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

Figure 11  Signaling pathway experiment. A: Protein bands; B-F: Protein expression of 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 treated with TNC inhibitory antibody or TNC recombinant protein with or without transfected with siRNA-TLR4. RMCs cultured with normal glucose concentrations (N, 5.5 mmol/L glucose) or high-glucose (H, 30 mmol/L glucose) medium for 24 h, RMCs were pretreated with 0.5 mg/mL TNC blocking peptide and cultured with high-glucose (Anti-TNC, 30 mmol/L glucose), RMCs were treated with 2.5 ug/mL recombinant TNC (r-TNC), RMCs were transfected with siRNA-TLR4 to silence TLR4 expression for 6 h, and the media were then replaced with normal-glucose (NG, 5.5 mmol/L glucose) and treated with 2.5 ug/mL r-TNC(r-TNC+siTLR4). All RMCs were treated for 24 h and collected for subsequent experiments. ^aP < 0.05 compared with RMCs cultured with normal glucose concentrations; ^cP < 0.05 compared with RMCs cultured with high glucose concentrations; ^eP < 0.05 compared with RMCs treated with r-TNC. TLR4, p-NF-κB p65, NF-κB p65, connective tissue growth factor, and fibronectin levels were all detected using Western blot. The results are presented as the mean ± SD of six independent experiments after normalization to GAPDH levels. TLR4: Toll-like receptor-4; p-NF-κB p65: Phosphorylated nuclear factor-κB p65 (Ser536); NF-κB p65: Nuclear factor-κB p65; CTGF: Connective tissue growth factor; FN: Fibronectin; N: Normal control; H: High-glucose.