HT (purity >98%) was purchased from Xi'an App-Chem Bio (Tech) Co., Ltd. (Shaanxi Sheng, China). Tumor necrosis factor-α (TNF-α) was an inflammatory induction factor obtained from PeproTech, Inc. (Rocky Hill, NJ, USA). The antibodies used in this experiment are included SIRT6, LC3, Beclin1, MCP-1, β-actin and horse radish peroxidase conjugated anti-rabbit secondary antibody. SIRT6 antibody (cat. no. 12486, Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a primary antibody. Polyclonal anti-microtubule-associated protein 1A/1B-light chain 3 (LC3; cat. no. 12741) and anti-Beclin1 antibodies (cat. no. 3495) were provided by Cell Signaling Technology, Inc. Antibodies against monocyte chemoattractant protein 1 (MCP-1; cat. no. NBP2-22115) were purchased from Novus Biologicals, LLC, Littleton, CO, USA. Antibodies against β-actin (cat. no. 4970) were purchased from Cell Signaling Technology, Inc. and horse radish peroxidase conjugated anti-rabbit secondary antibody was provided by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Lipofectamine^® 2000 was obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNAiso Plus, PrimeScript RT Master mix and SYBR Premix Ex Taq II were purchased from Takara Bio, Inc. (Otsu, Japan). A rat IL-1β ELISA kit was obtained from Cusabio Biotech Co., Ltd (cat. no. KET9001). (Newark, DE, USA). The mRNA primers were synthesized by Generay Biotech Co., Ltd. (Shanghai, China). 3-methyladenine (3-MA) was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Rat IL-1β and IL-6 enzyme-linked immunosorbent assay (ELISA) kit was obtained from Cusabio Biotech Co., Ltd. (cat. no. KET9001). All other chemicals were of high purity and were obtained from commercial sources.

The approval for the use of the animals in this study was granted by the Animal Ethics Committee of Xi'an Jiaotong University (Xi'an, China). Adult male Sprague-Dawley rats were provided by the Experimental Animal Center of Xi'an Jiaotong University (Animal certificate no. SYXK (shan) 2014–003). A total of 2 Male Sprague-Dawley rats (200–300 g) were euthanized as previously described immediately upon receipt (21). The knee cartilages were separated under microscopy and digested with 0.25% trypsin-EDTA for 30 min and collagenase II for 4 h at 37°C, and then the chondrocytes were filtered to produce a single cell suspension. Chondrocytes were cultured in high-glucose (4.5 g/l) Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) with antibiotics (1% penicillin and streptomycin) and 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences) in an incubator containing 5% CO₂ at 37°C, Passages between 2 and 4 were used for the experiments.

A Cell Counting kit (CCK)-8 assay (Roche Applied Science, Penzberg, Germany) was performed to determine cell viability. Briefly, chondrocytes were seeded at a density of 2×10³ cells well in 96-well microplates. Following incubation for 24 h, HT was added to the culture medium at different concentrations (0, 12.5, 25, 50, 100, 200 and 400 µM) and then treated with TNF-α (5 ng/ml) for 24 h at 37°C. The cells were then cultured with fresh media for an additional 48 h at 37°C. CCK-8 solution was added (10 µl well), and the cells were further incubated for 3 h at 37°C in a 5% (v/v) CO₂ atmosphere. The optical density was measured using a microplate reader (Thermo Fisher Scientific, Inc.) at a wavelength of 450 nm, with a background control of media and CKK-8 solution as a blank.

Following different treatment conditions, cell culture supernatants were collected. The sample was centrifuged at a rate of 200 × g for 10 min at 4°C. Concentrations of pro-inflammatory cytokines IL-1β and IL-6 were measured using a high-sensitive ELISA, according to the manufacturer's protocol.

Rat small interfering RNA (siRNA) and negative control siRNA (NC siRNA) were chemically synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Chondrocytes were seeded in 6-well plates at the density of 5.0×10⁵/ml and then were transfected with 20 µM synthesized siRNA targeting rat SIRT6. The siRNA and Lipofectamine 2000 were separately diluted in serum-free DMEM and incubated for 5 min at room temperature. Then the two solutions were gently mixed and incubated for 20 min at room temperature, prior to addition to the cells. The chondrocytes were transfected with siRNA-Lipofectamine complexes and incubated for 48 h at 37°C in a CO₂ incubator, and then used in subsequent experiments.

Cellular proteins were extracted with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing protease inhibitor cocktail. The sample concentration was determined by BCA kit (Beyotime Institute of Biotechnology), after that, 25 µml of samples were added to each well and electrophoresis was performed in 10% of the separation gel. Protein samples were separated by SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk for 4 h at room temperature and then incubated with antibodies against rabbit anti-SIRT6 (1:1,000), anti-LC3II/I (1:1,000), anti-Beclin1 (1:1,000), anti-MCP-1 (1:400) and β-actin (1:1,000) at 4°C overnight. The membranes were then incubated with a goat anti-rabbit IgG-horseradish peroxidase antibody (1:5,000 or 1:10,000) for 2 h at room temperature and subsequently washed three times with TBST buffer solution (1% Tween-20). The relative intensity of protein bands was quantified by Quantity One Analysis Software (version 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and β-actin was used as an internal control.

