Objective: To explore the regulative role of decorin on the ECM gene-expression in diabetic nephropathy, recombinant adenovirus expressing rat decorin (Ad-decorin) was constructed to further investigate the effects of decorin overproduction on the expression of TGFbeta1 and ECM in rat mesangial cells (RMCs) in high glucose condition.
Methods: The recombinant decorin adenovirus and lacz adenovirus(Ad-lacz), as a control, were constructed. RT-PCR, restriction enzyme digestion, western blot and gene sequence were used for validating correctness of Ad-decorin. MTT was used to examine the biological function of decorin (decorin expressed by Ad-decorin transduced CHO cells was used to interact with TGFbeta1 which can inhibit the proliferation of Mv1Lu cells). Then Ad-decorin was transferred into rat mesangial cells cultured in high-glucose (450 mg/dL) media and Ad-lacz was as the control transducer. TGFbeta1, decorin, collagen IV, fibronectin, laminin and tenascin mRNA in RMCs at 24, 48 and 72 hours after Ad-decorin infection were determined with RT-PCR. The distribution and expression of TGFbeta1 protein was detected in RMCs at 96 hours after Ad-decorin infection by immunoperoxidase cell staining.
Results: RT-PCR, restriction enzyme digestion, western blot and gene sequence all confirmed that Ad-decorin could express correct decorin mRNA and protein. MTT showed that decorin protein expressed by Ad-decorin-transfected CHO cells abrogated the inhibitive effect of TGFbeta1 on the proliferation of Mv1Lu cells. Decorin mRNA significantly increased in Ad-decorin transduced RMCs at all the observed time points, reached the peak at 24 hours(2.2-fold, P<0.05) and the overexpression lasted to the end of the observation at 72 hours(1.7-fold, P<0.05) compared to that in Ad-lacz transduced RMCs. Meanwhile, TGFbeta1 mRNA level began to fall at 48 hours (-20%, P<0.05) in Ad-decorin transduced RMCs and went to the valley at 72 hours (-46, P<0.05). ECM components, such as tenascin, laminin, fibronectin and collagen IV, were reduced notably in the Ad-decorin transduced RMCs from the 48 hours to the end of study versus those in the Ad-lacz transduced RMCs. Cellular immunohistochemistry further confirmed that the Ad-decorin transduced RMCs produced much less TGFbeta1 compared with the Ad-lacz transduced RMCs.
Conclusion: The constructed recombinant decorin adenovirus can highly efficiently express biologically active decorin. Overexpression of decorin down-regulates the expression of TGFbeta1 and ECM components from RMCs. These results suggest that overexpression of decorin may be one of the therapeutic approaches to diabetic nephropathy.