股骨组织的代谢组学分析揭示知母对骨质疏松大鼠代谢途径的影响

doi:10.5246/jcps.2025.02.011

中国药学(英文版) ›› 2025, Vol. 34 ›› Issue (2): 135-149.DOI: 10.5246/jcps.2025.02.011

股骨组织的代谢组学分析揭示知母对骨质疏松大鼠代谢途径的影响

温雨馨¹, 蒋琪¹, 黄壮¹, 陈鹏宇¹, 洪星¹, 汪琼²^,³, 黄韬⁴^,^*(), 韩林涛¹^,²^,^*()

^1. 湖北中医药大学药学院, 湖北武汉 430065
^2. 湖北时珍实验室, 湖北武汉 430060
^3. 湖北中医药大学基础医学院, 湖北武汉 430065
^4. 武汉市红十字会医院骨科, 湖北武汉 430015

收稿日期:2024-10-10 修回日期:2024-11-21 接受日期:2024-12-04 出版日期:2025-03-01 发布日期:2025-03-02
通讯作者: 黄韬, 韩林涛

Metabolomics analysis of femoral tissue reveals the impact of Anemarrhenae Rhizoma on metabolic pathways in osteoporotic rats

Yuxin Wen¹, Qi Jiang¹, Zhuang Huang¹, Pengyu Chen¹, Xing Hong¹, Qiong Wang²^,³, Tao Huang⁴^,^*(), Lintao Han¹^,²^,^*()

¹ College of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, Hubei, China
² Hubei Shizhen Laboratory, Wuhan 430060, Hubei, China
³ Basic Medical College, Hubei University of Chinese Medicine, Wuhan 430065, Hubei, China
⁴ Orthopaedic Department, Wuhan Red Cross Hospital, Wuhan 430015, Hubei, China

Received:2024-10-10 Revised:2024-11-21 Accepted:2024-12-04 Online:2025-03-01 Published:2025-03-02
Contact: Tao Huang, Lintao Han
Supported by:
Wuhan Health Research Fund (Grant No. WZ21A06); National Natural Science Foundation of China (Grant No. 81903815).

摘要/Abstract

摘要：

本研究旨在评估知母(AR)对骨质疏松症大鼠的治疗潜力, 并阐明知母缓解骨质疏松症(OP)的代谢途径。通过卵巢切除术(OVX)诱导大鼠骨质疏松症, 然后口服高剂量或低剂量的AR以及戊酸雌二醇, 为期14周。采用显微计算机断层扫描(Micro-CT)检查大鼠股骨组织形态, 并采用酶联免疫吸附试验(ELISA)检测大鼠血清中PINP和CTX-I的水平, 以评价AR治疗OP的疗效。此外, 还利用气相色谱-质谱法(GC-MS)对股骨组织进行了代谢组学分析。通过UPLC-MS/MS和在线数据库搜索, 确定了AR的生物活性成分及其对OP的治疗靶点, 并通过整合差异代谢物和潜在靶点建立了代谢网络。此外, Western blot分析确认了关键的分子靶点。研究结果表明, AR 治疗能明显减轻大鼠因OVX引起的OP。代谢组学分析表明, AR通过调节10种关键代谢物(如亚油酸和肌醇)的水平以及影响5种关键代谢途径(包括亚油酸代谢和磷脂肌醇信号系统)来发挥其作用。其中, 亚油酸代谢途径是进一步研究的重点。Western blot分析表明, AR可逆转CYP1A2和CYP2C9(与亚油酸代谢途径相关的两个靶点)在OP大鼠体内的上调。总之, AR可能通过调节 OVX 大鼠体内的代谢物水平来改善OP, 其作用机制可能以调节亚油酸代谢途径为中心。

关键词: 知母, 骨质疏松症, 大鼠, 代谢组学

Abstract:

