CAJ | 학술논문

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0.1 mg/kg LPS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 800 mg/kg和LPS 8 t上g/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OX40 mRNA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12 h升高最明显,之后逐渐下降.ETT组大鼠肝组织OX40 mRNA表达水平虽高于正常对照组,但明显低于ALF组,与ALF组相比,差异有统计学意义(6、24、48 h时间点比较,F值分别为5.027、5.539、5.011,均P＜0.05;12 h F值为36.688,P＜0.01).ALF组OX40蛋白的表达12 h达峰值,24 h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.
목적 관찰내독소내수(ETT)급정상대서접수D-안기반유당(D-GalN)/지다당(LPS)주사후간조직기자격분자OX40표체적변화,탐토ETT발생적가능궤제.방법 웅성SD대서54지,안수궤수자표법분위정상대조조6지、급성간공능쇠갈(ALF)조화ETT조각24지.ETT조급ALF조선분별이0.1 mg/kg LPS화0.9％NaCl용액복강주사,매일1차,련속5차,우제6천동시복강주사D-GalN 800 mg/kg화LPS 8 t상g/지,분별재주사후6、12、24화48h4개시간점류취대서혈액급간장표본.RT-PCR검측대서간조직OX40 mRNA표체,Western인적법측정간조직OX40단백표체,ELISA검측혈청TNF-α、IL-6수평.수거분석채용단인소방차분석、LSD법、Dunnett T3검험.결과 ETT조대서혈청TNF-α、IL-6수평수고우정상대조조,단명현저우ALF조.ALF조대서간조직OX40 mRNA표체상승,우조모후12 h승고최명현,지후축점하강.ETT조대서간조직OX40 mRNA표체수평수고우정상대조조,단명현저우ALF조,여ALF조상비,차이유통계학의의(6、24、48 h시간점비교,F치분별위5.027、5.539、5.011,균P＜0.05;12 h F치위36.688,P＜0.01).ALF조OX40단백적표체12 h체봉치,24 h개시하강.ETT조OX40단백적표체교ALF조명현하강(6hF치위8.658,P＜0.05; 12、24、48hF치분별위34.611、28.176、16.747,균P＜0.01).결론 ALF조대서간조직OX40표체수평승고,ETT조대서출현OX40적억제,TNF-α、IL-6석방감소,제시OX40재ETT과정중가능발휘중요작용.
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 ％NaC1 (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-galactosamine (D-GalN) 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-GalN/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; 12 h: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-α and IL-6 are lower in ETT group,which suggested that OX40 may play an important role in the process of ETT.