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目的:探讨乌司他丁(ulinastatin,UTI)预处理对大鼠肝脏缺血再灌注(ischemia reperfusion,IR)损伤后炎性反应的影响,并研究高迁移率蛋白B1(high mobility group box 1,HMGB1)在UTI预处理机制中的作用。方法:采用SD大鼠70%肝脏IR损伤模型,即在肝脏缺血45min后再灌注2h。将40只大鼠随机为4组,分别为0.9%氯化钠对照组(对照组,n=10)、UTI预处理组(UTI组,n=10)、UTI+HMGB1拮抗剂组(UTI+Anti-HMGB1组,n=10)和乌司他丁+HMGB1诱导剂组(UTI+rHMGB1组,n=10)。比较各组大鼠在IR后血清中丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)的水平;采用酶联免疫吸附试验检测各组大鼠血清中肿瘤坏死因子α(tumornecrosis factor-alpha,TNFα)和白细胞介素1(interleukin 1,IL1)的水平;对各组大鼠的肝组织进行HE染色观察其病理学改变;采用免疫组织化学染色方法检测各组大鼠肝组织中HMGB1蛋白的表达水平。结果:与对照组相比,UTI组在IR后血清中ALT、AST均显著降低(P<0.05)。UTI+rHMGB1组IR后血清TNFα、IL1水平与对照组相比无显著差异(P>0.05);UTI组和UTI+Anti-HMGB1组IR后血清TNFα、IL1水平与对照组相比均显著降低(P<0.05)。肝组织病理显示,UTI组和UTI+Anti-HMGB1组的肝细胞坏死和中性粒细胞浸润显著轻于对照组和UTI+rHMGB1组。免疫组织化学染色显示,UTI组和UTI+Anti-HMGB1组肝细胞HMGB1表达程度显著低于对照组(P<0.05)。结论:UTI预处理可以显著抑制大鼠肝IR损伤后肝细胞中HMGB1的表达水平以及下游炎症因子的水平,从而减轻IR损伤;HMGB1诱导剂可逆转UTI的肝保护作用。
OBJECTIVE: To investigate the effect of ulinastatin (UTI) pretreatment on the inflammatory response after liver ischemia-reperfusion (IR) injury in rats, and to investigate the effect of high mobility group box 1 HMGB1) in UTI pretreatment mechanism. Methods: The model of IR injury in 70% of the liver of SD rats was reperfusion for 2 hours after the ischemia of the liver for 45 minutes. Forty rats were randomly divided into four groups: 0.9% sodium chloride control group (n = 10), UTI pretreatment group (n = 10), UTI + HMGB1 antagonist group Anti-HMGB1 group, n = 10) and ulinastatin + HMGB1 inducer group (UTI + rHMGB1 group, n = 10). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of rats in each group were compared. The levels of serum ALT and AST were detected by enzyme-linked immunosorbent assay The levels of tumor necrosis factor-alpha (TNFα) and interleukin 1 (IL-1) in serum were determined. The pathological changes of the liver tissues of each group were observed by HE staining. Immunohistochemical staining Methods The expression of HMGB1 protein in liver tissue of each group was detected. Results: Compared with the control group, the serum levels of ALT and AST in the UTI group were significantly decreased (P <0.05). The levels of TNFα and IL1 in serum of UTI + rHMGB1 group were not significantly different from those of control group (P> 0.05). The levels of TNFα and IL1 in serum of UTI + UTI + Anti-HMGB1 group were significantly lower than those of control group P <0.05). Liver histopathology showed that liver cell necrosis and neutrophil infiltration in UTI group and UTI + Anti-HMGB1 group were significantly lower than those in control group and UTI + rHMGB1 group. Immunohistochemical staining showed that the expression of HMGB1 in UTI group and UTI + Anti-HMGB1 group was significantly lower than that in control group (P <0.05). CONCLUSION: UTI pretreatment can significantly inhibit the expression of HMGB1 in liver cells and the level of downstream inflammatory cytokines in rats after IR injury, so as to reduce the IR injury. HMGB1 induces the liver protective effect of UTI.