The ATDC5 murine chondrogenic cell line (Riken Cell Bank, Tsukuba, Japan), were plated at a density of 1.2×10⁴ cells/cm² in 6-well plate (Corning, Inc., Corning, NY, USA). The cells were cultured with maintenance medium, which was Dulbecco's modified Eagle's medium/F12 (11320–033; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 5% fetal bovine serum (10099–141; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (SV30010; GE Healthcare Life Sciences, Logan, UT, USA), at 37°C in a humidified atmosphere of 5% CO₂. The maintenance medium and it was exchanged every other day. The differentiation medium, where 1% Insulin-Transferrin-Selenium (41400–045; Gibco; Thermo Fisher Scientific, Inc.) was added to the maintenance medium, which was used when the cells became confluent. The differentiation medium was changed every other day.

ATDC5 cells were cultured with differentiation medium for 7, 14 and 21 days. Total RNA was extracted using TRIzol reagent (15596026, Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was synthesized using PrimeScript RT Master mix (RR036A; Takara Bio, Inc., Otsu, Japan) Three or more different samples were used. qPCR reactions were performed using SYBR Fast qPCR mix (RR430A, Takara Bio, Inc.) according to the manufacturer's protocol. Amplification curves of samples were converted into relative expression values according to the curves of standard controls. Relative quantification of each gene was normalized against GAPDH. The 2^−∆∆Cq method was used to calculate relative gene expression levels (20). The sequences of the following primers were used: GAPDH forward (F) 5′-AGCTTCGGCACATATTTCATCTG-3′ and reverse (R) 5′-CGTTCACTCCCATGACAAACA-3′; Col II F 5′-ACGAAGCGGCTGGCAACCTCA-3 and R 5′-CCCTCGGCCCTCATCTCTACATCA-3; Col X F 5′-TGCCCGTGTCTGCTTTTACTGTCA-3 and R 5′-TCAAATGGGATGGGGGCACCTACT-3. Amplification cycle was set as 94°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 10 sec, and finally a melt curve was inserted with 65°C to 95°C.

The toxicity of HF on chondrocytic ATDC5 cells was observed using the CCK-8 method. After 14 days of chondrogenic differentiation, ATDC5 cells were seeded in 96-well plates with a density of 4×10³ cells/well and incubated for 24 h. The cells were treated with different concentrations of HF (0, 6.25, 12.5, 25, 50, 100 and 200 ng/ml). CCK-8 (CK04; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was applied to monitor the cell viability after 24, 48 and 72 h and the percentage of cell viability was calculated based on the absorbance at 450 nm after 3 h incubation using a microplate reader (51119300; Thermo Fisher Scientific, Inc.).

After 14 days of chondrogenic differentiation, ATDC5 cells were treated with HF (17395–31-2; Watson Noke Scientific Ltd., Kunshan, China) at different doses 0, 6.25, 12.5 and 25 ng/ml for 6 h, and were treated with 2 ng/ml rhTGF-β1 (240-B-002; R&D Systems, Inc., Minneapolis, MN, USA) for 30 min and processed for western blotting. Then the time-course experiment was performed. After 14 days of chondrogenic differentiation, ATDC5 cells were treated without HF for 0 h, or with 25 ng/ml HF for 4, 8 and 24 h. Then cells were treated with either 2 ng/ml rhTGF-β1 or the reagent used to reconstitute rhTGF-β1 (sterile 4 mM HCl containing 1 mg/ml bovine serum albumin) in an equal volume for 30 min and processed for western blotting. Samples (20 µg) were subjected to 10% SDS-PAGE (4561083; Bio-Rad Laboratories Inc., Hercules, CA, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (ISEQ00010; Merck KGaA, Darmstadt, Germany). Membranes were blocked in 5% bovine serum albumin (AD0023; Sangon Biotech Co., Ltd., Shanghai, China) for 1 h, incubated with primary antibodies against Smad2/3 (1:1,000; cat. no. 3102S; Cell Signaling Technology Inc., Danvers, MA, USA), phosphorylated (p)-Smad2 (1:1,000; cat. no. 3108; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and GAPDH (1:1,000; cat. no. 2118S; Cell Signaling Technology Inc.) at 4°C overnight, followed by incubation with secondary antibody (peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L); 1:5,000; cat no. ZB-2301; OriGene Technologies, Inc., Beijing, China) for 2 h. Protein was visualized using Pierce Fast Western Blot kit, enhanced chemiluminescence substrate (cat. no. 35055; Thermo Fisher Scientific, Inc.).

C57BL/6 male mice (3-month old, 24–25 g, 22 in total) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were maintained in an animal room on a 12-h light/dark cycle with a temperature and humidity of 25±2°C and 55%, respectively. The mice were provided with food and water ad libitum. The anterior cruciate ligament of the right knee was transected to establish a destabilized OA animal model. Sham operation was performed on independent mice by opening the knee joint capsule and suturing the incision in the right knee joint. Mice were divided into Sham, vehicle-treated anterior cruciate ligament transection (ACLT) group and HF-treated ACLT group (n=6–8 per group). Either HF (0.25 mg/kg) or distilled water of equivalent volume (0.2 ml) was administered by oral gavage every other day for 30 days since the second day post-operation. Mice were sacrificed at day 30 post-operatively. All animal experiment protocols were reviewed and approved by the Institutional Animal Care and Use Committee of First Affiliated Hospital of Xinjiang Medical University (Xinjiang, China).

