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

OBJECTIVE: To explore whether electroacupuncture(EA could alleviate osteoarthritis(OA) through affecting the DNA methylation regulated transcription of miR-146a and mi R-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(qR T-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 ChI P 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.