大黄游离蒽醌抑制NLRP3/Caspase-1通路改善大鼠重症急性胰腺炎肝损伤的研究

Free Anthraquinones in Dahuang(大黄)Improves Liver Injury in Rats with Severe Acute Pancreatitis by Inhibiting NLRP3/Caspase-1 Pathway

目的:探究大黄游离蒽醌通过抑制NLRP3/Caspase-1通路对大鼠重症急性胰腺炎(SAP)肝损伤的保护作用。方法:雄性SD大鼠随机分为正常对照组、SAP模型对照组、大黄游离蒽醌22.5、45、90 mg/kg组和生大黄900 mg/kg组。除正常对照组外,3.5%牛磺胆酸钠逆行注射到胰胆管内制备SAP大鼠模型,正常对照组注射等体积生理盐水。各药物组灌胃给药,12 h/次,共3次,末次给药2 h后处死动物取材。另按照方案给药3次后,观察大鼠7 d死亡率。HE染色观察胰腺和肝脏的病理变化;检测血清淀粉酶(AMS)、脂肪酶(LPS)、丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)活力;ELISA法检测肝组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-6和IL-18的水平;蛋白免疫印迹法检测肝组织TLR4、NLRP3、Pro-caspase-1和Caspase-1的蛋白表达。结果:SAP模型对照组大鼠7 d死亡率为41.67%,大黄游离蒽醌45、90 mg/kg组大鼠能够降低SAP模型大鼠的死亡率。与正常对照组相比,SAP模型对照组大鼠胰腺、肝脏病理评分显著增高,血清AMS、LPS、ALT和AST活力以及肝组织TNF-α、IL-1β、IL-6和IL-18含量显著上升(P<0.01),肝组织TLR4、NLRP3、Pro-caspase-1和Caspase-1蛋白表达显著上调(P<0.01)。与SAP模型对照组相比,大黄游离蒽醌45、90 mg/kg组可明显降低胰腺和肝脏病理学评分,明显降低血清AMS、LPS、ALT和AST活力及肝组织TNF-α、IL-1β、IL-6和IL-18含量(P<0.05或P<0.01),肝组织TLR4、NLRP3、Pro-caspase-1和Caspase-1蛋白表达均显著下调(P<0.01)。结论:大黄游离蒽醌对SAP继发肝损伤具有保护作用,其机制可能与抑制NLRP3/Caspase-1通路的表达,减少肝组织促炎细胞因子的产生有关。

Objective:To explore the protective effect of free anthraquinones in Dahuang(大黄)against liver injury in rats with severe acute pancreatitis(SAP)from the perspective of the NLRP3/Caspase-1 pathway.Methods:Male SD rats were randomly divided into the normal control group,SAP model group,22.5 mg/kg,45 mg/kg,and 90 mg/kg free anthraquinone groups,and 900 mg/kg raw Dahuang(大黄)group.Rats in all groups except for the normal control group were injected with 3.5%sodium taurocholate into the pancreatic duct for inducing SAP,while those in the normal control group were injected with the same amount of saline water.Rats in each medication group were intragastrically administrated with the corresponding drugs,once every 12 h,for three times in total.Two hours after the last administration,the rats were sacrificed.Followed by another three times of drug administration according to the regimen,the 7-day mortality was then observed.HE staining was used to observe the pathological changes in the pancreas and liver.The activities of serum amylase(AMS),lipase(LPS),alanine aminotransferase(ALT),and aspartame aminotransferase(AST)were detected.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),and interleukin-18(IL-18)were measured by ELISA,and the protein expression levels of TLR4,NLRP3,Pro-caspase-1,and Caspase-1 in liver tissues by Western blotting.Results:The 7-day mortality rate in the SAP model group was 41.67%.The free anthraquinones in Dahuang(大黄)at 45 and 90 mg/kg reduced the mortality of SAP model rats.Compared with the normal control group,the SAP model group exhibited significantly increased pathological scores in rat pancreas and liver,enhanced serum AMS,LPS,ALT,and AST activities,elevated TNF-α,IL-1β,IL-6,and IL-18 in liver tissue(P<0.01),and up-regulated protein expression of TLR4,NLRP3,Pro-caspase-1,and Caspase-1 in liver tissue(P<0.01).Compared with the SAP model group,the free anthraquinones in Dahuang(大黄)at 45 mg/kg and 90 mg/kg significantly reduced the pathological scores of pancreatic and liver tissues,weakened the serum activities of AMS,LPS,ALT,and AST,lowered the contents of TNF-α,IL-1β,IL-6,and IL-18 in liver tissue(P<0.05 or P<0.01),and down-regulated the protein expression levels of TLR4,NLRP3,Pro-caspase-1,and Caspase-1(P<0.01).Conclusion:The free anthraquinones in Dahuang(大黄)protect against the liver injury secondary to SAP,which may be related to their inhibition of the NLRP3/Caspase-1 signaling pathway and reduction of the production of pro-inflammatory cytokines in liver tissue.