Notch inhibitor DAPT ameliorates steatosis of L02 cells in vitro
Objective The purpose of this experiment was to investigate the effects of Notch inhibitor(DAPT)on cellular inflammatory factors and lipid related genes in L02 cells in vitro.Methods The steatosis of L02 cells was established by palmitic acid(PA)incubation in vitro,and also intervened by DAPT at 0μM,1μM,2μM,5μM and 10 μM concentration.The cell survival rate was detected by CCK-8,the cell lipid droplets was observed by nile red staining,and the cellular TNF-α,IL-1 β and IL-6 mRNA and fat related factors[sterol regulatory element-binding protein 1c(SREBP1c),FASN and ACACA]mRNA loads were detected by RT-qPCR.The cell p65 and SREBP1c expression was detected by Western blot.Results The cell survival rates of L02 cell without PA intervention at 1μM,2μM,5μM and 10 μM DAPT incubation for 24 h were all decreased compared to in the control(85.2±5.3%,84.6±2.9%,84.4±6.0%and 84.5±3.2%vs.100.0%,respectively,P<0.05);in2 μMand5μM DAPT-intervened cells,the TNF-α mRNA levels were(0.6±0.01)and(0.5±0.09),significantly lower than in the control[(1.0±0.0),P<0.01],the IL-1 β mRNA levels were(0.7±0.2)and(0.4±0.0),significantly lower than[(1.1±0.1),P<0.01]in the control,and the IL-6 mRNA levels were(0.8±0.1)and(0.6±0.1),significantly lower than[(1.0±0.0),P<0.05]in the control group;the p65 expression showed also remarkably decreased(P<0.05);the lipid droplets in 2 μM,5 μM and10 μM DAPT-intervened cells were significantly weaker than in the control group[(0.2±0.1),(0.3±0.0),(0.1±0.0)vs.(0.73±0.0),P<0.001],the FASN mRNA loads in2 μM and 5 μM DAPT-intervened cells were(0.7±0.0)and(0.4±0.1),much lower than[(1.0±0.0),P<0.001]in the control group,and the ACACA mRNA load in 2 μM and 5 μM DAPT-intervened cells were(0.6±0.1)and(0.3±0.0),much lower than[(1.0±0.0),P<0.001]in the control group;the expression of SREBP1c protein at 5 μM and 10 μM DAPT-intervened cells was significantly weaker than in the control group(P<0.01).Conclusion The DAPT could effectively inhibit the expression of inflammatory factors and ameliorate the formation of intracellular lipid droplets in L02 cells with PA-induced steatosis in vitro,hinting the mechanism might be related to the regulation of fat-related factors.Our findings suggest that the inhibition of Notch signal pathway might have a potential to alleviate the occurrence and progression of cell steatosis.
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