A total of 72 adult male Sprague-Dawley rats, (380–400 g; 14 weeks) were purchased from the Guangdong Medical Laboratory Animal Center (Foshan, China) were used in the present study. The rats were randomly assigned into three groups: Control group (n=24), immobilization group (IM group, n=24), and immobilization+anti-C5a antibody group (IM+anti-C5a group, n=24). The rats in the control group did not undergo any treatment, while rats in the IM and IM+anti-C5a groups underwent the following procedure: A degreasing cotton patch was placed in the popliteal fossa of unilateral (50% right and 50% left in each group) knee joints to immobilize the joint at 180° and fixed with a plaster bandage (Anji Wande Medical Products Co., Ltd., Anji, China) for 6 weeks. Saline (1 ml) or anti-C5a antibody (0.1 µg/ml; Wuhan Boster Biological Technology, Ltd., Wuhan, China; cat. no. PB0184) were given every week for 6 weeks. A total of four rats/group for 6 weeks were sacrificed at the end of each week for the subsequent assays. All procedures were approved by the Guizhou Medical University (Guiyang, China) Animal Care and Use Committees and were in accordance with the National Institutes of Health Guide (1996) for the care and use of laboratory animals (21).

The femur was truncated at 5 cm above the femoral condyle, and tibia was truncated at 3 mm below the tibial plateau. Soft tissue such as muscle, fat, periosteum surrounding knee joint were removed carefully. Patellar ligament, medial and lateral collateral ligament and anterior cruciate ligament was cut off, and the aponeurotic laminae on both sides were cut open. The patella is flipped down. The knee is opened and the articular cartilage is cut with a 15# scalpel blade. The cartilage on each joint surface is cut off. Do not accidentally bring the subchondral bone and periosteum.

Cut the skin longitudinally along the median knee joint until an area of about 3×3 cm centered on the knee joint is exposed. Lift the patella with a tweezers. femur was truncated at 0.13~0.14 cm above the upper edge of patella. Split down to the tibia along both sides of the patella. After the knee joint cavity was opened, a layer of smooth and shiny yellowish synovial tissue adhered to the lower edge of the patella can be seen.

The specimens were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, at pH 7.4 overnight at 4°C. The tissues were dehydrated through a series graded ethanol baths (100, 95, 90, 80 and 70%), and the specimens were embedded in paraffin. The embedded tissue was cut into 4 µm sections. The center of the scanned region was set for histological evaluation.

TUNEL staining of fractured double-stranded DNA in apoptotic cells was performed. Paraffin sections (4 µm) of synovial and cartilage tissues were used for the evaluation of apoptosis by TUNEL staining. The In Situ Cell Death Detection kit-POD (Roche Diagnostics, Basel, Switzerland; cat. no. 11684817910) was used to determine the number if apoptotic cells, according to the manufacturer's protocol. Counting of TUNEL-positive cells was conducted on ×200-magnified sections for each group. A H&E Staining kit (Shanghai Yeasen Biotechnology Co., Ltd., Shanghai, China, cat. no. 60524ES60) was used for staining the cardiac muscle, according to the manufacturer's protocols. Briefly, slides were stained for 5 min with hematoxylin, washed with diluted water three to five times at room temperature and subsequently stained with eosin solution for 20 sec at room temperature.

Total RNA was extracted from the synovial fluid and cartilage tissue by using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA). cDNA was synthesized using 1 µg total RNA via an RT kit (DBI Bioscience, Newark, DE, USA). The RNA solution was incubated at 65°C for 5 min, and the temperature protocol for the reverse transcription reaction was 37°C for 15 min, followed by 98°C for 5 min. The primers used in qPCR were as follows: C5a sense, 5′-CTCACCTTCCTCACCAATGC-3′ and antisense, 5′-GCTTGTTCTTCCACTTTCTGAT-3′; GAPDH sense, 5′-CCTCGTCTCATAGACAAGATGGT-3′ and antisense, 5′-GGGTAGAGTCATACTGGAACATG-3′. SYBR-Green qPCR master Mix (DBI Bioscience) was used for qPCR amplification. Cycling conditions included denaturation at 95°C for 2 min followed by 40 cycles of annealing at 94°C for 20 sec and extension at 58°C for 20 sec. On the basis of exponential amplification of the target gene relative to the housekeeping gene as an internal reference, the quantity of amplified molecules at the quantification cycle was given by 2^−ΔΔCq (22).

Expression levels of IL-1β, C5a, TNF-α and IL-17A in the synovial fluid and cartilage tissue were measured using an antigen-based sandwich ELISA. The Rat IL-1β ELISA kit (cat. no. E-EL-R0012c), Rat C5a ELISA kit (cat. no. E-EL-R0257c), Rat TNF-α ELISA kit (cat. no. E-EL-R0019c) and Rat IL-17A ELISA Kit (cat. no. E-EL-R0566c; all Elabscience, Houston, TX, USA) were used. The absorbance was measured at 450 nm using a microplate spectrophotometer (Multiskan GO; Thermo Fisher Scientific, Inc.).

