Effect Of Intermittent Hypoxia On Lipid Metabolism In Liver Cells And Its Underlying Mechnism

Background and ObjectiveNonalcoholic fatty liver disease (NAFLD) is a kind ofclinicopathological syndrome characterized with excessive deposition oflipids in liver cells, in addition to alcohol and other specific factors.Recently it has been reported that abnormal lipid deposition exists in thehepatocytes of patients with sleep apnea hypopnea syndrome（SAHS） andintermittent hypoxia induced by chronic sleep apnea syndrome hasrelationship with disorders of lipid metabolism in hepatocyte. However,there were few studies on intermittent hypoxia (IH) and hepatocyte lipidmetabolism. reactive oxygen species(ROS)、hypoxia inducible factors（HIFS）are important factors for the regulation of mammals’ reaction tolow oxygen, adipose differentiation-related protein （ADFP）、 sterolregulatory element binding protein-1c （SREBP-1c）、fatty acid synthase(FAS）are the key regulators for transport and synthesis of fatty acids inhepatocytes. Our study is to use three gas incubators、normal humanhepatocytes (L02) and Hepatoma cells (HepG2) for establishing intermittent hypoxic cell model, in order to explore the effect of IH on liverlipid metabolism and its relevant components，with the aim of providing anew theoretical basis for the treatment and prevention of NAFLD.Methods1.Grouping：control group (21%02), experimental group (group ofintermittent hypoxia)2.Establishment of intermittent hypoxia cell model: L02and HepG2cells were cultured with different oxygen concentrations（1%02、2%02、21%02）in the three gas incubators for8hours everyday and were treatedfor1、2、3、4、5、6days respectively. Cell morphology was observed bymicroscope, cell survival rate was detected by MTT, intracellulartriglyceride content was detected by triglyceride test kit， and lipiddeposition was observed by oil red O staining.3.The protein expressions of HIF-1α、HIF-2α、ADFP、SREBP-1c、FAS were detected by Western Bloting.4.The ROS content in hypatic cells were tested by ROS detection kits.ResultsPart1Intermittent hypoxia-induced abnormal lipid deposition inhepatocytes1. Screening of oxygen concentration: the cell survival rates of L02and HepG2cells cultured in the condition of1%02、2%02、3%02、21%02were evaluated by MTT. The cell survival rate was less than50%after6 days under the condition of1%02, cell survival rate was up to about100%after6days under the condition of2%02、3%02. Pursuant to the relevantreference[3], we chose2%02and3%02for the subsequent experimnet.2. Selection of the optimal time point：The effects of2%02、3%02onTG content in liver cells were detected by TG test kits. Result：with thetime extension，the TG content was up to the peak after exposed to2%02and3%02for five days, while the2%02group was much more prominent(p<0.05). Consequently,2%02and1、2、3、4、5days were selected as ourexperimental conditions.3. The detection of hepatocellular lipid(1) The TG contents in hypatocytes had not changed significantly inthe control group with the time extension （FL02=0.6， P=0.67>0.05;FHepG2=0.82，P=0.53>0.05）, while they gradually increased in2%02groupswith significantly differences (（FL02=61.83，P<0.01；FHepG2=104.19，P<0.01）).(2) Oil red O staining: Compared with the control group, muchmore visible orange lipids accumulations and increased integration wereobserved in L02and HepG2cells after exposed to intermittent hypoxia.Part2Possible mechanism about the effect of intermittent hypoxia onhepatic lipid metabolism.1. The expressions of interest proteins in L02and HepG2cells：afterexposed to2%02for8h/d and lasted for1、2、3、4、5days respectively， compared to the control group, the expressions of HIF-1α、HIF-2αweremore in the experimental group1st day（p＜0.05）. With the extension oftime, the expression of HIF-1αgradually increased（FL02=371.19, FHepG2=150.84, P<0.05）,while HIF-2αdecreased. After4days exposed to IH,HIF-2αwas less than the control group（tL02=3.18, tHepG2=2.54，P<0.05），the difference is significantly.2.The ROS content in L02and HepG2： ROS content of theexperimental group increased than the control group (tL02=22.06, tHepG2=7.2,P<0.05)，and time-dependent boost (FL02=1021.84, FHepG2=49.89, P<0.01)，the difference is significantly.3.The expression of SREBP-1c、FAS、ADFP factors in liver cells：ADFP, SREBP-1c and FAS interest proteins，expression in the experimentgroup increase compared with the control group respectively，（L02:tSREBP-1c=5.83, tFAS=9.84, tADFP=19.99, P<0.05; HepG2:tSREBP-1c=8.4,tFAS=9.18, tADFP=5.46, P<0.05）, also time-dependent boost, with timepassing(FL02,SREBP-1c=204.49, FL02,FAS=38.2, FL02,ADFP=154.31, P<0.05;FHepG2,SREBP-1c=107.35, FHepG2,FAS=279.71, FHepG2,ADFP.=352.06, P<0.05), thedifference is significantly.Conclusion1. Intermittent hypoxia can induce abnormal lipid deposition in L02and HepG2cells2. ROS induced by IH may influence HIF-1α-SREBP-1c-FAS and ADFP pathway via regulating the expressions of hypoxia inducible factorsand adipose differentiation-related protein, which leads to the abnormalhepatic lipid metabolism，HIF-2may be involved in intermittent hypoxiainduced-hepatocyte fatty degeneration at the early stage.3. ROS and HIFs might be the new targets for the studies ofnon-alcoholic fatty liver disease.