Urotensin Ⅱ在急性肝衰竭小鼠肝组织中的表达及损伤作用

Expression and role of Urotensin Ⅱ in lipopolysaccharide/D-galactosamine-induced acute liver failure in mice

目的:探讨Urotensin Ⅱ(UⅡ)在急性肝衰竭(acute liver failure,ALF)小鼠肝组织中的表达及损伤作用.方法:♂Balb/c小鼠随机分成4组(每组6只):正常对照组(A组)、预处理对照组(B组)、模型组(C组)和预处理模型组(D组).模型动物以脂多糖(lipopolysaccharide,LPS)/D-半乳糖胺(D-galactosamine,D-GalN)腹腔注射,预处理动物在造模前30min,用UⅡ受体拮抗剂Urantide0.6mg/kg尾静脉注射.LPS/D-GalN攻击12h后,采集血清和肝组织标本,并观察24h小鼠存活情况;采用Reitman-Frankel法检测血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天冬氨酸氨基转移酶(aspartate amino-transferase,AST)活性水平;采用HE染色显微镜观察肝组织损伤程度;RT-PCR法检测UⅡ及其受体UTmRNA的表达;ELISA法检测血清UⅡ多肽分泌水平;免疫组织化学方法检测肝组织UⅡ多肽及其UT受体蛋白质表达.结果:C组小鼠死亡率为66.7%,A、B和D组所有动物均存活;LPS/D-GalN攻击引起C和D组小鼠血清ALT和AST水平显著升高(P<0.01),而D组较C组显著降低(2271.09U/L±102.24U/Lvs1160.67U/L±258.32U/L,1569.42U/L±204.04U/Lvs1030.31U/L±108.09U/L,P<0.01);C组小鼠肝组织结构破坏明显,见大片出血性坏死及炎症表现,D组肝组织结构保持完整,仅有局灶性出血坏死,炎症明显减轻;C和D组小鼠血清UⅡ多肽水平较A和B组高(P<0.01),但D组较C组明显降低(3.73g/L±0.52g/Lvs1.90g/L±0.27g/L,P<0.01);LPS/D-GalN诱导了C和D组小鼠肝组织UⅡ和UT的mRNA及蛋白质高水平表达,而D组的表达水平较C组显著降低(P<0.01).结论:LPS/D-GalN可诱导ALF小鼠肝组织表达和分泌UⅡ,并促进肝组织UT受体的表达;UⅡ的表达与分泌可能存在正反馈调控机制;UⅡ/UT受体介导了LPS/D-GalN诱导的ALF的发生.

AIM： To investigate the expression and role of Urotensin Ⅱ （UⅡ） in lipopolysaccharide （LPS）/ D-galactosamine （D-GalN）-induced acute liver failure （ALF） in mice. METHODS： Male Balb/c mice were randomly and equally divided into four groups： normal control group （group A）, pre-treatment control group （group B）, ALF model group （group C）, and pre-treatment model group （group D）. ALF were induced in mice by intraperitoneal injection of LPS （50 g/kg body weight）/D-GalN （800 mg/kg body weight）. The pre-treatment mice were intravenously injected with Urantide （0.6 mg/kg body weight） 30 min before model induction. Serum and liver tissues were sampled 12 h after LPS/D-GalN injection. Mortality was calculated 24 h after attack. Serum levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were detected using the Reitman-Frankel method. Histopathological changes were observed by hematoxylin and eosin （HE） staining. Serum UⅡ levels were assessed by ELISA, and the expression of UⅡ and UT was detected by RT-PCR and immunohisto-chemistry. RESULTS： A mortality of 66.7% was observed in group C, while all mice of groups A, B and D survived. Serum ALT and AST levels had a dramatic increase in groups C and D, but were significantly lower in group D than in group C （2 271.09 U/L ± 102.24 U/L vs 1 160.67 U/L ± 258.32 U/L, 1 569.42 U/L ± 204.04 U/L vs 1 030.31 U/L ± 108.09 U/L, both P 0.01）. Wide- spread destruction of liver architecture, hemorrhagic necrosis, and neutrophil infiltration were noted in group C, whereas liver architecture was completely preserved, and focal necrosis and fewer neutrophil infiltrates were observed in group D. After LPS/D-GalN challenge, serum UⅡ levels increased sharply in groups C and D, but were lower in group D than in group C （3.73 g/L ± 0.52 g/L vs 1.90 g/L ± 0.27 g/L, both P 0.01）. Overexpression of liver U Ⅱ and UT mRNAs and proteins was induced by the injection of LPS/D-GalN in groups C and D. Compared to group C, group D had lower levels of UⅡ and UT in the liver. CONCLUSION： UⅡ expression and secretion can be induced by LPS/D-GalN challenge in the liver tissue of ALF mice via a positive feedback mechanism associated with promoting the expression of its receptor UT. UⅡ/UT receptor may be a vital mediator of LPS/D-GalN-induced ALF.