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目的研究积雪草苷对增生性瘢痕成纤维细胞结缔组织生长因子(CTGF)的影响及RhoA/ROCK-Ⅰ信号通路在其中的作用。方法分离培养人增生性瘢痕组织来源的成纤维细胞,应用积雪草苷300μg.mL-1及RhoA的下游效应器Rho激酶的特异抑制剂Y-27632对实验细胞进行干扰实验。分为对照组、积雪草苷组、Y-27632组、积雪草苷+Y-27632组。运用荧光定量PCR(SYBR Green)及免疫荧光细胞化学的方法检测各组瘢痕成纤维细胞中RhA、ROCK-Ⅰ、CTGF mRNA与蛋白表达。结果积雪草苷300μg.mL-1能抑制RhoA、ROCK-Ⅰ、CTGF的mRNA与蛋白表达,与对照组比较具有统计学意义(P<0.05);Y-27632能阻碍积雪草苷的作用,积雪草苷(300μg.mL-1)+Y-27632组,CTGF的mRNA与蛋白表达明显高于单纯积雪草苷组(P<0.05),与对照组及Y-27632组比较无显著性差异(P>0.05);Y-27632组与对照组比较RhoA、ROCK-Ⅰ、CTGF的mRNA与蛋白表达没有统计学意义。结论积雪草苷300μg.mL-1能抑制RhoA、ROCKⅠ-、CTGF的mRNA与蛋白表达;而且可通过RhoA/ROCKⅠ-信号通路抑制瘢痕成纤维细胞中CTGF mRNA与蛋白表达。这可能是积雪草苷治疗瘢痕的作用机制之一。
Objective To study the effects of asiaticoside on CTGF in hypertrophic scar fibroblasts and its role in RhoA / ROCK-Ⅰ signaling pathway. Methods Human fibroblasts derived from human hypertrophic scar were isolated and cultured. The interference experiments were carried out on the experimental cells using asiaticoside 300 μg.mL-1 and Y-27632, a specific inhibitor of RhoA downstream effector of RhoA. Divided into control group, asiaticoside group, Y-27632 group, asiaticoside + Y-27632 group. The mRNA and protein expressions of RhA, ROCK-Ⅰ and CTGF in each group of scar fibroblasts were detected by SYBR Green and immunofluorescence cytochemistry. Results Asiaticoside 300 μg.mL-1 could inhibit the mRNA and protein expression of RhoA, ROCK-Ⅰ and CTGF, which were significantly different from those of the control group (P <0.05). Y-27632 could inhibit asiaticoside , Asiaticoside (300μg.mL-1) + Y-27632 group, CTGF mRNA and protein expression was significantly higher than that of asiaticoside alone group (P <0.05), compared with the control group and Y-27632 group (P> 0.05). The mRNA and protein expressions of RhoA, ROCK-Ⅰ and CTGF in Y-27632 group were not significantly different from those in control group. Conclusion Asiaticoside 300μg.mL-1 can inhibit the mRNA and protein expression of RhoA, ROCKⅠ- and CTGF, and inhibit the expression of CTGF mRNA and protein in scar fibroblasts through RhoA / ROCKⅠ-signaling pathway. This may be one of the mechanisms of asiaticoside treatment of scars.