驻春胶囊通过调控miR-935促进成骨细胞分化的机制  

Mechanism of Zhuchun Capsule Promoting Osteoblast Differentiation by Regulating miR-935

作　　者：张宁[1,2] 董一平 刘曼 骆聪聪 袁强 张颖 ZHANG Ning;DONG Yiping;LIU Man;LUO Congcong;YUAN Qiang;ZHANG Ying(Hunan University of Chinese Medicine,Changsha Hunan China 410208;Luoyang Orthopaedic-Traumatological Hospital of Henan Province(Henan Provincial Orthopedic Hospital),Luoyang Henan China 471002;Henan University of Chinese Medicine,Zhengzhou Henan China 450046)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]河南省洛阳正骨医院(河南省骨科医院),河南洛阳471002 [3]河南中医药大学,河南郑州450046

出　　处：《中医学报》2024年第6期1131-1137,共7页Acta Chinese Medicine

基　　金：国家自然科学基金项目(81774348,81874477);河南省卫生健康中青年学科带头人资助项目(HNSWJW-2020028);河南省科技攻关项目(202102310152)。

摘　　要：目的:探讨驻春胶囊对成骨细胞的影响及驻春胶囊通过调控miR-935促进成骨细胞分化的作用机制。方法:体外培养小鼠胚胎成骨细胞前体细胞MC3T3-E1,采用CCK-8法检测驻春胶囊的细胞毒性和半最大效应浓度(concentration for 50%of maximal effect,EC50)。将MC3T3-E1细胞分为无诱导组、诱导组、诱导+驻春胶囊组、无诱导+驻春胶囊组,诱导14 d和21 d后,茜素红染色观察细胞成骨诱导情况。miR-935转染成骨细胞,将细胞分为miR-935 inhibitor和inhibitor NC组。qRT-PCR和Western blot检测细胞miR-935、信号转导与转录激活因子1(signal transducer and activator of transcription 1,STAT1)、Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)基因和蛋白表达水平。结果:驻春胶囊对MC3T3-E1细胞的EC50值为4684 mg·L^(-1)。茜素红染色结果显示,与无诱导组比较,诱导组、诱导+驻春胶囊组、无诱导+驻春胶囊组细胞均可见不同程度的红色结节和钙沉积。qRT-PCR结果显示,诱导14 d和21 d时,与无诱导组比较,诱导组细胞的ALP mRNA、RUNX2 mRNA、OCN mRNA、miR-935 mRNA水平升高,STAT1 mRNA水平降低(P<0.05),无诱导+驻春胶囊组细胞的STAT1 mRNA水平降低(P<0.05)。与诱导组比较,诱导+驻春胶囊组细胞的RUNX2 mRNA、OCN mRNA水平升高,STAT1 mRNA水平降低(P<0.05)。WB结果显示,诱导14 d和21 d时,与无诱导组比较,诱导组细胞的ALP、RUNX2、OCN水平升高,STAT1水平降低(P<0.01),无诱导+驻春胶囊组细胞的ALP、RUNX2、OCN水平升高,STAT1水平降低(P<0.05)。与诱导组比较,诱导+驻春胶囊组细胞的ALP、RUNX2、OCN水平升高,STAT1水平降低(P<0.05)。qRT-PCR结果显示,与正常对照组比较,驻春胶囊组细胞miR-935 mRNA水平升高(P<0.01)。与inhibitor NC组比较,miR-935 inhibitor组细胞OCN mRNA、ALP mRNA、miR-935 mRNA水平降低,STAT1 mRNA水平升高(P<0.05)。WB结果显示,与inhibitorObjective:To investigate the effect of Zhuchun Capsule on osteoblast and the mechanism of Zhuchun capsule promoting osteoblast differentiation by regulating miR-935.Methods:Mouse embryonic osteoblast progenitor cells MC3T3-E1 were cultured in vitro,and the cytotoxicity and concentration for 50%of maximal effect(EC50)of Zhuchun capsule were detected by CCK-8 method.MC3T3-E1 cells were divided into non-induction group,induction group,induction+Zuchun capsule group,and induction+Zhuchun capsule group.After 14 and 21 days of induction,bone formation induction of Mc3t3-e1 cells was observed by alizarin red staining.miR-935 was transfected into osteoblasts and the cells were divided into miR-935 inhibitor and inhibitor NC groups.miR-935,signal transducer and activator of transcription 1 were detected by qRT-PCR and Western blot.STAT1,Runt-related transcription factor 2(RUNX2),alkaline phosphatase(ALP),osteocalcin(OCN)gene and protein expression levels.Results:The EC50 value of Zuchun capsule against MC3T3-E1 cells was 4684 mg·L^(-1).Alizarin red staining showed that different degrees of red nodules and calcium deposition were observed in the induced group,the induced+Zuchun capsule group and the non-induced+Zuchun capsule group compared with the non-induced group.qRT-PCR results showed that after 14 and 21 days of induction,compared with the non-induction group,the levels of ALP mRNA,RUNX2 mRNA,OCN mRNA and miR-935 mRNA in the induction group were increased,and the levels of STAT1 mRNA were decreased(P<0.05).The STAT1 mRNA level of cells in the non-induction+Zuchun capsule group was decreased(P<0.05).Compared with induction group,RUNX2 mRNA and OCN mRNA levels in induction+Zuchun capsule group were increased,while STAT1 mRNA levels were decreased(P<0.05).WB results showed that after 14 and 21 days of induction,compared with the non-induction group,the levels of ALP,RUNX2 and OCN in the induction group were increased,and the level of STAT1 was decreased(P<0.01);the levels of ALP,RUNX2 and OCN in the non-induction+Zuchu

关 键 词：驻春胶囊 miR-935 骨质疏松症 成骨细胞分化 STAT1 RUNX2 

分 类 号：R285.5[医药卫生—中药学]