急性肝衰竭早期尾加压素Ⅱ表达及其与前炎症细胞因子TNF-α和IL-1β的关系

Expression of urotensin Ⅱ and its relationship with pro-inflammatory cytokines of TNF-α and IL-1β in early stage of acute liver failure

目的探讨小鼠急性肝衰竭(ALF)早期尾加压素Ⅱ(UⅡ)表达和分泌的动态变化及其对前炎症细胞因子的影响。方法选择6周龄雄性BALB/c小鼠60只,随机分为模型组和预处理组,每组30只。两组均建立ALF动物模型,即采用脂多糖(LPS)50μg/kg联合右旋半乳糖胺(D-GalN)800 mg/kg(LPS/D-GalN)腹腔注射;预处理组在LPS/D-GalN注射前0.5 h,以UⅡ受体拮抗剂urantide 0.6 mg/kg尾静脉注射。两组均在LPS/D-GalN注射后0、0.5、1、2和6 h处死动物(每一时间点各6只小鼠),其中0 h作为对照(仅腹腔内注射0.2 mL无菌生理盐水);采集动物血清和肝组织标本。采用RT-PCR及ELISA方法分别检测UⅡ、肿瘤坏死因子α(TNF-α)及白介素1β(IL-1β)mRNA和蛋白质的表达。结果 LPS/D-GalN注射后0.5 h,肝组织和血清中UⅡ的表达和分泌即快速上升并达峰值,且峰值水平持续至LPS/D-GalN攻击2 h后,至6 h时UⅡ的表达与分泌虽有所降低,但仍显著高于正常水平(P<0.05);而TNF-α水平在LPS/D-GalN攻击后0.5 h内无明显升高(P>0.05),直到1 h后才快速上升达峰值(P<0.05),至6 h时开始下降(P<0.05);IL-1β的表达与分泌则直到6 h后才有明显上升(P<0.05)。Urantide的应用不仅显著抑制了LPS/D-GalN攻击诱导的UⅡ高表达,也使上调的TNF-α和IL-1β表达和分泌明显受抑(P<0.05)。结论 UⅡ可刺激TNF-α的表达,有可能作为炎症级联效应的触发者在ALF的发生或启动中发挥关键性影响。

Objective To investigate the alteration of expression and secretion of urotensinⅡ（UⅡ） and its effect on pro-inflammatory cytokines in early stage of acute liver failure（ALF） in mice.Methods Sixty male BALB/c mice aged 6 weeks were randomly divided into model group and pre-treatment group,with 30 mice in each group.ALF animal models were established in model group by challenging with 50 μg/kg lipopolysaccharide（LPS） and 800 mg/kg D-galactosamine（D-GalN）（LPS/D-GalN） via intraperitoneal injection,and mice in pre-treatment group were administered with 0.6 mg/kg urantide（UⅡ receptor antagonist） via caudal vein injection 0.5 h before injection of LPS/D-GalN.Mice in both groups were sacrificed 0 h,0.5 h,1 h,2 h and 6 h（6 mice at each time point in each group） after injection of LPS/D-GalN,with those sacrificed 0 h after injection as controls（intraperitoneal injection of 0.2 mL normal saline）,and the samples of serum and liver tissues were taken.The expression of UⅡ,tumor necrosis factor-α（TNF-α） and interleukin-1β（IL-1β） mRNA and protein was detected by RT-PCR and ELISA respectively.Results The expression and secretion of UⅡ in serum and liver tissues rapidly increased and reached the peak 0.5 h after injection of LPS/D-GalN,lasted till 2 h after injection,decreased 6 h after injection,while still higher than the normal levels 6 h after injection（P0.05）.The level of TNF-α did not significantly increase 0.5 h after injection of LPS/D-GalN（P0.05）,reached the peak 1 h after injection（P0.05）,and began to decrease 6 h after injection（P0.05）.The expression and secretion of IL-1β did not significantly increase until 6 h after injection of LPS/D-GalN（P0.05）.The application of urantide significantly inhibited the increased expression of UⅡ and expression and secretion of TNF-α and IL-1β induced by LPS/D-GalN challenge（P0.05）.Conclusion UⅡ can stimulate the expression of TNF-α,and may play a key role in the pathogenesis and priming of ALF as a trigger of inflammatory cascade.