二仙汤含药血清通过BK通道对H_(2)O_(2)诱导的MC3T3-E1细胞增殖和成骨分化的影响  

Effect of Erxian Decoction-containing serum on H_(2)O_(2)-induced proliferation and osteogenic differentiation of MC3T3-E1 cells via BK channels

作　　者：任明诗 丁羽 李子涵 吴雨蒙 黄思敏 罗兰兰 张宇静 施旻[3] 夏循礼[3] 刘波[1,2] REN Ming-shi;DING Yu;LI Zi-han;WU Yu-meng;HUANG Si-min;LUO Lan-lan;ZHANG Yu-jing;SHI Min;XIA Xun-li;LIU Bo(School of Pharmacy,Jiangxi University of Chinese Medicine,Nanchang 330004,China;Key Laboratory of Traditional Chinese Medicine Prevention and Treatment of Senile Disease,Jiangxi Provincial Administration of Traditional Chinese Medicine,Nanchang 330004,China;College of Life Sciences,Jiangxi University of Chinese Medicine,Nanchang 330004,China)

机构地区：[1]江西中医药大学药学院,江西南昌330004 [2]江西省中医药管理局中药防治老年性疾病重点研究室,江西南昌330004 [3]江西中医药大学生命科学学院,江西南昌330004

出　　处：《中国中药杂志》2023年第9期2522-2529,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81860802);江西省自然科学基金项目(20202BABL206140);江西中医药大学校级科技创新团队发展计划项目(CXTD22007);江西中医药大学校级研究生创新基金项目(JZYC21S29)。

摘　　要：探讨二仙汤(Erxian Decoction,EXD)含药血清通过BK通道(BK channel)对氧化应激的MC3T3-E1细胞增殖及成骨分化的影响。构建H_(2)O_(2)诱导MC3T3-E1细胞氧化应激模型,使用3 mmol·L^(-1)的氯化四乙基铵(tetraethylamine chloride,TEA)阻断MC3T3-E1细胞BK通道。将MC3T3-E1细胞分为对照(control)组、模型(model)组、EXD组、TEA组和TEA+EXD组。MC3T3-E1细胞按照分组分别处理2 d后,加入700μmol·L^(-1)H_(2)O_(2)继续培养2 h,CCK-8法检测细胞增殖活性,碱性磷酸酶(alkaline phosphatase,ALP)试剂盒法检测细胞ALP活力,蛋白免疫印迹法检测细胞蛋白表达,实时荧光定量PCR检测细胞mRNA表达,茜素红染色法检测成骨细胞矿化区域。结果显示,与control组相比,model组细胞增殖活性和ALP活力显著降低,BK通道α亚基(BK channelαsubunit,BKα)、一型胶原(collagenⅠ,COL1)、骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)、骨保护素(osteoprotegerin,OPG)和磷酸化丝氨酸-苏氨酸激酶(serine-threonine kinase,Akt)表达水平下降,Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、BMP2和OPG mRNA表达水平降低,钙结节面积显著缩小;EXD含药血清干预可以显著提高细胞增殖活性和ALP活力,上调BKα、COL1、BMP2、OPG和磷酸化的Akt、叉头框转录因子O亚族1(forkhead box protein O1,FoxO1)蛋白表达,促进RUNX2、BMP2和OPG mRNA表达,增大钙结节面积;而TEA阻断BK通道后,EXD含药血清促进BKα、COL1、BMP2、OPG和磷酸化Akt、FoxO1蛋白表达,提高RUNX2、BMP2和OPG mRNA表达,增大钙结节面积的作用被逆转。EXD含药血清能改善氧化应激的MC3T3-E1细胞增殖活性、成骨分化和矿化能力,其机制与调控BK通道以及下游Akt/FoxO1信号通路有关。This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels.The oxidative stress model was induced in MC3T3-E1 cells by H_(2)O_(2),and 3 mmol·L^(-1)tetraethylammonium(TEA)chloride was used to block the BK channels in MC3T3-E1 cells.MC3T3-E1 cells were divided into a control group,a model group,an EXD group,a TEA group,and a TEA+EXD group.After MC3T3-E1 cells were treated with corresponding drugs for 2 days,700μmol·L^(-1)H_(2)O_(2)was added for treatment for another 2 hours.CCK-8 assay was used to detect cell proliferation activity.The alkaline phosphatase(ALP)assay kit was used to detect the ALP activity of cells.Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR)were used to detect protein and mRNA expression,respectively.Alizarin red staining was used to detect the mineralization area of osteoblasts.The results showed that compared with the control group,the model group showed significantly blunted cell proliferation activity and ALP activity,reduced expression of BK channelαsubunit(BKα),collagenⅠ(COL1),bone morphogenetic protein 2(BMP2),osteoprotegerin(OPG),and phosphorylated Akt,decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2),BMP2,and OPG,and declining area of calcium nodules.EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity,up-regulate the protein expression of BKα,COL1,BMP2,OPG,and phosphorylated Akt,and forkhead box protein O1(FoxO1),promote the mRNA expression of RUNX2,BMP2,and OPG,and enlarge the area of calcium nodules.However,BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα,COL1,BMP2,OPG,and phosphorylated Akt and FoxO1,increasing the mRNA expression of RUNX2,BMP2,and OPG,and enlarging the area of calcium nodules.EXD-containing serum could improve the proliferation activity,osteogenic differe

关 键 词：二仙汤 氧化应激 BK通道 成骨细胞 成骨分化 骨形成 

分 类 号：R285[医药卫生—中药学]