusion injury of liver in rat models of donation after cardiac death

摘要:

目的 探讨TLR4特异性抑制剂调控HMGB1/TLR4信号通路对大鼠心脏死亡器官捐献供肝缺血再灌注（IR）损伤的作用。方法 将8周龄Balb/c雄性大鼠分为对照组［术前30 min经腹腔注射二甲基亚砜（DMSO）无菌生理盐水］、对照抑制组（术前30 min经腹腔注射含TLR4抑制剂的DMSO无菌生理盐水）、模型组（术前30 min经腹腔注射DMSO无菌生理盐水）、模型抑制组（术前30 min经腹腔注射含TAK242的DMSO无菌生理盐水），每组6只。收集肝脏组织标本，采用苏木精-伊红染色检测肝脏细胞形态结构，Suzuki评分评估肝脏细胞损伤情况；TUNEL染色法检测细胞凋亡率，Western blotting检测肝细胞HMGB1/TLR4及下游炎症因子相对表达量；免疫荧光和Western blotting测定TLR4与HMGB1的共表达作用。结果 病理结果显示，模型组肝脏损伤情况较对照组严重，模型抑制组肝脏损伤情况优于模型组；模型组Suzuki评分高于对照组（P <0.05），模型抑制组Suzuki评分低于模型组（P <0.05）。模型组、模型抑制组HMGB1、TLR4、IL-1β、IL-6蛋白相对表达量高于对照组（P <0.05）；模型抑制组HMGB1、TLR4、IL-1β、IL-6蛋白相对表达量低于模型组（P <0.05）。模型组肝脏组织细胞凋亡率高于对照组和模型抑制组（P <0.05）。模型组HMGB1蛋白相对表达量高于对照组和模型抑制组（P <0.05）。免疫荧光结果显示，模型组大鼠肝细胞HMGB1的免疫活性显著增加，且主要位于细胞浆内；但对照组HMGB1免疫活性无显著变化。结论 TLR4特异性抑制剂可显著下调HMGB1/TLR4信号通路及相关信号分子，减轻供肝氧化应激和炎症反应，对供肝IR损伤具有保护作用。

Abstract:

Objective To investigate the effects of the selective Toll-like receptor 4 (TLR4) inhibitor TAK-242, involved in regulating high mobility group box 1 (HMGB1)/TLR4 signaling pathway, on ischemia reperfusion (IR) injury of liver after cardiac death in rats.Methods Eight-week-old Balb/c male rats were divided into control group [intraperitoneal injection of sterile normal saline containing dimethyl sulfoxide (DMSO) 30 min before the operation], control inhibition group (intraperitoneal injection of sterile normal saline containing DMSO and TAK-242 30 min before the operation), model group (intraperitoneal injection of sterile normal saline containing DMSO 30 min before the operation), and model inhibition group (intraperitoneal injection of sterile normal saline containing DMSO and TAK-242 30 min before the operation), each with 6 rats. Liver tissues were collected, and HE staining was used to detect the liver histology. The Suzuki score was applied to evaluate the damage of liver cells, while the TUNEL staining was applied to detect the apoptosis rate. In addition, Western blotting was used to detect the expression of HMGB1/TLR4 and downstream inflammatory factors in liver cells, and the co-expression of TLR4 and HMGB1 was determined via immunofluorescence and Western blotting.Results The liver damage of the model group was greater compared with the control group and the model inhibition group, with a higher Suzuki score in the model group as well (P < 0.05). The protein levels of HMGB1, TLR4, interleukin (IL)-1β and IL-6 in the model group and model inhibition group were higher than those in the control group (P < 0.05), while the protein levels of these molecules in model inhibition group were lower than those in model group (P < 0.05). The apoptosis rate was higher in the model group relative to the control group and the model inhibition group (P < 0.05). In addition, the immunofluorescence intensity of HMGB1 was increased significantly in the liver cells of rats in the model group rather than the control group, and the staining was primarily observed in the cytoplasm.Conclusions Selective TLR4 inhibitor can significantly down-regulate the HMGB1/TLR4 signaling pathway and related signaling molecules, and reduce the oxidative stress and inflammatory response in the liver of rats after cardiac death, thereby playing a role in protecting against the IR injury of the donor liver.