摘要
目的研究雷公藤甲素诱导细胞色素P450 3A4(CYP3A4)表达下调在其引发肝损伤中的作用。方法将L-02细胞和C57BL/6雄性小鼠随机分为4组,分别为正常组(C)、雷公藤甲素(TP)组、雷公藤甲素(TP)+卡马西平(CBZ)组、雷公藤甲素(TP)+氨氯地平(AML)组, 24 h后收集培养基,C57BL/6雄性小鼠2周后安乐死,取血和肝脏。流式细胞仪观察用药后各组细胞的凋亡率,小鼠肝脏进行组织染色观察肝细胞损伤情况。试剂盒检测细胞上清液和小鼠血清的丙氨酸氨基转移酸(alanine transaminase,ALT)、天冬氨酸氨基转移酶(aspartate transaminase,AST)。通过Western blot法和RT-PCR法检测细胞和小鼠肝脏CYP3A4的mRNA和蛋白表达水平,应用高效液相色谱法检测细胞培养基中和小鼠血清中雷公藤甲素的含量。结果流式细胞仪检测的细胞凋亡结果和小鼠肝脏病例照片结果显示,TP组明显比C组严重,加入P450 3A4诱导剂的TP+CBZ组则明显减轻,而加入P450 3A4抑制剂的TP+AML组的细胞进一步加剧。体内外ALT和AST检测结果显示,TP组明显高于C组,TP+CBZ组则明显降低,TP+AML组进一步增加。Western blot法和RT-PCR结果显示,TP组CYP3A4表达量明显低于C组,TP+CBZ组的CYP3A4表达量则明显升高,TP+AML组的CYP3A4表达量更低。高效液相检测结果显示,TP+CBZ组中TP的含量低于TP组,而TP+AML组中TP的含量则相反。结论 TP通过抑制其主要代谢酶CYP3A4来减慢自身代谢,可能是其产生肝损伤的机制之一。
Objective To investigate the role of triptolide-induced down-regulation of cytochrome P450 3 A4(CYP3 A4)expression in causing liver injury.Methods L-02 cells and C57 BL/6 male mice were randomly divided into 4 groups respectively, including normal group(C), triptolide(TP) group, triptolide(TP)+carbamazepine(CBZ) group and triptolide(TP)+amlodipine(AML) group. After 24 h, the medium was collected, and C57 BL/6 male mice were euthanized and taken blood and liver 2 weeks later. The apoptosis rate of each group was detected by flow cytometry, while the damage of mice liver cells was observed by hematoxylin-eosin staining. The kit was uesd to detectethe level of ALT and AST in cell supernatant and mouse serum. The mRNA and protein expression levels of CYP3 A4 in cells and mouse liver were tested by Western blot and RT-PCR. The content of triptolide in cell culture medium and mouse serum was detected by HPLC.Results The results showed that the TP group significantly had more apoptosis and tepatocyte injury than the normal group. However, compared to the TP group, the apoptosis and liver cell injure of the TP+CBZ group significantly relieved, while the TP+AML group aggravated. The levels of ALT and AST in vitro and in vivo of the TP group were significantly higher than the normal group, and the lower level was observed in the TP+CBZ group and higher level was observed in the TP+AML group. The expression of CYP3 A4 in TP group and TP+AML group significantly declined conapared with group C, while the expression of in the TP+CBZ group significantly higher. The results of HPLC showed that the TP content in the TP+CBZ group was lower than that in the TP group, while it was opposite in the TP+AML group.Conclusion TP can slow down cells metabolism by inhibiting its main metabolic enzyme CYP3 A4, which may be one of the underlying mechanisms of liver damage.
作者
杨新华
夏宏光
金涌
Yang Xinhua;Xia Hongguang;Jin Yong(College of Pharmacy,Anhui Medical University,Hefei 230032;harmact Dept,The 8th People's Hospital of Hefei,Hefei 238000)
出处
《安徽医科大学学报》
CAS
北大核心
2020年第4期496-502,共7页
Acta Universitatis Medicinalis Anhui
基金
安徽省自然科学基金(编号:1808085MH284)。
