摘要
背景:骨关节炎现已被认为是一种代谢性疾病,既往研究表明糖酵解在骨关节炎的发生发展中起重要作用。Compound 3k作为一种新型糖酵解小分子抑制剂,具有抗炎及抗肿瘤等功效,因此可靶向糖酵解,有望为骨关节炎治疗提供新的思路。目的:基于缺氧诱导因子1α/活性氧的氧化应激通路探究Compound 3k在糖酵解过度活跃所导致的骨关节炎中的作用机制。方法:取对数生长期的ATDC5成软骨细胞,用10 ng/mL白细胞介素1β作用24 h诱导骨关节炎体外细胞模型,以CCK-8法检测不同浓度(0.25,0.5,1,2.5,5,10,15μmol/L)Compound 3k的细胞毒性,选出合适浓度进行后续实验。将软骨细胞随机分为对照组、模型组、治疗组,模型组以10 ng/mL的白细胞介素1β诱导,治疗组以Compound 3k预刺激2 h后与白细胞介素1β共培养,用CCK-8法检测各组细胞增殖情况;用ELISA试剂盒检测各组细胞炎症水平;用试剂盒检测各组细胞活性氧、细胞外乳酸脱氢酶及葡萄糖含量;qRT-PCR及Western blot检测相关炎症因子白细胞介素6、肿瘤坏死因子α及糖酵解相关基因葡萄糖转运蛋白1、甘油醛3-磷酸脱氢酶、单羧酸转运蛋白1和缺氧诱导因子1α的表达水平。结果与结论:①与对照组相比,模型组细胞增殖活性下降、糖酵解水平活跃,表现为细胞外乳酸脱氢酶含量增加(P<0.001),葡萄糖含量减少(P<0.001),相关炎症因子白细胞介素6(P<0.0001)及肿瘤坏死因子α(P<0.001),糖酵解相关基因葡萄糖转运蛋白1(P<0.001)、甘油醛3-磷酸脱氢酶(P<0.001)、单羧酸转运蛋白1(P<0.001)及缺氧诱导因子1α(P<0.001)的表达水平均上调,并伴随氧化应激,活性氧过量产生。②与模型组相比,Compound 3k的治疗有效提高细胞增殖活性,抑制过度活跃的糖酵解水平的同时,抑制了骨关节炎软骨细胞炎症(P<0.001)及糖酵解相关基因的表达(P<0.001),且抑制氧化应激,缺氧诱导因子1α的表达水平下调(P<0.0001),活性氧水平下降。③上述结果证实,Compound 3k抑制了白细胞介素1β诱导的软骨细胞炎症,其机制可能与糖酵解及缺氧诱导因子1α/活性氧介导的氧化应激有关。
BACKGROUND:Osteoarthritis is now considered a metabolic disease.Previous studies have shown that glycolysis plays an important role in the occurrence and development of osteoarthritis.Compound 3k,as a novel small molecule inhibitor of glycolysis,has anti-inflammatory and anti-tumor effects.Therefore,it can target glycolysis and is expected to provide new ideas for the treatment of osteoarthritis.OBJECTIVE:To explore the role of Compound 3k in osteoarthritis caused by glycolytic overactivity based on the hypoxia-inducible factor 1 alpha(HIF-1α)/reactive oxygen species(ROS)pathway.METHODS:ATDC5 chondroblasts at logarithmic growth phase were taken to induce osteoarthritis in an in vitro cellular model by the action of 10 ng/mL interleukin-1βfor 24 hours.The cytotoxicity of Compound 3k at different concentrations(0.25,0.5,1,2.5,5,10,15μmol/L)was detected by cell counting kit-8 assay,and the appropriate concentrations were selected for the subsequent experiments.The chondrocytes were randomly divided into control,model and treatment groups.The model group was induced with 10 ng/mL interleukin 1β,and the treatment group was pre-stimulated with Compound 3k for 2 hours and then co-cultured with interleukin 1β.The proliferation of the cells in each group was detected by the cell counting kit-8 assay;the inflammatory level of the cells in each group was detected by the ELISA kit;the ROS,extracellular lactate and glucose contents were detected using the kit;qRT-PCR and western blot were used to detect the levels of related inflammatory factors,interleukin-6 and tumor necrosis factor-α,glycolysis-related genes glucose transporter protein-1,glyceraldehyde 3-phosphate dehydrogenase,monocarboxylate transporter protein-1 and HIF-1α.RESULTS AND CONCLUSION:Compared with the control group,the model group showed a decrease in cell proliferative activity,active glycolysis level,manifested by an increase in extracellular lactate content(P<0.001)and a decrease in glucose content(P<0.001),interleukin-6(P<0.0001)and tumor necrosis factor-α(P<0.001).The expression levels of glycolysis-related genes glucose transporter protein-1(P<0.001),glyceraldehyde 3-phosphate dehydrogenase(P<0.001),monocarboxylic acid transporter protein-1(P<0.001)and HIF-1α(P<0.001)in the model group were all up-regulated,accompanied by oxidative stress and overproduction of ROS.Compared with the model group,Compound 3k treatment effectively increased cell proliferation activity and inhibited the level of overactive glycolysis(P<0.001),while suppressing the expression of genes related to inflammation(P<0.001)and glycolysis in osteoarthritic chondrocytes,inhibiting oxidative stress,downregulating the expression level of HIF-1α(P<0.0001)and decreasing the content of ROS.To conclude,Compound 3k inhibits interleukin-1βinduced chondrocyte inflammation,and its mechanism may be related to glycolysis and HIF-1α/ROS mediated oxidative stress.
作者
郭素蓉
曹士盛
穆星彤
杨青
张娟
Guo Surong;Cao Shisheng;Mu Xingtong;Yang Qing;Zhang Juan(School of Stomatology,Tianjin Medical University,Tianjin 300041,China;Department of Stomatology,Tianjin Medical University Stomatological Hospital,Tianjin 300041,China)
出处
《中国组织工程研究》
CAS
北大核心
2025年第2期363-370,共8页
Chinese Journal of Tissue Engineering Research
基金
天津市卫生科技健康项目(TJWJ2022MS015),项目负责人:张娟。
