Research On Regulation Mechanism Of Chronic Hypoxia On Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver disease,NAFLD is one of the most common liver diseases characterized by chronic and metabolic alterations.NAFLD,including a variety of benign and reversible pathological changes from steatosis（fatty liver）,can also progress to more serious and irreversible pathological changes.It has been estimated that approximately 24%of patients with nonalcoholic steatohepatitis（NASH）and cirrhosis;andabout 15%may ultimately develop from liver cirrhosis,and eventually hepatocellular carcinoma（HCC）.The progressive stage of NASH can develop into fibrosis and even hepatocellular carcinoma,which is seriously life-threatening.It is generally agreed that the pathogenesis of NAFLD progression to NASH is the theory of"multiple hitting".Oxidative stress,lipid peroxidation and inflammation caused by lipid accumulation are one of the key factors.A large amount of lipid accumulates in hepatocytes,causes a series of cytotoxic reactions,which eventually lead to inflammation and fibrosis in NAFLD.However,clinically,the occurrence and development of NASH is complex and diverse,the"multiple strikes"theory can not fully explain,and even the latest research shows that fibrosis is a compensatory way to protect the liver.In addition,the effects of oxidative stress and other factors from different levels and sources on the occurrence and development of NASH need to be further studied.Hypoxia is the most common natural and pathological condition,and many evidence confirmed that chronic hypoxia is closely related to the pathogenesis of NASH.The most common systemic chronic hypoxia is obstructive sleep apnea（OSAS）,which can lead to liver injury,cardiac metabolic syndrome and NAFLD.In addition,Excessive high-fat diet intake with sedentary habits combined withchronic intermittent hypoxia,which can lead to lipid accumulation,inflammation and lipid peroxidation.In addition,a variety of factors suggested that the progression of liver injury and fibrosis in NAFLD seems to be related to hypoxia.Numerous studies have confirmed that the level of mitochondrial oxidation in muscle,liver and other tissues in high-altitude habitants might be higher than that of sea level dwellers.As one of the most important organs with abundant blood supply,the liver is extremely sensitive to hypoxia stimulation.Hypoxia can not only cause microcirculatory disturbance,hepatocyte injury,hyperemia,edema and steatosis,but also cause hypoxic damage to important organelles such as mitochondria,and eventually lead to apoptosis.Long-term exposure to hypoxia is one of the important factors leading to the high prevalence of fatty liver at high altitude.the possible main reason is that hypoxia suppresses part of the cyclic oxidation of tricarboxylic acid and reduces theβoxidation of fatty acids at the same time.Increase or decrease synthesis,which may cause liver lipid accumulation and induce the formation of fatty liver.In addition,the accumulation of lipids in the liver can not effectively produce catabolism,which can cause lipid oxidative stress.We believe that chronic hypoxia and high-fat diet,mitochondrial oxidative stress and other factors may aggravate liver inflammation and fibrosis,making it develop into NASH,or even hepatocellular carcinoma.Hypoxia inducible factor（HIFs）is the most critical regulator of the response of cells and tissues in organisms to hypoxia stimulation.So far,there are few studies on acute and chronic hypoxia and the role of HIFs in NAFLD,but the morphological evidence of our group shows that liver hypoxia and NAFLD-induced steatosis occur at the same time,especially around the vein,which is like ethanol-induced fatty liver in pathologically.HIFs are the main regulatory factor for cells to sense hypoxia and trigger hypoxic profoundeffects.HIF is composed ofαandβsubunit heterodimers,in whichαcan be divided into 1αand 2αsubunits with respecting to their differences in structure and function.Hypoxia inducible factor-2α（HIF-2α）is a protein molecule widely expressed in many cells under hypoxia.Although its function is not as widespread and significant as HIF-1α,it is highly expressed in liver,intestine and kidney,and also plays a regulatory role in the key genes