Triptolide Enhances Liver Injury By Inhibiting Its Metabolic Enzyme Cytochrome P450 3A4

Tripterygium wilfordii hook F(TWHF)is a vine plant that is mainly produced in southern China,including Zhejiang,Hunan,Anhui,Yunnan,Fujian,and Taiwan.The earliest record of its medicinal use appeared in "Southern Yunnan Materia Medica" by Lan Mao in 1476.It began to attract worldwide attention in the 1960 s,and its extracts proved to be effective in treating rheumatoid arthritis.Triptolide(TP),also known as triptolide,is a complex triepoxide diterpene isolated from the crude extract of TWHF.As one of the main biologically active components of TWHF,it has unique biological activity in vitro and in vivo.However,before triptolide can reach its clinical potential,it still needs to overcome many problems,such as poor water solubility,narrow treatment window and multiple organs.Toxicity,long-term use can lead to serious adverse events,such as hepatomegaly,liver injury.The exact mechanism of TP-induced liver injury is unknown.The liver is the main site of drug metabolism,and can be biotransformed through a variety of enzyme systems in liver microsomes,the most important of which is cytochrome P450(CYP450).CYP450 is a group of structurally and functionally related isoenzymes.It mainly exists in the endoplasmic reticulum of organisms,and is the most important enzyme system of mixed-function oxidases.The metabolism of TP in rat liver microsomes is mainly mediated by CYP3 A,and adult CYP3 A enzyme are mainly CYP3A4.This article focuses on the role of TP-induced down-regulation of CYP3A4 expression in liver injury.The main methods are as follows:1.Cell experiment: L-02 cells were randomly divided into 4 groups: normal group(C),triptolide(TP)group,triptolide(TP)+ carbamazepine(CBZ)group,and triptolide Rattanin(TP)+ amlodipine(AML)group.After 24 h,the culture medium was collected,and the apoptosis rate of cells in each group was observed by flow cytometry.The kit detected the ALT and AST of the cell supernatant.The m RNA and protein expression levels of CYP3A4 in cells were detected by Western blot and RT-PCR.The content of triptolide in cell culture medium was detected by high performance liquid chromatography.Apoptosis results detected by flow cytometry showed that the TP group was significantly more severe than the C group,the TP + CBZ group with the P450 3A4 inducer was significantly reduced,and the TP + AML group with the P450 3A4 inhibitor was further intensified.The results of ALT and AST in vivo and in vitro showed that the TP group was significantly higher than the C group,the TP + CBZ group was significantly reduced,and the TP + AML group was further increased.Western blot and RT-PCR results showed that the expression of CYP3A4 in the TP group was significantly lower than that in the C group,the expression of CYP3A4 in the TP + CBZ group was significantly increased,and the expression of CYP3A4 in the TP + AML group was lower.The HPLC results showed that the content of TP in the TP + CBZ group was lower than that in the TP group,while the content of TP in the TP + AML group was the opposite.2.Animal experiments: C57 BL / 6 male mice were randomly divided into 4 groups: normal group(C),triptolide(TP?0.6mg/kg/day)group,triptolide(TP?0.6mg/kg/day)+ carbamazepine(CBZ?24mg/kg/day)group,and Triptolide(TP?0.6mg/kg/day)+ amlodipine(AML?1.0mg/kg/day)group.After 2 weeks,C57 BL / 6 male mice were euthanized and blood and liver were taken.Mouse liver was stained to observe the damage of liver cells.The kit detected ALT and AST of mouse serum.The m RNA and protein expression levels of CYP3A4 in mouse liver were detected by Western blot and RT-PCR,and the triptolide content in mouse serum was detected by high performance liquid chromatography.The results of mouse liver photos showed that the TP group was significantly more severe than the C group,and the TP + CBZ group with the P450 3A4 inducer was significantly reduced,while the TP + AML group with the P450 3A4 inhibitor was further exacerbated.The results of ALT and AST in vivo and in vitro showed that the TP group was significantly higher than the C group,the TP + CBZ group was significantly reduced,and the TP + AML group was further increased.Western blot and RT-PCR results showed that the expression of CYP3A4 in the TP group was significantly lower than that in the C group,the expression of CYP3A4 in the TP + CBZ group was significantly increased,and the expression of CYP3A4 in the TP + AML group was lower.The HPLC results showed that the content of TP in the TP + CBZ group was lower than that in the TP group,while the content of TP in the TP + AML group was the opposite.The experimental results of this study show that TP can significantly inhibit the expression of CYP3A4 in L-02 cells and C57 BL / 6 male mice,and at the same time,TP can damage the liver of L-02 cells and C57 BL / 6 mice.After the addition of CYP3A4 inhibitor AML,the expression of CYP3A4 was further reduced,and the content of TP was significantly increased,suggesting that the metabolism of TP was reduced.As a result,the damage of TP to L-02 cells and mouse liver was increased;in contrast,with the addition of CYP3A4 inducer CBZ,the expression of CYP3A4 increased,and the content of TP decreased,suggesting that the metabolism of TP was accelerated,and the result was that the damage of TP to L-02 cells and mouse liver was significantly reduced.From the above results,it may be concluded that TP slows its metabolism by inhibiting the expression of CYP3A4,thus causing the liver damage,but whether it is the main mechanism of liver damage remains to be experimentally proven in the next step.