Zhao Xin, Hu Yanping, Xiong Heran, Dai Bo, Liu Wei, Chen Meiling, Zhou Fan, Xiang Chao, Wang Song
Department of Tuina, Wuhan Hospital of Traditional Chinese Medicine, No.49 Lihuangpi Road, Wuhan, 430017 Hubei P.R. China.
Hubei University of Chinese Medicine, Wuhan, 430065 Hubei P.R. China.
Cytotechnology. 2025 Jun;77(3):110. doi: 10.1007/s10616-025-00773-z. Epub 2025 May 28.
This study mainly explored the potential mechanism of Huangqi Guizhi Wuwu Decoction (HGWD) in the treatment of osteoarthritis (OA) based on network pharmacology, and to investigate whether HGWD promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by upregulating ESR1 based on in vitro experiments. The core target genes and potential signaling pathways related to HGWD in OA treatment were analyzed using network pharmacology analysis. BMSCs were isolated from rats to induce for adipogenic/osteogenic differentiation, which were assessed by Oil Red O staining and Alizarin Red staining, respectively. ESR1 knockdown lentivirus was transfected into BMSCs and different concentrations of rat HGWD-containing serum was prepared to treat BMSCs. Cell proliferation activity was measured by CCK-8 assay to select the optimal concentration for further experiments. Cells were treated with 10% HGWD-containing serum and transfected with ESR1 knockdown lentivirus. Osteogenic differentiation was assessed by ALP staining, ALP activity measurement, and Alizarin Red staining. The expression of osteogenic differentiation-related genes OCN, RUNX2, and COL1A1 was detected by qRT-PCR and Western blot. Network pharmacology analysis revealed that ESR1 is one of the core targets of HGWD in OA treatment. HGWD-containing serum promoted proliferation and osteogenic differentiation ability of BMSCs, and also increased ESR1 expression. The promoting effects of HGWD-containing serum on proliferation and osteogenic differentiation were partially polished in response to ESR1 knockdown in BMSCs. Based on the collective evidence, the therapeutic effects of HGWD in OA may be achieved by promoting osteogenic differentiation of BMSCs via upregulating ESR1 expression.
The online version contains supplementary material available at 10.1007/s10616-025-00773-z.
本研究主要基于网络药理学探讨黄芪桂枝五物汤（HGWD）治疗骨关节炎（OA）的潜在机制，并通过体外实验研究HGWD是否通过上调ESR1促进骨髓间充质干细胞（BMSCs）的成骨分化。采用网络药理学分析方法分析HGWD治疗OA的核心靶基因和潜在信号通路。从大鼠分离BMSCs并诱导其向脂肪/成骨分化，分别通过油红O染色和茜素红染色进行评估。将ESR1基因敲低慢病毒转染至BMSCs，并制备不同浓度含大鼠HGWD血清处理BMSCs。采用CCK-8法检测细胞增殖活性，以选择进一步实验的最佳浓度。用10%含HGWD血清处理细胞并转染ESR1基因敲低慢病毒。通过碱性磷酸酶（ALP）染色、ALP活性测定和茜素红染色评估成骨分化。采用qRT-PCR和蛋白质免疫印迹法检测成骨分化相关基因OCN、RUNX2和COL1A1的表达。网络药理学分析显示ESR1是HGWD治疗OA的核心靶点之一。含HGWD血清促进了BMSCs的增殖和成骨分化能力，同时也增加了ESR1的表达。在BMSCs中敲低ESR1后，含HGWD血清对增殖和成骨分化的促进作用部分被消除。综合证据表明，HGWD治疗OA的作用可能是通过上调ESR1表达促进BMSCs的成骨分化来实现的。
在线版本包含可在10.1007/s10616-025-00773-z获取的补充材料。
