| Objective:Progression of liver fibrosis,as a critical period of liver disease,can come from a variety of primary liver diseases.First of all,due to the improvement of living standards in China,the incidence of alcoholic and non-alcoholic hepatitis has increased significantly,both of which are important basic diseases of liver fibrosis;secondly,the population of hepatitis B and C patients in China is huge.Due to the medical care has improved,the survival time of patients with viral hepatitis has prolonged,and eventually more and more patients are going to liver fibrosis stage.Although liver fibrosis is adaptable in the short term,that is,it can maintain the integrity of the tissue and is reversible;but when the damage factors exist for a long time,the fibrosis will secondary to the formation of substantial scars and eventually lead to liver cirrhosis and even liver cancer.Therefore,liver fibrosis is a critical period of liver disease.Studying the occurrence and development process of liver fibrosis is particularly important for improving people's overall health and happiness.Tissue damage and inflammation are important causes of fibrosis.According to previous studies,the formation of liver fibrosis mainly includes three stages: the first stage,target cells can be hit by primary liver injury factors and release damage signals;the second stage,the internal inflammatory cells including macrophages are activated to initiate the inflammatory response;in the third stage,stellate cells are activated to produce extracellular matrix.The three stages are not independent of each other,but jointly catalyze the progress of liver fibrosis.If the damage factors persist and other risk factors added in,the dynamic deposition of extracellular matrix will promote the development of fibrosis and eventually become irreversible.Nuclear factor erythroid-derived 2-like 2(NFE2L2,also known as Nrf2)is an important transcription factor that regulates the oxidative stress response.Studies have shown that Nrf2 is related to the progress of inflammation and fiber generation.Previously,the research on Nrf2 in liver fibrosis was divided into two categories: the global Nrf2 knockout mice for research and the application of Nrf2 activators for intervention,but the lack or activation of global Nrf2 cannot fully explain the effect or the mecanism of Nrf2 on liver fibrosis.The parenchymal cells of the liver are hepatocytes,and non-parenchymal cells mainly include macrophages and vascular endothelial cells.The three cell types are closely related to liver oxidative stress and inflammation.In this paper,three cell-specific Nrf2 knock-out mouse models were established,and the carbon tetrachloride-induced liver fibrosis model was used to analyze the occurrence and development of liver fibrosis in detail based on Nrf2.In the first part,three cell-specific Nrf2 knockout mouse models were used to confirm the phenotypes related to liver fibrosis;the second part was for the mechanism of liver injury and inflammation in hepatocyte-or monocyte-specific Nrf2 knockout mice;the third part analyzes the specific molecular mechanism of Nrf2 affecting oxidative stress and inflammation in hepatocytes.The three parts step by step were designed to fill the role and mechanism of Nrf2 in different cells for liver fibrosis caused by carbon tetrachloride,and provide a new theoretical basis for the prevention and treatment of liver fibrosis.Methods:1.Cell-specific Nrf2 knockout mice were given 8 weeks of carbon tetrachloride(CCl4)exposure to induce liver fibrosis.The Nrf2-Lox P and Albumin-Cre mice was used to obtain hepatocyte-specific Nrf2 knockout(Nrf2(L)-KO)mice and littermate control(Nrf2-Lox P)mice.Male 13-week-old mice were used,one group was the solvent control group(Veh),and the other group was the model group(CCl4): Nrf2-Lox P,Veh group,Nrf2(L)-KO,Veh group,Nrf2-Lox P,CCl4 group,Nrf2(L)-KO,CCl4 group,and each group of 6-10 mice.The body weight of the mice was recorded twice a week,and according to the body weight,an intraperitoneal injection of olive oil control or carbon tetrachloride was performed.Mouse plasma and various important organ tissue samples were collected at the end of the experiment.In the same way as above,use Nrf2-Lox P and Lysozyme2-Cre mouse or Cdh5-Cre mouse to obtain monocyte-specific Nrf2 knockout(Nrf2(M)-KO)mice,vascular endothelial cell specific Nrf2 knockout(Nrf2(E)-KO)mice and their littermate-born controls.The grouping,monitoring and treatment were the same as above,with 7-9 animals per group.2.Cell-specific Nrf2 knockout mice were given acute exposure to CCl4 within 48 hours.Male 13-week-old male mice of different genotypes were chosen,one group is the solvent control group(Veh),and the other group is the model group(CCl4): Nrf2-Lox P,Veh group,Nrf2(L/M)-KO,Veh group,Nrf2-Lox P,CCl4 group,Nrf2(L/M)-KO,CCl4 group,each group of 6-10 mice.Mice were intraperitoneally injected with olive oil or carbon tetrachloride according to their body weight.Within 48 hours exposure,tail vein serum was collected at different time points,and mouse plasma and organ tissue samples were collected at the end of observation.3.In vivo sample processing.Plasma aspartate aminotransferase,alanine aminotransferase,chemokine MCP-1 and MIP-1? levels were detected using the corresponding kit;the content of malondialdehyde(MDA)and hydroxyproline were determined in liver tissues;H&E staining,Masson staining,and immunohistochemical staining of liver tissue were performed after 4% polymer formaldehyde fixation;the transcription