白术提取物对IEC-6细胞迁移过程Rho GTPaes及肌球蛋白II表达的影响

Effect of Methanol Extracts from Rhizoma Atractylodis Macrocephalae on Expression of Rho GTPaes and Myosin Ⅱ During Intestinal Epithelial Cell Migration

目的观察白术提取物对小肠上皮细胞(IEC-6)迁移多胺介导钾通道激活信号通路指标Rho GTPaes(Rho A、Rac1和Cdc42)表达的影响,对肌球蛋白II(myosinⅡ)分布和表达的影响,以探讨益气健脾中药白术对胃肠黏膜损伤修复的作用机制。方法荧光定量PCR法检测Rho A、Rac1和Cdc42 mRNA表达;Western blot法检测Rho A、Rac1和Cdc42蛋白表达;免疫荧光法检测myosinⅡ蛋白分布和表达。结果白术提取物能提高细胞迁移过程Rho A、Rac1、Cdc42 mRNA和蛋白表达,逆转多胺合成抑制剂二氟甲基鸟氨酸(DFMO)所致的Rho A、Rac1、Cdc42 mRNA和蛋白表达抑制;提高细胞迁移过程myosinⅡ表达,从而增加细胞骨架应力纤维形成,改善DFMO所致的myosinⅡ蛋白表达和分布的抑制。结论白术提取物促进IEC-6细胞迁移的作用机制,与其影响多胺介导钾通道激活信号通路有关。

Objective To observe the effects of methanol extracts from Rhizoma Atractylodis Macroeephalae（RAM） on the expression of Rho GTPaes（RhoA, Racl, Cdc42） which are the markers of polyamine-inediated potassium channel activating signaling pathway, and the on distribution and expression of myosinⅡduring intestinal epithelial cell （IEC-6） migration, thus to explore the mechanism of spleen-invigorating and qi-replenishing RAM on promoting gastrointestinal mueosal repair after wounding. Methods The expression levels of RhoA, Rael, Cde42 mRNA and protein were assessed by real-time fluorescence quantitative RT-qPCR and Western blot methods, respectively. The distribution and expression of myosin Ⅱ was detected by immunofluoreseence assay. Results Methanol extracts from RAM could increase RhoA, Rael, Cdc42 mRNA and protein levels, reversed the inhibition of RhoA, Rael, Cdc42 mRNA and protein expression induced by polyamine inhibitor alpha-difluoromethyl ornithine （DFMO）, improved the expression of myosin Ⅱduring cell migration, which resulted into the increase of the formation of cytoskeleton stress fiber, and the improvement of DFMO-induced inhibition of myosin Ⅱprotein expression and distribution. Conclusion The mechanism of RAM in promoting IEC-6 cell migration is probably related to its effect on polyamine-induced potassium channel activating signaling pathway.