摘要
目的通过网络药理学和体外实验,探讨左归丸治疗绝经后骨质疏松症的作用机制,验证左归丸促进MC3T3-E1细胞增殖与成骨分化的能力。方法使用网络药理学方法,收集左归丸和绝经后骨质疏松症相关靶点,取交集靶点,构建药物-成分-靶点-疾病交互网络。将20只大鼠分为给药组和空白组,制作左归丸含药大鼠血清和空白大鼠血清用于体外实验,采用MTT法测试左归丸对细胞增殖能力的作用,采用微板法检测细胞中碱性磷酸酶的含量,采用茜素红S染色法检测成骨作用,采用qRT-PCR法检测ESR1、ETS1、PPARγ、RAS、STAT3、MAPK1、MAPK3、OSTERIX、RUNX2和OPG基因的转录水平,采用Western blot法分析RAS、OPG、ERK、P-ERK、ETS1、PPARγ蛋白的表达。结果(1)左归丸与绝经后骨质疏松症相关化合物有102个、交集靶点189个,MAPK1、PPARγ、ETS1等为关键靶点;(2)与空白血清组相比,左归丸含药血清组细胞活性更高;(3)茜素红S染色显示左归丸含药血清组成骨能力均优于空白血清组(P<0.05),碱性磷酸酶水平高于空白血清组(P<0.05);(4)左归丸含药血清组MAPK1、RAS、ETS1、STAT3、OSTERIX、OPG和RUNX2基因转录水平升高(P<0.05),ESR1、PPARγ和MAPK3基因转录水平下降(P<0.05);(5)与空白血清组相比,左归丸含药血清组的p-ERK、RAS、ETS1和OPG蛋白表达上调(P<0.05),PPARγ蛋白表达下调(P<0.05),ERK无显著差异。结论左归丸含药血清可促进MC3T3-E1细胞增殖和成骨分化,其机制可能与ERK/ETS1/PPARγ信号通路改变有关。
Objective To explore the mechanism of action of Zuogui Wan in treating postmenopausal osteoporosis through network pharmacology and in vitro experiments,and to verify its ability to promote MC3T3-E1 cell proliferation and osteogenic differentiation.Methods Network pharmacology was used to collect targets related to Zuogui Wan and postmenopausal osteoporosis,and intersecting targets were taken to construct a drug component target disease interaction network.In vitro experiments were conducted to test the effect of Zuogui Wan on cell proliferation ability using MTT assay.The content of alkaline phosphatase in cells was detected using microplate assay.Osteogenesis was detected using Alizarin Red S staining.The transcription levels of ESR1,ETS1,PPARγ,RAS,STAT3,MAPK1,MAPK3,OSTERIX,RUNX2,and OPG genes were detected using qRT PCR.The expression of RAS,OPG,ERK,P-ERK,ETS1,and PPARγproteins was analyzed using Western blot.Results①Zuo Gui Wan has 102 compounds related to postmenopausal osteoporosis,with 189 intersecting targets,including MAPK1,PPARγ,ETS1,and other key targets.②Compared with the blank serum group,the cell activity of the Zuogui Wan drug containing serum group was higher.③Alizarin red S staining showed that the bone ability of the Zuogui Wan drug containing serum group was superior to that of the blank serum group(P<0.05),and the alkaline phosphatase level was higher than that of the blank serum group(P<0.05).④The transcription levels of MAPK1,RAS,ETS1,STAT3,OSTERIX,OPG,and RUNX2 genes in the serum containing Zuogui Wan were increased(P<0.05),while the transcription levels of ESR1,PPARγ,and MAPK3 genes were decreased(P<0.05).⑤Compared with the blank serum group,the expression of p-ERK,RAS,ETS1,and OPG proteins was upregulated(P<0.05)and PPARγprotein was downregulated(P<0.05)in the Zuo Gui Wan drug containing serum group,while ERK showed no significant difference.Conclusion The drug containing Zuo Gui Wan can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells,and its mechanism may be related to changes in the ERK/ETS1/PPARγsignaling pathway.
作者
顾金光
董永丽
李辰华
秦伟凯
魏戌
杨国华
GU Jinguang;DONG Yongli;LI Chenhua;QIN Weikai;WEI Xu;YANG Guohua(Wangjing Hospital,China Academy of Chinese Medical Sciences,Beijing 100102,China)
出处
《中国骨质疏松杂志》
北大核心
2025年第8期1142-1151,共10页
Chinese Journal of Osteoporosis
基金
国家自然科学基金(82305278)
中国中医科学院优秀青年科技人才培养专项(ZZ17-YQ-012)
第七批全国老中医药专家学术经验继承工作项目(202276)。
关键词
左归丸
绝经后骨质疏松症
成骨分化
网络药理学
Zuo Gui Wan
postmenopausal osteoporosis
osteogenic differentiation
network pharmacology
