Role of STAT3 in the regulation of autophagy in the glomerular mesangial cells of diabetic nephropathy / 中华肾脏病杂志

ABSTRACT

Objective To observe the changes of STAT3 signaling transduction pathway and autophagy activity in human glomerular mesangial cells cultured in high glucose, as well as the effect of STAT3 on autophagy, exploring whether SAT3 further influence extracellular matrix proteins type IV collagen secretion through the regulation of autophagy. Methods Culture human renal mesangial cells under different conditions, STAT3 pathway was inhibited with specific blocking agent S3I?201 and siRNA respectively. The experiment was divided into (1) Control group normal glucose concentration; (2) High glucose group divided into 12 h, 24 h, 48 h, 72 h incubation group. (3) High glucose+S3I?201 group pretreated cells with 30 μmol/L S3I?201 (Selleck S1155) for 1 h, then incubation with high glucose for another 24 hours. (4) High glucose+STAT3?siRNA group siRNA transfection firstly, then incubation with high glucose for 24 hours. (5) High glucose+S3I?201+3?MA group pretreated cells with 2 mmol/L 3?MA (Selleck S2767) and 30 μmol/L S3I?201 for 1 h, then incubation with high glucose for another 24 hours. Western blot was employed to detect the protein of STAT3, p?STAT3 and autophagy related protein LC3, p62 expressions. The changes of autophagosome quantity was observed with transmission electron microscope. The extracellular matrix protein collagen IV expression was measured with ELISA. Results Compared with the control group, glomerular mesangial cells cultured with high glucose for 24h, the expressions of STAT3 and p?STAT3 increased (P<0.01), while the expression of autophagy related proteins LC3II/LC3I decreased. The expression of p62 increased and the number of autophagosome reduced under transmission electron microscope, which all indicated the decrease of autophagy activity (P<0.05). Blocking STAT3 signaling pathway with S3I?201 and STAT3?siRNA respectively, compared with high glucose group, LC3II/LC3I was up?regulated and p62 was down?regulated, and the number of autophagosome was increased significantly, which all indicated the increase of autophagy activity (P<0.05). Extracellular matrix proteins collagen IV expression of cells cultured with high glucose was higher than the control group (P<0.05), and the application of S3I?201 blocking STAT3 pathway caused type IV collagen expression to decrease (P<0.05). The application of the autophagy inhibitor 3?MA could convert the result and lead to an increase of type IV collagen expression (P<0.01). Conclusions High glucose could active STAT3 signaling pathway of human renal mesangial cell and increase STAT3, p?STAT3 expression. High glucose could inhibit autophagy activity of human renal mesangial cells. Inhibition of STAT3 pathway activation may reduce the inhibitory effect of high glucose on autophagy of human renal mesangial cells. High glucose leads to an increase of type IV collagen secretion of human glomerular mesangial cells. The activation of STAT3 pathway may increase type IV collagen secretion through negative regulation of autophagy, which eventually leads to diabetic nephropathy.