Reagents and instruments

Reagents: Hyaluronic acid (HA, 924474, Merck, Shanghai, China); Chondroitin sulfate (CS, Y0000593, Merck, Shanghai, China); Phorbol 12-myristate 13-acetate (PMA, P8139, Merck, Shanghai, China); Pentobarbital sodium (P3761, SIGMA, America); 10% Fetal bovine serum (FBS, 10100147 C, Thermo Fisher, Waltham, MA, USA); 1% penicillin-streptomycin (15140148; Thermo Fisher, Waltham, MA, USA); DMEM/F12 medium (D0697, Merck, Shanghai, China); TNF-α ELISA kit (A11534, Abclonal, Wuhan, China); IL-1β ELISA kit (A16288, Abclonal, Wuhan, China); IL-6 ELISA kit (A11114, Abclonal, Wuhan, China); Hematoxylin Eosin staining solution (G1005, Servicebio, Wuhan, China); Safranine O-Fast Green Stain Kit (R21845, Saint-Bio, Shanghai, China); CCK8 kit (C0037, Beyotime, Shanghai, China); Phosphate-buffer saline (PBS, P1020, Solarbio, Beijing, China).

Instruments: electrophoresis (WIX-EP600, wix, Beijing, China); Protein electrophoresis tank (WIX-miniPRO4, wix, Beijing, China); protein transfer groove (WIX-miniBLOT4, wix, Beijing, China); Chemiluminescent imaging system (SH-523, shenhuabio, Hanzhou, China); enzyme marker (Multiskan FC, ThermoFisher, Waltham, MA, USA); short-speed centrifuge (S1010E, Scilogex, C-6 Rocky Hill, CT, USA); High-speed freezing centrifuge (H1-16KR, kechengyiqi, Changsha, China); Decolourisation shaker (SLA-O3000-S, Scilogex, C-6 Rocky Hill, CT, USA); orthostatic microscope (MZ62, mshot, Guangzhou, China); fluorescence inverted microscope (MF52, mshot, Guangzhou, China); ultrapure water system (Smart-N15VF, healforce, Hong Kong, China); Electronic ice box (EICE4, eastwin, Suzhou, China); ice machine (IMS-20, csckdq, Changshu, China); Tissue Dehydrators (TS-C20, taiho, Hefei, China); Paraffin embedding machine (ES-300, bio-hisure, Jinhua, China); Paraffin Slicer (YD-335, zjyidi, Jinhua, China).

Animal modeling and drug administration

Forty 6-week-old SD male rats (200–240 g) were purchased from Hunan Hu Jinda Laboratory Animal Co. Ltd, Production License No.: SCXK (Hunan) 2016-0002. Before the beginning of the experiment, the rats underwent a 3-day acclimatization period in a constant temperature (18–26 °C) and humidity (40–70% RH) environment. The study strictly followed the 3R principles and was approved by the Animal Committee (Ethics No. XZ202411). Rats were randomly divided into 6 groups, Sham group, OA group, OA + HA group, OA + CS group, OA + HA + CS group, and OA + HA + CS + PMA group, 5 rats in each group. anterior cruciate ligament transection (ACLT) was used in the OA, OA + HA, OA + CS, and OA + HA + CS + PMA groups to establish the OA rat model [12]. In the Sham group, the knee joint of the left hind limb was completely exposed and then the joint cavity and skin were sutured. The rats in the OA + HA group were intraperitoneally injected (IP) with 0.2mL HA (10 mg/ml dissolved in saline) [13], and the rats in the OA + CS group were IP with CS (100 mg/kg) [14]. The amount of IP of HA and CS in the OA + HA + CS group and the OA + HA + CS + PMA group was the same as that in the previous two groups, where the amount of HA and CS in the OA + + HA + CS + PMA group was the same as that in the previous two groups. In the OA + HA + CS + PMA group, PMA (10 mg/kg) was also injected, and 50 µL of sterile saline was injected intraperitoneally in the Sham group and the OA group as a control, and the frequency of injection was twice a week in both groups. The rats were anesthetized and executed by IP of sodium pentobarbital (140 mg/kg) at the end of the 8th week, and serum and bone tissues were collected.

