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Journal of Traditional Chinese Medicine >

2023 , Vol. 43 >Issue 5: 983 - 990

DOI: https://doi.org/10.19852/j.cnki.jtcm.2023.05.004

Electroacupuncture stimulating Neixiyan (EX-LE5) and Dubi (ST35) alleviates osteoarthritis in rats induced by anterior cruciate ligament transaction via affecting DNA methylation regulated transcription of miR-146a and miR-140-5p

  • Luobin DING ,
  • Huajun WANG ,
  • Yao LI ,
  • Jia LI ,
  • Ling LI ,
  • Yangping GAO ,
  • Jian GUAN ,
  • Weiqiang GENG
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  • 1 Department of Orthopedics, the Third Hospital of Shijiazhuang, Shijiazhuang 050011, China
    2 Department of Orthopedic Surgery and Sports Medicine Center, the First Affiliated Hospital, Jinan University, Guangzhou 510630, China
    3 Division of Rheumatology, Guangdong Provincial People’s Hospital; Guangdong Academy of Medical Sciences, Guangzhou 510080, China
    4 Department of TCM Orthopedics and Traumatology, the Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, China
    5 Department of Orthopedic Surgery, No. 980 Hospital, Joint Logistics Support Force People's Liberation Army (PLA), Handan Campus of Bethune International Hospital, Handan 050082, China

Received date: 2022-06-22

  Accepted date: 2022-09-07

  Online published: 2023-08-28

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Abstract

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 via affecting the DNA methylation regulated transcription of miR-146a and miR-140-5p.

Key words： osteoarthritis, knee; electroacupuncture; DNA methylation; MicroRNAs

Cite this article

Luobin DING , Huajun WANG , Yao LI , Jia LI , Ling LI , Yangping GAO , Jian GUAN , Weiqiang GENG . Electroacupuncture stimulating Neixiyan (EX-LE5) and Dubi (ST35) alleviates osteoarthritis in rats induced by anterior cruciate ligament transaction via affecting DNA methylation regulated transcription of miR-146a and miR-140-5p[J]. Journal of Traditional Chinese Medicine, 2023 , 43(5) : 983 -990 . DOI: 10.19852/j.cnki.jtcm.2023.05.004

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