其刺激分子OX40在内毒素耐受大鼠肝组织中的表达及意义

Expression of OX40 in the livers of endotoxin tolerance rats and its significance

目的观察内毒素耐受（ETT）及正常大鼠接受D-氨基半乳糖（D—GalN）／脂多糖（LPS）注射后肝组织其刺激分子OX40表达的变化，探讨ETT发生的可能机制。方法雄性SD大鼠54只，按随机数字表法分为正常对照组6只、急性肝功能衰竭（ALF）组和ETT组各24只。ETT组及ALF组先分别以0．1mg／kgLPS和0．9％NaCl溶液腹腔注射，每日1次，连续5次，于第6天同时腹腔注射D-GalN 800mg／kg和LPS8μg／只，分别在注射后6、12、24和48h4个时间点留取大鼠血液及肝脏标本。RT—PCR检测大鼠肝组织OX40mRNA表达，Western印迹法测定肝组织OX40蛋白表达，ELISA检测血清TNF-α、IL-6水平。数据分析采用单因素方差分析、LSD法、Dunnett T3检验。结果ETT组大鼠血清TNF-α、IL-6水平虽高于正常对照组，但明显低于ALF组。ALF组大鼠肝组织OX40 mRNA表达上升，于造模后12h升高最明显，之后逐渐下降。ETT组大鼠肝组织0X40 mRNA表达水平虽高于正常对照组，但明显低于ALE组，与ALF组相比，差异有统计学意义（6、24、48h时间点比较，F值分别为5．027、5．539、5．011，均P〈0．05；12hF值为36．688，P〈0．01）。ALF组OX40蛋白的表达12h达峰值，24h开始下降。ETT组OX40蛋白的表达较ALF组明显下降（6hF值为8．658，P〈0．05；12、24、48hF值分别为34．611、28．176、16．747，均P〈0．01）。结论ALF组大鼠肝组织OX40表达水平升高，ETT组大鼠出现OX40的抑制，TNF-α、IL-6释放减少，提示OX40在ETT过程中可能发挥重要作用。

Objective To investigate the mechanism of endotoxin tolerance （ETT） through observing the expression of OX40 in liver tissues of ETT rats. Methods SD male rats were randomly divided into three groups： normal group （n= 6）, acute liver failure （ALF） group （n= 24） and ETT group （n=24）. Lipopolysacharide （LPS） 0.1 mg/kg （ETT groups） or 0.9%NaCl （ALF groups） was administered by five consecutive intraperitoneal injections at 24 h intervals, and at the sixth day, all animals were treated with intraperitoneal injections of D-galaetosamine （D-GaIN） 800 mg/kg and LPS 8 μg/rat. Blood and liver tissue were collected at 6, 12, 24 and 48 hours after the injection of D-GaIN/ LPS. The gene expression of OX40 in the liver was measured by reverse transcription-polymerase chain reaction （RT-PCR）. The protein expression of OX40 was estimated by Western blot. The tumor necrosis factor （TNF）-α and interleukin （IL）-6 levels were determined by enzyme linked immunosorbent assay （ELISA）. The data analysis was performed by one-way ANOVA, lease significant difference （LSD） and Dunnett＇s T3 test. Results The expressions of TNF-α and IL-6 were both significantly lower in ETT group compared to ALF group, but still higher than that of control group. The gene expressions of OX40 peaked at 12 hours and decreased gradually in ALF group. The gene expressions of OX40 were significantly lower in ETT group compared to ALF group （6 h：F =5.027, 24 h：F=5.539, 48 h：F=5.011, all P〈0.05; 12h：F=36.688; P〈0.01）,but still higher than that of normal group. The tendency of OX40 protein expression in ALF group was peaked at 12 hours and decreased at 24 hours. In ETT group, the expression of OX40 was lower, and the difference between ETT group and ALF group had statistical significance （6 h：F=8. 658, P〈0.05; 12 h：F 34. 611, 24 h：F=28. 176, 48 h：F=16. 747; all P〈0.01）. Conclusions The level of OX40 is increased in ALF group, while the expressions of OX40, TNF a and IL-6 are lower in ETT group, which suggested that OX40 may play an important role in the process of ETT.