The Experimental Study Of Inhibition Effect Of PPARγ Agonist Rosiglitazon On HIV-1Tat-induced Inflammatory Gene Expression

PART ⅠThe preliminary study of hCMEC/D3cell culture, growthcharacteristics and the impact of HIV-1Tat on the cell viability【Objective】To set up a suitable culture system of hCMEC/D3(humancerebral microvascular endothelial cells), clarify the cell growth characteristicsand explore the impact of HIV-1Tat(HIV-1transactivator of transcription) onthe hCMEC/D3cell viability for subsequent researches.【Method】hCMEC/D3cells were cultured in EBM-2medium withsupplement,inverted phase contrast microscope was applied for cell morphologyobservation and then the growth curve of hCMEC/D3was plotted. MTT wasused to measure hCMEC/D3activity after treatment with HIV-1Tat.【Results】hCMEC/D3were active and stable with continuously passagingand the growth characteristics were clarified. The cell viability of hCMEC/D3treated with indicated concentrations of HIV-1Tat for24h (250ng/ml,500ng/ml,1000ng/ml) were97.60%,91.57%and87.15%, respectively, the cell viability of hCMEC/D3treated with1000ng/ml HIV-1Tat for various times(6h,12h,18hand24h) were96.86%,92.31%,88.06%and86.79%, respectively.【Conclusion】We successfully set up a suitable culture system ofhCMEC/D3which grew in a good manner. MTT results indicated that theactivation of hCMEC/D3was not affected by treatment with HIV-1Tat at≦1000ng/ml (time at≦24h). PART Ⅱ The effect of PPARγagonist on HIV-1Tat-induced proteinexpressions of ICAM-1and VCAM-1【Objective】 To investigate the effect of PPARγ (peroxisomeproliferator-activated receptor gamma) agonist rosiglitazone on HIV-1Tat-induced protein expressions of ICAM-1(intercellular adhesion molecule-1)and VCAM-1(vascular cell adhesion molecule-1).【Method】 hCMEC/D3were treated with/without HIV-1Tat and/oragonist in four groups, the protein expressions of ICAM-1and VCAM-1inhCMEC/D3were assessed by Western blot at12h after treatment.【Results】HIV-1Tat induced upregulation of the protein expression ofICAM-1and VCAM-1in hCMEC/D3(P﹤0.05and P<0.01, respectively),PPARγ agonist rosiglitzone inhibited HIV-1Tat-induced protein expression ofICAM-1(P﹤0.05) and VCAM-1(P>0.05).【Conclusion】 PPARγ agonist rosiglitazone partly inhibited HIV-1Tat-induced inflammatory gene expression. PARTⅢ The effect of PPARγ agonist on HIV-1Tat-induced mRNAexpressions of ICAM-1and VCAM-1and the possible signaling mechanism【Objective】To evaluate the effect of PPARγ agonist rosiglitazone onHIV-1Tat-induced mRNA expressions of ICAM-1and VCAM-1as well as thepossible signaling mechanism.【Method】 hCMEC/D3were treated with/without HIV-1Tat and/oragonist or antagonist in eight groups, the mRNA expressions of ICAM-1andVCAM-1in hCMEC/D3were determined by Real-time RT-PCR at6h aftertreatment. hCMEC/D3were devided by four groups, the protein expressions ofAkt in hCMEC/D3were assessed by Western blot at1h after treatment.【Results】HIV-1Tat induced upregulation of the mRNA expression ofinflammatory genes of ICAM-1and VCAM-1in hCMEC/D3(P﹤0.001,respectively),and PPARγ agonist rosiglitazone inhibited HIV-1Tat-inducedmRNA expression of ICAM-1and VCAM-1(P﹤0.01, respectively).This effectwas abolished by the addition of PPARγ antagonist GW9662(P<0.01,respectively) and Akt inhibitor KP3721(P<0.01, respectively). HIV-1Tatinduced upregulation of the p-Akt expression in hCMEC/D3(P﹤0.01)，and thetreatment with rosiglitazone dramatically inhibited HIV-1Tat-inducedinflammatory activation of Akt phosphorylation (P﹤0.01).【Conclusion】PPARγ agonist rosiglitazone inhibited HIV-1Tat-inducedinflammatory gene expression of ICAM-1and VCAM-1via Akt signaling pathway.