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

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.

1. INTRODUCTION

Knee osteoarthritis (OA) is the most common joint degenerative disease over the world. It is characterized by progressive degeneration of articular cartilage tissue, formation of osteophyte at the edge of the joint and reactive changes of subchondral bone.¹ It is mainly manifested as knee pain, weakness and difficulties in walking, which seriously affects the patients’ daily life.² Although many studies have been carried out for OA investigation, the detailed mechanisms for the pathogenesis of OA is not completely understood, and no therapy about directly disease-modifying is available currently.

Accumulating evidences have implicated that DNA methylation and microRNAs (miRNAs) play both important roles as regulators of in the onset and development of OA.³

Firstly, DNA methylation at cytosine-p-guanine (CpG) sites has been identified as an important epigenetic mechanism in OA progression. Many methylated CpG sites in connection with OA associated genes were observed by the studies for genome-wide DNA methylation.^4,5 Existed studies demonstrated that the expression of the genes which associated with OA was modulated by the status of methylation in their regulatory regions through affecting the binding of transcription factors on their target sequences.^6,7

Secondly, as one type of the non-coding RNAs, microRNAs (miRNAs) in these years also serve as pivotal epigenetic roles on regulating gene expressions during OA progression. Among them, two protecting miRNAs including miR-146a and miR-140-5p, have become the most widely studied miRNAs in OA related research. The expression of miR-140-5p is specifically up-regulated in articular cartilage during the process of chondrogenesis.⁸ A series studies have demonstrated that lower miR-140-5p expression has been detected in knee OA patients,⁹ and overexpression of miR-140-5p may benefit for OA treatment.^10,11 On the other hand, expression and correlation of miR-146a with the inflammatory responses were observed in OA synovium tissues.¹² Meanwhile, impaired secretions of cytokines, including macrophage colony-stimulating factors and inflammatory mediators induced by interluekin-1, were observed after transfection of miR-146a into human synovial compared to the controls, indicating the therapeutic values of miR-146a expression for OA in clinic.¹³ Meanwhile, the correlation between the specific hypermethylated CpG sites in the regulatory regions of miR-146a and miR-140-5p and decreased miR-146a and miR-140-5p expression was observed in OA by previous studies.¹⁴ In addition, hypermethylation status of the regulatory regions of miR-146a and miR-140-5p was also correlated with the reduced binding for transcription factors, like nuclear factor-kappa B (NF-kB) and signal transducer and activator of transcription 3 (SMAD-3), to these hypermethylated regions of miR-146a and miR-140-5p.¹⁴

Because of the safety,¹⁵ affordability,¹⁶ and excellent effect on pain relief,¹⁷ acupuncture therapy is conditionally recommended and applied for different types of musculoskeletal pain disorders based on the American College of Rheumatology guidelines on knee OA.¹⁸ Moreover, the therapeutic effect of acupuncture on functional recovery and control of pain in patients with knee OA was demonstrated by many studies.^19,20 Electroacupuncture (EA) is a novel type of acupuncture techniques which using the stimulation of electricity. Effectively pain and stiffness alleviation were observed in the patients with OA after EA treatment.^21,22 Additionally, inhibition of cartilage degeneration and anterior cruciate ligament transection (ACLT) in ovariectomized rabbits and rats respectively were observed after EA treatment in vivo.^23,24

The current study was carried out to investigate whether EA alleviates OA via affecting the DNA methylation regulated transcription of miR-146a and miR-140-5p.

2. MATERIALS AND METHODS

2.1. Ethics statement

All the experiments and procedures enrolled in animals in this study were carried out according to the Chinese Guidelines of Animal Care and Welfare. All the designs, procedures, and protocols involved in the animal experiments were submitted, reviewed and approved by the Animal Care and Use Committee of Jinan University (Guangzhou, China) (Approval ID: 20200047).

