Objective: To construct a plasmid expressing peroxisome proliferator-activated receptor gamma1 (PPARgamma1) and to study its antifibrotic effects on transfected mesangial cells under the condition of high glucose.
Methods: Wild type full length of mouse PPARgamma1 (mPPARgamma1/WT) cDNA was ligated into an expressing vector pIRES-EGFP. This constructed pIRES-EGFP-mPPARgamma1/WT was then transfected into cultured glomerular mesangial cells facilitated by lipofectin. The transfection efficiency was determined by RT-PCR, Western bloting for the expression level of PPARgamma1, as well as by specific PPRE binding activity. The effects of over-expressed PPARgamma1 on the expressions of TGF-beta1, PAI-1 and FN in the mesangial cells stimulated by high glucose (30 mmol/L) was examined by RT-PCR and ELISA. A vector expressing dominant negative type of mPPARgamma1, pIRES-EGFP-mPPARgamma1/DN, lacking the biological activity of PPARgamma1, was also constructed and transfected by using the same technology to serve as a control throughout the study.
Results: The accuracy of constructed and selected plasmids was confirmed by restriction enzymatic analyses and DNA sequencing. The expression level and activity of PPARgamma1 in the mesangial cells reached the peaks 48 hours after transfection. Over-expressed PPARgamma1 significantly inhibited the high glucose-induced increases in TGF-beta1, PAI-1 and fibronectin syntheses (all P < 0.05).
Conclusion: The constructed expressing vector pIRES2-EGFP-mPPARgamma1/WT provides a useful tool for further study on the effects and mechanisms of PPARgamma1. Over-expressed PPARgamma1 may protect the mesangial cells from fibrosis caused by high glucose.