When the mice were sacrificed, the right knee joints were resected and fixed in 10% buffered formalin for 24 h. The specimen was decalcified in 10% EDTA (pH 7.3) for 21 days and embedded in paraffin sagittally. Serial sections at 4-µm thickness of the medial compartment of the knee joint were processed for Safranin O and Fast Green staining. The sections were incubated with primary antibodies against MMP-13 (1:100; cat. no. ab39012; Abcam; Cambridge, UK), Col X (1:100; cat. no. ab58632; Abcam), TGF-β1 (1:100; cat. no. ab92486; Abcam) and p-Smad2/3 (1:40; cat. no. sc-11769; Santa Cruz Biotechnology Inc.) overnight at 4°C. Subsequently a horseradish peroxidase-streptavidin detection system (1:1,000; cat no. ZB-2301 and ZB-2306; OriGene Technologies, Inc., Beijing, China) was used to detect the immunoactivity at room temperature for 2 h, followed by counterstaining with hematoxylin (OriGene Technologies, Inc.). The number of all chondrocytes and positively stained ones were calculated by eye in the entire articular cartilage using an optical microscope (version 510_UMA_cellSens19-Krishna-ch_00_01August 2013, Olympus Corporation, Tokyo, Japan). The Osteoarthritis Research Society International (OARSI)-Modified Mankin score was calculated as previously described by Pritzker et al (21).

Data are presented as the mean ± standard deviation. One-way analysis of variance followed by the Least-Significant Difference post hoc test was used to determine if the difference among different groups was statistically significant. SPSS version 22.0 (IBM Corporation, Armonk, NY, USA) was used for data analysis. P<0.05 was considered to indicate a statistically significant difference.

To examine the chondrogenic differentiation of ATDC5 cells, expression of Col II and Col X were analyzed by RT-qPCR. Expression of Col II increased from day 1 and peaked at day 14, indicating the early-stage differentiation of chondrocytes. Col X expression gradually increased from day 7 to day 21, which indicated hypertrophic and calcified chondrocytes (Fig. 1).

In order to determine the appropriate dosage of HF for the subsequent in vitro study, toxicity of HF on chondrocytic ATDC5 cells was tested using CCK-8. Following incubation with HF of different concentrations for 24, 48 and 72 h, a dose-dependent and time-dependent decrease in cell viability was observed. Cell viability was >84% after exposure to HF of 6.25, 12.5 and 25 ng/ml for 24 h. However, it was reduced to 63% following treatment with 50 ng/ml HF and it was even lower in groups treated with 100 and 200 ng/ml HF (Fig. 2A). From 24 to 72 h of exposure, 6.25, 12.5 and 25 ng/ml HF had a negligible effect on the cell viability. As for HF concentrations of 50, 100 and 200 ng/ml, the cell viability decreased in a time-dependent manner (Fig. 2B). These findings suggested that HF of 6.25, 12.5 and 25 ng/ml did not affect chondrocytic ATDC5 cell viability.

To determine whether HF has a role in regulating TGF-β1 signaling in chondrocytic ATDC5 cells, ATDC5 cells that underwent differentiation for 14 days were used. Dose and time-dependent experiments were performed followed by western blotting. The level of p-Smad2 protein was reduced in a does dependent manner, whereas the level of total Smad2/3 remained unchanged (Fig. 3A). Next, the present study determined the effect of HF pretreatment of chondrocytic ATDC5 cells at three different time points (4, 8 and 24 h) on the phosphorylation of Smad2 protein in response to TGF-β1. The level of p-Smad2 was reduced with time, whereas the level of total Smad2/3 remained unchanged (Fig. 3B).

To investigate whether HF can attenuate OA progression by way of oral gavage an ACLT mice model was established and HF was administered every other day. Safranin O Fast Green staining was applied to assess the severity of articular cartilage degeneration. Deparaffinization of the slides was performed in xylene three times for 5 min, followed by hydration in 100% alcohol twice for 5 min, 95% alcohol for 5 min and 80% alcohol for 5 min. Hematoxylin was added to slides for 1 min prior to hydrating the slides gently in running water for 10 min. Slides were then stained with 0.2% Fast Green for 5 min, and then subjected to 1% acetic acid for 10 sec, 0.1% Safranin O for 2 min and rinsing in water for 30 sec. Slides were hydrated in 95% alcohol for 15 sec, 100% alcohol twice for 15 sec, followed by 3 changes in xylene prior to cover-slipping the slides. Proteoglycan loss in HF-treated group was reduced compared with the vehicle-treated group (Fig. 4A). Furthermore, the severity of articular cartilage degeneration was quantitatively evaluated using OARSI-Modified Mankin score. HF-treated group had a lower score compared with the vehicle-treated group, indicating reduced articular cartilage damage (Fig. 4B). Additionally, the expression of MMP-13 and Col X was lower in the HF-treated group when compared with the vehicle-treated group (Fig. 5A and B), indicating that HF had a protective role in articular cartilage degeneration. The present study aimed to investigate whether TGF-β1 had a role in the onset of OA. It was determined that the protein expression of TGF-β1 in articular cartilage was higher in the vehicle-treated group compared with the Sham group, and HF reduced its expression (Fig. 5C). Additionally, similar results were obtained for the expression of p-Smad2/3 in articular cartilage. HF-treated group had reduced expression of p-Smad2/3 compared with the vehicle-treated group (Fig. 5D). Altogether, these findings suggest HF attenuated articular cartilage degeneration by inhibiting elevated TGF-β1 signaling in articular cartilage.