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| LC-MS联合网络药理学分析探讨壮骨强肌方治疗POP作用机制 |
| Study on the mechanism of POP treatment with Zhuanggu Qiangji formula by LC-MS combined with network pharmacological analysis |
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| DOI：10.3969/j.issn.1006-7108.2025.05.014 |
| 中文关键词: 原发性骨质疏松症 壮骨强肌方 MAPK 液相色谱-质谱联用技术 网络药理学 |
| 英文关键词:primary osteoporosis Zhuanggu Qiangji formula MAPK liquid chromatography-mass spectrometry network pharmacology analyses |
| 基金项目:国家自然科学基金（82274551）；广州中医药大学“双一流”与高水平大学学科协同创新团队重点项目（2021XK21） |
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| 中文摘要: |
| 目的 利用液相色谱-质谱联用技术（LC-MS）和网络药理学分析壮骨强肌方（ZGQJ）的潜在治疗靶点，进而通过细胞实验验证其对成骨细胞分化的影响和作用机制。方法 利用LC-MS鉴定ZGQJ化学成分；通过知网、PubMed及Web of Science数据库筛选ZGQJ关键化合物；Swiss Target Prediction数据库获得化合物作用靶点；GeneCards、DISEASE和CTD数据库获得POP疾病相关靶点；ZGQJ与POP靶点取交集，继而进行潜在治疗靶点的功能富集分析；制作ZGQJ含药血清并干预成骨分化诱导过程，予以ALP和ARS染色；Western blot检测RUNX2、OSX、P-ERK/ERK及P-P38/P38蛋白表达水平。结果 ZGQJ共鉴定到607个化合物，筛选出16个关键化合物并匹配到459个靶点，与POP靶点取交集，最终获得50个ZGQJ治疗POP的潜在作用靶点。功能富集分析显示：主要涉及细胞对脂质反应及磷酸化等生物过程；磷脂酰肌醇3-激酶复合物Ⅰ类及细胞外基质等细胞组分；磷酸转移酶活性及磷脂酰肌醇激酶活性等分子功能；VEGF、PI3K-Akt、自噬及MAPK等信号通路。细胞实验验证结果显示：ZGQJ-M及ZGQJ-H含药血清组ALP染色及ARS染色效果优于空白血清组（P<0.05），两组诱导成骨分化关键蛋白RUNX2和OSX表达增高（P<0.05），时间梯度ZGQJ-H含药血清干预后P-P38和P-ERK蛋白表达均呈现先升后降的激活趋势（P<0.05）。结论 ZGQJ能够促进成骨分化并激活P38/ERK MAPK信号通路，其促成骨的机制可能由P38/ERK MAPK信号通路介导。 |
| 英文摘要: |
| Objective Liquid chromatography-mass spectrometry (LC-MS) and network pharmacology analyses were utilized to predict the potential therapeutic targets of Zhuanggu Qiangji formula (ZGQJ). Its effects and mechanisms on osteoblast (OB) differentiation were further verified with cellular experiments. Methods The chemical composition of ZGQJ was identified using LC-MS. The key compounds of ZGQJ were screened from CNKI, PubMed, and Web of Science databases. The potential targets of the key compounds were obtained with the Swiss Target Prediction database. GeneCards, DISEASE, and CTD databases were used to obtain POP disease-related targets. The intersection of ZGQJ and POP disease targets was taken as a potential target of ZGQJ for the treatment of POP. Functional enrichment analysis was performed for potential therapeutic targets. ZGQJ-containing serum was prepared and intervened in the process of osteogenic differentiation. Alkaline phosphatase staining (ALP) and Alizarin red staining (ARS) were applied. The protein expression levels of RUNX2, OSX, P-ERK/ERK, and P-P38/P38 were detected using Western blotting. Results A total of 607 compounds were identified with LC-MS in ZGQJ. Sixteen key compounds were screened out. The key compounds were matched to 459 targets, which were intersected with POP disease-related targets. Finally 50 potential targets of ZGQJ for POP treatment were obtained. Further functional enrichment analyses showed that the results were related to biological processes such as cellular response to lipid and phosphorylation, cellular components such as phosphatidylinositol 3-kinase complex class I and extracellular matrix, molecular functions such as phosphotransferase activity and phosphatidylinositol kinase activity, signaling pathways such as VEGF, PI3K-Akt, autophagy, and MAPK. The results of cellular experiments showed that ALP staining and ARS staining in ZGQJ-M and ZGQJ-H groups were better than that in the control group (P<0.05). The expressions of RUNX2 and OSX proteins increased in both groups (P<0.05). Both P-P38 and P-ERK protein expressions were activated after time gradient ZGQJ-H intervention (P<0.05). Conclusion ZGQJ is able to promote osteogenic differentiation, which may be mediated by the P38/ERK MAPK signaling pathway. |
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