Effects Of COX-2on Triglyceride Metabolism And The Expression Of Genes PPAR-γ And CPT-1in Steatosis BRL-3A Cell

PurposeTo study Effects of COX-2over-expression or silencing on steatosis BRL-3Aand the mRNA expression of lipid metabolism-related genes PPAR-γ and CPT-1.Methods1. Selecting the optimal concentration of medical fat emulsion prepare the Modelof the hepatocyte steatosis:different concentrations of fat emulsion(1%,2%,4%,6%,8%,10%) foster BRL-3A,after24h,confirm the optimal concentrationof fat emulsion by oil red and hematoxylin stain detect the cell morphology and theformation of intracellular lipid droplets as well as ELISA detect the intracellular TGcontent and MTT detect cell proliferative activity.2. This experiment is divided into seven groups: normal BRL-3A group steatosisBRL-3A control group,pYr-1.1-hU6-EGFP-COX-2shRNA group,pYr-1.1-hU6-EGFP-HK group,pcDNA3.1(+)-rCOX-2group,pcDNA3.1(+)-HK group,nimesulide200ug/ml positive control group.Except the normal BRL-3A group,6%fat emulsionfoster other groups24h,The corresponding plasmid respectively transfected intosteatosis BRL-3A by lipofectamineTM2000.After24h,ELISA detect the intracellularTG content,understanding silence or overexpress COX-2effect on the TG levels insteatosis BRL-3A;RT-PCR detect the expression of COX-2and lipid metabolism-related genes PPAR-γ and CPT-1mRNA.Results1.With the fat emulsion concentration increasing,observed by oil red andhematoxylin stain,the red vesicles lipid droplets is increasing in BRL-3A.Theintracellular triglycerideis content is increasing by ELISA detect.OD values isdecreasing by MTT detect.After6%fat emulsion solution inducted,the intracellularTG content is significantly more than normal hepatocyte blank control group,the cell survival rate is more than90%.6%is determined the optimal concentration whichinduce steatosis in this experiment.2. After transfected,ELISA detect the intracellular TG content,RT-PCR detectedCOX-2and PPAR-γ as well as CPT-1mRNA expression in seven groups:(1)Compared with normal BRL-3A group,the intracellular TG content increasesignificantly,COX-2mRNA expression increase,PPAR-γ and CPT-1mRNAexpression decrease in steatosis hepatocyte blank control group;(2)Compared with steatosis hepatocyte blank control group,the intracellular TGcontent decrease,COX-2mRNA expression decrease,PPAR-γ and CPT-1mRNAexpression increase in pYr-1.1-hU6-EGFP-COX-2shRNA group and nimesulide200ug/ml positive control group.the former is more obvious;(3)Compared with steatosis hepatocyte blank control group,the intracellular TGcontent increase,COX-2mRNA expression increase,PPAR-γ and CPT-1mRNAexpression decrease in pcDNA3.1(+)-rCOX-2group;(4)Compared with steatosis BRL-3A control group,pYr-1.1-hU6-EGFP-HKgroup and pcDNA3.1(+)-HK group are not statistically significant.Conclusions1.6%fat emulsion induce BRL-3A to successfully establish model of hepaticsteatosis in vitro.2. After BRL-3A steatosis,intracellular TG content increase,COX-2expressionincrease,lipid metabolism-related genes PPAR-γ and CPT-1expression decrease.3. COX-2silence or inhibition,COX-2expression decrease,intracellular TGcontent decrease,PPAR-γ and CPT-1expression increase.4. COX-2overexpression,COX-2expression increase,intracellular TG contentincrease,PPAR-γ and CPT-1expression decrease.