2.2. Induction of OA in rats

Eight-week-old Sprague-Dawley rats [male, weight: (200 ± 20) g, n = 60] were kept under a 12-h light-dark cycle condition at (24 ± 2) ℃ temperature and 50% ± 5% humidity. To construct the KOA model, ACLT method was employed. Briefly, after general anesthesia using pentobarbital sodium, a medial longitudinal incision (2 cm long) was taken at the right knee joint. And then opened the joint capsule, exposed the medial ligament, and finally caused a lateral dislocation of the knee. The anterior cruciate ligament was cut off. Cefotiam hydrochloride (1.0-1.3 mg/kg) was used to protect the rats from infections for 3 d. All rats were sacrificed and samples were collected 8 weeks after surgery. The successful establishment of knee OA modeling was assessed based on the followings: (a) Behavioral indicators: alternation of the gait and rest posture, decreased activity frequency and flexibility, and the time of eating and the total amount of eating before and after modeling; (b) Knee Joints are swelling, severely inflamed and unable to bear weight; (c) Direct pathological observation by modified Mankin scores: Mankin's score is divided into four levels from the cellularity, cell cloning, safranine-O staining and surface integrity, with a maximum of 23 points and a minimum of 0 point. Higher points indicate severer OA.²⁵

The rats were randomized to three groups through random number table method by using Microsoft EXCEL version 2010. The 60 rats were randomly divided into three groups with 20 in each group: control group, OA group, and OA + electroacupuncture (EA) group. A number mark was created on each rat’s back according to group to avoid any mismatching.

2.3. EA Treatment

The Neixiyan (EX-LE5) and Dubi (ST35) were used to treat the knee acupoints of rats with OA. According to the National Standard of the People's Republic of China, the name and location of acupoints, issued in 2006 (GB/T 12346-2006), Dubi (ST35) was located in a depression lateral of the patellar ligament. Opposite Dubi (ST35), Neixiyan (EX-LE5) is located in the center of the depression. After cleaning a rat’s skin with alcohol swabs, one investigator held the animal while the second swiftly inserted disposable acupuncture needles, with electrodes soldered to their handles, approximately 1/2 in deep into each flank at the acupoints. The needles and electrodes were stabilized with adhesive tape. The procedure typically lasted less than 20 s and caused little distress to the animal. After the insertion of acupuncture needle (0.25 mm diameter × 40 mm length) into the acupoint and connection of it to electrical stimulator (Type SDZ-Ⅱ, Suzhou Medical Appliance Factory Co., Ltd., Suzhou, China), low-frequency EA (2 Hz) was chosen to stimulate the acupoint electrically. For EA stimulation, 100 Hz burst frequency (duration 2.2 s) and square-wave burst pulses (duration 1.1 s) with alternating polarity were set. The intensity of the output voltage was 17.3 V and the pulse width was shorter than 1 ms. The stimulation was performed 30 minutes of every day for 8 weeks. No any treatment of the model and normal group rats was given during the stimulation. The EA procedure was conducted under anesthesia status by continuous inhalation of 2% isoflurane. The condition of breath, heart beating and body temperature was monitored. Following treatment, all rats were allowed unrestricted activity in cage.

2.4. Tissue sampling

The animals were weighed after completion of the final EA treatment. Under deep anesthesia using an intraperitoneal overdose (80 mg/kg) of 2% sodium pentobarbital, the rats were euthanized by cervical dislocation. The cartilage from tibias tibial plateau from each group was isolated for next procedure.

2.5. Analysis and assessment of cartilage damage

The harvested cartilage tissues were fixed by in 4% paraformaldehyde. After 2-d fixation, the decalcification was performed using 10% ethylene diamine tetraacetic acid (EDTA) for another 8 weeks. After cutting into small sections (1.2 cm × 1.2 cm × 0.5 cm), the medial femoral condyle was embedded in paraffin and cut into 4-μm paraffin sections. After dewaxing with xylene and rehydration, the sections were stained with Safranin O. A microscope (Olympus Corporation, Tokyo, Japan) was employed to observe the morphological changes of cartilage and capture images (× 100). To evaluate the degeneration of each femoral condyle, the modified Mankin's score was recorded to score the stained tissues.

2.6. RNA extraction and quantitative real time polymerase chain reaction (qRT-PCR)

The TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was chosen to extract the total mRNA from tissues of synovium and cartilage. Then, the SuperScript Ⅲ reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) was used to reverse 1 μg mRNA to cDNA. ABI 7300 real-time PCR instrument (Applied Biosystems, Waltham, MA, USA) was employed to determine the expression of miR-146a and miR-140-5p. The 7300 system SDS software was used to analyze the results obtained from qRT-PCR assay. miRNA expressions were normalized to U6 as internal control by using 2^−ΔΔCt. The primer sequences were listed below: miR-140-5p: forward 5’-AGTGGTTTTACCCTATGG-3’, miR-140-5p reverse: 5’-AACTGGTGTCGTGGAG-3’; miR-146a forward: 5’-GTGAGAACTGAA TTCC-AT-3’; miR-146a reverse: 5’-AACTGGTG-TCGTGGAG-3’. U6 forward: 5’-CTCGCTTCGG-CAGCACA-3’, reverse: 5’-AACGCTTCACGAATT-TGCGT-3′.

