高迁移率族蛋白B1表达水平与大鼠脓毒症严重程度及预后关系的实验研究

Relation between level of expression of high mobility group protein B1 in hepatic tissue with the severity and prognosis of sepsis in rat

目的探讨高迁移率族蛋白B1（HMGB1）与脓毒症严重程度及预后间的关系，寻找致死性脓毒症的可靠监测及治疗指标。方法90只SPF级雄性SD大鼠（体重250～300g）被随机分为假手术组（A组）、盲肠结扎穿孔术（CLP）组（B组）及丙酮酸乙酯（EP）治疗组（C组），每组30只，分别施以假手术（A组）或CLP（B组和C组）。术后立即腹腔注射生理盐水（Ns）2ml（A组和B组）或EP2ml（130mg／kg，C组），12h重复1次。以术后0、6、12、24、48和72h为观察点，观察各组大鼠术后活动、进食、竖毛、腹泻、眼球凹陷、呼吸等情况；活杀并观察腹腔肠管扩张、充血、腹水、病灶包裹、肺表面充血等情况。另取20只大鼠以B、C两组同样的模型及处理方式观察生存时间。采用逆转录-聚合酶链反应（RT—PCR）技术检测肝组织HMGBl mRNA表达水平，酶联免疫吸附法（ELISA）检测血浆白细胞介素-1β（IL-1β）、IL-6及肿瘤坏死因子-α（TNF-α）水平；绘制各组生存曲线，并进行相关分析。结果B组大鼠术后脓毒症表现最强，C组明显减轻。而A组没有相关表现或表现轻微。B组血浆IL-1β、IL-6和TNF-α水平于术后6h显著升高，至12h已明显下降；而C组上升的幅度明显低于B组，但明显高于A组。B组肝脏HMGBl mRNA表达水平在12h开始升高，24h达高峰，并维持至48h后开始下降，且HMGBl表达水平与IL-6水平呈正相关（r=0．91）。C组生存时间较B组明显延长（P〈0．01），HMGBl表达水平与脓毒症严重程度及动物生存率显著相关。结论脓毒症晚期释放的HMGBl是脓毒症致死效应的关键炎症介质，其表达水平与实验动物的脓毒症严重程度及预后高度相关。

Objective To investigate whether the levels of expression of high mobility group protein B1 （HMGBl） m hepatic tissue are related with seventy and prognosis of sepsis m rat, m order to look for a dependable marker for monitoring, and evaluating the efficiency of the treatment of sepsis. Methods Ninety SD rats （specific-pathogen free, male, 250 -300 g） were randomly divided into sham operation group （group A）, cecal ligation and puncture （CLP） group （group B） and CLP followed by ethyl pyruvate （EP） treatment group （group C）. Laparofury was done aseptically, and the cecum was returned to the abdomen （group A） or CLP was carried out （group B and C）. The abdominal wall was then closed layer by layer. Normal saline （NS） 2 ml （group A and B） or EP 2 ml （130 mg/kg, group C） was intraperitoneally injected and repeated every 12 hours. Five animals were sacrificed at 0, 6, 12, 24, 48 and 72 hours after the operation, blood samples and 100 grams of hepatic tissue were harvested before the animal died. Before sacrifice, activity, food taking, piloerection, diarrhea, enophthalmia, respiration, distention of intestine, hyperemia, ascites, and omental adhesion to the cecum, and hyperemia of lung were observed. Another group of 20 rats were used to observe the survival rate after CLP. The level of HMGB1 mRNA expression in the hepatic tissue was assessed by reverse transcriptase- polymerase chain reaction （RT- PCR）, and the levels of interleukin- 1β （IL -1β）, IL -6 and tumor necrosis factor-α （TNF -α） in plasma were determined by enzyme linked immunoadsorbent assay （ELISA）. Results The mortality of sepsis was higher in group B than other groups （both P〈0.01）, that of group C was significantly lower than B but higher than A （both P〈0.01）. The levels of IL - 1β, IL - 6 and TNF -α in plasma rose to the peak at 6 hours after CLP in all groups, and group B〉group C〉group A （P〈0.01）, and they descended at 12 hours. The HMGBl mRNA expression level in hepatic tissue was measurable at 6 hours and rose at 12 hours in group B, and maintained a high level up to 48 hours after CLP. The HMGBl expression was positive correlated with IL - 6 （r= 0. 91）. The value in group C was significantly lower than group B at all time points （all P〈0.01）. The degree of severity and survival rate of sepsis were highly correlated with HMGBl levels. Conclusion As a late-released inflammatory mediator, HMGBl plays a key role in lethal effect of sepsis, the surviving time and the severity degree of sepsis are highly correlated with HMGBl expression level in hepatic tissue.