Total RNA was extracted from cells using RNAiso Plus reagent. cDNAs were synthesized from total RNA using PrimeScript RT Master mix following the manufacturer's protocol. cDNA was amplified using the SYBR Premix Ex Taq™ kit. Primers are given in Table I. The relative gene expression was quantitatively analyzed by the 2^ΔΔCq method (22). Data were normalized to the levels of GAPDH mRNA.

Data are presented as the mean ± standard error from three independent experiments. Statistical significance was determined using one-way analysis of variance and the Fisher's least significant difference post hoc test, using SPSS software, version 19.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Initial experiments were performed in an anti-inflammatory cell model using HT in TNF-α-treated chondrocytes. It is hypothesized that high concentrations of HT may exhibit cytotoxicity on chondrocytes. Cultured chondrocytes were exposed to increasing concentrations of HT (12.5, 25, 50, 100, 200 and 400 µM) for 24 h. Cell viability was examined using CCK-8 assay. As presented in Fig. 1, cell viability of chondrocytes was significantly reduced following HT treatment at concentrations of 200 and 400 µM. At 50 µM HT treatment, there was ~100% cell viability and no significant cytotoxicity observed, and therefore, dosages around this concentration were used in subsequent experiments to determine the anti-inflammatory effect of HT on TNF-α-induced cell inflammation.

HT has been demonstrated to exert anti-inflammatory properties, as verified by in vitro studies (17,19). HT modulates inflammatory responses in murine peritoneal macrophages and human hepatoma HepG2 and Hep3B cell lines. These previous observations encouraged the authors of the present study to determine the effect of HT on inflammatory responses in TNF-α-induced chondrocytes.

As demonstrated in Fig. 2A-C, TNF-α (5 ng/ml) alone markedly increased the levels of IL-1β, IL-6 compared with the control, and the MCP-1 protein level was also slightly elevated compared with the control, and exposure of chondrocytes to increasing concentrations of HT (25 to 100 µM) resulted in a reduction in expression of these proteins, suggesting that HT exhibited an anti-inflammatory effect in a dose-dependent manner. Therefore, HT had a concentration-dependent inhibitory effect on the release levels of IL-1β, IL-6 and MCP-1 proteins in TNF-α-stimulated chondrocytes.

To investigate the effect of HT on autophagy in chondrocytes, the conversion of LC3I to LC3II and the expression of the Beclin1 protein were measured using western blotting and the results were demonstrated in Fig. 3A and B. Chondrocytes treated with HT exhibited statistically significant increased expression levels of LC3-II (50 and 100 µM HT) and cleaved Beclin1 (25, 50 and 100 µM) compared with chondrocytes cultured with TNF-α alone, suggesting that HT may promote autophagy in chondrocytes under TNF-α-induced inflammatory conditions.

Based on these findings, HT may inhibit inflammatory responses and promote autophagy in a dose-dependent manner. However, whether the anti-inflammatory effects of HT are dependent on activation of autophagy remains unclear. To determine whether the inflammatory response is mediated by autophagy induction in TNF-α-treated chondrocytes, the effect of 3-MA, an autophagy inhibitor, on TNF-α-induced chondrocyte inflammation was evaluated. It was demonstrated in Fig. 3C, D and E that the anti-inflammatory effect of HT was significantly blocked by pretreatment of cells with 3-MA. These results suggested that the inflammatory response of TNF-α-induced chondrocytes was inhibited through autophagy.

The aim was to determine the regulatory role of SIRT6 on the inflammatory response in chondrocytes in the presence of HT. To do this, the expression levels of SIRT6 in chondrocyte cells were determined. The protein and mRNA expression levels of SIRT6 were determined by western blot analysis (Fig. 4A) and RT-qPCR (Fig. 4B). Fig. 4A and B revealed that HT enhanced the expression of SIRT6 at the protein and mRNA level in chondrocytes compared with cells stimulated with TNF-α alone.

Next, it was investigated whether the anti-inflammatory effect of HT may be regulated by SIRT6 expression in chondrocytes, and it was demonstrated that the expression of SIRT6 protein and mRNA were reduced to ~30% by SIRT6-specific siRNA in chondrocytes, compared with chondrocytes treated with NC siRNA (Fig. 4C and D, respectively). In support with the hypothesis that SIRT6 regulates the anti-inflammatory effect of HT in chondrocytes, siRNA knockdown of SIRT6 was associated with significantly increased release of the proinflammatory cytokines IL-1β and IL-6 (P<0.01; Fig. 4E and F). The results also revealed that knockdown of SIRT6 increased the protein expression levels of MCP-1 compared with the NC siRNA+HT+TNF-α group (P<0.01; Fig. 4G). Taken together, these findings suggested that HT inhibited the TNF-α-induced inflammatory response through SIRT6 activation in chondrocytes.

The final aim of the present study was to determine whether HT regulates autophagy through SIRT6 in TNF-α-induced chondrocytes. To assess this, chondrocytes were transfected with SIRT6-specific siRNA to decrease SIRT6 expression and subsequently, western blotting was performed to determine the effect on LC3-1/II and Beclin1 expression levels. As presented in Fig. 5A and B, knockdown of SIRT6 reduced the levels of Beclin-1 and LC3-II in chondrocytes compared with the NC siRNA group. These results suggested that knockdown of SIRT6 significantly suppressed the expression of LC3 and Beclin1 (P<0.01). Therefore, it was concluded that HT regulated the inflammatory response through SIRT6-meditated autophagy in TNF-α-induced chondrocytes.