2.7. Quantitative methylation specific polymerase chain reaction (qMSP) analysis

To analyze the methylation of specific regions in the regulatory elements of miR-146a and miR-140-5p, qMSP assay was conducted by using real-time PCR kit (ABI 7300, Applied Biosystems, Foster, CA, USA) as described previously. Briefly, the Methyl Primer Express software was employed to design the primers targeted with methylated or unmethylated DNA sequences. 2× EpiTect Master Mix (Qiagen, Valencia, CA, USA) was used to amplify the bisulfite-treated genomic DNA. The formular DNA methylation = CtU - CtM (CtU and CtM represent the cycle threshold of unmethylated and methylated primers respectively) was used to calculate the values of DNA methylation. 0%, 10%, 25%, 50%, 75%, 90% and 100% methylated DNA mixed with human unmethylated control were used to generate a standard curve.

2.8. Analysis of Bisulfite DNA sequencing (BDS)

Methlyl Primer Express (software v1.0) was employed to design the primers for Bisulfite-treated DNA amplification by using PCR (Table 1). QIAquick PCR Purification kit (Qiagen, Valencia, CA. USA) was used to purify the products of PCR amplification, and subsequently a Bigdye terminator v3.1 cycle sequencing kit (Applied Biosystems, Waltham, MA, USA) was employed to sequence the purified PCR products. The results obtained from sequencing were analyzed by using ABI 3130 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). The percentage of methylation for each CpG site in the miR-140-5p regulatory region and miR-146 promoter was calculated by the formular (C/[C + T] × 100), C and T represents the ratio between peak height values of cytosine (C) and thymine (T) respectively.

2.9. Chromatin immunoprecipitation (ChIP) assay

ChIP was carried out using a ChIP assay kit (Abcam, Cambridge, UK) on cartilage and synovium from control, OA and EA treated group. Samples were taken at this point as positive controls in the PCR reaction (input chromatin) and were incubated with monoclonal antibody against SMAD-3 or NF-kB (Abcam, Cambridge, UK) overnight at 4 ℃. Human purified immunoglobulin G was selected as control antibody (Abcam, Cambridge, UK). DNA-protein complexes were collected using Salmon Sperm DNA/Protein a Agarose beads, followed by washing, elution and reverse cross-linking. DNAs were extracted with phenol-chloroform and precipitated with ethanol. Recovered DNAs were re-suspended in Tris-Ethylene-diaminetetraacetic acid buffer and were later analyzed by PCR. PCR products were fractionated on 3% agarose gels and were stained with ethidium bromide. The relative signal intensity of each band was quantified using the National Institutes of Health Scion Image (Scion Inc, Salt Lake City, UT, USA) and normalized to the Input chromatin of the same sample.

2.10. Western blot analysis

After lysating of the tissues of rat cartilage and synovial by using radio Immunoprecipitation Assay buffer (Beyotime, Haimen, China). Bicinchoninic acid protein assay kit (Beyotime, Haimen, China) was used to determine the concentration of proteins. Then, after the separation of the protein samples by using an 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel, the protein bands were transferred and blocked with 5% fatty-free milk no less than 1 h at room temperature, then the protein was incubated with indicated primary antibodies 4 ℃ overnight or 2 h at room temperature. Antibodies used in this study as follows: anti-DNA Methyltransferase 1(DMNT1) (1 : 200, Abcam, UK), anti- DMNT3a (1 : 200, Abcam, Cambridge, UK), anti-DMNT3b (1 : 200, Abcam, UK), anti-NF-κB (1 : 1000, Abcam, Cambridge, UK), anti-signal transducer and activator of transcription 3( SMAD3) (1 : 1000, Abcam, Cambridge, UK) and anti-H3 (1 : 1000, Abcam, Cambridge, UK). Finally, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and analyzed by using enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA) after three times washing with 0.1% tween-20 solution in phosphate buffer solution. The Molecular Image, ChemiDoc XRS Image System (Bio-Rad Laboratories, Hercules, CA, USA) was employed to collect and capture the images of the membrane and the Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA) was used to calculate the density value of protein bands which normalized with H3 was as an internal control.

