The Effect Of NADPH Oxidase On The Matrix Deposition In Diabetic Nephropathy

[OBJECTIVE]To investigate the alteration of NADPH oxidase in high glucose, and study the effect of NADPH oxidase on the matrix deposition in mesangial cell, expecting to elucidate the preliminary impact of the oxidative stress inducing by NADPH oxidase in diabetic nephropathy.[METHODS] 1. Mesangial cell was cultured in high glucose for different time. The mRNA of p22phox, which was the primary subunit of NADPH oxidase, was detected by semi-quantitative reberse transcription polymerase chain reaction (RT-PCR). The Reactive Oxygen Species (ROS) in cells was measured by Flow cytometry(FCM), using DCFH-DA and HE label. 2.High glucose cultured Mesangial cell, FN, PAI-1, MMP-2 reflected the matrix deposition, specific inhibitors of NADPH oxidase, DPI, and the ROS scavenger, such as superoxide dismutase (SOD), catalase , N-Acetylcysteine (NAC), were added to inhibit NADPH oxidase and the oxidative stress, in order to investigate the oxidase's effect on the matrix deposition. [RESULTS] 1.The activity of NADPH oxidase was up-regulated with glucose concentration and exposure time increasing, but the expression of p22phox mRNA was not increased until cultured in high glucose for 5 days. 2.The up-regulation of NADPH oxidase's activity could induce FN, PAI-1 mRNA increasing, but MMP-2 mRNA decreasing, inhibition of NADPH oxidase could normalize them. Taking SOD, catalase, NAC to inhibit NADPH oxidase in different levels, results indicated that NADPH oxidase showed its effect on matrix deposition through O₂^- and H₂O₂. [CONCLUSIONS] 1. High glucose increased the expression and activity of NADPH oxidase. 2. NADPH oxidase effected on matrix deposition through O₂ and H₂O₂.