白藜芦醇对急性肝损伤小鼠NLRP3炎性体表达的影响

The effects of resveratrol on NLRP3 inflammasome expression in a mouse model of acute liver injury

目的观察白藜芦醇对急性肝损伤(acute liver injury,ALI)小鼠Nod样受体家族3(Nod-like receptor 3,NLRP3)炎性体表达的影响,探讨白藜芦醇对ALI的保护作用及其机制。方法本实验采用四氯化碳制作ALI小鼠模型。将雄性ICR小鼠随机分成正常对照组、模型组、白藜芦醇低、中、高剂量组及阳性对照组,每组7只。白藜芦醇低、中、高剂量组及阳性对照组于造模前24 h及1 h分别腹腔注射剂量为10、20及30 mg/kg的白藜芦醇或剂量为100 mg/kg的乙酰半胱氨酸,对照组及模型组在相应时间点腹腔注射等量生理盐水,造模时模型组及各药物干预组采用腹腔注射5%四氯化碳,对照组腹腔注射等量橄榄油。采用蛋白质印迹(Western blot,WB)法测定小鼠肝组织NLRP3、凋亡相关微粒蛋白(apoptosis-associated speck-like protein contain,ASC)、炎性半胱天冬酶-1(caspase-1)蛋白,酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)法检测炎症因子白介素(interleukin,IL)-1β及IL-18,全自动生化分析仪测定小鼠肝功能,病理组织学观察肝脏损伤情况及其程度。结果模型组小鼠谷丙转氨酶(alanine aminotransferase,ALT)及谷草转氨酶(aspartate aminotransferase,AST)水平高于正常对照组(P<0.01);白藜芦醇各剂量组及阳性对照组小鼠ALT及AST水平均低于模型组(P<0.01)。模型组小鼠肝脏炎症积分及损伤面积均高于正常对照组(P<0.01);白藜芦醇各剂量组及阳性对照组小鼠肝脏炎症积分及损伤面积均低于模型组(P<0.05或P<0.01)。模型组小鼠NLRP3、ASC、caspase-1、IL-1β及IL-18表达高于正常对照组(P<0.01);白藜芦醇各剂量组及阳性对照组小鼠NLRP3、ASC、caspase-1、IL-1β及IL-18表达均低于模型组(P<0.05或P<0.01)。病理组织切片显示,模型组小鼠肝细胞结构表现为胞浆疏松,小叶内坏死灶较多,坏死灶中可见中性粒细胞浸润;白藜芦醇各剂量组及阳性对照组小叶内坏死灶及中性粒细胞浸润等改变较模型组减少,肝细胞的受损面积较小。结论白藜芦醇可以显著减轻四氯化碳诱导的ALI,其机制可能与抑制NLRP3炎性体活化及其下游炎症级联反应有关。

Objective To observe the effects of resveratrol on the expression of Nod like receptor 3(NLRP3)in mice with acute liver injury(ALI),and explore the protective effect and mechanism of resveratrol on ALI.Methods The study induced mice ALI by carbon tetrachloride.The male ICR mice were randomly divided into normal control group,model group,resveratrol low-dose group,resveratrol medium-dose group,resveratrol high-dose group,and positive control group,7 in each group.The low,medium and high dose resveratrol group and the positive control group were intraperitoneally injected with 10,20 and 30 mg/kg resveratrol or 100 mg/kg acetylcysteine respectively 24 hours and 1 hour twice before the establishment of the mouse model.The control group and the model group were intraperitoneally injected with the same amount of normal saline at the corresponding time point.The model group and the drug intervention groups were intraperitoneally injected with 5%carbon tetrachloride.The control group was intraperitoneally injected with 5%carbon tetrachloride equal amount of olive oil.Western blot(WB)was used to determine the NLRP3,apoptosis-associated specklike protein contain(ASC),and inflammatory caspase-1(caspase-1)in the liver of mice.Enzyme linked immunosorbent assay(ELISA)was used to examined the inflammatory(interleukin,IL)-1βand IL-18.The liver function was measured by automatic biochemical analyzer and the degree of liver injury was observed by histopathology.Results The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the model group were higher than that of the normal control group(P<0.01),and those in each resveratrol dose groups and positive control group were lower than that in the model group(P<0.01).The inflammatory score and damage area of liver in model group were higher than those in normal control group(P<0.01),and those in each resveratrol dose groups and positive control group were lower than those in the model group(P<0.05 or P<0.01).The expression of NLRP3,ASC,caspase-1,IL-1βand IL-18 in the model group was higher than that in the normal control group(P<0.01),and that in each resveratrol dose groups and positive control groups was lower than that in the model group(P<0.05 or P<0.01).Pathological sections showed that the structure of hepatocytes in the model group showed cytoplasmic looseness,more necrotic foci in lobules and neutrophil infiltration in necrotic foci.The necrosis and neutrophil infiltration in various dose groups of resveratrol and positive control group were less than those in the model group,and the damaged area of hepatocytes was smaller.Conclusion Resveratrol could significantly reduce acute liver injury induced by carbon tetrachloride via suppression of NLRP3 inflammasome activation and downstream effects.