This study aimed to assess the therapeutic potential of Anemarrhenae Rhizoma (AR) in osteoporotic rats and to elucidate the metabolic pathways involved in AR’s role in alleviating osteoporosis (OP). OP was induced in rats through ovariectomy (OVX), followed by oral administration of either high or low doses of AR, as well as estradiol valerate, over a 14-week period. Micro-computed tomography (Micro-CT) was employed to examine the femur tissue morphology, while enzyme-linked immunosorbent assay (ELISA) was adopted to measure serum levels of PINP and CTX-I to evaluate AR’s efficacy in treating OP. Additionally, metabolomic profiling of femur tissues was conducted using gas chromatography-mass spectrometry (GC-MS). The bioactive components of AR, along with its therapeutic targets for OP, were identified through UPLC-MS/MS and online database searches, and metabolic networks were established by integrating differential metabolites and potential targets. Furthermore, Western blotting analysis confirmed key molecular targets. The findings revealed that AR treatment significantly mitigated OVX-induced OP in rats. Metabolomic analysis indicated that AR exerted its effects by modulating the levels of 10 key metabolites (such as linoleic acid and inositol) and influencing five crucial metabolic pathways, including linoleic acid metabolism and the phosphoinositide signaling system. Among these, the linoleic acid metabolic pathway emerged as a pivotal focus for further investigation based on the constructed interaction network of differential metabolites and targets. Western blotting analysis demonstrated that AR reversed the up-regulation of CYP1A2 and CYP2C9, two targets associated with the linoleic acid metabolic pathway, in OP rats. In conclusion, AR appeared to ameliorate OP by modulating metabolite levels in OVX rats, with its mechanism of action likely centered on regulating the linoleic acid metabolic pathway.

Key words: Anemarrhenae Rhizoma, Osteoporosis, Rats, Metabolomics

Supporting:

温雨馨, 蒋琪, 黄壮, 陈鹏宇, 洪星, 汪琼, 黄韬, 韩林涛. 股骨组织的代谢组学分析揭示知母对骨质疏松大鼠代谢途径的影响[J]. 中国药学(英文版), 2025, 34(2): 135-149.

Yuxin Wen, Qi Jiang, Zhuang Huang, Pengyu Chen, Xing Hong, Qiong Wang, Tao Huang, Lintao Han. Metabolomics analysis of femoral tissue reveals the impact of Anemarrhenae Rhizoma on metabolic pathways in osteoporotic rats[J]. Journal of Chinese Pharmaceutical Sciences, 2025, 34(2): 135-149.

图/表 6

Table 1. Results of active components of AR by UPLC-MS/MS.

Figure 1. UPLC-MS/MS Analysis of AR. (A) Extraction ion chromatogram of AR (positive ions); (B) Extraction ion chromatogram of AR (negative ions). The abscissa is the retention time of metabolite determination, and the ordinate is the ion current intensity of ion determination.

Figure 2. (A) Representative μCT images; (B–H) Quantitative analyses of parameters regarding bone microstructure, including BMD, BS/TV, BV, BV/TV, Tb.N, Tb.Pf, and SMI (n = 3); (I–J) The changes in serum CTX-I and PINP were observed among the groups (n = 7). All bar graphs are presented as the mean ± SD. #P < 0.05 and ##P < 0.01 vs. sham group; *P < 0.05 and **P < 0.01 vs. OVX group. BMD, Bone mineral density; bone BS/TV, surface tissue volume; BV, bone volume; BV/TV, bone mass percentage; Tb. N, trabecular number; Tb. Pf, trabecular pattern factor; SMI, structural model index.

Figure 3. (A) PCA score plots; (B) OPLS-DA score plots; (C) The corresponding validation plots based on 200 times permutation tests; (D) Heat map of the differential metabolites; (E) Metabolic pathways of the significant metabolites.

Figure 4. (A) AR-OP intersection target Venn diagram; (B) Cytoscape software was used to visualize the metabolite data and potential targets; (C) Linoleic acid metabolic sub-network (Red hexagons, pink hexagons, gray diamonds, green rounded rectangles, blue circles, and purple circles represent key compounds, other compounds, reactions, proteins, key targets, and other targets, respectively).

Figure 5. (A) Western blotting representative images of CYP1A2 and CYP2C9; (B–C) Protein expression of CYP1A2 and CYP2C9 (#P < 0.05 and ##P < 0.01 vs. sham group; *P < 0.05 and **P < 0.01 vs. OVX group). (D) Effect of linoleic acid metabolism on osteogenesis.

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股骨组织的代谢组学分析揭示知母对骨质疏松大鼠代谢途径的影响

Metabolomics analysis of femoral tissue reveals the impact of Anemarrhenae Rhizoma on metabolic pathways in osteoporotic rats

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