体外脂肪变L02细胞Notch家族和脂质代谢变化研究

Notch family and lipid metabolism changes in LO2 cells with adipogenesis in vitro

目的探讨脂肪变肝细胞Notch基因水平变化及其对脂质代谢的影响。方法使用棕榈酸(PA)处理人L02细胞,构建体外脂肪变性细胞模型,采用不同浓度(0 mM、0.1 mM、0.25 mM和0.5mM)PA处理,并在0.25mM PA处理细胞,给予不同浓度(0μM、1μM、2μM、5μM和10μM)的γ分泌酶抑制剂(DAPT)干预L02细胞24 h,分别以0mM PA为对照组和0μM DAPT为模型组,采用实时定量PCR法检测Notch家族中Notch1、-2、-3及其下游Hes-1和Hey-1mRNA水平,采用比色法测定细胞谷草转氨酶(AST)、谷丙转氨酶(ALT)、甘油三酯(TG)、胆固醇(TC)、游离脂肪酸(FFA)水平。结果在0.25 mM和0.5 mM PA处理组,细胞Notch3 mRNA水平分别为(0.6±0.2)和(0.4±0.1),显著低于对照组【(1.0±0.1),P<0.01】,Hes-1 mRNA水平分别为(1.7±0.2)和(1.5±0.1),显著高于对照组【(1.0±0.1),P<0.01】;培养上清AST水平分别为(6.7±2.6)U/L和(12.4±1.6)U/L,显著高于对照组【(2.4±0.1)U/L,P<0.05】,ALT水平分别为(5.7±0.8)U/L和(10.3±0.7)U/L,显著高于对照组【(2.2±0.3)U/L,P<0.01】;细胞TG水平分别为(0.4±0.04)mmol/g和(0.4±0.04)mmol/g,显著高于对照组【(0.2±0.02)mmol/g,P<0.01】;在0.5mM PA处理组,细胞Notch1 mRNA、TC和FFA水平分别为(1.9±0.1)、(1.2±0.4)mmol/g和(0.07±0.004)mmol/g,显著高于对照组【分别为(1.0±0.1)、(0.2±0.02)mmol/g和(0.02±0.001)mmol/g,P<0.01】;采用不同浓度DAPT干预0.25mM PA处理细胞24 h,其中在2μM DAPT处理组,细胞Hes-1 mRNA、Hey-1 mRNA、培养上清AST和细胞TG水平分别为(0.5±0.1)、(0.6±0.3)、(6.4±2.9)U/L和(0.03±0.01)mmol/g,在5μM DAPT处理组分别为(0.4±0.2)、(0.6±0.1)、(7.3±1.3)U/L和(0.04±0.01)mmol/g,在10μM DAPT处理组分别为(0.3±0.2)、(0.4±0.1)、(4.9±0.7)U/L和(0.02±0.01)mmol/g,均显著低于模型组【分别为(1.0±0.1)、(1.0±0.04)、(12.2±0.3)U/L和(0.07±0.01)mmol/g,P<0.01】;在10μM DAPT干预细胞,TC水平为(0.2±0.03)mmol/g,显著高于模型组【(0.1±0.02)mmol/g,P<0.01】。结论通过抑制Notch信号能有效降低体外培养的脂肪变细胞TG和培养上清AST水平,提示Notch信号通路可能参与了肝细胞脂质代谢过程,值得进一步的研究。

Objective The aim of this experiment was to investigate Notch family and lipid metabolism changes in LO2 cells with adipogenesis in vitro.Methods Palmitic acid(PA)was used to intervene human hepatocytes(L02 cells)to construct an in vitro steatosis cell model.The L02 cells were cultured with different concentrations(0 mM,0.1 mM,0.25 mM and 0.5 mM)of PA and different concentrations 0μM,1μM,2μM,5μM and 10μM)ofγsecretase inhibitors N-[N-(3,5-difluorophenacetyl)-L-alnyl]-S-phenylycine t-butyl ester(DAPT)in 0.25mM PA-intervened cells for 24 h.The cells at 0 mM PA culture was used as the control group and those with 0μM DAPT culture was defined as the model group.The Notch family(Notch 1,2 and 3)and its downstream genes(Hes-1 and Hey-1)mRNA levels were detected by real-time quantitative PCR.The aspartate aminotransferase(AST)and alanine transaminase(ALT)levels in supernatants,the cell triglyceride(TG),cholesterol(TC)and free fatty acid(FFA)levels were measured by colorimetric method.Results In 0.25 mM and 0.5 mM PA-intervened cells,the Notch3 mRNA levels were(0.6±0.2)and(0.4±0.1),respectively,both significantly lower than[(1.0±0.1),P<0.01]in the controlled cells,and the Hes-1 mRNA levels were(1.7±0.2)and(1.5±0.1),significantly higher than[(1.0±0.1),P<0.01]in the control;the AST levels in the supernatants were(6.7±2.6)U/L and(12.4±1.6)U/L,significantly higher than[(2.4±0.1)U/L,P<0.05]in the control,and the ALT levels were(5.7±0.8)U/L and(10.3±0.7)U/L,significantly higher than[(2.2±0.3)U/L,P<0.01]in the control;the cell TG levels were(0.4±0.04)mmol/g and(0.4±0.04)mmol/g,significantly higher than[(0.2±0.02)mmol/g,P<0.01]in the control;the Notch1 mRNA,TC and FFA levels in cells at 0.5mM PA intervention were(1.9±0.1),(1.2±0.4)mmol/g and(0.07±0.004)mmol/g,significantly higher than[(1.0±0.1),(0.2±0.02)mmol/g and(0.02±0.001)mmol/g,respectively,P<0.01]in the control;after 24 hour treatment with 2μM DAPT,the Hes-1 mRNA,Hey-1 mRNA,AST and TG levels were(0.5±0.1),(0.6±0.3),(6.4±2.9)U/L and(0.03±0.01)mmol/g,with 5μM DAPT intervention were(0.4±0.2),(0.6±0.1),(7.3±1.3)U/L and(0.04±0.01)mmol/g,and with 10μM DAPT culture were(0.3±0.2),(0.4±0.1),(4.9±0.7)U/L and(0.02±0.01)mmol/g,all significantly lower than[(1.0±0.1),(1.0±0.04),(12.2±0.3)U/L and(0.07±0.01)mmol/g,P<0.01]in model cells;the cell TC level was(0.2±0.03)mmol/g in 10μM DAPT intervention,significantly higher than[(0.1±0.02)mmol/g,P<0.01]in the model.Conclusion Inhibition of Notch signaling could effectively improve cell injures in adipogenic cells,suggesting that the Notch signaling pathway might be closely related to lipid metabolism in hepatocytes intro,which is worthy of further study.