2.11. Statistical analysis

The GraphPad Prism version 8.0 for windows (GraphPad Software, San Diego, CA, USA) software was used to analyze all the data obtained from this study. The statistical comparisons of modified Mankin’s scores, miR-140 and miR-146a expressions, rate of Cytosine methylation, band intensity and DNA methylation related proteins among normal group, OA group and EA group were analyzed by one-way analysis of variance (ANOVA). For multiple comparisons, Bonferroni’s post hoc tests were conducted. All the values in this study were shown as mean ± standard deviation. Statistical significance was characterized by P < 0.05.

3. RESULTS

3.1. Effect of EA on the changes of Cartilage Morphology

Compared to the normal group, OA caused significantly decreased Safranin O staining (Figure 1A) and partially replicated or defected tide mark (Figure 1B) in OA group. However, EA treatment obviously restored the reduced Safranin O staining and defected tide mark which caused by OA (Figure 1C). Overall, compared to the normal group, OA induction dramatically increased the Mankin’s score in OA group (17.6 ± 0.3 vs 1.7 ± 0.3, t = 16.11, P < 0.01). While, these elevated scores by OA induction were obviously reduced by EA treatment (11.3 ± 0.2 vs 17.6 ± 0.3, t = 8.45, P < 0.05).

Figure 1. EA Impacts on cartilage morphology by Safranin O staining (x 100).

A: normal cartilage stained by Safranin O staining; B: OA cartilage stained by Safranin O staining Samples were collected 8 weeks after surgery C: EA treated OA cartilage. The stimulation was performed 30 min of every day for 8 weeks. OA: osteoarthritis; EA: electroacupuncture.

3.2. EA significantly upregulated miR-146a and miR-140-5p expressions

Our findings showed that the expression of miR-140-5p was drastically reduced in OA cartilage in comparison with normal cartilage (1.1 ± 0.2 vs 2.8 ± 0.3, t = 4.45, P < 0.05). In EA treated cartilage, miR-140-5p expression level were significantly higher than that in OA group (2.0 ± 0.3 vs 1.1 ± 0.2, t = 3.318, P < 0.05).

On the other hand, in comparison to the synovium of normal rats, downregulated miR-146a expression was found in OA synovium (1.0 ± 0.2 vs 2.4 ± 0.2, t = 7.66, P < 0.05). However, this suppressed miR-146a expression was dramatically elevated by the EA administration (1.7 ± 0.3 vs 1.0 ± 0.2, t = 3.16, P < 0.05).

3.3. EA changed the status of methylation in the regulatory regions of miR-140-5p in OA cartilage

The binding site of SMAD-3 on the regulatory sequence of miR-140-5p at position−120 bp has been previously illustrated.³³ In another study, Papathanasioua et al ²⁰ showed the presence of CpG sites in miR-140 regulatory sequence and demonstrated that eleven CpG sites are enrolled.

Then, the methylation of DNA in the regulatory region of miR-140-5p was accessed by using qMSP in three groups. We found that it was significantly hypermethylated of the miR-140-5p regulatory region in OA cartilage compared with normal group (52% ± 6% vs 26% ± 3%, t = 8.25, P < 0.05). After EA treatment, DNA methylation in the regulatory sequences of miR-140-5p significantly decreased in comparison with OA group (36% ± 4% vs 52% ± 6%, t = 6.72, P < 0.05).

Next BDS assay showed that from the eleven CpG sites, nine CpG sites were methylated in OA cartilage in comparison with normal cartilage (Figure 3C). And methylation levels at seven CpG sites were significantly decreased following EA treatment when compared to OA group.