Cell culture and grouping

Neonatal (within 24–72 h of birth) male SD rats were purchased from Hunan Hu Jinda Laboratory Animal Co. Ltd, Production License No.: SCXK (Hunan) 2016-0002. This study strictly followed the 3R principle and was approved by the Animal Ethics Committee of Hunan Evidence-based Biotechnology Co., Ltd (Ethics No. XZ202411). Rats were anesthetized by IP of sodium pentobarbital (40 mg/kg) and then cervical vertebrae were dislocated to execute the rats. Primary rat knee chondrocytes were isolated, and the isolated chondrocytes were cultured in DMEM/F12 medium containing 10% FBS and 1% penicillin-streptomycin, and passaged at 37℃ with 5% CO2. The cultured 1–2 generations of chondrocytes were equally divided into 6 groups, Control group, OA group, OA + HA group, OA + CS group, OA + HA + CS group, and OA + HA + CS + PMA group. The chondrocytes of the five groups except the Control group were exposed to a medium containing IL-1β (20ng/mL) and incubated for 36 h to establish the OA cell model. The OA + HA group, the OA + CS group, the OA + HA + CS group, and the OA + HA + CS + PMA group were added to the medium with HA (20 mg/mL), CS (100 mg/mL), and PMA ( 10 mg/mL), and equal amounts of DMEM/F12 medium were added to the Control and OA groups.

HE staining

The rat bone tissue was fixed in 10% neutral buffered formalin, dehydrated through a graded series of ethanol, cleared with xylene, and embedded in paraffin for sectioning. The paraffin sections of bone tissue were deparaffinized with xylene and rehydrated through graded ethanol to water. Hematoxylin and eosin (H&E) staining was performed, followed by clearing in xylene and dehydration through a graded ethanol series. Finally, the sections were mounted with neutral resin and observed under a standard optical microscope for imaging.

Saffron O-fast green staining

The bone tissue was decalcified by soaking in 5% calcium acetate for 1 day, followed by paraffin embedding to prepare paraffin sections. The paraffin sections of bone tissue were deparaffinized with xylene and rehydrated through graded ethanol to water. Iron hematoxylin staining was followed by an acidic differentiation solution, the solid green staining solution was added to fix the green, the sections were washed with a weak acid solution, followed by immersion staining using senna staining solution, and then dehydrated in xylene and ethanol sequentially, and the sections were sealed and then viewed under an ordinary light microscope.

OARSI score

Specimens to be analyzed were coronal sectioned after routine embedding, stained, and then observed and scored microscopically. Two main aspects were included: articular cartilage damage and osteochondrogenesis. The articular cartilage was scored on the following scale: 0 = normal; 0.5 = very minimal degeneration. Small loss of blue (or other cationic dye) staining (loss of proteoglycans), but no structural changes; 1 = very small degeneration: small surface abrasion and fibrosis below the cartilage surface are seen, with no loss of chondrocytes or cartilage matrix, and the area of damage is less than 5% of the total area; 2 = mild degeneration: the damage is vertically and below the surface of the cartilage, but seldom extends deeper into the cartilage layer. There is partial loss of cartilage surface matrix or localized areas of chondrocyte/proteoglycan loss are seen, but there is still good collagen preservation, and the area of cartilage damage is about 5–10% of the total cartilage surface; 3 = Moderate Degeneration: the injury extends vertically and downward into the calcarine layer of cartilage, with localized areas of chondrocyte/proteoglycan loss, and about 10–24% of articular cartilage is involved; 4 = Marked Degeneration: Vertical fissure/damage extending below the calcified cartilage, more than 25–50% of the articular surface of the cartilage, or loss of localized areas of chondrocytes/proteoglycans, involving 25–50% of the thickness of the articular cartilage; 5 = Severe Degeneration: vertical fissure/damage extending below the calcified cartilage of the articular, with 50–75% of the surface of the cartilage compromised or localized areas of chondrocytes/proteoglycans missing, with approximately 50–75% of cartilage thickness is compromised; 6 = very severe degeneration: vertical fissures/destruction of cartilage extending below the calcified cartilage, greater than 75% of the articular surface is compromised, and there may be only small areas of cell-free collagen remaining. Osteochondral scoring and measurement: largest osteochondral (tibial or femoral) measured using an eyepiece, 1 = small (< 150 μm); 2 = medium (151 to 300 μm); 3 = large (> 300 μm).

