Zhao Xu, Li Yun, Lin Xiaowen, Wang Junnan, Zhao Xuejun, Xie Juntian, Sun Tao, Fu Zhijian
Department of Pain Management, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, Shandong Province 250021, P.R. China,
J Pain Res. 2018 Nov 27;11:3003-3017. doi: 10.2147/JPR.S183594. eCollection 2018.
Ozone injection is generally used for the management of pain in diseases such as osteoarthritis (OA). Recent studies have shown that reduced autophagy in chondrocytes plays an important role in the development of OA. The purpose of this study was to determine whether ozone treats OA by inducing autophagy in OA chondrocytes.
In this study, primary chondrocytes were stimulated with IL-1β for 24 hours to simulate an OA chondrocyte model, followed by treatment with ozone (30 µg/ mL) or pretreatment with 3-methyladenine or compound C before ozone treatment. Then, cell viability was detected by a CCK-8 kit, and the AMPK/mTOR signaling pathway and autophagy were detected by Western blotting and immunofluorescence. The mRNA expression levels of IL-6, TNF-α, MMP-13 and TIMP-1 were measured by quantitative real-time PCR. Finally, autophagosomes in chondrocytes were observed by transmission electron microscopy.
Ozone improved cell viability in chondrocytes stimulated by IL-1β. The decreased level of autophagy in IL-1β-stimulated chondrocytes improved with ozone treatment through activation of the AMPK/mTOR signaling pathway. In addition, the mRNA expression levels of IL-6 and TNF-α were suppressed by ozone treatment in chondrocytes stimulated with IL-1β. Ozone increased the mRNA level of TIMP-1 and decreased the mRNA level of MMP-13 in chondrocytes stimulated with IL-1β.
These results suggested that ozone improved the decreased level of autophagy in chondrocytes stimulated with IL-1β through activation of the AMPK/mTOR signaling pathway. Moreover, ozone treatment suppressed inflammation and helped maintain metabolic balance in chondrocytes stimulated with IL-1β.
臭氧注射通常用于治疗骨关节炎（OA）等疾病中的疼痛。最近的研究表明，软骨细胞中自噬的减少在OA的发展中起重要作用。本研究的目的是确定臭氧是否通过诱导OA软骨细胞中的自噬来治疗OA。
在本研究中，用白细胞介素-1β（IL-1β）刺激原代软骨细胞24小时以模拟OA软骨细胞模型，然后用臭氧（30μg/mL）处理或在臭氧处理前用3-甲基腺嘌呤或化合物C进行预处理。然后，用CCK-8试剂盒检测细胞活力，并用蛋白质免疫印迹法和免疫荧光法检测AMPK/mTOR信号通路和自噬。通过定量实时PCR测量IL-6、肿瘤坏死因子-α（TNF-α）、基质金属蛋白酶-13（MMP-13）和金属蛋白酶组织抑制因子-1（TIMP-1）的mRNA表达水平。最后，通过透射电子显微镜观察软骨细胞中的自噬体。
臭氧提高了IL-1β刺激的软骨细胞的活力。通过激活AMPK/mTOR信号通路，臭氧处理改善了IL-1β刺激的软骨细胞中自噬水平的降低。此外，臭氧处理抑制了IL-1β刺激的软骨细胞中IL-6和TNF-α的mRNA表达水平。臭氧增加了IL-1β刺激的软骨细胞中TIMP-1的mRNA水平，降低了MMP-13的mRNA水平。
这些结果表明，臭氧通过激活AMPK/mTOR信号通路改善了IL-1β刺激的软骨细胞中自噬水平的降低。此外，臭氧处理抑制了炎症，并有助于维持IL-1β刺激的软骨细胞中的代谢平衡。
