Glycolytic enzyme PKM2 regulates cell senescence but not inflammation in the process of osteoarthritis.

Chondrocyte senescence is an important mechanism underlying osteoarthritis in the senile population and is characterized by reduced expressions of the extracellular matrix proteins. The involvement of glycolysis and the tricarboxylic acid cycle in the development of osteoarthritis is inclusive. The present study aims to investigate the role of the glycolytic enzyme M2 isoform of pyruvate kinase (PKM2) in chondrocytes in senescence and inflammation. Primary chondrocytes are isolated from the knee joints of neonatal mice. Small interfering RNAs (siRNAs) against PKM2 are transfected using lipofectamine. RNA sequencing is conducted in primary chondrocytes with the PKM2 gene deleted. Cell apoptosis, autophagy, reactive oxygen species measurement, and senescent conditions are examined. The glycolytic rate in cells is measured by Seahorse examination. Interleukin 1-β (IL-1β) increases the protein expressions of matrix metallopeptidases (MMP)13 and PKM2 and reduces the protein expression of collagen type II (COL2A1) in primary chondrocytes. Silencing of PKM2 alters the protein expressions of MMP13, PKM2, and COL2A1 in the same pattern in quiescent and stimulated chondrocytes. RNA sequencing analysis reveals that PKM2 silencing reduces senescent biomarker p16INK4a expression. Compared with low-passage chondrocytes, high-passage chondrocytes exhibit increased expression of p16INK4a and reduced expression of COL2A1. Silencing of PKM2 reduces SA-β-Gal signals and increases COL2A1 expression in high-passage chondrocytes. Seahorse assay reveals that PKM2 deletion favors the tricarboxylic acid cycle in mitochondria in low- but not in high-passage chondrocytes. In summary, the glycolytic enzyme PMK2 modulates chondrocyte senescence but does not participate in the regulation of inflammation. To understand the role of PKM2 in chondrocytes, RNA sequencing was conducted in primary mouse chondrocytes with siRNA-mediated Pkm2 silencing.

软骨细胞衰老（chondrocyte senescence）是老年人群骨关节炎（osteoarthritis）的核心发病机制之一，其典型特征为细胞外基质蛋白（extracellular matrix proteins）表达量下调。糖酵解（glycolysis）与三羧酸循环（tricarboxylic acid cycle）在骨关节炎发生发展中的参与机制尚未阐明。本研究旨在探讨糖酵解关键酶丙酮酸激酶M2亚型（PKM2, pyruvate kinase M2 isoform）在软骨细胞衰老与炎症反应中的调控作用。实验提取新生小鼠膝关节组织中的原代软骨细胞，采用脂质体转染试剂（Lipofectamine）将靶向PKM2的小干扰RNA（siRNA, small interfering RNA）转染至细胞内，并对PKM2基因敲除的原代软骨细胞开展RNA测序（RNA-seq）分析。检测指标包括细胞凋亡（cell apoptosis）、自噬（autophagy）、活性氧（ROS, reactive oxygen species）水平及细胞衰老状态；采用Seahorse细胞能量代谢分析仪检测细胞糖酵解速率。结果显示：白细胞介素1-β（IL-1β, Interleukin 1-β）可上调原代软骨细胞中基质金属蛋白酶13（MMP13, matrix metallopeptidases 13）与PKM2的蛋白表达水平，并下调Ⅱ型胶原（COL2A1, collagen type II）的蛋白表达；沉默PKM2可在静息与炎症刺激后的软骨细胞中，以相同模式调控MMP13、PKM2及COL2A1的蛋白表达。RNA测序分析表明，沉默PKM2可降低衰老标志物p16INK4a的表达水平。与低代次软骨细胞相比，高代次软骨细胞的p16INK4a表达量上调，而COL2A1表达量下调；在高代次软骨细胞中，沉默PKM2可减弱衰老相关β-半乳糖苷酶（SA-β-Gal）的阳性信号，并上调COL2A1的表达。Seahorse能量代谢分析显示，PKM2敲除可促进低代次软骨细胞线粒体中的三羧酸循环，但对高代次软骨细胞无此调控效应。综上，糖酵解酶PKM2可调控软骨细胞衰老，但不参与炎症反应的调控。为进一步明确PKM2在软骨细胞中的功能，本研究对经siRNA介导沉默PKM2的原代小鼠软骨细胞开展了RNA测序分析。