3.4. EA changed the status of methylation in the promoter of miR-146a in OA synovium

Previous researches have shown that two NF-kB binding sites were found at position－440 bp in miR-146a promoter.³⁴ Furthermore, previous study also observed the exist of ten CpG sites around the binding sites of NF-kB at position −440 bp within miR-146a promoter.²⁰

We secondly accessed the levels of DNA methylation at the regulatory region of miR-146a in three groups through qMSP method. Compared to the normal synovium, significantly higher methylation was observed in OA synovium (54% ± 6% vs 29% ± 3%, t = 8.30, P < 0.05). While, in comparison to the OA group, this elevated methylation in the promoter sequence of miR-146a in OA synovium was significantly decreased by EA treatment (39% ± 4% vs 54 ± 6%, t = 7.37, P < 0.05).

BDS analysis revealed that, half of the CpG sites in OA synovium were significantly hypermethylated compared with the normal synovium. And methylation levels at four CpG sites were significantly decreased following EA treatment when compared to OA group.

3.5. EA changed the binding of SMAD-3 and NF-κB to the regulatory sequences of miR-140-5p and miR-146a respectively

In comparison to the normal cartilage, we observed weaker binding of SMAD-3 in OA cartilage (0.53 ± 0.14 vs 1.00 ± 0.05, t = 6.32, P < 0.05). After EA treatment, stronger binding was observed compared to OA group (0.80 ± 0.14 vs 0.53 ± 0.14, t = 3.37, P < 0.05) (supplementary Figure 1).

Additionally, compared to normal synovium, a decreased NF-κB binding was observed from the results of ChIP assay (0.64 ± 0.14 vs 1.00 ± 0.05, t = 5.17, P < 0.05), however, this decreased binding affinity was enhanced by EA treatment compared to the OA group (0.79 ± 0.14 vs 0.64 ± 0.14, t = 2.33, P < 0.05) (supplementary Figure 2).

3.6. EA downregulated DMNT levels, SMAD3 and NF-κB protein expressions

To further evaluate the effect of EA on DNA methylation, several DMNT protein levels including DMNT1, DMNT3a, and DMNT3b were examined. The SMAD3 expressions were detected in cartilage tissues and NF-κB protein expressions were investigated in synovium tissues. We found all DMNT1, DMNT3a, and DMNT3b were significantly increased in both OA cartilage and synovium tissues in comparison with normal group (Cartilage: DMNT1: 0.47 ± 0.02 vs 0.14 ± 0.01, t = 5.12; DMNT3a: 0.67 ± 0.03 vs 0.22 ± 0.02, t = 9.57; DMNT3b :0.40 ± 0.03 vs 0.19 ± 0.04, t = 2.64, all P < 0.05; Synovium: DMNT1: 0.51 ± 0.03 vs 0.16 ± 0.03, t = 5.32; DMNT3a: 0.71 ± 0.03 vs 0.20 ± 0.02, t = 10.17; DMNT3b: 0.44 ± 0.01 vs 0.19 ± 0.02, t = 4.33, all P < 0.05). Accordingly, both SMAD3 protein levels in cartilage and NF-κB protein levels in synovium were also markedly increased than normal group (SMAD3: 0.56 ± 0.02 vs 0.10 ± 0.02, t = 12.14; NF-κB: 0.58 ± 0.03 vs 0.10 ± 0.01, t = 13.23, all P < 0.05). After EA treatment, DMNT1, DMNT3a, and DMNT3b expressions in OA cartilage were dramatically suppressed in comparison to those in OA group (Cartilage: DMNT1: 0.33 ± 0.01 vs 0.47 ± 0.02, t = 1.61; DMNT3a: 0.38 ± 0.01 vs 0.67 ± 0.03,t = 7.13; DMNT3b: 0.30 ± 0.01 vs 0.40 ± 0.03, t = 1.22, all P < 0.05; Synovium: DMNT1: 0.31 ± 0.04 vs 0.51 ± 0.03, t = 2.68; DMNT3a: 0.36 ± 0.02 vs 0.71 ± 0.03 t = 8.33; DMNT3b : 0.35 ± 0.01 vs 0.44 ± 0.01, t = 1.11, all P < 0.05). On the other hand, DMNT1 and DMNT3a expressions were also drastically reduced in OA synovium tissues. In addition, SMAD3 protein levels in cartilage and NF-κB protein levels in synovium were also drastically reduced (SMAD3: 0.18 ± 0.03 vs 0.56 ± 0.02, t = 9.75; NF-κB: 0.22 ± 0.03 vs 0.58 ± 0.03, t = 9.68, all P < 0.05) (Figure 2).

