DOI: https://doi.org/10.21203/rs.2.18601/v1
Osteoarthritis (OA) acts as a prevalent chronic age-related disease joint disease featuring cartilage degradation, osteophyte formation, and joint stiffness, which seriously affects the health of the elderly people[1]. There is an increase in the incidence rate of OA with the aging of population, thus it is necessary to find an effective strategy for the treatment of OA[2].
During the progression of OA, the increased expression of interleukin–1β (IL–1β) in the articular cartilage and synovium leads to the destruction of cartilage, downregulation of type II collagen[3]. Furthermore, IL–1β has the function of inducing the chondrocyte release of prostaglandin E2 (PGE2) and nitric oxide (NO). Thus, reducing the IL–1β in articular cavity could delay the development of OA[4].
Resveratrol is a common polyphenolic compound in the skin of red grapes[5]. Resveratrol exhibits anti-ageing, anti-inflammatory, and anti-oxidant activities, and has cardio-, neuro-, and chondroprotective effects as proved by many studies[6]. Nuclear factor-kappa B (NF-κB) signaling pathway greatly affects the OA progress[7]. NF-κB is the central potential pathway that could regulating inflammatory factors. Normally, NF-κB is combined with IκB and located in the cytoplasm. Chondrocytes stimulated by IL–1β would lead to the phosphorylation and degradation of IκB, and then the NF-κB p65 is dissociated from IκB.
However, the effects of resveratrol in treatment of OA are still unknown. This study focuses on investigating whether Resveratrol has a beneficial role in inhibiting inflammatory response induced by IL–1β in the human osteoarthritis chondrocyte. Besides, the underlying mechanisms of cell inflammatory response mediated by Resveratrol is also explored.
Resveratrol (purity>99%) was bought from the Sigma Chemical Co. (St. Louis, MO, USA). IL–1β was purchased from PeproTech (Rocky Hill, NJ, USA). Primary antibodies against collagen-II, iNOs, and COX–2, and those against p65, p-p65, p-IkBα and IkBα 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).
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.
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 (5×105 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.
Total RNA from the human OA chondrocytes (1×107 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.
RIPA lysis buffer (Solarbio, Beijing, China) was used to lyse chondrocytes and the BCA Protein Quantitation Kit (Boster, Wuhan, China) helped to measure the concentration of the protein. The same amounts of protein from every group received SDS-PAGE treatment, followed by being transferred to nitrocellulose membranes. The membranes were blocked by using 5% skim milk, and received incubation with the primary antibodies against COX–2 (1:500), iNOS (1:1000), p-p65 (1:1000), p65 (1:1000), IkB (1:1000), and p-IkB (1:1000), followed by incubation with proper HRP-conjugated secondary antibodies. The bands on PVDF membranes were observed with the exposure machine Amersham Imager 600.
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% BSA was used to block the chondrocytes for 30 minutes at 37°C. Then, chondrocytes were washed by PBS for three times. Then the samples underwent overnight incubation with rabbit anti-human collagen II antibody (1:500; Santacruz, USA) at 4°C. The chondrocytes were then received 30 min of incubation with FITC-labeled goat anti-rabbit secondary antibody (1:800; Boster, Wuhan, China) at room temperature. The nuclei were stained for one min with DAPI at room temperature and then washed by PBS for 3 times (5 min each time).
Data were analyzed with the SPSS 19.0 software (version 19.0, SPSS Corp., Chicago, American). Data are represented as the mean± standard deviation (SD) of the three independent experiments. The one-way analysis of variance (ANOVA) was performed to analyze the statistical significance and Tukey’s post-hoc test helped to compare different groups. GraphPad Prism software (Version 6.0; GraphPad Software, San Diego, CA, USA) was employed to graph. P<0.05 was considered with a statistically significant difference.