Immunohistochemistry

The paraffin sections of cartilage tissue were deparaffinized with xylene, rehydrated through graded ethanol to water, and underwent heat-induced antigen retrieval. Endogenous enzymes were eliminated using a 3% aqueous hydrogen peroxide solution. and sealed in a PBS solution containing 10% BSA (JK-E1344, Jkbio, Shanghai, China) for 1 h. Subsequently, the sections were exposed to specific primary antibodies Collagen-II (ab307674, 1:1000, abcam, Shanghai, China) and Aggrecan (MA3-16888, 1:1000, ThermoFisher, Waltham, MA, USA), and incubated overnight at 4 °C before coupling with HRP of the primary antibody secondary antibody was incubated for 30 min at room temperature. Color development was performed using DAB colour development kit (G1212, Servicebio, Wuhan, China), followed by counterstaining with hematoxylin. Imaging was captured using digital confocal microscopy.

ELISA

Chondrocytes were centrifuged and the supernatant was collected, and cartilage tissue was added to RIPA buffer to extract tissue stock. The supernatant and tissue stock were collected and the levels of TNF-α, IL-6, and IL-1β in chondrocytes and cartilage tissues were determined using ELISA kits.

Western blot

Total protein lysates within chondrocytes and cartilage tissues were obtained using RIPA lysate (P0013B, Beyotime, Shanghai, China) and protein concentrations were determined using the BCA protein quantification kit (P0009, Beyotime, Shanghai China), followed by upsampling of protein samples onto polyacrylamide gels (SDS-PAGE, G2003-50T, Servicebio, Wuhan, China) for electrophoretic separation. After transferring the samples to a PVDF membrane (WJ001S, epizyme, Shanghai China), the membrane was closed with 5% skimmed milk powder, and the samples were incubated with anti-IκBα (A11168, 1:1000, Abclonal, Wuhan, China), p-IκBα (AP0614, 1:1000, Abclonal, Wuhan, China), p65 (A22684, 1:1000, Abclonal, Wuhan, China), and p-65 (AP0123, 1:1000, Abclonal, Wuhan, China). Shanghai China), Collagen-II (A24863, 1:1000, Abclonal, Wuhan, China), Aggrecan (MA3-16888, 1:1000, ThermoFisher, Waltham, MA, USA) and GAPDH (AC001, 1:1000, Abclonal, Wuhan, China) primary antibodies were incubated at 4 °C overnight. Then it was incubated with HRP-coupled secondary antibody (1:2000, Proteintech) at room temperature for 2 h. It was washed with TBST solution for 5 min and developed using BeyoECL Star (G2020, Servicebio, Wuhan, China) assay for 30 s. The gray values of the bands were analyzed using ImageJ software (V1.8.0.112, NIH, Madison, WI, USA).

CCK-8

Chondrocytes (1 × 104/well) from each group were inoculated into 96-well plates separately, and CCK-8 reagent (10 µL) was added to the inoculated cells in each well, and then incubated for 2 h at 37 °C with 5% CO2. The absorbance of each well was measured at 450 nm using a microplate spectrophotometer, and the absorbance values were positively correlated with the proliferative activity of chondrocytes.

Immunofluorescence

Chondrocytes were fixed in 4% paraformaldehyde for 15 min and washed three times with PBS. The chondrocytes were subsequently closed using normal goat serum (S9070, solarbio, Beijing, China) at room temperature for 30 min. Chondrocytes were incubated with primary antibodies against Aggrecan (MA3-16888, 1:500, Thermo Fisher, Waltham, MA, USA) and Collagen-II (ab307674, 1:500, abcam, Shanghai, China) for 24 h at 4 °C, and then incubated with secondary antibodies for 1 h at room temperature. After three rinses of PBS, the membrane was observed under a fluorescence microscope and images were captured. Calculate the fluorescence positivity rate using ImageJ software (V1.8.0.112, NIH, Madison, WI, USA).

Statistical analysis

Data were expressed as mean ± standard deviation. For data obeying normal distribution, differences were tested using analysis of variance one-way analysis of variance (ANOVA), and multiple comparisons were performed using Bonferroni’s test. Statistical analyses were performed using SPSS 22.0 (IBM, Corp., Armonk, NY, USA) software, and data were plotted using GraphPad Prism (v. 9.0, La Jolla, CA), and differences were considered significant when P < 0.05.