The Effect And Potential Mechanism Of Oxygen Therapy On Improvement Of Hepatic Steatosis

Aims:Nonalcoholic fatty liver disease（NAFLD）,one of the most important chronic liver disease,is manifested by hepatic steatosis and lack of secondary causes of hepatic fat accumulation such as significant alcohol consumption.There is currently no effective treatment for NAFLD.Hypoxia is an important pathogenic factor in the development and progression of NAFLD and hypoxia-inducible factor 2α（HIF-2α）mediates this effect.This study investigated the effect of oxygen therapy（OT）on hepatic hypoxia and hepatic steatosis and related mechanisms.Methods:C57BL/6J mice were divided into control（CON）group,high-fat diet（HFD）group and oxygen therapy（OT）group.The HFD group fed 60%HFD for 12 weeks,and the OT group was given HFD combined OT for 8 weeks after given HFD for 4 weeks of high-fat diet.The oxygen therapy was twice daily with 1 atmosphere（atm）88%O₂ for1 hour.Glucose tolerance test and insulin tolerance test were used to assess glucose tolerance and insulin resistance,oxygen concentration in the inferior vena cava near the liver outlet was detected by oxygen electrode,serum lipids and liver triglycerides（TG）levels,liver H&E and Oil red O staining were determined,and genes involved in lipid metabolism were detected by real-time polymerase chain reaction（RT PCR）.In vitro,primary hepatocytes were intervened with palmitic acid（PA）,and there were a control group（conventional culture）,PA group（0.25 m M PA,24 h）,and oxygen supply group(PA 0.25 m M,24 h,and placed in 100%O2+1 atm oxygen chamber at last one hour.Hepatocytes were stained with Oil red O and TG was measured,genes and protein levels of glucose and lipid metabolism were also detected.Hep G2 cells were divided into CON group,PA group（0.5m M PA 24h）perfluorocarbon（PFC）group（PA 24 h,and added oxygen perfluorocarbon nanoparticle particles at last 0.5,1,1.5,or 2 h supply environment）,and then Oil red O staining and TG were determined.The liver function and oxidative stress levels of mice,as well as the proliferation ability and oxidative stress of primary hepatocytes were detected.In the second part of the present study,the expression of hepatic HIF-1αand HIF-2αwere detected by RT PCR and western blot;liver biopsy samples from three groups of patients with non-NAFLD,simple fatty liver and steatohepatitis were subjected to immunohistochemical staining and RT PCR to detect the expression of HIF-1αand HIF-2α;the gene expression of hepatic HIF-2αon HFD at different times（HFD8W,HFD12W）were determined by RT PCR;Lentivirus（LV）was used to overexpress HIF-2α（LV-HIF-2α）in hepatocytes,and Oil red O staining,TG levels and the expression of lipid synthesis genes were determined after PA intervention and OT therapy.Hepatocytes were divided into given or no-given OT group after cycloheximide（CHX）intervention for 0,15,30,45 and 60 minutes to detect the protein synthesis of HIF-2α,and were divided into given or no-given OT group after MG132 intervention to detect the protein degradation of HIF-2α.In the third section,carboxylesterase 1f（Ces1f）,downstream signaling molecules of HIF-2αand OT therapy,were screened by whole-genome sequencing.Hepatic ces1f levels of mice treated were detected.Gene expression of HIF-2αand Ces1f were detected after primary hepatocytes infected by HIF-2αsmall interfering RNA（si RNA）or LV-HIF-2α;Hepatocellular lipid deposition were determined by Oil red O staining after infection with si RNA Ces1f;TG and glucose output levels of primary hepatocytes were detected after si RNA HIF-2αor si RNA Ces1f;TG levels of hepatocytes were detected after intervenion with si RNA HIF-2αand then LV-Ces1f.Results:The first section showed that OT improved oxygen flow in the liver,and decreased the body weight and liver/body weight ratios in of HFD mice（P<0.05）.Hepatic steatosis due to the HFD was improved by OT,as evidenced by decreased hepatic TG levels and reduced lipid deposition based on H&E-and Oil Red O-stained hepatic tissue sections（P<0.05）,which was accompanied by the alleviation of dyslipidaemia（P<0.05）.Furthermore,OT significantly improved hepatocellular steatosis and reduced intracellular TG levels in PA-treated primary hepatocytes and Hep G2 cells and was accompanied by improved genes and proteins levels of glucose（P<0.05）.OT improved cell proliferation ability,oxidative stress indicators,reduced the obvious swelling of mitochondria and mitochondrial cleavage in PA-treated primary hepatocytes and was not associated with liver damage（P<0.05）,OT downregulated genes that are associated with hepatic de novo lipogenesis and lipid uptake in HFD mice and PA-treated hepatocytes（P<0.05）.The second section showed that OT significantly reduced the hepatic gene and protein expression of HIF-2αdue to HFD and PA in vivo and in vitro（P<0.05）.The hepatic gene and protein expression of HIF-2αwas associated with the progression of NAFLD.The gene expression of hepatic HIF-2αincreased with the prolongation of HFD（P<0.05）.In vitro,the improvement in hepatic steatosis mediated by OT and suppressive effect of OT on genes associated with hepatic de novo lipogenesis in primary hepatocytes treated with PA was diminished by the overexpression of HIF-2α.OT was confirmed to decrease HIF-2αlevels by reducing the synthesis of HIF-2αin CHX test and promoting the ubiquitination/degradation of HIF-2αin primary hepatocytes in MG132 test.The third part of the present study showed that the heptocellar gene expression of Ces1f changed synchronously when the primary hepatocytes were infected with LV-HIF-2αor si RNA HIF-2α.The hepatic genes of Ces1f and HIF-2αincreased synchronously with the prolongation of HFD and decreased by OT（P<0.05）.si RNA Ces1f improves hepatocellular lipid accumulation;si RNA HIF-2αincreased glucose output and genes expression related to gluconeogenesis,and si RNA Ces1f decreased glucose output and genes expression related to gluconeogenesis（P<0.05）.When primary hepatocytes infected with si RNA HIF-2αand LV-Ces1f,the improvement of hepatocellular lipid accumulation by inhibition of HIF-2αwas weakened（P<0.05）.Genes expression associated with hepatocellular de novo lipogenesis in PA treated hepatocytes were downregulated by si RNA Ces1f or si RNA HIF-2α.,LV-Ces1f weakened inhibition of si RNA HIF-2αon gene expression of hepatocellular de novo lipogenesis in PA treated hepatocytes.Conclusion:Oxygen therapy improves hepatic steatosis through inhibition of HIF-2α/Ces1f/hepatic de novo lipogenesis pathway.Oxygen therapy might be a new potential treatment for NAFLD.