The Mechanistic Study Of Hepatotoxicity Induced By Diclofenac In Humanized TgCYP3A4/hPXR Mice

Diclofenac, a non-steroidal anti-inflammatory drug, have an effect of anti-inflammatory, and had been used in clinic all over the world. But diclofenac has noticeable adverse reactions, especially the risk of hepatotoxicity, including liver cell toxicity such as inflammation, necrosis, and even death.The undergoing heptoxicity mechanism induced by diclofenac had not been completely understanded. Diclofenac is mainly metablized by the cell metabolism enzyme CYP2C9 and CYP3A4 in the human body, mostly is metablized by CYP2C9 to 4 ’-hydroxy diclofenac, and less percentage is metablized by CYP3A4 to 5- hydroxy diclofenac, which has high bio-chemical activity, can bond directly to cell macromolecules in liver(protein enzymes, biological membrane structure, lead to direct cell dysfunction of liver. Due to the species difference of metabolic enzymes, the toxicity mechanism of diclofenac in wild-type animal can not fμLly represent the mechanism in the human body.Therefore, this humanized Tg CYP3A4 / h PXR double transgenic mice is used as animal models to further explore the liver toxicity mechanism. The mice Human PXR gene and CYP3A4 gene had been transferred to the mice had the typical characteristics of CYP3A4 metabolism, it is a valuable model in the study of CYP3A4 transcription and function.The present study will be conducted to explore the toxicity mechanism of diclofenac by Tg3A4/h PXR mice. And the study was conducted from the following aspects:1. The introduction breeding, reproduction and gene identification for theTg CYP3A4/h PXR double transgenic miceFirstly, the newly introduced Tg CYP3A4 / h PXR double transgenic mice were performed to reproduction and gene identification by PCR method. Results showed that 95% mice expressed the CYP3A4 and PXR gene, and the transferred gene had not escaped, can be stably passaged. The method can be used to establish stable genetic strain for the Tg CYP3A4 / h PXR double transgenic mice. 2. Study on the pharmacokinetics, stablility and inhibitary potentials for the microsomeof Tg CYP3A4 / h PXR double transgenic micePharmacokinetic is critical for preclinical studies in new drug research and development, it plays an important role in the screening of drug absorption, distribution, metabolism and excretion(ADME) and toxicity. In clinical studies, 39% of the candidate compounds to be eliminated due to poor pharmacokinetic properties. Attributed species difference between the preclinical research object(animals) and clinical research object(person), many mistakes had occurred during the new drug research.Therefore, it is important to understand the species similarity and variation in the metabolic characteristics between animal and human.In this part of study, pharmacokinetic study in Tg CYP3A4 / h PXR double transgenic mice had been conducted. The linear relationship between doses and blood drug concentration was investigated and the correlation of blood drug concentration and toxicity of phenotype had been confirmed.The enzyme kinetics parameters, such as activity, apparent Km and Vmax in liver microsomal of the Tg CYP3A4 / h PXR double transgenic mice had been assayed. Also, inhibitary potentials had been assayed in this study.These results will help to understand the metabolic stability of diclofenac in the Tg CYP3A4 / h PXR double transgenic mice and promote its application in preclinical drug evaluation. 3. The establishment of hepatotoxicity phenotype in the g CYP3A4/h PXR double transgenic miceAs the mice was identified to carry positive cyp3a4 and h PXR gene, the female mice were selected into the experiment group, and the wild-type Balb/c mice served as the control. All the mice were administrated intraperitoneally to 80 mg/kg diclofenac, 80 mg/kg diclofenac and 10mg/kg rifampicin,160 mg/kg diclofenac, 160 mg/kg diclofenac and 10mg/kg rifampicin, 10mg/kg rifampicin and vehicle.Transaminase(ALT) and aspertate aminotransferase(AST) levels were determined and liver tissue pathological slices were conducted.Results showed that ALT and AST level were significantly higher than pre-administration for the group of Tg CYP3A4 / h PXR double transgenic mice, especially for the mice induced by rifampicin. In contrast, the ALT and AST level have no significant change for the group of wild type Balb/c mice.The liver pathological confirmed that the same phenomenon.Therefore, we concluded that the stable hepatotoxicity model induced by diclofenac in Tg CYP3A4 / h PXR double transgenic mice had been established successfully. 4. Metabolomics research on diclofenac hepatotoxicityMetabonomics technology was used to research the change of endogenous and exdogenous compounds in the Tg CYP3A4/h PXR double transgenic mice after intraperitoneally injection of diclofenac. The metabolites profile of diclofenac were analyzed between pre-dose and post-dose by PLS- DA and S-plot, and then the differential compounds were identified and some biomarker was confirmed through the database.The metabolic profile showed that group in pre-dose and post-dose can be differentiated, most components were concentrated together, and only a minority of compounds deviated from the strong accumulation point.Total 10 compounds be clarified from database analysis.These compounds mainly refered to glutathione(GSH), tryptophan metabolism, purine metabolism and inflammation pathway. 5. The preliminary study on the hepatotoxicity mechanism induced by 5-hydroxy diclofenac mediated by inflammatory cytokinesThe mechanism of liver toxicity was further studied through the direct toxic effect on liver cells and the proliferation of inflammation factors induced by diclofenac. Firstly, primary liver cell of Tg CYP3A4 / h PXR double transgenic mice administrated to diclofenac, hydroxy 4 ’- diclofenac and 5 hydroxy. And then MTT method was used to assay the inhibitory rate.Diclofenac and its metabolite 4 ’- hydroxy diclofenac and 5- hydroxy diclofenac were administrated intraperitoneally to Tg CYP3A4 / h PXR double transgenic mice. The peripheral lymphocytes were sperated and the content of IL-1beta, IL-6, IL-17 and TGF-beta inflammation factors were determined. The role of immune stimulation induced by diclofenac and its metabolites was studied on the mice, and the possible mechanisms of inflammatory cytokines mediated hepatotoxicity was discussed.The results showed that 5-hydroxy diclofenac can significantly prolife IL-6 and IL-17 in the mice. However, diclofenac and 4’-hydroxy diclofenac cann’t stimulate the proliferation of inflammation cytokines. Therefore, the contribution of 5-hydroxy diclofenac on stimulating immune response and causing the liver toxicity had been confirmed primarily.In conclusion, present study had primarily illuminated the hepatotoxicity mechanism induced by diclofenac on the Tg CYP3A4/ h PXR double transgenic mice, the hepatotoxicity phenotype had been established, toxicity of metabolites and pathways mediated by inflammatory cytokines had been explored. This study can provide basic data and direction for the further research.