Figure 2. Expression of DMNT proteins and SMAD3 and NF-κB among normal, OA and EA treated group.

A: cartilage tissue; B: synovium tissue. Samples were collected 8 weeks after surgery. EA stimulation was performed 30 min of every day for 8 weeks DMNT: DNA Methyltransferase; SMAD3: signal transducer and activator of transcription 3; NF-κB:nuclear factor-kappa B; OA: osteoarthritis; EA: electroacupunture.

4. DISCUSSION

Based on the in vitro studies conducted by Papathanasioua by using chondrocytes in vitro,¹⁴ we found EA significantly upregulated miR-146a and miR-140-5p expressions in OA cartilage and synovium in rats. qMSP analysis showed that EA also markedly decreased methylation levels of miR-140-5p regulated region and miR-146a promoter in OA cartilage and synovium. BDS and ChIP analysis showed that EA significantly increased the binding of NF-kB and SMAD3 on the miR-146a promoter and hypermethylated miR-140 regulatory region. 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.

In recent years, researchers paid more attentions to the epigenetics as a new and important mechanism for regulation of the pathogenesis and progression of OA.²⁶ Compared to the regulatory mechanisms by genetics, the regulation of epigenetics was characterized by developmental stages and environmental stimuli dependent, highly dynamic, as well as tissue-specific.²⁷ Epigenetics is a kind of regulatory mechanism for gene expression through direct modification of DNA, RNAs, and histones.²⁸ Currently, there are three mechanisms for the regulation of epigenetic were identified, such as the modification of DNA (like methylation), histones, and non-coding regulatory RNAs-mediated regulation.²⁹ Accumulated evidences indicated that OA was not only associated with aging and loss of cartilage, but also affected by genetic regulations, such as epigenetic regulation.³⁰

Even though the effect of EA treatment on OA is usually explained by placebo effects, the mechanism remains partially understood. Previous works implicated that EA showed protective effects on cartilage and pain relieving.^31,32 Our present study revealed a novel mechanism about the effect of EA treatment on OA from an epigenetic role- DNA methylation and microRNA interaction concept.

EA has been shown to affect DNA methylation in many diseases. For example, EA has an effect on hypothalamic DNA methylation in polycystic ovary syndrome rats and decreased the global DNA methylation levels.³³ In another study by Yu et al,³⁴ EA reduces cognitive deficits partially through increasing the DNA methylation level in the promoter region of the glycogen synthase kinase-3β gene. However, whether EA could perform DNA methylation effects on OA remains unknown.

Transcription factors binding to microRNA regulation area serves as an important mechanism to control gene expression. A series of transcription factors including NF-κB,³⁵ AP1,³⁶ SMAD3³⁷ and FoxO3³⁸ have been proved to be involved in regulating gene expressions involved in OA progression. In our current study, EA significantly increased the SMAD3 binding on the hypermethylated and regulatory region of miR-140, as well as enhanced the NF-kB binding on the promoter sequences of miR-146a.

In this study, we enrolled two acupoints Neixiyan (EX-LE5) and Dubi (ST35) for EA treatment. Neixiyan (EX-LE5)³⁹ and Dubi (ST35)⁴⁰ are the most commonly used acupuncture points in the treatment of knee OA. Meanwhile, the two acupuncture points are also the most common tenderness acupoints of knee OA. Many studies have enrolled these two acupoints for knee OA intervention.^41,42

There were several limitations that should be taken into account. Secondly, the subchondral bone is reported to play a vital role in the pathogenesis of OA and is commonly associated with articular cartilage defects. However, we only examined changes in cartilage and synovium in this study. Further research is needed to conduct the epigenetic changes on subchondral bone.

The biggest highlights of this study were: (a) EA markedly decreased methylation levels of miR-140-5p regulated region and miR-146a promoter in OA cartilage and synovium. (b) EA significantly increased the binding of NF-kB and SMAD3 on the miR-146a promoter and hypermethylated miR-140 regulatory region. (c) EA also significantly decreased expressions of methylation related proteins- DMNT1, DMNT3a, and DMNT3b as well as NF-κB and SMAD3.

In conclusion,our findings added a new concept that EA relieving OA cartilage and controlling synovium inflammation from the epigenetic role of transcription factor and microRNA interaction.

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.com.