电针刺激内膝眼（EX-LE5）和犊鼻（ST35）缓解前交叉韧带切断术诱导的骨关节炎，影响 miR-146a 和 miR-140-5p 的 DNA 甲基化调控转录。
Electroacupuncture stimulating Neixiyan (EX-LE5) and Dubi (ST35) alleviates osteoarthritis in rats induced by anterior cruciate ligament transaction affecting DNA methylation regulated transcription of miR-146a and miR-140-5p.
机构信息
Department of Orthopedics, the Third Hospital of Shijiazhuang, Shijiazhuang 050011, China.
Department of Orthopedic Surgery and Sports Medicine Center, the First Affiliated Hospital, Jinan University, Guangzhou 510630, China.
出版信息
J Tradit Chin Med. 2023 Oct;43(5):983-990. doi: 10.19852/j.cnki.jtcm.2023.05.004.
OBJECTIVE
To explore whether electroacupuncture (EA) could alleviate osteoarthritis (OA) through affecting the DNA methylation regulated transcription of miR-146a and miR-140-5p.
METHODS
Sixty male eight-week-old Sprague-Dawley rats were divided into three groups: normal group (normal healthy rats; no treatment), model group (OA rats; no treatment) and EA group (OA rats treated with EA). Safranin O staining and modified Mankin's score were performed to evaluate the histopathological alterations and degeneration of cartilage 8 weeks after 8 consecutive weeks of treatment. Quantitative real time polymerase chain reaction (qRT-PCR) assay was employed to evaluate the expression of miR-146a in the cartilage tissue and miR-140-5p in the synovium tissue, respectively. The bisulfite sequencing analysis and quantitative methylation specific PCR (qMSP) were used to analyze the status of methylation in the regulatory regions of miR-146a and miR-140-5p. Chromatin immunoprecipitation (ChIP) assay were performed to assess the binding of nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (SMAD-3) in the regulatory regions of miR-146a and miR-140-5p. Western blot analysis was performed to detect the expressions of DNA Methyltransferase 1 (DMNT1), DNA Methyltransferase 3A (DMNT3A), and DNA Methyltransferase 3A (DMNT3b), NF-κB, SMAD3 levels.
RESULTS
Our results showed that EA treatment significantly upregulated miR-146a and miR-140-5p expressions. qMSP analysis showed that EA significantly decreased methylation levels of miR-140-5p regulated region and miR-146a promoter in OA cartilage and synovium. Bisulfite DNA sequencing (BDS) and ChIP analysis showed that EA significantly increased binding affinity of SMAD3 and NF-kB on the hypermethylated miR-140 regulatory region and miR-146a promoter, respectively. Western Blot analysis demonstrated that EA also significantly decreased expressions of methylation related proteins- DMNT1, DMNT3a, and DMNT3b as well as NF-κB and SMAD3.
CONCLUSIONS
Electroacupuncture stimulating Neixiyan (EX-LE5) and Dubi (ST35) may alleviate OA affecting the DNA methylation regulated transcription of miR-146a and miR-140-5p.
目的
探讨电针对骨关节炎（OA）的缓解作用是否通过影响 miR-146a 和 miR-140-5p 的 DNA 甲基化调控转录来实现。
方法
将 60 只 8 周龄雄性 Sprague-Dawley 大鼠分为三组：正常组（正常健康大鼠；无治疗）、模型组（OA 大鼠；无治疗）和电针组（OA 大鼠接受电针治疗）。治疗 8 周后，通过番红 O 染色和改良 Mankin 评分评估软骨的组织病理学改变和退变。采用实时定量聚合酶链反应（qRT-PCR）法分别检测软骨组织中 miR-146a 和滑膜组织中 miR-140-5p 的表达。采用亚硫酸氢盐测序分析和定量甲基化特异性 PCR（qMSP）分析 miR-146a 和 miR-140-5p 调控区的甲基化状态。采用染色质免疫沉淀（ChIP）实验评估 miR-146a 和 miR-140-5p 调控区中核因子-κB（NF-κB）和信号转导和转录激活因子 3（SMAD3）的结合情况。采用 Western blot 分析检测 DNA 甲基转移酶 1（DMNT1）、DNA 甲基转移酶 3A（DMNT3A）和 DNA 甲基转移酶 3B（DMNT3b）、NF-κB、SMAD3 水平。
结果
结果表明，电针治疗可显著上调 miR-146a 和 miR-140-5p 的表达。qMSP 分析显示，电针可显著降低 OA 软骨和滑膜中 miR-140-5p 调控区和 miR-146a 启动子的甲基化水平。亚硫酸氢盐 DNA 测序（BDS）和 ChIP 分析显示，电针可显著增加 SMAD3 和 NF-κB 与高甲基化 miR-140 调控区和 miR-146a 启动子的结合亲和力。Western blot 分析表明，电针还可显著降低甲基化相关蛋白-DMNT1、DMNT3a 和 DMNT3b 以及 NF-κB 和 SMAD3 的表达。
结论
电针刺激内膝眼（EX-LE5）和犊鼻（ST35）可能通过影响 miR-146a 和 miR-140-5p 的 DNA 甲基化调控转录来缓解 OA。
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