Metformin regulates inflammation and fibrosis in diabetic kidney disease through TNC/TLR4/NF-κB/miR-155-5p inflammatory loop

Figure 6 Silencing Toll-like receptor-4 protein expression inhibits the expression of tenascin-C and fibrosis factors (connective tissue growth factor and fibronectin), as well as nuclear factor-κB p65 phosphorylation. A: Protein bands; B-F: Protein expression of tenascin-C (TNC), Toll-like receptor-4 (TLR4), phosphorylated nuclear factor-κB p65 (Ser536) (p-NF-κB p65), connective tissue growth factor (CTGF), and fibronectin (FN). Rat mesangial cells (RMCs) were transfected with siRNA-TLR4 for 6 h, and the media were then replaced with normal-glucose (NG, 5.5 mmol/L glucose) or high-glucose (HG, 30 mmol/L glucose) medium for 24 h (NG, 5.5 mmol/L glucose), (HG, 30 mmol/L glucose). RMCs were transfected with siRNA-TLR4 and siRNA-NC for 6 h, and the media were then replaced with normal-glucose (NG, 5.5 mmol/L glucose) or high-glucose (HG, 30 mmol/L glucose) medium for 24 h. ^aP < 0.05 compared with RMCs cultured with normal glucose concentrations; ^cP < 0.05 compared with RMCs cultured with high glucose concentrations. TNC, TLR4, p-NF-κB p65, NF-κB p65, CTGF, and FN levels were detected using Western blot. The results are presented as the mean ± SD of six independent experiments after normalization to GAPDH levels. TNC: Tenascin-C; TLR4: Toll-like receptor-4; p-p65: Phosphorylated nuclear factor-κB p65 (Ser536); p65: Nuclear factor-κB p65; CTGF: Connective tissue growth factor; FN: Fibronectin; N: Normal control; NC: Negative control; H: High glucose.