[Effects of inhibiting RhoA by Stealth RNA on high glucose-induced RhoA/ROCK signaling pathway in human mesangial cells].

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.