| Objective:To investigate the effect of 1,25(OH)2D3 on free fatty acid(FFA)-induced nonalcoholic steatohepatitis(NASH)and its mechanism of improving lipid peroxidation and inflammation through cytochrome P450(CYP450)pathway in vitro.Methods:The first part:Human normal liver cell line HL-7702(LO2)cells were selected and treated with FFA of 500 μmol/L(according to palmitic acid:oleic acid:1:2).Meanwhile,different concentrations of 1,25(OH)2D3 were added into the medium,which were divided into:Normal group,solvent control group,FFA group,FFA+solvent control group,FFA+VD3 low-dose group(15ng/mL VD3,VD3-1 group),FFA+VD3 medium-dose group(30ng/mL VD3,VD3-2 group),FFA+VD3 high-dose group(60ng/mL VD3,VD3-3 group),cultured for 72h.1.Cell viability was determined by Trypan blue staining.2.Lipid deposition in cells was observed by oil-red O staining,and the optical density(OD)of cells in each group was determined by adding isopropyl alcohol to dissolve the dye.3.Cell supernatant was collected and enzyme-linked immunosorbent assay(ELISA)was used to detect thiobarbituric acid reactive substance(TBARS),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),monocyte chemotactic protein 1(MCP-1),interleukin-10(IL-10)and interleukin-4(IL-4);4.The dose effect curve of 1,25(OH)2D3 was made and statistically analyzed.The second part:Plasmid was introduced into cultured LO2 cells by Lipo3000,and FFA of 500 μmol/L was adopted(according to palmitic acid:oleic acid was 1:2)NASH cell model was established after 72h treatment,or siRNA gene EPHX2 of sEH,a key enzyme of CYP450 pathway,was constructed by adding 1,25(OH)2D3(60 ng/mL)simultaneously.FFA group,FFA+ empty plasmid group,FFA+empty plasmid+VD3 solvent group,FFA+empty plasmid+VD3 group,FFA+si-RNA group,FFA+si-RNA+VD3 group were established.Cell supernatant was collected and the levels of lipid peroxidation markers(TBARS)and inflammatory markers(IL-1β,TNF-α,MCP-1,IL-10 and IL-4)were determined by ELISA.Results:The first part:Compared with normal group,the cell viability of FFA group was significantly decreased(P<0.001),the OD value of intracellular lipid droplets was significantly increased(P<0.01),and lipid peroxidation index TBARS was significantly increased(P<0.001).The pro-inflammatory cytokines IL-1β(P<0.001),TNF-α(P<0.001)and MCP-1(P<0.001)were significantly increased,while the anti-inflammatory cytokines IL-4(P<0.001)and IL-10(P<0.001)were significantly decreased.Compared with FFA+VD3 solvent control group,cell viability and intracellular lipid drop OD values in FFA+VD3 group had no significant changes(P>0.05),lipid peroxidation index TBARS decreased significantly(P<0.05).The pro-inflammatory factors IL-1β(P<0.001),TNF-α(P<0.05)and MCP-1(P<0.05)were significantly decreased,while the anti-inflammatory factors IL-4(P<0.05)and IL-10(P<0.01)were significantly increased.Compared with FFA+VD3 solvent control group,the cell viability of FFA+VD3 group was significantly decreased(P<0.01),the OD value of intracellular lipid droplets had no significant change(P>0.05),and the lipid peroxidation index TBARS was significantly decreased(P<0.001).The pro-inflammatory cytokines IL-1β(P<0.001),TNF-α(P<0.001)and MCP-1(P<0.001)were significantly decreased,while the anti-inflammatory cytokines IL-4(P<0.001)and IL-10(P<0.001)were significantly increased.Compared with the FFA+VD3 solvent control group,the cell viability of FFA+VD3 group was significantly decreased(P<0.001),the OD value of intracellular lipid droplets had no significant change(P>0.05),and the lipid peroxidation index TBARS was significantly decreased(P<0.001).The pro-inflammatory cytokines IL-1β(P<0.001),TNF-α(P<0.001)and MCP-1(P<0.001)were significantly decreased,while the anti-inflammatory cytokines IL-4(P<0.001)and IL-10(P<0.001)were significantly increased.Dose-response curve analysis indicated that 1,25(OH)2D3 had anti-lipid peroxidation and anti-inflammatory effects on NASH cells,and there was a significant dose-response relationship.The second part:Compared with FFA group:Lipid peroxidation index TBARS(P<0.001),pro-inflammatory factors IL-1β(P<0.001),TNF-α(P<0.001)and MCP-1(P<0.001)in FFA+ si-EPHX2 group were significantly decreased.Anti-inflammatory cytokines IL-4(P<0.001)and IL-10(P<0.001)were significantly increased.Compared with FFA+empty plasmid+VD3 solvent group,Lipid peroxide index TBARS in FFA +empty plasmid+VD3 group were significantly decreased(P<0.01).pro-inflammatory factors IL-1β(P<0.001).TNF-α(P<0.05)and MCP-1(P<0.05).Anti-inflammatory factors IL-4(P<0.001)and IL-10(P<0.05)were significantly increased.Compared with FFA+si-EPHX2 group,Lipid peroxidation index TBARS(P<0.01),pro-inflammatory factors IL-1β(P<0.001).TNF-α(P<0.01)and MCP-1(P<0.01)in FFA+ si-EPHX2+VD3 group were significantly decreased.Anti-inflammatory factors IL-4(P<0.01)and IL-10(P<0.05)were significantly increased.Conclusion:1,25(OH)2D3 has anti-lipid peroxidation and anti-inflammatory effects on FFA-induced NASH cells through CYP450 pathway. |
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