Objective: To observe the effect of small interference RNA (Stealth RNAiTM siRNA) of RhoA on the inflammatory response and fibrosis in human mesangial cell (HMC) and explore the role of RhoA/ROCK signaling pathway in the process of diabetic nephropathy.
Methods: Synchronized HMC were divided into several groups. Lipofectamine(TM)2000 was employed to transfect RhoA-siRNA and RhoA-negative siRNA into the above cells. RhoA-siRNA could inhibit the expression of RhoA. The expressions of RhoA, ROCK-I, fibronectin (FN), connective tissue growth factor (CTGF) and tumor necrosis factor-alpha (TNF-α) were detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) and ELISA (enzyme-linked immunosorbent assay).
Results: (1) The expressions of RhoA, ROCK-I and CTGF mRNA were inhibited by RhoA siRNA transfection in high glucose-induced HMC. The expression of each mRNA was reduced 26% - 60% as compared with the high glucose-induced group (P < 0.05); (2) After RhoA siRNA transfection and culturing with high glucose for 48 h, FN, the secretions of CTGF and TNF-α significantly declined [FN: (1.99 ± 0.04) mg/L vs. (4.31 ± 0.13) mg/L, CTGF:(4.98 ± 0.17) mg/L vs. (6.06 ± 0.09) mg/L; TNF-α: (61.17 ± 2.59) ng/L vs.(91.76 ± 2.27) ng/L, all P < 0.05]. The levels of FN and CTGF almost decreased to those of normal glucose-induced HMC.
Conclusion: The levels of FN, CTGF and TNF-α in high glucose-induced HMC may be lowered by inhibiting RhoA through RNA interference and reducing the accumulation of extracellular matrix, glomerular fibrosis and inflammation. Thus it provides a new intervention target for the prevention of diabetic nephropathy.