Fu Dapeng, Lu Jianmin, Yang Sheng
Department of Orthopedic Surgery, Affiliated Zhongshan Hospital of Dalian University, Dalian, China
Department of Orthopedic Surgery, Affiliated Zhongshan Hospital of Dalian University, Dalian, China.
Ann Clin Lab Sci. 2016 Jul;46(4):353-9.
The association between obesity and reactive oxygen species (ROS) production in osteoarthritis (OA) patients has already been identified. However, the specific mechanism underlying ROS production and OA progression has never been elucidated. Osteoarthritic cartilage was obtained from patients undergoing total hip arthroplasty, and chondrocytes were isolated from these tissues. The cells were treated with varying concentrations (10,100,500 μM, and 5 mM) of oleic/palmitate (O/P) mixture at different times, that is at 6, 24, and 48 h. Cell viability was determined using MTT assay. ROS production was detected using immunofluorescence and flow cytometry. The protein levels of NOX4 and cleaved-caspase3 were detected using Western blot assay. O/P significantly decreased cell viability at 10, 100, 500 μM, and 5 mM in a dose-dependent manner. Furthermore, the cell viability was reduced by 500 μM O/P mixture at 6, 24, and 48 h in a time-dependent manner. Pretreatment with 500 μM O/P significantly enhanced ROS production and cell apoptosis in chondrocytes. Furthermore, treatment with O/P mixture significantly enhanced the expression of NOX4 and caspase3 activation in a dose- and time- dependent manner. More importantly, inhibition of NOX4 could partially eliminate O/P-induced chondrocytes apoptosis by reducing ROS production. To conclude, O/P treatment enhances ROS production and cell apoptosis mainly by upregulating the protein levels of NOX4 and caspase3 activation in chondrocytes, indicating a potential therapeutic target of OA in obesity patients.
肥胖与骨关节炎（OA）患者体内活性氧（ROS）生成之间的关联已得到确认。然而，ROS生成与OA进展背后的具体机制从未被阐明。从接受全髋关节置换术的患者身上获取骨关节炎软骨，并从这些组织中分离软骨细胞。在不同时间点（即6小时、24小时和48小时）用不同浓度（10、100、500μM和5 mM）的油酸/棕榈酸酯（O/P）混合物处理细胞。使用MTT法测定细胞活力。使用免疫荧光和流式细胞术检测ROS生成。使用蛋白质印迹法检测NOX4和裂解型半胱天冬酶3的蛋白水平。O/P在10、100、500μM和5 mM时以剂量依赖性方式显著降低细胞活力。此外，500μM O/P混合物在6小时、24小时和48小时时以时间依赖性方式降低细胞活力。用500μM O/P预处理显著增强软骨细胞中的ROS生成和细胞凋亡。此外，用O/P混合物处理以剂量和时间依赖性方式显著增强NOX4的表达和半胱天冬酶3的激活。更重要的是，抑制NOX4可通过减少ROS生成部分消除O/P诱导的软骨细胞凋亡。总之，O/P处理主要通过上调软骨细胞中NOX4的蛋白水平和半胱天冬酶3的激活来增强ROS生成和细胞凋亡，表明肥胖患者OA的潜在治疗靶点。
