骨质疏松肾阳虚和肾阴虚证型下左归丸含药血清干预成骨细胞ERK1/2,Wnt/β-catenin信号通路的研究  

Intervention effects of Zuoguiwan containing serum on osteoblast through ERK1/2 and Wnt/β-catenin signaling pathway in models with kidney-Yang-deficiency,kidney-Yin-deficiency osteoporosis syndromes

作　　者：章建华[1] 邢婧[2] 范连霞[2] 尹华[2] ZHANG Jian-hua XING Jing FAN Lian-xia YIN Hua(The First Affiliated Hospital, Zhejiang University of Chinese Medical, Hangzhou 310006, China Standardization Laboratory in Pharmacy College, Zhejiang University of Chinese Medicine, Hangzhou 310053, China)

机构地区：[1]浙江中医药大学附属第一医院,浙江杭州310006 [2]浙江中医药大学药学院中药标准化研究实验室,浙江杭州310053

出　　处：《中国中药杂志》2017年第20期3983-3989,共7页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81273772);浙江省自然科学基金项目(LY17H270011);浙江省重中之重一级学科--中药学学科科研开放基金项目(Yao2016005);浙江中医药大学校级科研基金项目(2015ZG09)

摘　　要：该文研究骨质疏松(OP)肾阳虚和肾阴虚证型下左归丸含药血清对成骨细胞增殖和碱性磷酸酶(ALP)表达及对成骨细胞ERK1/2,Wnt/β-catenin信号通路相关因子β-catenin,ERK1,ERK2 mRNA及蛋白表达的作用。采用手术去卵巢法造成大鼠骨质疏松模型,并在手术10周后,分别注射氢化可的松、灌胃甲状腺素造成大鼠OP肾阳虚及OP肾阴虚模型;用新生24h乳鼠颅骨体外培养得成骨细胞,以碱性磷酸酶和茜素红染色进行鉴定,分别用OP肾阳虚、OP肾阴虚及OP组(对照组)所得的左归丸含药血清、空白血清干预成骨细胞,采用MTS法检测细胞增殖情况,ELISA法检测ALP表达,RT-PCR法检测ERK1/2,β-catenin mRNA的表达,Western blot法检测ERK1/2,β-catenin蛋白表达。结果表明:OP肾阴虚证型下左归丸含药血清在促进成骨细胞增殖、ALP表达、成骨细胞ERK1/2,Wnt/β-catenin信号通路相关因子β-catenin,ERK1,ERK2 mRNA及蛋白表达方面比OP肾阳虚证型下左归丸含药血清的作用更强。这与"左归丸滋肾阴"的中医理论相吻合,为临床辩证治疗骨质疏松症提供科学依据。左归丸通过ERK1/2和Wnt/β-catenin信号通路调控成骨细胞的增殖与分化,可能是左归丸防治骨质疏松肾阴虚证的机制之一。To clarify the effects of Zuoguiwan containing serum on osteoblast proliferation and alkaline phosphatase （ALP） expres- sion and its effects on the expression offl-catenin, ERK1, ERK2 mRNA and protein of osteoblast through ERK1/2, Wnt/β-catenin sig- naling pathway in models with osteoporosis （OP） kidney-Yang-deficiency, osteoporosis（OP） kidney-Yin-deficiency syndrome. Rat os- teoporosis models were established by ovariectomy surgery, and 10 weeks after surgery, hydrocortisone was injected and thyroxine was administered by intragastric administration to establish OP kidney-Yang-deficiency rat model, and OP kidney-Yin-deficiency rat model. Osteoblasts were obtained from 24 h newborn rat skull and were identified by alkaline phosphatase and alizarin red staining. Zuoguiwan containing serum of OP, OP kidney-Yang-deficieney, and OP kidney-Yin-defieieney, as well as the blank serum were used to intervene the osteoblast, and the cells proliferation was detected by MTS. ELISA assay was used to detect ALP expression. RT-PCR assay was used to detect the mRNA expression of ERK1, ERK2, fl-catenin and protein expression levels were detected by Western blot. The re- sults showed that Zuoguiwan containing serum in OP kidney-Yin-defieieney model had stronger effect than OP kidney-Yang-deficiency in promoting osteoblast proliferation, ALP expression, osteoblast ERK1/2, Wnt/β-catenin signaling pathway related factors fl-catenin, ERK1, ERK2 mRNA and protein expression levels. This was consistent with the TCM theory of ＂Zuoguiwan nourishes kidney Yin＂ , providing a scientific basis for the clinical and dialectical treatment of osteoporosis. Zuoguiwan could regulate the proliferation and dif- ferentiation of bone cells by ERK1/2 and Wnt/β-catenin signaling pathway, which may be one of the mechanisms of Zuoguiwan for the prevention of osteoporosis.

关 键 词：左归丸 成骨细胞 含药血清 骨质疏松肾阳虚 骨质疏松肾阴虚 WNT/Β-CATENIN信号通路 ERK1/2信号通路 

分 类 号：R285[医药卫生—中药学]