Resveratrol affects human OA chondrocyte viability
Chondrocytes was stimulated with different concentrations of resveratrol (6, 12, 24, 48μM) by CCK-8 method. According to Figure. 1, 48μM resveratrol remarkably lowered the viability of cell after 24h of treatment. There was no significant difference between the 6, 12, 24 μM relative to the control group (P>0.05).
Effect of resveratrol on NO, PGE2, iNOS and COX-2 production in human OA chondrocytes
Based on Figure. 2 and Figure. 3, IL-1β could lead to an obvious increase in the PGE2, NO, COX-2 and iNOS production compared with the control group. Nevertheless, following resveratrol administration at higher concentrations (24μM), the PGE2, NO, COX-2 and iNOS production was significantly decreased.
Resveratrol affects the expression of MMP-1, MMP-3 and MMP-13 in human OA chondrocytes
Figure. 4, displays an obvious increase in the expression of MMP-1, MMP-3 and MMP-13 mRNA due to IL-1β. Resveratrol significantly reduced the MMP-1, MMP-3 and MMP-13 due to IL-1β, and the optimal dose was 24μM.
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 and B, treatment with IL-1β in chondrocytes increased the expression of p-IkB or p-p65. Nevertheless, groups under the co-treatment with resveratrol still exhibited a lower level of p-IkB or p-p65 relative to the IL-1β group. The total IkB and p65 presented an unchangeable expression in different treatment groups respectively. As demonstrated by these data, resveratrol could restore the activation level exhibited by p-IkB and p-p65 signaling cascades (Figure. 7).
We firstly identify the optimal dose of resveratrol in inhibiting inflammation of OA. Resveratrol significantly decreased the overproduction of NO, PGE2, COX–2, MMP–1, MMP–3, MMP–13 and iNOS stimulated by IL–1β. These effects was mainly regulated the NF-κB signaling pathway.
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.
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.
Not Applicable.
Requests for the raw data may be sent to the corresponding author.
OA = Osteoarthritis; IL–1β = interleukin–1β; CCK–8 = cell counting kit–8; COX–2 = cyclooxygenase–2; MMP–1 = matrix metalloproteinase–1; MMP–3 = matrix metalloproteinase–3; MMP–13 = matrix metalloproteinase–13; NF-κB = nuclear factor-kappa B; PGE2 = prostaglandin E2; NO = nitric oxide;
The authors declare that there are no conflicts of interest in the paper.
The authors declare that they have no competing interests.
XFS, YC and QRD conducted experiments; MQZ analyzed data; QRD and XFS wrote manuscript; YC revised manuscript; XFS and YC designed the experiment and edited manuscript.
1 Department of Orthopedics, the Second Affiliated Hospital of Soochow University, Suzhou 215004, P. R. China (Xiao-Fei Shen, [email protected]); 2 Department of Orthopedics, Yancheng City No.1 People’s Hospital, Yancheng 224005, P. R. China (Yi Cheng, [email protected]; Qi-Rong Dong, [email protected]; Min-Qian Zheng, [email protected]);
Corresponding to: Qi-Rong Dong, Department of Orthopedics, the Second Affiliated Hospital of Soochow University, No.1055, Sanxiang Road, Suzhou 215004, Jiangsu Province, P. R. China, E-mail: [email protected].
Not applicable (No images, videos or details associated with an individual person).
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Not applicable.
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Gene |
Forward primer |
Reverse primer |
MMP-1 |
5′-GGGAATAAGTACTGGGCTGTTCAG-3′ |
5′-CCTCAGAAAGAGCAGCATCGATATG-3′ |
MMP-3 |
5′-CTGGCCTGCTGGCTCATGCTT-3′ |
5′-GCAGGGTCCTTGGAGTGGTCA-3′ |
MMP-13 |
5′-CCAGAACTTCCCAACCAT-3′ |
5′-ACCCTCCATAATGTCATACC-3′ |
GAPDH |
5’-CGACAGTCAGCCGCATCTT-3′ |
5’-CCAATACGACCAAATCCGTTG-3′ |
Table 1: Primers used in this study.