Contributed equally
Osteoarthritis (OA) is a common degenerative joint disease. Inflammation may exaggerate the catabolism and degeneration in the pathogenesis of OA. Hydroxytyrosol (HT) has been used in the management of inflammatory diseases. In addition, reports have revealed that autophagy was a therapeutic target of diseases caused by inflammation. Sirtuin 6 (SIRT6) has also been demonstrated to prevent OA development by reducing both the inflammatory response and chondrocyte senescence. However, the roles of SIRT6 and autophagy in cartilage and its underlying anti-inflammatory mechanism are unknown. Therefore, the present study aimed to determine the effects of HT on autophagy and inflammation in chondrocytes, and clarify whether HT regulates the inflammatory response through SIRT6-mediated autophagy. The expression of protein and mRNA were determined by western blot analysis and reverse transcription-quantitative polymerase chain reaction. The production of cytokines was detected by ELISA. It was demonstrated that HT inhibited the levels of interleukin (IL)-1β and IL-6 in tumor necrosis factor (TNF)-α-stimulated chondrocytes in a concentration-dependent manner. In addition, HT promoted cell autophagy and increased the mRNA and protein expression levels of SIRT6 in chondrocytes stimulated with TNF-α. Autophagy inhibitor 3-methyladenine or knockdown of SIRT6 decreased the inhibitory effects of HT on the inflammatory response in chondrocytes. In addition, knockdown of SIRT6 attenuated the expression of microtubule-associated protein 1A/1B-light chain 3 and Beclin1 in chondrocytes. Overall, these findings suggested that HT inhibits the inflammatory response of chondrocytes through SIRT6-mediated autophagy. The present study provided a new drug target for the clinical treatment of inflammatory diseases.
Osteoarthritis (OA) is a common degenerative joint disease which is predominantly characterized by irreversible loss of the cartilage and inflammation of synovium (
Autophagy, an evolutionarily conserved self-protective mechanism (
Hydroxytyrosol (HT) is a phenol molecule derived from olive leaves and olive oil and is part of the traditional Mediterranean diet (
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 (
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
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
HT has been demonstrated to exert anti-inflammatory properties, as verified by
As demonstrated in
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
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
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 (
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 (
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
Pro-inflammatory cytokines serve critical roles in the development of inflammation (
A previous study revealed that members of the Sirtuin family participate in inflammation (
To the best of the author's knowledge, this may be the first study to demonstrate that HT prevents TNF-α-induced chondrocyte inflammation by enhancing SIRT6 mediated autophagy activation. The present study provided a potential mechanism associating autophagy with the onset and progression of OA (
In conclusion, the results of the present study suggested that HT inhibits the inflammatory response of chondrocytes through SIRT6-mediated autophagy. This may provide novel insights into the association between HT, autophagy and inflammation in chondrocytes. Therefore, modulating autophagy represents an attractive future therapeutic target for treating diseases caused by inflammation. However, the present study did not confirm whether HT directly regulates SIRT6, which can affect autophagy to inhibit inflammatory response. Further investigation will allow better characterization of the molecular mechanisms underlying these effects. A further study will investigate which receptors or signal molecules are involved in the regulatory process of HT on SIRT6.
Effect of HT on TNF-α-induced chondrocyte viability. Chondrocytes were incubated with different concentrations of HT (0, 12.5, 25, 50, 100, 200 and 400 µM) for 24 h. The cell viability was determined using a Cell Counting kit-8 assay. The results were expressed as the mean ± standard error (n=3). **P<0.01 vs. 0 µM HT. HT, hydroxytyrosol; TNF-α, tumor necrosis factor-α.
HT inhibits the TNF-α-induced inflammatory response in chondrocytes. The effect of HT on the production of (A) IL-1β and (B) IL-6 in chondrocytes was measured by ELISA assay. Chondrocytes were pretreated with various concentrations (25, 50 and 100 µM) HT for 1 h, then stimulated with TNF-α (5 ng/ml) for 24 h. The release of IL-1β and IL-6 was measured using high-sensitive ELISA kits. (C) The expression of MCP-1 was detected by western blot analysis. The results were expressed as the mean ± standard error (n=3). **P<0.05 vs. control group. #P<0.05; ##P<0.01 vs. TNF-α group. HT, hydroxytyrosol; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein 1.
HT promotes autophagy in TNF-α-induced chondrocytes. Chondrocytes were pretreated with HT at different concentrations (25, 50 and 100 µM) for 1 h and then stimulated with TNF-α (5 ng/ml) for 24 h. The protein expression of (A) LC3II and (B) Beclin1 were assessed by western blot analysis. Next, the chondrocytes were pretreated with an autophagy inhibitor, 3-MA (5 mM), for 1 h, and then incubated with HT (50 µM) for 1 h, followed by stimulation with TNF-α for 24 h. The concentration of (C) IL-1β and (D) IL-6 in cell supernatants were determined by ELISA. (E) The expression levels of MCP-1 were detected by western blot analysis. The results are expressed as the mean ± standard error (n=3). **P<0.01 vs. control group. ##P<0.01 vs. TNF-α group. ++P<0.01 vs. HT+TNF-α group. HT, hydroxytyrosol; TNF-α, tumor necrosis factor-α; 3-MA, 3-methyladenine; IL-1β, interleukin-1β; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein 1.
HT upregulates SIRT6 expression in chondrocytes stimulated with TNF-α. Chondrocytes were pretreated with HT (25, 50 and 100 µM) for 1 h, and then stimulated with TNF-α for 24 h. The expression of SIRT6 (A) protein and (B) mRNA were determined by western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. Chondrocytes were transfected with SIRT6 siRNA or NC siRNA for 48 h, and then the expression of SIRT6 (C) protein and (D) mRNA was assessed. The chondrocytes transfected with SIRT6 siRNA or NC siRNA were pretreated with HT (50 µM) for 1 h and then stimulated with TNF-α (5 ng/ml) for 24 h. The release of (E) IL-1β and (F) IL-6 were measured using an ELISA kit. (G) The expression of MCP-1 was detected by western blot analysis. The results were expressed as the mean ± standard error (n=3). **P<0.01 vs. control group. #P<0.05, ##P<0.01 vs. TNF-α group. ++P<0.01 vs. NC siRNA+HT+TNF-α group. HT, hydroxytyrosol; TNF-α, tumor necrosis factor-α; SIRT6, sirtuin-6; NC, negative control.
HT regulates autophagy through SIRT6 in TNF-α-induced chondrocytes. The chondrocytes transfected with SIRT6 siRNA or NC siRNA were pretreated with HT (50 µM) for 1 h and then stimulated with TNF-α (5 ng/ml) for 24 h. Expression of (A) LC3II and (B) Beclin1 were determined by western blot analysis. The results were expressed as the mean ± standard error (n=3). **P<0.01 vs. control group. ++P<0.01 vs. NC siRNA + HT + TNF-α group. HT, hydroxytyrosol; TNF-α, tumor necrosis factor-α; SIRT6, sirtuin-6; NC, negative control; LC3, microtubule-associated protein 1A/1B-light chain 3.
Primer sequences used for reverse transcription-quantitative polymerase chain reaction.
Gene | Primer | Sequence (5′-3′) |
---|---|---|
Sirtuin 6 | Forward | 5′-GCAGTCTTCCAGTGTGGTGT-3′ |
Reverse | 5′-CCATGGTCCAGACTCCGT-3′ | |
GAPDH | Forward | 5′-GGCACAGTCAAGGCTGAGAATG-3′ |
Reverse | 5′-ATGGTGGTGAAGACGCCAGTA-3′ |