level of fibrosis indicators,oxidative stress and classic genes of inflammation were detected in liver tissues;fibrosis pathway proteins and NRF2,NF-?B and other signal pathways were detected in liver tissues;fresh liver tissue was ground,and flow cytometry was used to detect cell surface protein expression.4.In vitro experiments to study the molecular mechanism of CCl4-induced hepatocyte damage and inflammation.Using the primary hepatocytes as experimental materials,the cells were exposed to carbon tetrachloride and LPS,and the expression of Nrf2 and its downstream genes and inflammation-related genes were detected;the culture medium of hepatocyte were collected,and the level of chemokines were detected after carbon tetrachloride or LPS.Using transwell to detect the ability to chemoattract macrophages of conditional medium.Human liver cell line L02 was used to confirm the key mechanism.Nrf2 stable knockdown cell line were generated(NRF2-KD),after carbon tetrachloride or LPS exposure,or pretreat the cells with reactive oxygen scavengers or Nrf2 agonists,we verified the relationship between Nrf2 and ROS,inflammation.The primary peritoneal macrophages were extracted from the same littermate,treated with carbon tetrachloride,and detected the protein and gene expression of Nrf2 and its downstream genes.Results:1.Hepatocyte-specific Nrf2 knockout aggravated the liver fibrosis in mice with 8weeks carbon tetrachloride exposure.Nrf2 knockout in vascular endothelial cells and monocytes had no effect on liver fibrosis.It was found that the lack of Nrf2 in hepatocyte elevated the pathological staining results of mouse fibrosis(H&E,MASSON staining,immunohistochemical staining).The levels of fibrosis-related biochemical indicators:hydroxyproline,protein,and genes were significantly increased(P <0.05),that is,compared with Nrf2-Lox P,Nrf2(L)-KO mice were sensitized to liver fibrosis.For mice lacking Nrf2 in monocytes or vascular endothelial cells,there was no significant difference in fibrosis-related levels compared with the Nrf2-Lox P group.2.Hepatocyte specific Nrf2 deficiency was more sensitive to liver oxidative damage and inflammation caused by acute carbon tetrachloride exposure.After acute carbon tetrachloride exposure,liver injury indicators and lipid peroxidation levels of Nrf2(L)-KO mice were significantly increased than those of Nrf2-Lox P.Nrf2 protein itself and its classical downstream genes m RNA and protein expression were decresed in Nrf2(L)-KO liver,compared with the Nrf2-Lox P group(P <0.05).According to the pathological results,the inflammatory response of Nrf2(L)-KO mice were increased,and also the levels of inflammation-related m RNA.Compared with the Nrf2-Lox P group,the expression of inflammatory factors,chemokines,and fibrosis-related genes in the liver and the expression of chemokines in the plasma of the Nrf2(L)-KO group were significantly increased.There was no significantly difference in the expression levels of pyrolysisrelated genes in liver tissues and NF-?B pathway proteins in the Nrf2(L)-KO group.After acute carbon tetrachloride exposure,there were not significantly difference in liver injury,inflammation,or fibrosis-related indicators between Nrf2-Lox P and Nrf2(M)-KO mice.3.Nrf2 can regulate the expression of downstream antioxidant genes and inflammation-related genes through transcription in hepatocyte,affect cell oxidative damage and macrophage chemotaxis,thereby changing the process of liver fibrosis caused by carbon tetrachloride.After the primary hepatocytes were exposed to carbon tetrachloride or LPS,the inflammation and chemokine indexes were increased,and was more obviously in the Nrf2(L)-KO group(P <0.05).After collecting the culture medium treated with carbon tetrachloride or LPS,it was found that the conditional medium of the Nrf2(L)-KO group had an improved chemotaxis ability to RAW cells.After carbon tetrachloride exposure,the expression of NRF2 downstream genes in the NRF2-KD group decreased,and the levels of reactive oxygen species(ROS)and MDA were significantly increased(P <0.05),and the expression of chemokines increased in L02 cells.After pretreated the cells with ROS inhibitor or Nrf2 activator,it was found that removing ROS could not,but activation of Nrf2 coud inhibit the increase of chemokine expression caused by carbon tetrachloride.Finally,we found that a certain dose of carbon tetrachloride could not activate the expression of Nrf2 and its downstream genes in primary mouse macrophages.Conclusions:1.Carbon tetrachloride can activate Nrf2 protein and downstream genes expression in liver tissue.Hepatocyte-specific Nrf2 knockout mice were more sensitive to liver fibrosis caused by carbon tetrachloride.The absence of Nrf2 in monocytes or vascular endothelial cells did not affect the liver fibrosis caused by carbon tetrachloride.The experimental results showed that Nrf2 in hepatocytes played an important role in liver fibrosis.2.Knockout of Nrf2 in hepatocytes sensitized the liver to lipid oxidative damage and inflammation caused by carbon tetrachloride,thereby affecting the process of fibrosis formation.The absence of Nrf2 in monocytes had no significant effect on oxidative stress and inflammatory response.3.After carbon tetrachloride exposure,the levels of oxidative damage and inflammation in primary hepatocytes and human liver cell lines lacking Nrf2 were upregulated,and can directly affect the chemotaxis of macrophages,thereby affecting the inflammatory response. |
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