基于转运体MRP2和P-gp研究甘草酸对雷公藤多苷所致肝损伤的保护作用及机制

Protective effect and mechanism of glycyrrhizic acid on Tripterygium wilfordii multiglycoside-induced hepatic injury based on transporter MRP2 and P-gp

目的基于药物转运体多药耐药相关蛋白2(MRP2)、P-糖蛋白(P-gp)探讨甘草酸对雷公藤多苷所致肝损伤的保护作用及机制。方法将40只雄性SD大鼠随机分为4组,即空白组、雷公藤多苷组、甘草酸组以及联合组(雷公藤多苷+甘草酸)。采用ELISA法测定大鼠血清及胆汁中谷草转氨酶(AST)、谷丙转氨酶(ALT)、总胆红素(TBIL)和总胆汁酸(TBA)的水平,通过HE染色判断大鼠肝脏病理情况。运用qRT-PCR法和Western blot法检测MRP2、P-gp基因和蛋白的表达情况。结果与空白组比较,雷公藤多苷组大鼠血清中AST、ALT、TBA、TBIL显著上升(P<0.01),胆汁中TBA、TBIL含量显著降低(P<0.01)。与雷公藤多苷组比较,联合组大鼠血清中AST、ALT、TBA、TBIL显著降低(P<0.05,P<0.01),胆汁中TBA、TBIL含量升高(P<0.05,P<0.01)。肝脏病理结果显示,雷公藤多苷组肝细胞排列紊乱,部分区域可见坏死灶;联合组中甘草酸能有效改变雷公藤多苷对肝细胞的损伤作用;空白组以及甘草酸组未见异常变化。与空白组比较,雷公藤多苷组大鼠肝脏Mdr1a、Mdr1b、MDR2 mRNA表达量以及P-gp蛋白表达均明显降低(P<0.05,P<0.01),同时MRP2 mRNA表达量和蛋白表达也显著下降(P<0.05,P<0.01)。联合组Mdr1a、Mdr1b、MDR2 mRNA以及P-gp蛋白表达显著上调(P<0.05,P<0.01),MRP2 mRNA表达量和蛋白表达也不同程度被上调(P<0.05,P<0.01)。结论雷公藤多苷所致肝损伤可能是抑制了肝脏MRP2和P-gp转运体,导致毒性物质外排减少,而甘草酸可以上调MRP2和P-gp基因和蛋白的表达,逆转雷公藤多苷对转运体的抑制作用,加快毒素排出,降低雷公藤多苷对肝脏的损伤。

Objective To determine the protective effect of glycyrrhizic acid on the liver injury induced by Tripterygium wilfordii multiglycoside based on drug transporter MRP2 and P-gp and related mechanism.Methods Forty male SD rats were randomly divided into 4 groups:a blank group,a Tripterygium wilfordii multiglycoside group,a glycyrrhizic acid group and a combination group (Tripterygium wilfordii multiglycoside+glycyrrhizic acid).Aspartate aminotransferase (AST),alanine aminotransferase (ALT),total bilirubin (TBIL) and total bile acid (TBA) in the serum and the bile were determined by ELISA.The pathological condition of the rat liver was judged by HE staining.The gene and protein expressions of MRP2 and P-gp were detected by qRT-PCR and Western blot.Results Compared with those of the blank group,the levels of AST,ALT,TBA and TBIL in the serum were significantly increased,while the content of TBA and TBIL in the bile was significantly decreased in the Tripterygium wilfordii multiglycoside group (P <0.01).Compared with the Tripterygium wilfordii multiglycoside group,the AST,ALT,TBA and TBIL values in the serum of the combination group were significantly lower (P <0.05,P <0.01),and the TBA and TBIL content in the bile of the combination group was significantly higher (P <0.05,P <0.01).Pathological results of the liver showed that hepatocytes in the Tripterygium wilfordii multiglycoside group were disordered and necrotic foci were seen in some areas,while glycyrrhizic acid effectively changed the damaging effect of tripterygium wilfordii multiglycoside on hepatocytes in the combination group.No abnormal changes were found in both the blank group and the glycyrrhizic acid group.Compared with the blank group,the mRNA expressions of Mdr1a,Mdr1b,MDR2 and P-gp protein expressions in the liver of rats in the Tripterygium wilfordii multiglycoside group were significantly decreased (P <0.05,P <0.01),and the gene and protein expressions of MRP2 were significantly decreased (P <0.05,P <0.01).After combining with glycyrrhizic acid,the mRNA expression of Mdr1a,Mdr1b,MDR2 and P-gp protein were significantly increased (P <0.05,P <0.01),and the expressions of MRP2 mRNA and protein were up-regulated to different degrees (P <0.05,P <0.01).Conclusion The hepatic injury induced by Tripterygium wilfordii multiglycoside may be due to the inhibition of MRP2 and P-gp transporters in the liver,resulting in the reduction of toxic substance efflux.Glycyrrhizic acid can up-regulate the expression of MRP2 and P-gp genes and proteins,reverse the inhibitory effect of Tripterygium wilfordii multiglycoside on transporters,accelerate toxin excretion and reduce the liver damage caused by Tripterygium wilfordii multiglycoside.