Resveratrol inhibit interleukin-1β induced inflammatory response in human osteoarthritis chondrocytes through NF-κB signaling pathway

Osteoarthritis (OA) is a common degenerative disease and affect millions of people. Resveratrol is a coumarin compound refined from traditional Chinese medicines with potential anti-inflammatory ability. This study aimed to evaluate protective anti-inflammatory effects of resveratrol in human OA chondrocytes. The chondrocytes were isolated from OA patients. CCK-8 method was used to explore the optimal dose of resveratrol for chondrocyte. Next, we used PCR and Western-blot method to assess the relative mRNA and protein expression in IL-1β group. We further explored relevant mechanism of resveratrol for anti-inﬂammatory in human OA chondrocytes by immunoﬂuorescence and Western-blot. The results showed that resveratrol blocked IL-1β-stimulated production of NO and PGE2. In addition, resveratrol inhibited the expression of COX-2, iNOs, MMP-1, MMP-3, MMP-13, and increased the levels of aggrecan and collagen-II. Mechanistically, resveratrol suppressed IL-1β-induced IκB-α degradation and NF-κB activation. In conclusion, our results demonstrate that resveratrol inhibits inflammation in OA via the regulation of NF-κB signaling pathway, and suggested that resveratrol may be a potential therapeutic agent for OA.

were acquired from the Abcam (Cambridge, MA, USA) and the Cell Signaling Technology (Beverly, MA, USA), respectively. Cell Counting Kit-8 (CCK-8) was provided by the Boster Biotechnology (Wuhan, China).

Primary human chondrocyte culture
We isolated the human chondrocytes from articular cartilages of four knee OA patients (classified as Kellgren-Lawrence score IV). All patients underwent total knee arthroplasty surgery at the Second Affiliated Hospital of Soochow University.
Briefly, cartilage tissues were firstly cut into small pieces, followed by being washed by Hank's for 3 times. Then, the small pieces underwent 30 min of digestion with 0.25 mg/mL trypsin, together with 8 h of digestion with 2 mg/mL collagenase type II at 37℃. Cells were first filtered by 180-µm filter, and then collected and centrifuged at 100g for 5min. After washing by Hank's for three times, the chondrocytes were cultured in DMEM added with 1% penicillin and 10% FBS.

Cell viability
We used CCK-8 kit to measure chondrocytes viability following the instruction of manufacturers. In brief, we seeded human OA chondrocytes in 96-well plates (5×10 5 cells/ml) and treated them with or without resveratrol (6, 12, 24, 48 μM) for 24h.
After that, we added 10 uL CCK-8 into the plates. All of the wells were incubated for 2-4 hours. Optical density of each wells was measured by Enzyme-linked immunodetector.
qRT-PCR Total RNA from the human OA chondrocytes (1×10 7 cells) was extracted using a Trizol reagent (Life Technologies, USA) following the instruction of manufacturers. A spectrophotometer was employed to measure the concentration and integrity exhibited by the total RNA considering the A260/A280 ratio. The iScript cDNA synthesis kit (Bio-Rad, USA) helped to reversely transcribe RNA into cDNA in a 20μl reaction system. Forward primers and reverse primers are listed in Table 1. The 2 − ΔΔCt method was adopted to calculate the relative mRNA expression changes.
Results are presented as the n-fold change compared with β-actin.

Immunofluorescence
The chondrocytes were seeded in confocal dish and treated as previously described.
We used PBS to rinse the confocal dish for three times at a 5-minute interval.
Subsequently, all samples of the cells were fixed by 4% paraformaldehyde for 15min. Then, PBS was used to wash these cells for 5 minutes for 3 times. Next, 0.5%

Resveratrol affects the expression of aggrecan and collagen-II in human OA chondrocytes
Immunofluorescence results were presented in Figure. 5. We found that IL-1β could remarkably decrease the collagen-II expression. The expression of collagen-II presented an obvious uptrend in resveratrol group than IL-1β group. We used RT-PCR to further confirm our immunofluorescence results. We measured Aggrecan and collagen-II relative expression in these groups, finding the obvious decrease in the expression of collagen-II and Aggrecan due to IL-1β. Resveratrol could rescue the degradation of Aggrecan and collagen-II in part ( Figure. 6).

