Effects of icariin-containing serum active ingredients on inflammatory chondrocytes: a metabolomics analysis

doi:10.12307/2026.907

Abstract

Abstract: BACKGROUND: Icariin, the main active component of the herb Epimedium, has been confirmed to possess cartilage-protective effects, but its in vivo metabolic transformation and mechanisms remain incompletely elucidated. In recent years, metabolomics technology has provided new tools for analyzing the complex in vivo metabolic processes of traditional Chinese medicine (TCM), while studies on medicated serum offer a more realistic reflection of the pharmacodynamic material basis of TCM.
OBJECTIVE: To analyze the pharmacodynamic material basis of icariin-containing serum using untargeted metabolomics.
METHODS: Untargeted metabolomics was employed to compare differential metabolites and related signaling pathways between icariin-containing serum and blank serum. The cell counting kit-8 assay was used to evaluate the effect of gentisic acid on the viability of C57BL/6J mouse chondrocytes and determine the optimal concentration. A lipopolysaccharide-induced inflammatory chondrocyte model was established. After 24 hours of intervention with 75 μmol/L gentisic acid, mitochondrial membrane potential and mRNA expression of ATP5F1A and ATP5F1B mRNA were detected.
RESULTS AND CONCLUSION: (1) Untargeted metabolomics revealed that the effects of icariin-containing serum might be associated with taurine and hypotaurine metabolism pathways, with the key metabolite gentisic acid influencing chondrocyte energy metabolism by regulating mitochondrial ATP synthase (ATP5F1A/ATP5F1B). (2) Gentisic acid promoted chondrocyte proliferation and restored mitochondrial membrane potential in inflammation-induced chondrocytes. (3) Gentisic acid significantly increased the expression levels of ATP5F1A and ATP5F1B in lipopolysaccharide-treated chondrocytes. These findings indicate that icariin-containing serum has a defined metabolic material basis for promoting cartilage repair, with the taurine and hypotaurine metabolism pathways serving as its core mechanism. This may involve promoting L-cysteine expression and modulating these metabolic pathways to exert anti-inflammatory effects, thereby maintaining cartilage homeostasis and facilitating repair. Gentisic acid may interact with ATP5F1A/ATP5F1B proteins to regulate chondrocyte energy metabolism, contributing to cartilage repair, and holds promise as a potential therapeutic agent for knee osteoarthritis.

Key words: icariin-containing serum, osteoarthritis, chondrocytes, inflammatory response, untargeted metabolomics, taurine, gentisic acid

CLC Number:

Chen Weinan, Li Yusheng, Li Linzhen, Jiao Hongzhuo, Zhang Mingzhe, Wang Jianlong, Zhang Juntao. Effects of icariin-containing serum active ingredients on inflammatory chondrocytes: a metabolomics analysis[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(36): 9429-9436.

Figures/Tables 6

2.1 非靶向代谢组数据分析主成分分析是通过正交变换，将一组可能相关的观测变量转换为线性不相关变量(即主成分)的统计方法[28-29]。该方法可揭示数据内在结构，从而更清晰地解释变量间关系。血清样本的主成分分析得分散点图如图1A所示。选用正交偏最小二乘法-判别分析(Orthogonalprojections to Latent Structures-Discriminant Analysis，OPLS-DA)的统计方法分析结果[30]。通过正交偏最小二乘法-判别分析，可过滤掉代谢物中与分类变量无关联的正交变量，之后分别对非正交变量和正交变量开展剖析，进而得到更可靠的代谢物组间差异和与淫羊藿苷含药血清相关程度的信息内容[30]。对数据进行UV格式化操作，针对第一主成分做正交偏最小二乘法-判别建模分析，开展200次置换检验，进一步检验模型有效性[30]。各个样本两两比较的正交偏最小二乘法-判别模型得分散点图和200次置换检验结果如图1B，C所示。鉴于UHPLC-Q/Orbitrap MS代谢组数据的固有特性，采用多元变量统计方法对数据进行分析。以|log2FC| > 1，P < 0.05， VIP > 0.6为标准筛选差异代谢物，共筛选差异代谢物130个，将筛选差异代谢物的结果以火山图的形式进行可视化(图1D)，其中脂肪酰类、苯及其衍生物和类固醇及其衍生物这3类代谢物得到了富集(图2A)，进一步分析差异代谢物，发现龙胆酸、牛磺胆酸等有机酸变化显著(图2B)。通过MetaboAnalyst网站进行通路富集分析，通过差异代谢物来筛选相关代谢通路，结果如图1E，F所示。结合淫羊藿苷等黄酮类物质在体内的代谢过程涉及龙胆酸等有机酚酸的转化，选取龙胆酸作为进一步研究的关键组分。 2.2 龙胆酸作用靶点分析将龙胆酸与氨基树脂交联，然后与软骨细胞裂解液共孵育。通过Pulldown实验[26]，根据差异条带分析龙胆酸可能的结合靶点(图3A)。质谱结果显示，龙胆酸与多"

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