抗中性粒细胞血清对D-氨基半乳糖诱导的急性肝衰竭大鼠的作用

Effect of anti-PMN serum treatment on D-GalN-induced ALF rats

目的研究D-氨基半乳糖(D-GalN)诱导的急性肝衰竭大鼠中性粒细胞的改变,探讨以中性粒细胞为靶点的干预手段对急性肝衰竭的治疗作用。方法构建D-GalN诱导的SD大鼠急性肝衰竭模型,观察造模后大鼠肝功能、炎性因子(TNF-α、IL-1β)及外周血与肝脏中性粒细胞水平随时间的变化。为观察中性粒细胞的治疗作用,将SD大鼠随机分为3组:对照组、肝衰组及治疗组(造模前24h尾静脉注射抗中性粒细胞血清)。生化及血液分析仪检测肝脏生化指标(ALT、AST、TBIL、BA)及外周血中性粒细胞的数量及比率;免疫组化染色和Tunel染色分别检测肝脏中性粒细胞数量及肝细胞凋亡水平;RT-PCR检测肝脏组织炎性因子mRNA水平。结果 D-GalN诱导SD大鼠急性肝衰竭后6h血清ALT、AST、TBIL及BA较造模前显著升高(P<0.05);肝脏组织中TNF-α、IL-1β的mRNA表达水平在造模后6h达到峰值(P<0.001),24h仍明显高于造模前水平(P<0.001);外周血中性粒细胞数量、比率在造模后12h显著增多(P<0.001),并至少持续至24h(P<0.001)。造模前24h尾静脉注射抗中性粒细胞血清后,大鼠外周血及肝脏中性粒细胞数量在造模后24h较肝衰组明显降低(P<0.001);同时,血清ALT、AST、TBIL及BA较肝衰组显著下降(P<0.05),肝脏组织凋亡细胞显著减少,肝脏TNF-α、IL-1β表达水平也在治疗后明显下降(P<0.05)。结论D-GalN诱导的急性肝衰竭大鼠可出现外周血及肝脏中性粒细胞聚集,抗中性粒细胞血清可有效改善急性肝衰竭大鼠的肝功能及肝脏免疫微环境。

Objective To investigate changes in thc neutrophils in rats with D-galactosaminc （D-GaIN）- induced acute liver failure （ALF） and to explore the therapeutic effect of interventions treatment of neutrophils on ALF. Methods Liver function, the exprcssions of inflammatory cytokines TNF-a and IL-1β, and thc changcs of neutrophils in the peripheral blood and the liver were observed in rats with D-GalN （intrapcritoncal injection）- induced ALF. SD rats were randomly divided into threc groups when trcated with intervention of neutrophils： control group, ALF group （intraperitoneal injection of D-GaIN）, and treatment group （intravenous injection of anti-PMN serum via tail vein 24 h before modeling）. Biochemical analysis was used to detect scrum ALT, AST, TBIL and blood ammonia. Hematology analyzer was applied to analyze the number and percentage of peripheral blood neutrophils. The number of neutrophils in the liver was evaluated by immunohistochemistry. Liver RT-PCR was adoptcd to detect the mRNA expression of inflammatory cytokines TNF-a and IL-1β. Results We found Ihat 6 h after D-GalN injection, serum ALT, AST, TBIL and blood ammonia in ALF rats were significantly increased （P〈0.05）. The mRNAexpression levels of inflammatory cytokincsTNF-aand IL-1β in thcliver reached the peak at 6 h after modeling （P〈0. 001）, and it was till notably higher at 24 h than before modeling （P〈0. 001）. The number and percentage of peripheral blood neutrophils and the number of neutrophils in the liver were all markedly increased 12 h after modeling （P〈0,001）, and the increase continued at least until 24 h （P〈0. 001）. 24 h after intravenous injection of anti-PMN serum via tail vein, ALF rats had a distinct decrease in the number of peripheral blood neutrophils and neutrophils in the liver 24 h after modeling （P〈0. 001）. Meanwhile, serum ALT, AST, TBIL and blood ammonia were all greatly decreased compared with those in ALF group （P〈0.05） a significant reduction of hepatocyte apoptosis was observed. Also, the expressions of TNF-a and IL-1β in the liver were remarkably decreased after treatment （P〈0.05）. Conclusion Neutrophils accumulated in peripheral blood and liver of rats with D-GaiN-induced ALF. The treatment of anti-PMN serum may have a therapeutic effect on liver function and immune microenvironment in ALF rats.