低氧状态下红景天苷经HIF-1α信号通路调节人成骨样MG-63细胞表型表达的研究  

Effect of salidroside on regulating phenotypic expression of human osteoblast-like MG-63 cells through HIF-1α signal passway

作　　者：祁琳[1] 王川[2] 梅国华[3] 温晓昶[4] 毛立群[3] 金鑫[5] 李玲[5] 王越[3] 

机构地区：[1]武警后勤学院附属医院药材科,天津300162 [2]武警后勤学院附属医院口腔科,天津300162 [3]武警后勤学院病原生物和免疫学教研室,天津300309 [4]武警后勤学院基础医学实验技术教研室,天津300309 [5]武警后勤学院药理学教研室,天津300309

出　　处：《中国药理学通报》2017年第6期836-843,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81273520;81572852);天津市自然科学基金资助项目(No 15JCYBJC28900;12JCZDJC26300);武警后勤学院科学技术研究项目(No WHJ201508;WHJ201509;WYKFM201609)

摘　　要：目的研究低氧状态下红景天苷对成骨细胞表型表达的调控作用及其作用机制。方法 MTT法检测细胞增殖能力;采用Annexin V/PI双染法分析低氧诱导的细胞凋亡情况;磷酸苯二钠法检测成骨细胞分化的早期标志性基因碱性磷酸酶(ALP)活性;RT-PCR技术检测低氧诱导因子(HIF)-1α、血管内皮生长因子(VEGF)、抑癌基因VHL蛋白(p VHL)、骨钙素(OC)、成骨细胞特异性转录因子Runx2及Osterix mRNA表达水平;采用ELISA法检测VEGF及OC的蛋白表达水平和I型胶原的含量;Western blot技术检测HIF-1α、p VHL、Osterix、Runx2的蛋白表达水平;免疫荧光共聚焦显微镜技术观察HIF-1α的核转位情况;荧光素酶报告基因检测技术检测HIF-1α的转录活性。结果低氧状态下红景天苷促进MG-63细胞增殖,抑制由低氧诱导的MG-63细胞凋亡,促进Osterix、Runx2表达及通过增加ALP活性、Ⅰ型胶原和OC含量促进成骨细胞分化成熟。进一步研究发现,低氧状态红景天苷可下调HIF-1α的表达及核转位,而上调其转录活性及下游靶基因VEGF的表达。结论低氧状态下红景天苷通过调节HIF-1α/VEGF信号通路促进成骨细胞增殖和分化,同时抑制由低氧诱导的成骨细胞凋亡。Aim To investigate the effect of salidroside on the phenotypic expression of osteoblast and the possible molecular mechanism in hypoxia environment.Methods MTT,Annexin V/PI double staining,alkaline phosphatase( ALP) activity assay and enzymelinked immnosorbent assay( ELISA) were performed respectively to detect the effect of salidroside on regulating phenotypic expression of MG-63 cells exposed to hypoxia. Then RT-PCR and Western blot were conducted to detect the expression levels of Osterix and Runx2 which were related to differentiation. At last,we investigated the expression levels of components of the HIF-1α pathway by RT-PCR,Western blot and ELISA,respectively. Immunofluorescence confocal microscopetechnology and luciferase reporter assay were performed to explore nuclear translocation and transcriptional activity of HIF-1α. Results Salidroside could markedly promote MG-63 cell proliferation,inhibit hypoxia-induced apoptosis,stimulate cell differentiation and promote expression levels of components of the HIF-1αpathway in hypoxia environment. Conclusion Salidroside could regulate phenotypic expression of MG-63 cells through HIF-1α/VEGF signaling pathway in hypoxia environment.

关 键 词：红景天苷 低氧诱导因子(HIF)-1α 成骨细胞 增殖 分化 凋亡 血管内皮生长因子(VEGF) 

分 类 号：R284.1[医药卫生—中药学] R329.24[医药卫生—中医学]