In the current study, twenty patients (10 men and 10 women) with diagnosed KOA and five control individuals (3 men and 2 female) were recruited from Hongqi Hospital Affiliated with Mudanjiang Medical University. These patients were scheduled for knee replacement surgery and able to walk at least forty-five meters independently (without the use of walking aids). Patients were excluded if they had uncontrolled systemic disease (non-musculoskeletal conditions that would make testing difficult and uncomfortable for the participants, such as chronic obstructive airway disease or congestive heart failure) or a preexisting neurologic or other orthopedic condition affecting walking. The study protocol was approved by the Human Research Ethics Committees of Hongqi Hospital Affiliated with Mudanjiang Medical University. All of the participants were informed about the nature of the study and signed a consent form prior to participation. The details for all participants are listed in Table I.

Primary human skeletal muscle cells were purchased from Procell (CP-H095, Wuhan, China, http://www.procell.com.cn/view/2244.html). The cells were cultured in specific complete medium for human skeletal muscle cells (CM-H095; Procell, Wuhan, China) supplemented with 10% heat-inactivated fetal calf serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/ml penicillin and streptomycin in 25-cm² culture flasks at 37°C in a humidified atmosphere with 5% CO₂.

The strength of the knee extensor muscle group was determined in the affected legs of the 20 patients with KOA and in the leg from which the muscle biopsy specimen was obtained in the 7 control subjects. A portable nonextendable strain gauge (load cell) was used to measure muscle strength for this study. The strain gauge was attached to the subject's leg using a webbing strap with a Velcro fastener. The subject sat in a tall chair with a strap around the lower leg 10 cm above the ankle joint, and the hip and knee joint angles were positioned at 90 degrees. The distance from the knee joint to the strap around the ankle was measured with a tape measure and was used for the calculation of torque [force (N) xdistance (m)]. Each subject exerted maximal force against the strap assembly for 3 sec. Three trials were recorded for each subject, and the highest score was used for the analysis.

Resting muscle samples were isolated from the vastus lateralis, as previously described (19). In brief, the muscle samples from KOA patients were collected during their knee replacement surgery ~5 cm proximal to the suprapatellar pouch. The biopsies were taken after the skin was incised and prior to knee joint capsule incision with no trauma to the muscle or the joint at that time (19).

Skeletal muscle (30 mg) was extracted using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and was collected following centrifugation at 12,000 × g for 30 min at 4°C. A bicinchoninic protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. A total of 15 µg protein was loaded per lane, separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 8% non-fat dry milk at 4°C overnight. Following three washes with PBS with Tween 20 (5 min/wash), the membranes were incubated with the following primary antibodies at 4°C overnight: p-p65 (#3033, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), p65 (#8242, 1:1,000; Cell Signaling Technology, Inc.), anti-IκBα (#4812, 1:1,000; Cell Signaling Technology, Inc.) USF1 (ab125020, 1:1,000; Abcam, Cambridge, MA, USA) and GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc.). Following several washes with TBST, the membranes were incubated with horseradish-peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) or HRP-conjugated mouse antigoat IgG (ZF-0311, all 1:5,000; Zhongshan Gold Bridge Biological Technology Co., Beijing, China) for 2 h at room temperature and then washed followed by detection with enhanced chemiluminescent substrate (EMD Millipore, Billerica, MA, USA). GAPDH was used as an internal control. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for density analysis.

The adenovirus vectors overexpressing USF1 (Ad-USF1) or negative control (NC) (Ad-NC) were constructed by GenChem (Shanghai, China). For the transfection of adenovirus vectors into primary human skeletal muscle cells, the cells were seeded at a density of 10⁶ cells/well in 6-well plate. At 80% confluence, Ad-USF1 and Ad-NC were transfected into primary human skeletal muscle cells at 30 multiplicity of infection (MOI) for 48 h. Then, the cells were collected for further study.

Muscle tissue or cell lysates were centrifuged at 16,000 × g for 15 min at 4°C, and supernatants were used to quantify the levels of TNF-α (cat no. DTA00C; Human TNF-α Quantikine ELISA kit), IL-6, (cat no. D6050; Human IL-6 Quantikine ELISA kit), IL-1β (cat no. DLB50; Human IL-1 beta/IL-1F2 Quantikine ELISA kit), and IL-8 (cat no. D8000C; Human IL-8/CXCL8 Quantikine ELISA kit) by way of a sandwich ELISA following the manufacturers' protocols (R&D Systems, Minneapolis, MN, USA). Samples were read at a 450 nm wavelength using a microplate reader (Model 3550; Thermo Fisher Scientific, Inc.).

