Zhang Guopeng, Zhang Hua, You Wulin, Tang Xiaochen, Li Xiufang, Gong Zhengfeng
Department of Orthopedics and Traumatology, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, P.R. China.
Department of Orthopedics and Traumatology, Wuxi Hospital of Chinese Medicine, Wuxi, Jiangsu 214001, P.R. China.
Exp Ther Med. 2020 Mar;19(3):2343-2352. doi: 10.3892/etm.2020.8471. Epub 2020 Jan 24.
The aim of the current study was to explore the role of Resveratrol (Res) in osteoarthritis (OA) and its underlying mechanism. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to determine the relative expression levels of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), microRNA-9 (miR-9), nuclear factor kappa B subunit 1 (NF-κB1), interleukin (IL)-6, matrix metallopeptidase 13 (MMP-13) and caspase-3 and in the model of OA, as well as examining the effect of Res on MALAT1, miR-9 and NF-κB1, IL-6, MMP-13 and caspase-3 expression levels. Immunohistochemical analysis was performed to examine NF-κB1 and MMP-13 protein levels in the model of OA. Dual-luciferase reporter assays were used to confirm the regulatory relationship between miR-9 and MALAT1 and NF-κB1, as well as examining the effect of Res on the transcriptional activation of MALAT1 promoter. Furthermore, the effect of Res on cell proliferation was examined by MTT assay. The relative mRNA expression levels of MALAT1 and NF-κB1 were significantly increased, while miR-9 expression was significantly decreased in the OA group compared with the sham group. Treatment with Res partially reversed the effects of OA on MALAT1, NF-κB1 and miR-9 expression. Similarly, the relative protein expression levels of NF-κB1, IL-6, MMP-13 and caspase-3 were significantly increased in the OA group compared with the sham group; however, treatment with Res partially reversed the effects of OA on the protein expression levels of NF-κB1, IL-6, MMP-13 and caspase-3. MALAT1 and NF-κB1 were identified as potential target genes of miR-9, and dual-luciferase assays were used to examine the effect of miR-9 on the luciferase activity of 3'UTR MALAT1 and NF-κB1. Treatment with Res suppressed the transcriptional activation of the MALAT1 promoter, thereby inhibiting MALAT1 expression. Additionally, the relative expression level of miR-9 significantly increased following treatment with Res in a dose-dependent manner, while the relative protein expression levels of NF-κB1, IL-6, MMP-13 and caspase-3 significantly decreased following treatment with Res compared with the control. Furthermore, treatment with Res significantly increased the growth rate of chondrocytes in a dose-dependent manner compared with the control. Taken together, these results suggest that direct targeting of the MALAT1/miR-9/NF-κB1/IL-6, MMP-13/caspase-3 axis may be a novel therapeutic strategy for the treatment of OA.
本研究的目的是探讨白藜芦醇（Res）在骨关节炎（OA）中的作用及其潜在机制。采用逆转录-定量聚合酶链反应和蛋白质印迹分析来测定转移相关肺腺癌转录本1（MALAT1）、微小RNA-9（miR-9）、核因子κB亚基1（NF-κB1）、白细胞介素（IL）-6、基质金属蛋白酶13（MMP-13）和半胱天冬酶-3在OA模型中的相对表达水平，以及研究Res对MALAT1、miR-9和NF-κB1、IL-6、MMP-13和半胱天冬酶-3表达水平的影响。进行免疫组织化学分析以检测OA模型中NF-κB1和MMP-13的蛋白水平。采用双荧光素酶报告基因检测法来确认miR-9与MALAT1和NF-κB1之间的调控关系，以及研究Res对MALAT1启动子转录激活的影响。此外，通过MTT法检测Res对细胞增殖的影响。与假手术组相比，OA组中MALAT1和NF-κB1的相对mRNA表达水平显著升高，而miR-9表达显著降低。Res处理部分逆转了OA对MALAT1、NF-κB1和miR-9表达的影响。同样，与假手术组相比，OA组中NF-κB1、IL-6、MMP-13和半胱天冬酶-3的相对蛋白表达水平显著升高；然而，Res处理部分逆转了OA对NF-κB1、IL-6、MMP-13和半胱天冬酶-3蛋白表达水平的影响。MALAT1和NF-κB1被确定为miR-9的潜在靶基因，并采用双荧光素酶检测法来研究miR-9对3'UTR MALAT1和NF-κB1荧光素酶活性的影响。Res处理抑制了MALAT1启动子的转录激活，从而抑制了MALAT1的表达。此外，Res处理后，miR-9的相对表达水平以剂量依赖性方式显著升高，而与对照组相比，Res处理后NF-κB1、IL-6、MMP-13和半胱天冬酶-3的相对蛋白表达水平显著降低。此外，与对照组相比，Res处理以剂量依赖性方式显著提高了软骨细胞的生长速率。综上所述，这些结果表明直接靶向MALAT1/miR-九/NF-κB1/IL-6、MMP-13/半胱天冬酶-3轴可能是治疗OA的一种新的治疗策略。