of lipid metabolism,fibrosis and inflammation.Previous studies have confirmed that the overexpression of HIF-2αis commonly in the liver with chronic hypoxia,which may be another potential cause of liver injury.Cell and animal experiments have confirmed that inhibiting the expression of HIF-2αcan reduce the liver injury caused by apoptosis.However,human data on the mechanism of HIF-2αin fatty liver and the progression of NAFLD are still few and the potential mechanism of HIF-2αregulating fibrosis and promoting hepatocyte metabolism during the development of NASH is still unclear.In order to explore the molecular mechanism that chronic hypoxia exposure at high altitude may increase the incidence of NASH,the expression of HIFs in human NASH liver was detected and analyzed.The effects of chronic hypoxia on metabolic phenotype（including body weight,liver index,blood glucose,serum lipid metabolism and liver injury index）and histopathological changes of NASH mice were dynamically observed by establishing a chronic hypoxia model of NASH mice.The expression levels of HIF and genes related to inflammation and fibrosis in the liver of chronic hypoxic NASH mice were detected,and the whole and local exocrine and local liver were used to further explain how chronic hypoxia at high altitude affects the molecular mechanism of NASH disease.It is hoped that the study of this experiment will provide some new ideas and intervention strategies for the pathogenesis of NASH.Part Ⅰ:Effects of chronic hypoxia on metabolic phenotyping of NASH in mice.Objective:To explore the effects of chronic hypoxia at high altitude on the overall metabolic characterization of NASH in mice.Methods:（1）The model of NASH（C57BL6J mice）was established by using high fat and high fructose diet water combined with CCL₄,and normal diet and water were used as ND control group.The mice were divided into four groups:normoxia NASH group（n=20）,chronic hypoxia NASH group（n=20）,normoxia ND group（n=20）and chronic hypoxia ND group（n=20）.The mice in the chronic hypoxia group were raised at an altitude of 4300m.The mice in normoxia group were raised at 36m above sea level.In order to dynamically observe the changes of various indexes,the samples were collected at the 4th week and 8th week respectively.（2）The chronic hypoxia was determined by blood hemoglobin concentrationsroutine examination.（3）Body weight,liver index,blood glucose,serum lipid metabolism（TG,T-CHO）,liver TG and T-CHO and liver injury（ALT,AST）were dynamically monitored.（4）The pathological changes of mouse liver were evaluated by H&E staining,oil Red O staining and Sirius Red staining.Results:（1）Compared with normoxia ND group,chronic hypoxia ND group lost weight,but there was no difference in liver index,blood glucose,serum lipid metabolism and liver injury.There were no obvious changes of steatosis,inflammation and fibrosis in liver pathology between the two groups.（2）Compared with ND group,the body weight,liver weight,blood glucose,serum lipid metabolism and liver injury index of NASH group were significantly higher than those of NASH group.Liver pathology showed that the liver of NASH group showed obvious steatosis.（3）Compared with the normoxia NASH group,the body weight and blood glucose of the chronic hypoxia NASH group decreased,but the liver index increased significantly.There was no significant difference in serum TG,T-CHO and liver T-CHO,but ALT,AST,and liver TG increased significantly.Pathology showed that the degree of steatosis,inflammatory cell infiltration and fibrosis in the liver of chronic hypoxia NASH group also increased significantly,and the above changes increased with the prolongation of hypoxia time.Conclusion:Chronic hypoxia improves the body weight and insulin resistance of NASH mice;chronic hypoxia may not affect the fatty degeneration of liver and the levels of serum TG and T-CHO in NASH mice;chronic hypoxia aggravates the levels of serum ALT and AST in NASH mice;chronic hypoxia aggravates liver index and TG deposition in NASH mice,but not T-CHO;chronic hypoxia promotes inflammatory cell infiltration and fibrosis in the liver of NASH mice.Part Ⅱ : Molecular mechanism of chronic hypoxia promoting the NASH in mice.Objective: To investigate the potential mechanism of chronic hypoxia in the development of NASH.Methods:（1）The model of NASH（C57BL6J mice）was established by using high fat and high fructose diet water combined with CCL4,and normal diet and water were used as ND control group.The mice were divided into four groups: normoxia NASH group（n = 20）,chronic hypoxia NASH group（n = 20）,normoxia ND group（n = 20）and chronic hypoxia ND group（n = 20）.The mice in the chronic hypoxia group were raised at an altitude of 4300 m.The mice in normoxia group were raised at 36 m above sea level.In order to dynamically observe the changes of various indexes.