Muscle tissues samples from KOA patients or control were cut into 5 µm. Then, the slices were fixed in 4% phosphate-buffered neutral formalin at room temperature for 20 min, embedded in paraffin and cut into 5-µm thick sections, followed by deparaffinizition, descending alcohol series of rehydration, and microwave-heating in sodium citrate buffer (Solarbio Science & Technology Co., Ltd.) at 100°C for 30 min for antigen retrieval. Sections were subsequently incubated with 0.3% hydrogen peroxide/phosphate-buffered saline for 30 min. The sections were incubated with a primary anti-p-p65 antibody (#3033; Cell Signaling Technology, Inc.,) or anti-IκBα (#4812, Cell Signaling Technology, Inc.) at a 1:50 dilution and 4°C overnight. Detection of the primary antibody was performed via incubation with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ZDR-5036, Zhongshan Gold Bridge Biological Technology Co.,) for 1 h at room temperature and visualized with a 3,3′-Diaminobenzidine substrate. Stained cells were counted in 5 random fields using light microscopy (magnification, 40×, Olympus CK40; Olympus Corporation, Tokyo, Japan).

Primary human skeletal muscle cells (~1×10⁶) cells were cultured in a 6-well plate for 24 h with glass coverslips. After that, the cells were transfected with Ad-NC or Ad-USF1 for 48 h. Then, the cells on the coverslips were fixed in 4% paraformaldehyde for 30 min at room temperature. The samples were washed three times in PBS for 5 min and fluorescence intensity was examined using a fluorescence microscope (Olympus Corporation) at a magnification of ×100.

Data are presented as the mean ± standard deviation. To compare the two groups, two-tailed unpaired Student's t-test was performed. For multiple group comparisons, one-way analysis of variance followed by Tukey's post hoc test were used. Statistical tests were performed using SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The basic physical characteristics are shown in Table I. No significance was found in the age, height, weight and BMI of patients with KOA or healthy controls. By contrast, lower muscle strength was identified in KOA patients than in healthy controls (Table I).

Next, we determined the inflammatory factors in the vastus lateralis muscle tissues of KOA patients and control individuals. ELISA showed that the levels of IL-6, MCP-1, IL-8 and TNFα were significantly increased in the muscle tissues of KOA patients compared with control individuals (Fig. 1).

p65 NF-κB signaling is a key signaling pathway that upregulates cytokine gene expression, including TNFα, IL-1β, IL-6, and MCP-1, in muscle tissues (7,9). Thus, we analyzed p65 NF-κB activation in the muscle tissues of KOA patients and control individuals. Western blot assays indicated that p65 NF-κB was significantly increased in the muscle tissues of KOA patients compared with control individuals, while IκBα, an inhibitor of NF-κB, was shown to be decreased in the muscle tissues of KOA patients (Fig. 2). We also analyzed histological changes of p-p65 and IκBα in skeletal muscle. In line with the findings of western blot, p-p65 was found to be enhanced in the muscle tissues of KOA patients compared with control individuals, but IκBα was decreased in the muscle tissues of KOA patients (Fig. 2B and C).

Furthermore, we evaluated the expression of USF1 in muscle tissues of KOA patients. Compared with control individuals, the protein levels of USF1 were significantly enhanced in the muscle tissues of KOA patients (Fig. 3).

To further explore whether USF1 activates NF-κB signaling in primary human skeletal muscle cells, adenovirus vectors overexpressing USF1 or NC were transfected into primary human skeletal muscle cells for 48 h. As shown in Fig. 4A, the transfection efficiency was similar between Ad-NC or Ad-USF1 in in primary human skeletal muscle cells. Western blot assays indicated that overexpression of USF1 significantly induced the transcription of p65 NF-κB signaling (Fig. 4B). Moreover, an ELISA assay revealed upregulation of inflammatory factors, including TNFα (Fig. 4C), IL-8 (Fig. 4D), IL-6 (Fig. 4E) and MCP-1 (Fig. 4F).