The Study On Effects Of Cyp2c29 In The Developing Of Chemically Induced Mouse Models Of Liver Injury

Hepatocellular carcinoma（HCC）is one of the most common and most lethal cancers in the world.Previous studies have shown that chronic inflammation plays an important role in the development of HCC,which promotes the malignant transformation and proliferation of hepatocytes,induces liver fibrosis and the invasion and metastasis of HCC.In this study,DEN combined with high fat diet was used to induce mouse HCC model.Through high-throughput sequencing,transcriptomics data were collected in different stages of HCC in mice.We focused on the gene expression changes,and tried to find the key genes and related signaling pathways in HCC development.Through the data analysis,we found that Cyp2c29（a member of the cytochrome P450 family,which is abundantly expressed in the liver of mice）expression is continuously decreased in the development of HCC.Further studies have shown that Cyp2c29 cauld inhibit the activation of NF-κB pathway in RAW264.7 cells and reduce the secretion of inflammatory cytokines by producing epoxy-eicosatetraenoic acid（EET）.In vivo experiments have shown that Cyp2c29 cauld slow down liver damage induced by acetaminophen（APAP）or carbon tetrachloride（CCl₄）,inhibit NF-κB and related inflammatory activation pathways in vivo,and reduce hepatocyte proliferation caused by inflammation.The main research contents and results of this study are as follows:（1）We esteblished a HCC tumorigenesis and progression model in mice by DEN combined with high fat diet.Pathological sections showed that in the course of 30 weeks,mice showed different pathological stages such as inflammation,steatosis,liver fibrosis and even HCC.Through RNA-seq sequencing and comparative analysis of mouse liver at different stages,we found that a series of cytochrome P450 enzyme genes showed significant changes in the different pathogenesis of mouse.In particular,Cyp2c29 was abundantly expressed in normal mice,but declined as the progression of HCC.This change mainly occured in liver parenchymal cells.Quantitative analysis of Cyp2c29 mRNA in liver tissue of mice at various stages by q RT-PCR also confirmed the result.In addition,we found the homologous genes CYP2C8 and CYP2C9 in human,which are highly similar to Cyp2c29 in mouse,were significantly associated with survival and HCC staging.These data suggest that the decrease of gene expression in the CYP2C family may be one of the important factors in the development of HCC,both in animal models and in human.（2）The effects of Cyp2c29 on hepatocytes,macrophages and hepatoma cells were studied by in vitro transfection experiments with Cyp2c29 fluorescent plasmid.In the experiment of transfecting Cyp2c29 plasmid into hepatocyte HL-7702,the production of EET was increased.EET had a significant anti-inflammatory effect,inhibited the activation of NF-κB pathway in RAW264.7 cells,and reduced interleukin-1（IL-1）and tumor necrosis factor（TNF）in cell supernatants.We used the supernatants of LPS-activated RAW264.7cells to mimic inflammatory mediators and found that a certain proportion of supernatants could cause proliferation of primary hepatocytes in mice,while 100 nmol/L 14,15-EET inhibited the proliferation.In vitro experiments showed that Cyp2c29-catalyzed EET production inhibited the activation of NF-κB pathway and inflammation,which may be responsible for the inhibition of hepatocyte proliferation by Cyp2c29.（3）We validated the effects of Cyp2c29 using the APAP and CCl₄-induced liver injury models.Similar to the DEN model,the expression of Cyp2c29 was down-regulated in mouse hepatocytes in the APAP model.By plasmid transfection,Cyp2c29 expression was increased in mouse liver.Compared with the empty vector group,the Cyp2c29 high expression group showed a lower degree of liver damage,a relatively significant decrease ALT and AST levels in serum,and a decrease in hepatocyte necrosis area in pathological sections.Western blotting results showed that the level of phosphorylated NF-κB in the liver of mice was down-regulated in the Cyp2c29 high expression group.Transcriptome sequencing revealed that the expression of genes reduced in the NF-κB,TNF and MAPK pathways.We also found that the expression of Cyp2c29 is negatively correlated with the expression of IL-1β,TNF,CCND1 and CCL2.In the CCl₄ model,the Cyp2c29 plasmid transfection group also showed decreased levels of ALT and AST,decreased inflammatory cells in the liver,and decreased liver injury score.Animal experiments have shown that Cyp2c29 may reduce liver damage by inhibiting the activation of NF-κB and subsequent inflammatory responses.（4）In the mouse liver injury model induced by CCl₄ or DEN,we verified the effects of liver-protecting traditional Chinese medicine Er-Zhi-Wan on the expression of Cyp2c29.The results demonstrated that Er-Zhi-Wan administration increased the level of Cyp2c29 in mouse liver.In both models,mice pre-treated with 600 mg/kg of Er-Zhi-Wan for four weeks had less liver injury,lower serum transaminase concentrations,than that in the model group.The activation of Kupffer cells was inhibited and the expression of inflammatory cytokines were decreased after Er-Zhi-Wan administration.It was confirmed that The Er-Zhi-Wan has liver protective effect on injury induced by CCl₄ and DEN.This effect may be caused by increasing the expression of Cyp2c29 in the liver,which inhibits the activation of Kupffer cells.The aforementioned results indicate that Cyp2c29 plays an important role in the development of HCC in mice.The expression of Cyp2c29 was significantly decreased in different mouse models of mouse liver injury induced by different factors,including the different stages of mouse HCC development in DEN model.We found that increasing the level of Cyp2c29 can increase cell EET production in vitro,inhibit LPS-induced NF-κB pathway activation in RAW264.7 macrophages,reduce phosphorylated NF-κB levels,and reduce the expression of inflammatory cytokines such as IL-1 and TNF.Furthermore,EET inhibited hepatocyte proliferation caused by inflammatory cytokines.In the model of liver injury induced by APAP and CCl₄,overexpression of Cyp2c29 can inhibit the activation of NF-κB-related pathway and reduce the expression of inflammatory cytokines,thereby reduced the damage of hepatocytes in mice and inhibited the compensatory proliferation of hepatocytes.In patients with HCC,the patient’s survival was positively correlated with the expression of CYP2C8 and CYP2C9（a homologous gene of Cyp2c29）.This study shows that CYP2C genes including Cyp2c29 may be important in the development of liver injury and HCC,and they are expected to be new targets for clinical diagnosis and treatment of liver disease.The Chinese medicine Er-Zhi-Wan can relieve liver injury and reduce inflammation in mice,and its mechanism may be related to the increase of Cyp2c29expression in mouse liver.