Ma Dong-wei, Wang Qiu-yue, Chen Qi-guang, Wu Dan, Wang Jiao, Hou Lu-lu
Department of Endocrinology, First Affiliated Hospital, China Medical University, Shenyang 110001, China.
Zhonghua Yi Xue Za Zhi. 2011 May 31;91(20):1417-21.
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.
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).
(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.
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.
观察RhoA的小分子干扰RNA（Stealth RNAiTM siRNA）对人肾小球系膜细胞（HMC）炎症反应及纤维化的影响，探讨RhoA/ROCK信号通路在糖尿病肾病发生过程中的作用。
将同步化的HMC分为若干组。采用Lipofectamine(TM)2000将RhoA-siRNA和RhoA阴性siRNA转染至上述细胞中。RhoA-siRNA可抑制RhoA的表达。采用实时逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）检测RhoA、ROCK-I、纤连蛋白（FN）、结缔组织生长因子（CTGF）和肿瘤坏死因子-α（TNF-α）的表达。
（1）RhoA siRNA转染可抑制高糖诱导的HMC中RhoA、ROCK-I和CTGF mRNA的表达。与高糖诱导组相比，各mRNA表达降低26% - 60%（P < 0.05）；（2）RhoA siRNA转染并与高糖共同培养48 h后，FN、CTGF和TNF-α的分泌显著下降[FN：(1.99 ± 0.04) mg/L对(4.31 ± 0.13) mg/L，CTGF：(4.98 ± 0.17) mg/L对(6.06 ± 0.09) mg/L；TNF-α：(61.17 ± 2.59) ng/L对(91.76 ± 2.27) ng/L，均P < 0.05]。FN和CTGF水平几乎降至正常糖诱导的HMC水平。
通过RNA干扰抑制RhoA，减少细胞外基质的积聚、肾小球纤维化和炎症，可降低高糖诱导的HMC中FN、CTGF和TNF-α的水平。从而为糖尿病肾病的预防提供了新的干预靶点。
