“大黄-桃仁”药对调控子宫腺肌病小鼠子宫内膜细胞动力学特征的机制研究

Regulation Mechanism of“Dahuang(Rhubarb)and Taoren(Peach Kernel)”on Dynamic Characteristics of Endometrial Cells in Adenomyosis Mice

目的探索活血化瘀药对“大黄-桃仁”(Rhubarb-Peach kernel,RP)对子宫腺肌病(adenomyosis,AM)小鼠子宫内膜细胞的动力学特征的调控作用。方法体外培养hEM-15A细胞,MTT实验及Transwell实验检测RP处理后细胞活力、增殖及迁移能力变化。采用同种异体垂体移植建立AM小鼠模型,随机分为空白对照组、模型组及药物治疗组,采用热板痛及机械痛觉过敏实验检测治疗疗效、HE染色观察病理浸润程度。透射电镜观察细胞动力学特征变化,免疫荧光染色检测微丝蛋白骨架调节酶Rho GTP酶成员A(ras homolog gene family,member A,RhoA)、Rho激酶(rhokinase 1,Rock1)、Ras相关的C3肉毒素底物1(ras-related C3 botulinum toxin substrate 1,Rac1)、细胞分裂周期蛋白42(cell division cycle 42,Cdc42)的表达水平及细胞定位。结果RP显著抑制hEM-15A细胞的异常增殖,并呈浓度依赖性;经1 mg/mL RP处理后,细胞迁移及侵袭能力显著下降。动物实验表明,与模型组比较,经RP治疗后小鼠热板痛及机械痛反应时间显著缩短,病理浸润程度减轻。电镜结果表明,与空白组比较,模型组小鼠子宫内膜上皮细胞排列紊乱、体积缩小,细胞形态由柱状变为梭形或不规则形;细胞间紧密连接、桥粒连接及细胞周边指状突起减少,细胞膜局部形成皱褶,经RP治疗后上述动力学特征变化改善。免疫荧光染色结果显示,RhoA、Rock1、Rac1、Cdc42可定位表达于子宫内膜腺上皮细胞、间质细胞及平滑肌细胞。与对照组比较,模型组小鼠Rho GTP酶的表达显著上调,经RP治疗后回调。结论AM子宫内膜上皮细胞动力学特征发生改变,有助于其从静止态向迁移状态转化。RP对AM的治疗机制与Rho GTP酶调控AM动力学特征,从而抑制细胞异常运动能力有关。

Objective To explore the potential mechanism of“Dahuang(Rhubarb)and Taoren(Peach Kernel)”(RP),a herb pair of activating blood circulation and removing blood stasis,in regulating the microfilament protein skeleton of endometrial cells in adenomyosis(AM)mice.Methods The hEM-15A cells were cultured in vitro,the MTT assay and transwell assay were used to detect the changes of cell viability,proliferation and migration ability after RP treatment.AM mouse model was constructed by allograft pituitary transplantation,and AM mice were randomly divided into blank control group,model group and drug treatment group.Heat pain and mechanical hyperalgesia tests were used to detect the therapeutic effect,and HE staining was used to observe the degree of pathological infiltration.The morphological changes of cytoskeleton were observed by electron microscopy,and the expression levels of Ras homolog gene family,member A(RhoA),Rhokinase 1(Rock1),Ras-related C3 botulinum toxin substrate 1(Rac1)and Cell division cycle 42(Cdc42)and cell localization were detected by immunofluorescence staining.Results RP inhibited the abnormal proliferation of hEM-15A cells in a concentration-dependent manner.After 1 mg/mL RP 151 treatment,the cell migration and invasion ability decreased significantly.AM mouse model was established successfully,and the reaction time of heat pain and mechanical pain in the model group was significantly shortened,which was improved after RP treatment.And the pathological infiltration degree of endometrial cells into muscle layer in the model group was alleviated after RP treatment.The results of electron microscopy showed that compared with those in the blank group,the epithelial cells in the model group were looser and reduced in size,and the cell morphology was changed from columnar to spindle or irregular.Tight intercellular junctions,desmosomes junctions and fingerlike processes around cells were reduced,and local folds of cell membranes were formed.The above dynamic characteristics were improved after RP treatment.Immunofluorescence staining showed that the expressions of RhoA,Rock1,Rac1 and Cdc42 could be localized and expressed in glandular epithelial cells.Compared with those of the control group,the expression of Rho GTP enzyme in the model group was significantly up-regulated,which was reversed after RP treatment.Conclusion AM endometrial epithelial cell dynamics were changed,which was helpful for its transformation from a stationary state to a migratory state.The treatment mechanism of AM by RP is related to the regulation of the dynamic characteristics of AM by Rho GTP enzyme and the inhibition of abnormal cellular motility.