| Background and ObjectiveDiabetic nephropathy is the main cause of end-stage renal disease and a major contributing cause of morbidity and mortality in patients with diabetes. Mesangial expansion with the accumulation of extracellular matrix (ECM) proteins such as collagen, fibronectin and laminin is the main feature of diabetic nephropathy. There is increasing evidence indicating that mesangial autocrine activation of transforming growth factor-β1(TGF-β1) mediates the effects of high glucose concentration , and that TGF-β lis a determinant in the development of human and experiment diabetic glomerulosclersis. Therapeutic approaches to down-regulate TGF-β1 level under high glucose conditions provide one strategy to inhibit the progression of diabetic nephropathy.Proteoglycan II(Decorin, DCN) is a secreted 37KDa proteoglycan witha core protein comprised primarily of leucine-rich repeats and adhered by a glucosaminoglyean strain. There is evidence that the core protein of DCN can bind to TGF- β1 and neutralize its activity.In the present study, we investigated the effect of DCN transferation on proliferation of mesangial cells and expression of TGF-β1, ECM components in high-glucose cultured rat mesangial cells.Experiment Design and MethodsRecombinant Adenovirus Vector of Rat Proteoglycan II ( AD-DCN ) and Recombinant Adenovirus Vector of Rat Lacz(AD-Lacz )were proliferated in AD-293 cell in vitro. The multiplicity of infection (MOI) of AD-DCN and AD-Lacz were tested . The rat mesangial cells (RMCs) were obtained from Institute of Cell Biology, Wuhan, were divided into 5 groups in this study: Groupl , all the cells were cultured in 4500mg/l glucose culture medium, AD-Lacz was added to the culture with M0I 50. Group 2 , all the cells were cultured in 4500mg/l glucose culture medium, AD-DCN was added to the culture with MOI 50. Group3, all the cells were cultured in 1000mg/l glucose culture medium, as a normal group. Group4, all the cells were cultured in 4500mg/l glucose culture medium. Group 5, in this group all the cells were cultured in 4500mg/l glucose culture medium , and treated with neutralizing rabbit anti-porcine TGF-β1 antibody(3ng/ml) as the positive control .All cells were cultured for 24 hours to 5days. The proliferation of rat mesangial cells in 5 different groups after 1 day, 2 days, 3 days, 4 days, 5 days culturing was tested by methyl thiazoleterazolium (MTT) . In each day, culture medium and cell lysis were collected and the expression of DCN, TGF-β1, collagen IV (C-IV) , fibronectin (FN) and laminin (LN)was analyzed by western blot analysis. β-actin protein was also analyzed by western blot as control. Bands of all proteins were analyzed by TOTALLAB(V2005). The ratio of the targetprotein and P -act in protein is used for the comparative quantifying of the target protein. Statistical analysis was carried by SPSS 10.0 for windows.Results:1. The proliferation of mesangial cells, increased secretion of DCN and TGF-3 1 as well as C-IV under high glucose environment occurred in first 24 hours, and continued throughout the whole study. FN and LN levels increased from the third day to the fifth day.2. AD-DCN inhibited the proliferation of rat mesangial cells caused by high glucose from the fourth day, which was slower than the inhibition of TGF-3 1 neutralizing antibody. In the fifth day, there is no statistically significant difference detected (p>0. 05) between AD-DCN and TGF-3 1 neutralizing antibody. Up-expression of DCN first occurred from the third day after adding AD-DCN . Expression of DCN in AD-DCN group was 1.41 fold and 1.67 fold compared to the high glucose group, and 1.34 fold and 1. 43 fold compared to AD-Lacz group in the third day and the fifth day, respectively. Down-expression of TGF-3 1 and collagen IV, FN and LN occurred from the third day after adding AD-DCN, and continued throughout the whole study and it is similar in that of TGF-3 1 neutralizing antibody group and normal control group. 3. The mild proliferation caused by AD-Lacz occurred in the first day and lasted throughout the whole study, but no statistically significant difference was detected compared to the high glocuse group (p>0. 05) . AD-Lacz had no statistically significant effect on the secretion of DCN compared with high glcouse group. Compared with high glucose group, in the first day after adding AD-Lacz, the expression of TGF-31, collagen IV, FN and LN had statistical significantly increase (p<0.05) , but in the third and fifth day , no statistically significant difference was detected (p>0. 05) .Conclusions:J. High glucose concentration stimulates proliferation of rat mesangial cells and increases the expression ofTGF-3 1, collagen IV, FN and LN in rat mesangial cells.2. The constructed recombinant DCN adenovirus can efficiently express biologically active DCN in rat mesangial cells. 3. Overproduction of DCN can inhibit the proliferation of rat mesangial cells and down-regulate TGF-ftl, collagen IV, FN and LN expression in high glucose conditioned rat mesangial cells.3. The function of AD-DCN on down-regulating TGF- fi, and ECM components expression is similar with the treatment of anti-TGF-p antibody.
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