Influence Of Hydrogen Sulfide On The Lipid Synthesis And Decomposition Of Mouse Primary Hepatocytes

Background: Non-alcoholic fatty liver disease(NAFLD) is one of a common disease included in metabolic syndrome which has become an important cause of chronic liver disease in developing and developed countries.There is no effective prevention and therapeutic drugs for this disease. The increase of lipid synthesis and deposition in the liver caused by insulin resistance and other factors play an important role in the pathogenesis of NAFLD. Hydrogen sulfide(H2S) is a gas molecules neurotransmitter which can be synthesised in the human and mammalian cells. H2 S can regulate a variety of physiological and pathophysiological processes. Some studies show that endogenous or exogenous H2 S supplement for the body can affect the liver lipid metabolism in normal physiological or pathophysiological conditions. The mechanisms by which H2 S shapes lipoprotein profile and controls liver lipid metabolism, however, remain unclear. It has been reported that in rat adipocytes, inhibiting cystathionine γ lyase(CSE) / H2 S system can through PKA-fat coat protein / hormone sensitive lipase(HSL) ways to enhance the role of lipolysis, increase lipid levels, so that the lipid load of liver cells increases and the risk of NAFLD risk increased. H2 S donor will be given to reduce fat cells lipolysis, so it is possible to reduce the risk of NAFLD. Thus, we speculate that the exogenous H2 S could affect the synthesis and catabolism of fat in liver cells. Fat accumulation in liver cells can Inhibite autophagy.Increase H2 S gas molecules can activate autophagy, but whether by changing the level of autophagy in liver cells to regulate fat metabolism is unclear.Objective: Exploring the influence of H2 S on synthesis and catabolism of fat and autophagy in hepatocyte, to provide a theoretical basis for the further understanding of the role of H2 S in NAFLD and development of H2 S donor treatment.Methods: Using two-step in situ perfusion way to isolation and culture C57BL/6 mouse primary hepatocytes. Oleic acid(oleic acid, OA) was used to induce hepatic steatosis models in vitro.To study of hydrogen sulfide on lipid synthesis of hepatocyte, cells were divided into four groups: control group incubated with normal medium; model group incubated with 1.2mmol/L of OA for 48h; H2 S group incubated with 1mmol/L GYY4137 plus 1.2 mmol/L of OA for 48h; PAG-treated group incubated with 200 μmol/L of PAG that can inhibit CSE’s activity plus 1.2 mmol/L of OA for 48 h. Oil red staining were used to detect intracellular lipid droplets changes, colorimetry was used to detect intracellular triglyceride(TG) content. Lipolysis experiments can be divided into four groups: the control group incubated with normal medium; model group incubated with 1.2 mmol/L of OA for 48h; hydrogen sulfide group incubated with 1.2 mmol/L of OA for 48 h followed by serum-free phenol red-free RPMI 1640 which contain 1mmol/L GYY4137 for 6h; PAG treatment group incubated with 1.2 mmol/L of OA for 48 hours followed by serum-free phenol red-free RPMI 1640 that contain 200 μmol/L of PAG for 6h. Culture medium was collected and colorimetry was used to detect glycerol content; cells were collected and Western blotting was used to detect the expression of HSL and p-HSL and autophagy-related genes LC3 A / B protein.Results: 1. when cells were incubated with induced fluid that containing 1.2 mmol/L of OA for 48 h, oil red staining showed that hepatocyte volume increases, the outline was vague and there were a large number of cytoplasmic lipid droplets which indicated that the model was successfully established.2. In observing fat synthesis experiments, compared with the control group, the triglyceride content in model group was significantly higher(P <0.05); compared with the model group, the TG content in hydrogen sulfide group and PAG treatment group showed no significant change.3. In observing lipolysis experiments, as compared with the control group, the glycerol release in model group was significantly increased(P <0.05), compared with the model group, the glycerol release in hydrogen sulfide group was significantly reduce(P<0.05).Western blotting results showed that: Compared with the control group, the HSL phosphorylation levels in model group were significantly increased(P<0.05); compared with the model group, the HSL phosphorylation levels in hydrogen sulfide group were significantly lower(P <0.05).4. LC3 immunofluorescence, western blotting results showed that hydrogen sulfide could promote the LC3 particles and protein fluorescence of steatosis hepatocytes increased, phase contrast microscopy showed an increase in intracellular vacuoles, electron microscopy showed increased number of autophagic lysosome. These results suggested that hydrogen sulfide could promote the lipolysis of steatosis in primary hepatocytes through enhance its autophagy levels.Conclusions:1. Exogenous H2 S donor GYY4137 can reduce the cytoplasm lipolysis of hepatocyte by reducing the phosphorylation level of HSL.2. Hydrogen sulfide can increase autophagy levels of steatosis hepatocyte, so as to accelerate the autophagy lipolysis.