基于Rho/ROCK信号通路探讨麻黄细辛附子汤对小鼠树突状细胞迁移的影响

Effect of Mahuang Xixin Fuzitang on Migration of Dendritic Cells in Mice by Regulating Rho/ROCK Signaling Pathway

目的:探讨麻黄细辛附子汤对小鼠树突状细胞(DCs)迁移的抑制作用及其潜在机制。方法:分离并培养小鼠骨髓源性DCs,显微镜观察不同时期细胞形态变化,流式细胞术检测CD11c+比例,鉴定DCs纯度;麻黄细辛附子汤(1、2、5、10、20、40、50、100 g·L^(-1))处理细胞24 h,细胞增殖与活性检测法(CCK-8)检测麻黄细辛附子汤对细胞增殖的影响,筛选给药浓度;脂多糖(LPS)造模后,将DCs分为空白组、模型组、麻黄细辛附子汤组(2、4、8 g·L^(-1)),流式细胞术检测各组细胞表面分子CD80、CD86、主要组织相容性复合物-Ⅱ(MHC-Ⅱ)分子表达;Transwell小室观察细胞迁移情况;酶联免疫吸附测定法(ELISA)检测细胞表面趋化因子C-C-基元受体7(CCR7)、趋化因子C-X-C-基元受体4(CXCR4)含量;免疫荧光法(IF)检测细胞微丝骨架中纤维肌动蛋白(F-actin)表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测Ras同源基因家族成员A(RhoA)、Rho关联含卷曲螺旋结合蛋白激酶1(ROCK1)mRNA表达;蛋白免疫印迹法(Western blot)检测RhoA、ROCK1蛋白表达。结果:与空白组比较,模型组CD80、CD86、MHC-Ⅱ表达显著升高(P<0.01),细胞迁移至下室数量显著增加(P<0.01),CCR7、CXCR4含量明显增加(P<0.05,P<0.01),F-actin表达显著升高(P<0.01),RhoA、ROCK1 mRNA和蛋白表达明显升高(P<0.05,P<0.01);与模型组比较,麻黄细辛附子汤组(2、4、8 g·L^(-1))处理24 h后,CD80、CD86、MHC-Ⅱ表达显著降低(P<0.01),细胞迁移至下室数量明显减少(P<0.05),CCR7、CXCR4含量明显减少(P<0.05,P<0.01),F-actin表达显著降低(P<0.01),RhoA、ROCK1 mRNA和蛋白表达明显降低(P<0.05,P<0.01)。结论:麻黄细辛附子汤可以抑制小鼠DCs迁移,其作用机制可能与降低Rho/ROCK信号通路活性影响细胞骨架改变有关。

Objective To investigate the inhibitory effect of Mahuang Xixin Fuzitang on the migration of dendritic cells(DCs)in mice and its underlying mechanism.Method Mouse bone marrow-derived DCs were isolated and cultured.The morphological changes of the cells at different stages were observed under a microscope,and the CD11c+proportion was detected by flow cytometry to identify DC purity.Cells were treated with Mahuang Xixin Fuzitang(1,2,5,10,20,40,50,100 g·L^(-1))for 24 hours,and the effect of Mahuang Xixin Fuzitang on cell proliferation was assessed using the cell counting kit-8(CCK-8)assay to determine the appropriate concentrations for treatment.After modeling by lipopolysaccharide(LPS)induction,DCs were divided into a blank group,a model group,and Mahuang Xixin Fuzitang groups(2,4,8 g·L^(-1)).The expression of surface molecules CD80,CD86,and major histocompatibility complex-Ⅱ(MHC-Ⅱ)were detected by flow cytometry.Transwell chamber assay was used to observe cell migration.The levels of chemokine C-C-primitive receptor 7(CCR7)and chemokine C-X-C-primitive receptor 4(CXCR4)on the cell surface were detected by enzyme-linked immunosorbent assay(ELISA).The expression of filamentous actin(F-actin)in the cell microfilament cytoskeleton was detected by immunofluorescence(IF)staining.Real-time quantitative polymerase chain reaction(Real-time PCR)was employed to determine the mRNA expression levels of Ras homolog family member A(RhoA)and Rho-associated coiled-coil containing protein kinase 1(ROCK1).Western blot analysis was performed to detect the protein expression of RhoA and ROCK1.Result Compared with the blank group,the model group exhibited significantly higher expression levels of CD80,CD86,and MHC-Ⅱ(P<0.01),a significantly increased number of cells migrating to the lower chamber(P<0.01),and significantly elevated levels of CCR7 and CXCR4(P<0.05,P<0.01).Additionally,F-actin expression was significantly increased(P<0.01),and both RhoA and ROCK1 mRNA and protein expression levels were significantly higher(P<0.05,P<0.01).Compared with the model group,treatment with Mahuang Xixin Fuzitang(2,4,8 g·L^(-1))for 24 hours resulted in significantly lower expression levels of CD80,CD86,and MHC-Ⅱ(P<0.01),a significantly reduced number of cells migrating to the lower chamber(P<0.05),and significantly decreased levels of CCR7 and CXCR4(P<0.05,P<0.01).Furthermore,F-actin expression was significantly reduced(P<0.01),and both RhoA and ROCK1 mRNA and protein expression levels were significantly decreased(P<0.05,P<0.01).Conclusion Mahuang Xixin Fuzitang can inhibit the migration of DCs in mice,and its mechanism of action may be related to reducing the activity of the Rho/ROCK signaling pathway,thereby affecting changes in the cell cytoskeleton.