基于蛋白组学探讨细辛水提物诱发肝损伤的作用机制

Mechanism of Asari Radix et Rhizoma water extract induced liver injury based on proteomics

目的基于蛋白组学探讨细辛水提物诱发肝损伤的作用机制。方法30只大鼠随机分为对照组和细辛高、低剂量(12、6 g/kg)组,每组10只,每日ig给药1次,连续28 d。末次给药后采取血清样本和肝脏样本,检测血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)活性;苏木素-伊红(hematoxylineosin,HE)染色法观察肝脏病理组织形态;串联质谱标签定量蛋白质组学法检测肝组织差异表达蛋白;免疫组化法测定大鼠肝脏组织细胞色素P4501A1(cytochrome P4501A1,CYP1A1)、金属硫蛋白-1(metallothionein-1,MT-1)、肌肉特异性烯醇化酶3(recombinant enolase 3,ENO3)、抗原转运蛋白(transporter associated with antigen processing 1,TAP1)蛋白表达。CCK-8法考察细辛水提物对小鼠肝实质AML-12细胞活力的影响,并采用qRT-PCR与Western blotting检测AML-12细胞中CYP1A1、MT-1、ENO3、TAP1蛋白及m RNA表达。结果与对照组比较,细辛组大鼠血清AST、ALT活性均显著升高(P<0.01);肝组织出现明显炎性改变;肝组织中存在179个差异蛋白,主要富集于次级代谢产物的生物合成途径、视黄醇代谢途径、吞噬体等;肝组织CYP1A1、MT-1蛋白表达显著增加(P<0.01),ENO3、TAP1蛋白表达显著减少(P<0.01)。体外实验结果显示,细辛组AML-12细胞CYP1A1、MT-1 m RNA与蛋白表达显著增加(P<0.05、0.01),ENO3、TAP1 m RNA与蛋白表达水平显著降低(P<0.05、0.01)。结论细辛水提物可诱发大鼠肝损伤,引起肝功能异常和肝组织病理改变,肝损伤机制与上调CYP1A1、MT-1 m RNA及蛋白表达及下调ENO3、TAP1 m RNA及蛋白表达有关。

Objective To investigate the mechanism of liver injury induced by Xixin(Asari Radix et Rhizoma)water extract based on proteomics.Methods A total of 30 rats were randomly divided into control group,Asari Radix et Rhizoma high-and low-dose(12,6 g/kg)groups,with 10 rats in each group,and rats were given intragastrically once a day for 28 d.Serum samples and liver samples were taken after the last administration to detect serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activities;Hematoxylin-eosin(HE)staining was used to observe the pathological morphology of liver tissue;Tandem mass spectrometry label quantitative proteomics method was used to detect differentially expressed proteins in liver tissue;Immunohistochemical method was used to determine the expressions of cytochrome P4501A1(CYP1A1),metallothionein-1(MT-1),muscle specific enolase 3(ENO3),and transporter associated with antigen processing 1(TAP1)proteins in liver tissue of rats.The CCK-8 method was used to investigate the effect of Asari Radix et Rhizoma water extract on viability of AML-12 cells,qRT-PCR and Western blotting were used to detect the protein and mRNA expressions of CYP1A1,MT-1,ENO3,TAP1 in AML-12 cells.Results Compared with control group,AST and ALT activities in serum of rats in Asari Radix et Rhizoma group were significantly increased(P<0.01).There were obvious inflammatory changes in liver tissue.There were 179 differential expressed proteins in liver tissues,mainly enriched in the biosynthesis pathway of secondary metabolites,retinol metabolic pathway,phagosomes,etc.The expressions of CYP1A1 and MT-1 proteins in liver tissue were significantly increased(P<0.01),while the expressions of ENO3 and TAP1 proteins were significantly decreased(P<0.01).The in vitro experimental results showed that the expression levels of CYP1A1 and MT-1 mRNA and protein in AML-12 cells treated with Asari Radix et Rhizoma were significantly increased(P<0.05,0.01),while the expression levels of ENO3 and TAP1 mRNA and protein were significantly reduced(P<0.05,0.01).Conclusion Asari Radix et Rhizoma water extract can induce liver injury in rats,leading to abnormal liver function and pathological changes in liver tissue.The mechanism of liver injury is related to the up-regulation of CYP1A1,MT-1 mRNA and protein expressions,as well as the down-regulation of ENO3,TAP1 mRNA and protein expressions.