Yang Bo, Huang Haisheng, He Qisong, Lu Wei, Zheng Lu, Cui Li
Department of Orthopedic Surgery, Shanghai TCM-Integrated Hospital Shanghai University of TCM, No. 230, Baoding Road, Hongkou, Shanghai, China.
Department of Acupuncture, Shanghai TCM-Integrated Hospital Shanghai University of TCM, No. 230, Baoding Road, Hongkou, Shanghai, China.
Evid Based Complement Alternat Med. 2021 Dec 8;2021:1905995. doi: 10.1155/2021/1905995. eCollection 2021.
Oxidative stress-induced chondrocyte apoptosis and degradation of the extracellular matrix (ECM) play an important role in the progression of osteoarthritis (OA). In addition, tert-butylhydroquinone (TBHQ) is an activator of the nuclear factor erythroid derived-2-related factor 2 (Nrf2). The present study aimed to determine the effectiveness of TBHQ in preventing the apoptosis of chondrocytes and degradation of the extracellular matrix, induced by oxidative stress, in vitro. Therefore, rat chondrocytes were exposed to 20 M tert-butyl hydroperoxide (TBHP) for 24 h to establish an oxidative damage model, in vitro. Thereafter, cell viability was evaluated using the Cell Counting Kit-8 assay. Moreover, the level of ROS was determined through 2',7'-dichlorofluorescein diacetate staining. The mitochondrial membrane potential of chondrocytes was also measured using JC-1. Furthermore, cell apoptosis was assessed through Annexin V-fluorescein isothiocyanate/propidium iodide staining. The study also performed Western blotting and qPCR to evaluate the expression of extracellular matrix components, matrix catabolic enzymes, and changes in signalling pathways. The results showed that 2.5 and 5 M of TBHQ reduced the TBHP-induced generation of excessive ROS and improved cell viability. Additionally, 2.5 and 5 M of TBHQ prevented mitochondrial damage and apoptosis in rat chondrocytes. Treatment with TBHQ also increased the mRNA and protein expression levels of aggrecan and collagen II. However, TBHQ reduced the mRNA and protein expression levels of matrix metalloproteinase 3 (MMP3) and matrix metalloproteinase 13 (MMP13) in rat chondrocytes. In addition, treatment with TBHQ enhanced the protein expression levels of Nrf2, NADPH quinone oxidoreductase 1 (NQO-1), and hemeoxygenase-1 (HO-1) in rat chondrocytes. The current study showed that TBHQ was not only effective in protecting against TBHP-induced oxidative stress but also inhibited the apoptosis of rat chondrocytes and degradation of the ECM by activating the Nrf2 pathway. The results therefore suggest that TBHQ holds potential for use in the treatment of OA.
氧化应激诱导的软骨细胞凋亡和细胞外基质（ECM）降解在骨关节炎（OA）进展中起重要作用。此外，叔丁基对苯二酚（TBHQ）是核因子红细胞2相关因子2（Nrf2）的激活剂。本研究旨在确定TBHQ在体外预防氧化应激诱导的软骨细胞凋亡和细胞外基质降解的有效性。因此，将大鼠软骨细胞暴露于20μM叔丁基过氧化氢（TBHP）24小时以建立体外氧化损伤模型。此后，使用细胞计数试剂盒-8测定法评估细胞活力。此外，通过2'，7'-二氯荧光素二乙酸酯染色测定ROS水平。还使用JC-1测量软骨细胞的线粒体膜电位。此外，通过膜联蛋白V-异硫氰酸荧光素/碘化丙啶染色评估细胞凋亡。该研究还进行了蛋白质印迹和qPCR，以评估细胞外基质成分、基质分解代谢酶的表达以及信号通路的变化。结果表明，2.5μM和5μM的TBHQ减少了TBHP诱导的过量ROS生成并提高了细胞活力。此外，2.5μM和5μM的TBHQ预防了大鼠软骨细胞的线粒体损伤和凋亡。用TBHQ处理还增加了聚集蛋白聚糖和胶原蛋白II的mRNA和蛋白质表达水平。然而，TBHQ降低了大鼠软骨细胞中基质金属蛋白酶3（MMP3）和基质金属蛋白酶13（MMP13）的mRNA和蛋白质表达水平。此外，用TBHQ处理增强了大鼠软骨细胞中Nrf2、NADPH醌氧化还原酶1（NQO-1）和血红素加氧酶-1（HO-1）的蛋白质表达水平。当前研究表明，TBHQ不仅有效地预防了TBHP诱导的氧化应激，还通过激活Nrf2途径抑制了大鼠软骨细胞的凋亡和ECM的降解。因此，结果表明TBHQ在OA治疗中具有应用潜力。
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