A total of 40 mg synovial fluid and cartilage tissue was placed in 120 µl radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) with phosphatase and protease inhibitors (BIOSS, Beijing, China) for 2 h at room temperature. Protein concentration was determined with bicinchoninic acid protein assay. Total protein (20 µl) was separated on 12% Tris-glycine polyacrylamide gels. Samples were subsequently transferred onto polyvinylidene fluoride membranes (Pall Life Sciences, Port Washington, NY, USA). Membranes were incubated in blocking buffer (cat. no. P0023B; Beyotime Institute of Biotechnology) for 8 h at 4°C, and subsequently incubated with rabbit anti-C5a (cat. no. ab11876; 1:2,000), rabbit anti-IL-1β (cat. no. ab200478; 1:2,000), rabbit anti-TNF-α (cat. no. ab9755; 1:2,000; all Abcam, Cambridge, UK;), rabbit anti-IL-17A (cat. no. SAB3701458-100UG; 1:2,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and rabbit anti-GAPDH (cat. no. KC-5G5; 1:5,000; Kangchen BioTech Co., Ltd., Shanghai, China) for 8 h at 4°C. Horseradish peroxidase (HRP) goat anti-rabbit immunoglobulin G secondary antibody (cat. no. BA1054; 1:5,000; Wuhan Boster Biological Technology, Ltd.) was added for 1 h at 25°C. Enhanced chemiluminescence substrate was used for protein visualization (Pierce; Thermo Fisher Scientific, Inc.). The specific protein band intensities were quantified by Image-Pro Plus 6.0 (Media Cybernetics, Inc, Rockville, MD, USA).

Slices were subjected to treatment using 3% hydrogen peroxide for 10 min at room temperature for endogenous peroxidase inactivation. The sections were subsequently blocked using 1% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 4 h to reduce non-specific staining. For immunohistochemical staining, the sections were incubated with primary antibodies against C5a (cat. no. ab11876; 1:1,000; Abcam) for 8 h at 4°C. After washing with PBS, the slides were incubated with goat anti-rabbit IgG biotinylated-modified secondary antibody (cat. no. A27035; 1:2,000; Thermo Fisher Scientific, Inc.), and conjugated HRP-labeled streptavidin (cat. no. K0609; 1:2,000; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) was added. Following staining, three high-magnification fields (×400) were randomly selected in each slide, and the images were recorded using an inverted light microscope (Zeiss AG, Oberkochen, Germany).

Paraffin sections underwent gradient dehydration by incubating the slices in 100% xylene and a graded ethanol series (100, 95, 90, 80 and 70%). Masson staining of Weigert's iron was performed with a Masson Trichome Staining kit, according to the manufacturer's protocol (cat no. M029; Shanghai Gefan Biotechnology Co., Ltd., Shanghai, China) for 10 min at room temperature. The stained slices underwent Ponceau S staining for 10 min at room temperature, and treatment using phosphomolybdic acid for 5 min at room temperature. Aniline blue staining was performed, and the slices subsequently underwent differentiation in 1% glacial acetic acid for 1 min. Slices were treated using dehydrated alcohol (95 and 100%) and 100% xylene. Slides were subsequently viewed under an inverted light microscope.

All statistical analyses were performed with SPSS 19.0 software (IBM Corp., Armonk, NY, USA). The data are presented as mean ± standard error of the mean from three separate experiments. Statistical significance was determined by paired or unpaired Student's t-test and one-way analysis of variance followed by Tukey's post-hoc test in cases of standardized expression data. P<0.05 was considered to indicate a statistically significant difference.

Morphological analysis was conducted to investigate the effect of knee joint IM on the synovial fluid and cartilage. H&E and Masson staining were used. As presented in Fig. 1, prolonged knee joint IM resulted in aggravation of the destruction of the synovial fluid and cartilage in the IM group, while anti-C5a antibody treatment reversed these alterations. Masson staining revealed marked fibroplasia in the synovial membrane and cartilage (Fig. 2). TUNEL staining demonstrated apoptosis in the synovial membrane and cartilage. As presented in Fig. 3, the staining intensity in the IM group was increased with prolonged treatment, indicating increased cellular apoptosis in the synovial membrane and cartilage, and treatment with anti-C5a mitigated these alterations.

The expression levels of C5a, IL-1β, TNF-α and IL-17A in the serum (Fig. 4A) and joint cavity fluid (Fig. 4B) were evaluated using ELISA. The results revealed that following 1 week of knee joint IM, the expression levels of C5a, IL-1β, TNF-α and IL-17A in the joint cavity fluid and serum were significantly increased compared with those in the control group (Fig. 4). These results indicated that knee joint IM significantly induced inflammation in the knee joint. With prolonged knee joint IM, the expression levels of C5a, IL-1β, IL-17A and TNF-α in the IM group were gradually increased during the experiment compared with the control and IM+anti-C5a groups. However, the levels of C5a, IL-1β, IL-17A and TNF-α in the joint cavity fluid of in IM+anti-C5a group were lower compared with those in the IM group.

An RT-qPCR assay, western blot analysis and immunohistochemistry were performed to assess the expression of C5a in the synovialis and cartilage. The expression patterns of C5a in the synovial fluid and cartilage in the rat knee joint following IM with different treatments were evaluated by immunohistochemistry. The results revealed that the expression of C5a was significantly decreased in the IM+anti-C5a group compared with the IM group (Fig. 5). In addition, the expression of C5a mRNA was increased following 1 week of knee joint IM in the IM and IM+anti-C5a groups in the synovial fluid (Fig. 6A, left) and cartilage (Fig. 6A, right), and western blotting demonstrated similar results in the synovial fluid (Fig. 6B) and cartilage (Fig. 6C). These results confirmed the findings that increased levels of C5a may serve an important role in the development of chronic inflammation in the immobilized knee joint, and dysfunction of the synovial fluid and cartilage induced by OA.