| Objective: To observe the effects of palmitic acid on ZAG and FXR gene expression in Hep G2 and Hepa1-6 cells and investigate the effects of overexpression of ZAG on deposition of triglyceride in Hep G2 cells induced by Palmitic acid.Methods: 1. Cultivation of Hep G2 and Hepa1-6 cells in vitro, then treated in serum-free conditions for 24 hours with PA at concentrations of 0, 0.2, 0.4 and 0.6m M. Cells were collected after 24 hours to detect protein level of ZAG and FXR gene by Western blot. 2. Hep G2 cells were cultured in vitro. Plasmid vector p IRES2-Zs Green1-h ZAG and empty vector were transfected into Hep G2 cells for 48 hours by Lipofectamine 2000-mediated gene transaction, then the cells were treated in serum-free conditions for 24 hours with PA at concentrations of 0.4m M. Intracellular lipid accumulation was examined using Oil Red O staining. Intracellular triglyceride was detected using the triglyceride measurement kit. The protein level of genes involved in metabolic nuclear receptors(FXR, PPARα, LXR and SREBP-1c) and fatty acids synthesis(FAS, ACC, SCD-1), intake(FATP4), β-oxidation(CPT1A) and adiponectin were detected by Western blot.Results: 1.The protein level of ZAG gradually reduced in a dose-dependent manner of PA(P<0.01). The protein level of ZAG was the lowest when cells were treated with PA at 0.6m M. The protein level of FXR was the lowest in Hep G2 cells when treated with PA at 0.4m M(P<0.01). In Hepa1-6 cells when treated with PA at 0.6m M, however, the protein level of FXR was the lowest(P<0.01). 2. Intracellular triglyceride level was significantly decreased and little lipid droplet was found in p IRES2-Zs Green1–h ZAG transfection group compared with the control group after treated with PA. 3. Compared with p IRES2-GFP group, the protein level of ZAG, Adiponectin, FXR, PPARα and CPT1 A were increased in p IRES2- h ZAG group(P<0.05). Moreover, the protein level of SREBP-1c, LXR, FAS, ACC and SCD-1 were significantly decreased(P<0.01), however, the protein level of FATP were not decreased significantly(P>0.05). In p IRES2-GFP+PA group, the protein level of ZAG, Adiponectin, FXR and PPARα were significantly decreased(P<0.01). However, the protein level of SREBP-1c, LXR, FAS, ACC, SCD-1 and CPT1 A were significantly increased(P<0.01). Compared with p IRES2-GFP+PA group, the protein level of ZAG, Adiponectin, FXR, PPARα and CPT1 A were increased in p IRES2-h ZAG+PA group(P<0.05). Moreover, the protein level of SREBP-1c, LXR, FAS, ACC, SCD-1 and FATP were significantly decreased(P<0.01).Conclusion: 1. Overexpression of ZAG could reduce the deposition of TG induced by palmitic acid in Hep G2 cells. 2. Over-expression of ZAG could reduce the deposition of TG in Hep G2 cells by up-regulated the expression of FXR, PPARα and CPT1 A, down-regulated the expression of LXRα, SREBP-1c, FAS, SCD-1, ACC and FATP.Objective: To construct a lentiviral vector carrying i RNA to silence ZAG, and observe its effects on deposition of triglyceride in Hep G2 cells induced by Palmitic acid.Methods: 1. Hep G2 cells were infected respectively with LV-AZGP1-RNAi and three kinds of LV-AZGP1-RNAi at MOI = 10. The LV-AZPG1-RNAi with best silencing effect was identified by RT-q PCR. 2. The LV-AZPG1-RNAi with best silencing effect was used to infect Hep G2 cells for 72 hours. Then the cells were treated with PA at concentrations of 0.4 m M for 24 hours. Intracellular lipid accumulation was examined using Oil Red O staining. Intracellular triglyceride was detected using the triglyceride measurement kit. The protein level of genes involved in metabolic nuclear receptors(FXR, PPARα, LXR and SREBP1c) and fatty acids synthesis(FAS, ACC, SCD-1), intake(FATP4), β-oxidation(CPT1A) and adiponectin were detected by Western blot.Results: 1. Compared to empty virus vector, RT-q PCR identified that three kinds of LV-AZGP1-RNAi reduced the expression of ZAG more than 70%(P<0.01), whereby, the second LV-AZGP1-RNAi have the best silence effect. 2. Intracellular triglyceride level was significantly increased and more lipid droplet was found in LV-AZPG1-RNAi infection group compared with the control group after treated with PA. 3. Compared with LV-GFP-RNAi group, the protein level of ZAG, Adiponectin, FXR, PPARα and CPT1 A were decreased in LV-AZGP1-RNAi group(P<0.05). Moreover, the protein level of SREBP-1c, LXR, FAS, ACC and SCD-1 were significantly increased(P<0.01), however, the protein level of FATP were not changed significantly(P>0.05). In LV-GFP-RNAi + PA group, the protein level of ZAG, Adiponectin, FXR and PPARα were significantly decreased(P<0.01). However, the protein level of SREBP-1c, LXR, FAS, ACC, SCD-1 and CPT1 A were significantly increased(P<0.01). Compared with LV-GFP-RNAi + PA group, the protein level of ZAG, Adiponectin, FXR, PPARα and CPT1 A were decreased in LV-AZGP1-RNAi + PA group(P<0.01). Moreover, the protein level of SREBP-1c, LXR, FAS, ACC, SCD-1 and FATP were significantly increased(P<0.01).Conclusion: 1. Silencing expression of ZAG could increase the deposition of TG induced by palmitic acid in Hep G2 cells.2. Silencing expression of ZAG could increase the deposition of TG in Hep G2 cells by down-regulated the expression of FXR, PPARα and CPT1 A, up-regulated the expression of LXRα, SREBP-1c, FAS, SCD-1, ACC and FATP.
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