All animal procedures conducted in the present study were approved by the Institutional Animal Care and Use Committee of the Fujian University of Traditional Chinese Medicine (Fuzhou, China). A total of 30 Sprague Dawley rats (age, 2 months; weight, 210±10 g) were purchased from the Shanghai Slack Laboratory Animal Co., Ltd. (Shanghai, China; certification no. SCXK: 2012-0002). All rats were housed in cages at a relative humidity of 55±5% and at room temperature (24±2°C) under a 12-h light/dark cycle. The rats were given ad libitum access to water and food.

Following 1 week of acclimation, the rats were randomly divided into three groups: The blank group and two experimental groups (n=10 rats/group). Rats in the two experimental groups were treated with EA using the bilateral Neixiyan (EX-LE4) and Waixiyan (ST 35) acupuncture points, which are located on the inside and outside of the depression of the patellar ligament, respectively. Disposable needles (diameter, 0.25 mm; length, 15 mm; Huatuo; Suzhou Medical Appliance Factory, Suzhou, China) were inserted to a depth of 4 mm at the acupuncture points. The acupuncture points of rats in experimental groups I and II were stimulated using the SDZ-II nerve and muscle stimulator (Huatuo; Suzhou Medical Appliance Factory) with an intensity of 2 mA and a frequency of 2 Hz for 15 and 30 min, respectively. The positively and negatively charged clips were connected to the right and left needles of the bilateral knee joints for 15 or 30 min each day for 3 days.

Arterial blood was collected from the abdominal aorta. Processing and storage of blood was conducted in accordance with a previously described method (16). The collected blood was placed at room temperature for 4 h and centrifuged at 1,500 × g for 15 min at room temperature. The serum fraction was isolated and was then filtered through a 0.22 µm filter. The resulting EAS was then aliquoted and stored at −20°C.

Chondrocytes were isolated (stepwise 0.2% collagenase type II digestion) from the knee cartilage of 4-week-old male Sprague Dawley rats (weight, 80±10 g), which were purchased from the Shanghai Slack Laboratory Animal Co., Ltd. (certification no. SCXK: 2012-0001). The chondrocytes were cultured and identified by collagen type II immunohistochemistry (17–19). Briefly, chondrocytes from the knee articular cartilage of 4-week-old rats were isolated using 0.2% type II collagenase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in magnesium- and calcium-free PBS (pH 7.4; HyClone; GE Healthcare, Logan, UT, USA) for 1 h at 37°C. The cells were then resuspended in Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 µg/ml streptomycin and 100 U/ml penicillin (HyClone; GE Healthcare), and were seeded in a monolayer at a density of 5×10⁵ cells/cm². Third generation chondrocytes were seeded on cover slips and cultured for 72 h, the cover slips were then rapidly washed in ethanol, dried and mounted for microscopic examination. Subsequently, the chondrocytes were incubated with rat monoclonal antibodies against type II collagen (BS1071; 1:100; Bioworld Technology, Inc., St. Louis Park, MN, USA) at 4°C overnight. The expression of type II collagen was observed using a phase-contrast microscope (BH2; Olympus Corporation, Tokyo, Japan). The primary chondrocytes were termed passage (P)1, and the P3 chondrocytes were used in subsequent experiments.

P3 chondrocytes were seeded into 6-well plates at a density of 1.3×10⁵/ml, and were incubated in DMEM (HyClone; GE Healthcare) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) for 72 h at 37°C in a 5% CO₂ incubator. The cells were divided into four groups: Control group, which was treated with 10% serum (100 µl/ml) obtained from the blank group; model group, which was cotreated with 10 µg/l TNF-α and 10% serum (100 µl/ml) obtained from the blank group; EAS I group, which was cotreated with 10 µg/l TNF-α and 10% serum (100 µl/ml) obtained from experimental group I; and EAS II group, which was cotreated with 10 µg/l TNF-α and 10% serum (100 µl/ml) obtained from experimental group II. All groups were treated for 24, 48, and 72 h at 37°C in a 5% CO₂ incubator.

