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.

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.

Association of articular cartilage YKL-40 levels with osteoarthritis / 实用医学杂志

Objective To determine the association of articular cartilage (AC) levels of YKL-40 with osteoarthritis (OA) at different stages of symptomatic severity in patients with knee OA.Methods 42 patients with knee OA were recruited into this study.The radiographic disease severity of OA was assessed by the Kellgren-Lawrence (K-L) grading system.11 patients with non knee OA were selected as normal control group.AC levels of YKL -40 were explored by immunohistochemical method.Symptomatic severity was determined using Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores from OA patients.Results YKL-40 levels in AC were positively related to WOMAC pain,function scores and total scores.WOMAC stiffness scores did not correlated with YKL-40 concentrations in AC.The correlation between AC YKL-40 levels and WOMAC scores was further analyzed by multinomial logistic regression.Multiple regression analysis showed that the correlation between AC YKL-40 levels and WOMAC scores was still significant after adjusting for other confounding factors.AC YKL-40 levels were significantly correlated with K-L grading in patients with knee OA.Conclusions YKL-40 in AC can be used as a potential biomarker for assessing the symptomatic severity of OA.