左归丸含药血清通过ERK/TGF-β/Smads信号级联调控MC3T3-E1细胞增殖与分化  

Effects of Zuogui pill-medicated serum on proliferation and differentiation of MC3T3-E1 cells via ERK/TGF-β/Smads signaling cascade

作　　者：蒿长英[1] 任艳玲[1] 刘立萍[1] 宋囡[2] 王智民[3] 

机构地区：[1]辽宁中医药大学基础医学院,辽宁沈阳110032 [2]辽宁中医药大学附属第二医院,辽宁沈阳110032 [3]辽宁中医药大学第一临床学院,辽宁沈阳110032

出　　处：《中国病理生理杂志》2012年第9期1670-1675,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30873226);教育部高等学校博士学科点专项科研基金资助项目(No.20102133110001);辽宁省高等学校优秀人才支持计划资助项目(No.LR201025)

摘　　要：目的:研究细胞外信号调节激酶(ERK)/转化生长因子β(TGF-β)/Sma和Mad相关蛋白(Smads)信号级联在左归丸含药血清干预成骨前体细胞系MC3T3-E1细胞增殖与分化中的作用。方法:以倍美力为阳性对照药,对Sprague-Dawley(SD)雌性大鼠灌服高、中、低剂量的左归丸混悬液,7 d后腹主动脉取血分离含药血清。采用噻唑蓝(MTT)法检测左归丸含药血清对MC3T3-E1细胞的增殖作用,采用改良钙钴染色法检测碱性磷酸酶(ALP)表达,采用茜素红染色法检测钙化结节,采用Western blotting法检测核结合因子α1(Cbfα1)和Ⅰ型胶原(ColⅠ)蛋白表达,采用real-time RT-PCR法检测TGF-β1、Smad4和Smad2 mRNA表达。结果:左归丸含药血清对MC3T3-E1细胞的促增殖作用呈剂量和时间相关性,其中以低剂量且体积分数为15%作用48 h后对MC3T3-E1的促增殖作用最大;左归丸含药血清能促进MC3T3-E1细胞ALP表达,增强细胞基质钙化,提高Cbfα1和ColⅠ蛋白分泌,上调TGF-β1、Smad4和Smad2 mRNA表达;加入ERK1/2信号通路特异性阻滞剂PD98059后,MC3T3-E1细胞增殖降低,ALP表达下降,细胞基质钙化减弱,Cbfα1和ColⅠ蛋白分泌降低,Smad4和Smad2 mRNA表达下调,TGF-β1mRNA表达进一步上调。结论:左归丸可能通过干预ERK/TGF-β/Smads信号级联而调控成骨细胞的增殖和分化,这可能是其防治骨质疏松症的机制之一。AIM：To study the effects of Zuogui pill（ZG）-medicated serum on the proliferation and differentiation of MC3T3-E1 cells via ERK/TGF-β/Smads signaling pathway. METHODS：Using Premarin（conjugated estrogens tablets） as a positive control, the SD female rats were fed with high-, medium- or low-dose of ZG suspension. ZG-medicated serum was separated from abdominal aortic blood 7 d after feeding of ZG. MTT assay was applied to test the effect of ZG-medicated serum on the viability of MC3T3-E1 cells. The production of alkaline phosphatase（ALP） was detected by a modified calcium and cobalt dyeing method. The calcified nodules were observed by the method of alizarin red staining. The levels of core binding factor α1（Cbfα1） and collagen type I（Col I） protein were analyzed by Western blotting. The mRNA expression of TGF-β1, Smad4 and Smad2 was measured by real-time RT-PCR. RESULTS：ZG-medicated serum promoted the proliferation of MC3T3-E1 cells in a dose-and time-dependent manner. Compared with other groups, treatment with 15% ZG（low dose） for 48 h increased the proliferation of MC3T3-E1 cells significantly. The protein levels of ALP, Cbfα1 and Col I,the calcified nodules, and the mRNA expression of TGF-β1, Smad4 and Smad2 in MC3T3-E1 cells were all significantly increased after treatment with ZG-medicated serum. After the addition of PD98059（a specific blocker of ERK1/2 signaling pathway）, all those were down-regulated except for mRNA expression of TGF-β1. CONCLUSION：ZG regulates MC3T3-E1 cell proliferation and differentiation via the intervention of ERK/TGF-β/Smads signaling cascade, which may be one of the mechanisms that ZG effectively prevents and treats osteoporosis.

关 键 词：左归丸 细胞外信号调节激酶类 转化生长因子β Smad蛋白类 MC3T3-E1细胞 

分 类 号：R363[医药卫生—病理学]