Following treatment with EAS, the chondrocytes were seeded into 6-well plates at a density of 1.3×10⁵/ml for 24 h. Following treatment with or without TNF-α and EAS for 24, 48 and 72 h, the medium was then discarded and 100 µl 0.5% MTT solution was added to replace the medium. Following a 4 h incubation at 37°C, the wells were emptied, and 150 µl dimethyl sulfoxide was added to the plate and agitated for 10 min. The absorbance was measured at 490 nm using an ELISA reader (BioTek Instruments, Inc., Winooski, VT, USA), and the means were calculated.

The cells were cultured in 6-well plates at a density of 1.3×10⁵/ml for 24 h. Following treatment with or without TNF-α and EAS for 48 h, cell morphology was observed using a phase-contrast microscope (BH2; Olympus Corporation).

The concentration of interleukin (IL)-1β in conditioned medium was measured using an ELISA kit (RLB00; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol. Briefly, following treatment with or without EAS for 48 h at 37°C in a 5% CO₂ incubator, the cell culture medium was collected and centrifuged at 1,500 × g for 15 min at room temperature to remove cell fragments, and IL-1β concentration was determined. Absorbance of the samples was measured using a microplate spectrophotometer (Omega Bio-Tek, Inc., Norcross, GA, USA). A standard curve was generated, from which IL-1β concentration was determined.

Treated chondrocytes were collected and fixed in 4% paraformaldehyde for 15 min. Subsequently, the cells were stained with 5 µg/ml DAPI for 5 min and washed three times with PBS. The cells were then observed under a Fluo-View confocal fluorescent microscope (Fluo-View FV10i; Olympus Corporation). A total of 10 visual fields in each group were randomly selected and the number of apoptotic chondrocytes in each of these fields was counted using ZEN 2009 Light Edition (Carl Zeiss AG, Oberkochen, Germany).

Total proteins were isolated from cells using radioimmunoprecipitation assay lysis buffer (P0013B; Beyotime Institute of Biotechnology, Shanghai, China), after which they were stored on ice for 30 min and the protein concentration was determined using the bicinchoninic acid assay (P0009; Beyotime Institute of Biotechnology). Protein (20 µg) was separated by 10 or 12% SDS-PAGE. Following electrophoresis, proteins were transferred to polyvinylidene fluoride membranes using a semidry blotting system, and the membranes were blocked with 5% w/v nonfat dry milk for 1 h at room temperature (20). The membranes were then incubated with rat monoclonal antibodies against Ras (ab52939; 1:5,000), Raf (ab33899; 1:2,000), MEK1/2 (ab178876; 1:5,000) and β-actin (ab6276; 1:1,000) (Abcam, Cambridge, UK), and ERK1/2 (4695s; 1:1,000) and p-ERK1/2 (4094s; 1:1,000) (Cell Signaling Technology, Inc., Beverly, MA, USA) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (ZB-2301; 1:5,000; OriGene Technologies, Inc., Beijing, China; or bs-0296G; 1:5,000; BIOSS, Beijing, China) for 1 h at room temperature. Blots were visualized using Pierce™ Enhanced Chemiluminescence Western Blotting Substrate (32106; Thermo Fisher Scientific, Inc.). The intensity of each band was semi-quantified using the Image Lab gel analyzing system (Image Lab 3.0™ Software; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and was normalized to the band intensity of β-actin.

Following treatment, the cells were fixed in ice-cold methanol and permeabilized with 1% Triton X-100 for 10 min, after which they were blocked with 5% bovine serum albumin (A8010; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 1 h at room temperature. The fixed cells were washed and incubated with rat monoclonal antibodies against matrix metalloproteinase (MMP)-3 (sc-30070; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and MMP-13 (PA5-16566; 1:100; Thermo Fisher Scientific, Inc.) at 4°C overnight, followed by incubation with Alexa Fluor 488 goat anti-rabbit immunoglobulin G (H+L) secondary antibodies (A-11008; 1:300; Thermo Fisher Scientific, Inc.) and DAPI (D1306; 1:100; Thermo Fisher Scientific, Inc.) for 5 min at room temperature in the dark. The signal was visualized and images were acquired using a laser confocal scanning microscope (Olympus Corporation).

