高迁移率族蛋白B1参与介导创伤后大鼠肝组织内质网应激

High mobility group box 1 contributes to the endoplasmic reticulum stress of liver in rats with trauma

目的探讨高迁移率族蛋白B1（HMGB1）在大鼠创伤后肝组织内质网应激（ERS）中的作用。方法SPF级SD大鼠60只，按随机数字表法分组，每组6只。采用15 kg标准重物挤压大鼠双后肢（持续挤压3 h后，解压30 min、挤压30 min重复3次）建立创伤应激致急性肝损伤模型。实验一分为对照组和挤压后6、18、30 h组；实验二分为对照组、挤压模型组（挤压后18 h）、HMGB1抑制剂正丁酸钠（SB）或丙酮酸乙酯（EP）对照组、SB或EP干预组（挤压3 h后腹腔注射SB溶液500 mg/kg或EP溶液40 mg/kg）。用全自动生化分析仪检测血清天冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）水平；常规苏木素-伊红（HE）染色后观察肝组织病理学改变；用蛋白质免疫印迹试验（Western Blot）检测肝组织HMGB1蛋白和ERS相关蛋白的表达；用免疫组化法检测肝组织HMGB1表达及其转位。结果① 与对照组比较，创伤应激后大鼠出现肝损伤病理学改变，血清AST、ALT水平明显增高，肝组织HMGB1及ERS相关蛋白葡萄糖调节蛋白78（GRP78）、天冬氨酸特异性半胱氨酸蛋白酶12（caspase-12）、肌醇需求酶1α（IRE1α）表达明显增高，均于18 h达峰值；C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白（CHOP）表达则呈时间依赖性升高，于挤压后30 h达峰值。免疫组化结果显示，挤压后6 h肝组织HMGB1表达增多，可见部分HMGB1从细胞核向细胞质移位，18 h表现较为明显。② 与挤压模型组比较，SB干预组和EP干预组HMGB1蛋白及ERS相关蛋白表达上调被抑制（HMGB1/β-actin：0.703±0.213、0.512±0.075比1.041±0.186，GRP78/β-actin：0.614±0.052、0.450±0.115比0.847±0.120，caspase-12/β-actin：0.636±0.066、0.812±0.142比1.086±0.130，CHOP/β-actin：0.314±0.046、0.621±0.123比0.996±0.764，IRE1α/β-actin：0.473±0.033、0.519±0.094比0.742±0.054，均P〈0.05），血清AST和ALT水平明显降低〔AST（U/L）：1？030.50±427.73、1？414.50±347.86比2？122.20±322.76，ALT（U/L）：285.75±11.30、368.50±80.58比473.80±33.54，均P〈0.01〕，急性肝损伤程度减轻。单纯SB或EP处理对各检测指标影响不大。结论HMGB1-ERS通路参与介导了创伤引起的大鼠急性肝损伤。

ObjectiveTo investigate the role of high mobility group box 1 （HMGB1） in hepatic endoplasmic reticulum stress （ERS） in rats with trauma.MethodsSixty SPF Sprague-Dawley （SD） rats were randomly divided into groups （n = 6）. The rat model of liver injury following traumatic stress was established by continuous compressing the bilateral hind-limbs of rats for 3 hours and then intermittent compressing and decompressing for 30 minutes respectively three times with standard weight of 15 kg. The experiment 1 was divided into two groups： control group and 6, 18, 30 hours after crush. The experiment 2 was divided into control group, crush model group （18 hours after crush）, HMGB1 inhibitor sodium butyrate （SB） or ethyl pyruvate （EP） groups, and SB or EP treatment groups （500 mg/kg SB solution or 40 mg/kg EP solution was injected intraperitoneally after 3 hours crush）. The levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in serum were measured with automatic biochemistry analyzer. Histopathological severity of liver injury was assessed by hematoxylin and eosin （HE） staining. The expressions of HMGB1 and ERS-related proteins were detected with Western Blot. The expression and translocation of HMGB1 in liver tissue were evaluated by immuno-histochemical technique.Results① Compared with the control group, the pathological changes of liver injury, the levels of AST and ALT in serum and protein expression of HMGB1 as well as ERS-related proteins such as glucose regulated protein 78 （GRP78）, caspase-12, and inositol-requiring enzyme 1α（IRE1α） in liver tissue were significantly increased after traumatic stress, and reached the peak at 18 hours. The expression of C/EBP-homologous protein （CHOP） was increased in a time-dependent manner and peaked at 30 hours after crush. Immunohistochemistry showed that HMGB1 expression increased at 6 hours after crush, some HMGB1 shifted from nucleus to cytoplasm, and the expression was more obvious at 18 hours. ② Compared with crush model group, the expressions of HMGB1 and ERS-related proteins were significantly decreased following the administration of HMGB1 inhibitors SB or EP （HMGB1/β-actin： 0.703±0.213, 0.512±0.075 vs. 1.041±0.186; GRP78/β-actin： 0.614±0.052, 0.450±0.115 vs. 0.847±0.120; caspase-12/β-actin： 0.636±0.066, 0.812±0.142 vs. 1.086±0.130; CHOP/β-actin： 0.314±0.046, 0.621±0.123 vs. 0.996±0.764; IRE1α/β-actin： 0.473±0.033, 0.519±0.094 vs. 0.742±0.054, all P 〈 0.05）, the levels of serum AST and ALT were significantly decreased [AST （U/L）： 1.030.50±427.73, 1.414.50±347.86 vs. 2.122.20±322.76; ALT （U/L）： 285.75±11.30, 368.50±80.58 vs. 473.80±33.54, all P 〈 0.01], the degree of acute liver injury was reduced. Only SB or EP could not affect the parameters mentioned above.ConclusionHMGB1-ERS pathway was involved in mediating traumatic stress-induced acute liver injury in rats.