单细胞蛋白质活性分析揭示了一种新型的软骨细胞亚群，以及与抗衰老和 OA 进展相关的相应关键主调控蛋白。

BACKGROUND: Osteoarthritis (OA) is a prevalent senescence-related disease with substantial joint pain, loss of joint function, and cartilage degeneration. Because of the paucity of single-cell studies of OA and the gene dropout problem of single-cell RNA sequencing, it is difficult to acquire an in-depth understanding of the molecular characteristics of various chondrocyte clusters. METHODS: Here, we aimed to provide new insights into chondrocyte senescence and a rationale for the development of effective intervention strategies for OA by using published single-cell RNA-sequencing data sets and the metaVIPER algorithm (Virtual Inference of Protein activity by Enriched Regulon). This algorithm was employed to present a proteome catalog of 62,449 chondrocytes from the cartilage of healthy individuals and OA patients at single-cell resolution. Furthermore, histopathologic analysis was carried out in cartilage samples from clinical patients and experimental mouse models of OA to validate above results. RESULTS: We identified 16 protein-activity-based chondrocyte clusters as well as the underlying master regulators in each cluster. By assessing the enrichment score of each cluster in bulk RNA-sequencing data, followed by gene-set variation analysis, we preliminarily identified a novel subpopulation of chondrocytes (cluster 3). This clinically relevant cluster was predicted to be the main chondrocyte cluster responsible for maintaining cellular homeostasis and anti-senescence. Specifically, we uncovered a set of the key leading-edge proteins of cluster 3 by validating the robustness of the above results using another human chondrocyte single-cell RNA-sequencing data set, consisting of 24,675 chondrocytes. Furthermore, cartilage samples from clinical patients and experimental mouse models of OA were used to evaluate the expression patterns of these leading-edge proteins, and the results indicated that NDRG2, TSPYL2, JMJD6 and HMGB2 are closely associated with OA pathogenesis and might play critical roles in modulating cellular homeostasis and anti-senescence in chondrocytes. CONCLUSION: Our study revealed a novel subpopulation of chondrocytes that are critical for anti-progression of OA and the corresponding master regulator proteins, which might serve as therapeutic targets in OA.

背景：骨关节炎（OA）是一种普遍的与衰老相关的疾病，其特征为关节疼痛剧烈、关节功能丧失和软骨退化。由于 OA 的单细胞研究较少，且单细胞 RNA 测序存在基因缺失问题，因此很难深入了解各种软骨细胞簇的分子特征。

方法：本研究旨在利用已发表的单细胞 RNA 测序数据集和 metaVIPER 算法（通过富集调控子虚拟推断蛋白质活性），为软骨细胞衰老提供新的见解，并为 OA 的有效干预策略的制定提供依据。该算法用于呈现健康个体和 OA 患者软骨中单细胞分辨率下 62449 个软骨细胞的蛋白质组目录。此外，对来自临床患者和 OA 实验性小鼠模型的软骨样本进行组织病理学分析，以验证上述结果。

结果：我们鉴定了 16 个基于蛋白质活性的软骨细胞簇，以及每个簇中的潜在主调控因子。通过评估每个簇在批量 RNA 测序数据中的富集分数，然后进行基因集变异分析，我们初步鉴定了一种新的软骨细胞亚群（簇 3）。该亚群具有临床相关性，预测为主要的软骨细胞簇，负责维持细胞内稳态和抗衰老。具体而言，我们通过验证另一个包含 24675 个软骨细胞的人类软骨单细胞 RNA 测序数据集，证实了上述结果的稳健性，从而鉴定出簇 3 的一组关键前沿蛋白。此外，我们还使用临床患者和 OA 实验性小鼠模型的软骨样本评估了这些前沿蛋白的表达模式，结果表明 NDRG2、TSPYL2、JMJD6 和 HMGB2 与 OA 发病机制密切相关，可能在调节软骨细胞的细胞内稳态和抗衰老中发挥关键作用。

结论：本研究揭示了一种对 OA 进展具有抗作用的新型软骨细胞亚群及其对应的主调控蛋白，它们可能成为 OA 的治疗靶点。