All data were collected from at least three independent experiments. Statistical analysis was performed using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). All data are presented as the mean ± standard deviation. The differences among the four groups were compared using one-way analysis of variance, and multiple comparisons were performed using the Student-Newman-Keuls-q test. P<0.05 was considered to indicate a statistically significant difference.

The present study examined whether EAS could promote the viability of TNF-α-treated chondrocytes using an MTT assay. The groups were treated with or without EAS for various durations. The results demonstrated that the viability of the model group was significantly lower than that of the control group (P<0.01), and the viability of the EAS I and II groups was significantly higher than that of the model group after 48 h (P<0.05; Fig. 1). Therefore, EAS treatment for 48 h was used in the subsequent experiments.

To determine the effects of EAS treatment on the morphology of cells, the morphological alterations of the cells in the various groups were observed by phase-contrast microscopy (Fig. 2). The morphology of the untreated control cells exhibited a healthy status, whereas TNF-α-treated chondrocytes presented more apoptotic cells that detached from each other and became bright, elongated and shrunken, or floated in the medium, as compared with cells in the EAS I and II groups. However, the TNF-α-induced alterations in cell morphology were not observed, or were less evident, in the TNF-α-stimulated chondrocytes treated with EAS.

To examine the effects of EAS on inflammation in TNF-α-treated chondrocytes, an ELISA was used to measure IL-1β concentration. As presented in Fig. 3, IL-1β concentration in the model group was significantly higher compared with in the control group (P<0.01); however, treatment with EAS significantly reduced IL-1β concentration compared with the model group (P<0.01). To further determine whether EAS inhibited inflammation via apoptotic processes, DAPI staining was used to assess chondrocyte apoptosis. Apoptotic cells exhibited typical alterations, including reduced cellular volume, bright blue staining, and condensed or fragmented nuclei. This phenomenon was more obvious in the model group compared with in the EAS I and II groups (Fig. 4). The percentage of apoptotic cells in the EAS groups was significantly lower than in the model group (P<0.05), thus suggesting that EAS may inhibit the apoptosis of TNF-α-treated chondrocytes via the regulation of IL-1β.

To gain insight into the mechanisms underlying the effects of EAS on the inflammation of TNF-α-treated chondrocytes, the protein expression levels of Ras, Raf and MEK1/2 were detected. The results demonstrated that the protein expression levels of Ras, Raf and MEK1/2 were lower in the EAS I and II groups compared with in the model group (P<0.05; Fig. 5). Furthermore, activation of the ERK1/2 signaling pathway has been reported to participate in chondrocyte inflammation induced by various stimuli. p-ERK1/2 is an intracellular signaling molecule that transduces extracellular responses, serves a well-known role in regulating chondrocyte inflammation and contributes to loss of the chondral matrix (21,22). Therefore, to understand the molecular mechanism by which EAS inhibits TNF-α-induced inflammation, the expression of p-ERK1/2 was detected. The results demonstrated that the protein expression levels of p-ERK1/2 were significantly reduced in the EAS groups compared with in the model group (P<0.01; Fig. 6). Taken together, these results indicated that EAS may inhibit TNF-α-mediated chondrocyte inflammation via the Ras-Raf-MEK1/2-ERK1/2 signaling pathway.

It is well known that MMPs serve a critical role in OA. Furthermore, a previous study reported that EA inhibited the expression of MMP-3 in chondrocytes (23). Therefore, the present study investigated the effects of EAS on the protein expression of MMP-3 and MMP-13 using immunofluorescent staining. Chondrocytes stimulated with TNF-α exhibited an increased release of MMP-3 and MMP-13 compared with the untreated controls. However, treatment of chondrocytes with EAS markedly inhibited the TNF-α-mediated release of MMP-3 and MMP-13 (Fig. 7), which indicated that EAS may inhibit the Ras-Raf-MEK1/2-ERK1/2 pathway, resulting in reduced MMP-3 and MMP-13 expression.