Resveratrol affects the activation of NF-κB in human OA chondrocytes
As demonstrated in Figure. 7A

Discussion
We firstly identify the optimal dose of resveratrol in inhibiting inflammation of OA.
Resveratrol was extracted from the root of Veratrum grandiflorum in 1940 for the first time by a Japanese [8] . Possessing a stilbene structure, resveratrol acts as a non-flavonoid polyphenol compound. It has been proven that resveratrol can make plants more resistant to pathogens as well as improve environment deterioration [6] .
Wei et al. [6] also revealed that resveratrol could improve the inflammatory damage as well as protect against OA in an OA rat model. Xu et al. [5] revealed that resveratrol could inhibit TLR4 through activating PI3K/Akt signaling pathways in obesity-related osteoarthritis, thus exerting anti-osteoarthritic effects. Hussain et al. [9] conducted a pilot interventional study and found that resveratrol was superior in terms of safety and efficacy to meloxicam alone for the treatment of pain and improvement of physical function in knee OA patients.
Firstly, we found that the optimal dose of resveratrol by CCK-8 methods. We found that when the dose of resveratrol larger than 48 μM, the cells viability was down with statistically significantly than control group and other dose of resveratrol. Kang et al. [10] revealed that reveratrol had no cytotoxic effect at 1 to 100 μM. Reason may be that they used rabbit chondrocyte and performed lactate dehydrogenase (LDH) assay to assess toxic effect.
It is known that articular chondrocytes-generated IL-1β has the function of stimulating the development of osteoarthritis. As reported, IL-1β could remarkably enhance the expressions exhibited by pro-inflammatory cytokines and MMPs in the chondrocyte; therefore, it enjoys a wide application in the stimulation of inflammatory response in the chondrocyte to simulate OA in vitro [11] . When we used IL-1β stimulate the chondrocytes, we found an obviously up-regulated MMP-1, MMP-3 and MMP-13 [12] . MMPs serve as the major enzymes regulating the remodeling of tissue as well as the degradation of extracellular matrix like collagens and aggrecan. Above results conformed previous findings [13] . Gu et al. [14] reported that resveratrol had the function of suppressing the MMP-13 and IL-6 expression in human articular chondrocytes induced by IL-1β through signaling cascades dependent and independent of TLR4/MyD88. Therefore, it is speculated that reveratrol could make MMPs less produced and activated during the progress of OA, thus exerting the anti-inflammation effect.
NF-κB signaling serves as an essential pathway related to the OA pathogenesis.
Rigoglou et al. [15] proposed that any strategies interfering NF-κB signaling pathway activation could provide novel potential treatment options for OA. When the NF-κB signaling pathway was activated, downstream protein such as NOS, COX-2 and MMPs were overexpression [16,17] . In normal cartilage, NF-κB is located in the cytoplasm and bonded with IκB. Stimulating with IL-1β and other stimulations would contribute to the phosphorylation and degradation of IκB, and then NF-κB p65 could be dissociated from IκB [18] , followed by being translocated into nucleus for activating genes related to inflammatory. Conforming the previously published study results, this study found the ability of IL-1β to facilitate p65 phosphorylation and IκBα degradation in the chondrocyte. Also, treating with resveratrol could weaken the NF-κB activation.
Moreover, resveratrol potently inhibited MMP-1, MMP-3 and MMP-13 expressions and aggrecan and collagen-II degradation, and the extracellular matrix was degraded. Besides, resveratrol exerted an obvious suppressing impact on the phosphorylation of NF-κB stimulated by IL-1β in human OA chondrocytes.

Conclusion
To sum up, according to the current study, resveratrol weakens the inflammatory response induced by IL-1β in human OA chondrocytes. As proved by other studies regarding the potential mechanism, inhibiting the activation of NF-κB could enhance the protective effect imposed by resveratrol. The study result confirmed eriodictyol as a novel preventative agent specific to OA. Nevertheless, the currently obtained results with regard to the in vitro situation might not be applicable for the in vivo situation, thus direct tests are needed. Table   Gene Forward primer Reverse primer  Effects of the resveratrol on the expression of iNOS and COX-2 in human OA chondrocytes in Figure 3 Effects of the resveratrol on the expression of iNOS and COX-2 in human OA chondrocytes in Effects of resveratrol on the expression of aggrecan and collagen-II in human OA chondrocyt Effects of resveratrol affects the activation of NF-κB in human OA chondrocytes.

Figure 7
Effects of resveratrol affects the activation of NF-κB in human OA chondrocytes.