（2）At the end of modeling at week 4 and week 8,liver tissue was collected and the lipid metabolismrelated genes（PPARα,CPT1,MTP,Apo B,FAT,FAS,ACC1,SCD,SREBP-1c）,NF-κB,inflammatory factors（TNF-α and IL-1β）and fibrosis genes（Collagen I,α-SMA,TGF-β）was detected by Western Blot or RT-qPCR.Results: In the chronic hypoxia NASH group:（1）The expression levels of the DNL pathway related genes（FAS,ACC1,SCD,SREBP-1c）of mice in NASH group were significantly up-regulated with the prolongation of hypoxia.（2）The expression levels of p-NF-κB was significantly up-regulated,and hypoxia exposure showed a temporal pattern.（3）The expression levels of inflammation-related genes and fibrosis related genes were significantly up-regulated with the prolongation.Conclusion: Chronic hypoxia mainly promotes the deposition of TG in liver by upregulating the expression of SREBP-1c-mediated DNL related gene（FAS,ACC1,SCD）.Chronic hypoxia could up-regulate the expression of inflammation-related genes TNF-α,IL-1β and p-NF-κB in NASH mice liver to promote the progression of inflammation.Chronic hypoxia activated the NF-κB pathway,and then promoted the up-regulation of fibrosis related genes Collagen I,α-SMA and TGF-β in the liver of NASH mice,thereby aggravating the progression of fibrosis.The upregulation of DNL related gene,TNF-α,IL-1β,p-NF-κB and fibrosis related gene expression interacts with hypoxia exposure.Part Ⅲ Regulation of HIF-2α in NASH.Objective: To investigate the potential mechanism of HIF-2α in NASH development.Methods :（1）Liver tissue samples from 7 NASH patients and 9 normal liver tissues were screened by pathological examination（H&E,Sirius Red）to evaluate the NAFLD activity score.Immunohistochemistry,Western Blot and RT-qPCR were performed to determine the expression level of HIFs in human and mouse NASH liver tissues（the mouse NASH modeling method was the same as the previous two parts）.（2）Lowexpression HIF-2α was constructed by lentivirus transfection HepG2 cells,empty lentivirus as control.HepG2 cells were induced by PAL to construct in vitro NASH model.HepG2 cells were treated with normoxia（21%O2）and hypoxia（5% O2）for 24 h,and lipid deposition was assessed by Oil red O.The expression levels of DNL related genes（FAS,ACC1,SCD,SREBP-1c）,NF-κB,inflammatory factors（TNF-α,IL-1β）and fibrosis genes（Collagen I,α-SMA,TGF-β）were detected by Western Blot or RTqPCR.Results:（1）NAS activity score confirmed that all 7 cases of fatty liver collected in this study were NASH.The expression of HIF-2α was significantly up-regulated in human NASH and mouse NASH livers,but not HIF-1α.（2）Chronic hypoxia could upregulated the expression of HIF-2α in NASH livers.The expression of HIF-2α in chronic hypoxic NASH group was significantly up-regulated with the prolonging of hypoxia time.（3）Inhibition of HIF-2α expression can down-regulated the SREBP-1c mediated DNL-related gene（FAS,ACC1,SCD）,inflammation related genes TNF-α and IL-1β,NF-κB and fibrosis related genes Collagen I,α-SMA and TGF-β.Conclusion: HIF-2α is upregulated in both human and mouse NASH liver tissues,and the role of HIF-2α in NASH is not determined by HIF-1α.Furthermore,HIF-2α protein expression may be related to NASH severity.Chronic hypoxia could up-regulate the transcription and protein levels of HIF-2α in mouse NASH liver tissue,suggesting that hypoxia could up-regulate HIF-2α.In vivo and vitro,hypoxia could promote the expression of SREBP-1c,mediated DNL associated genes（FAS,ACC1,SCD）,inflammation related genes TNF-α and IL-1β,NF-κB and fibrosis related genes Collagen I,α-SMA and TGF-β by up-regulating the expression of HIF-2α.Interfering with HIF-2α expression could alleviate the lipid accumulation,inflammation and fibrossi of NASH.