Quercetin prevents osteoarthritis progression possibly via regulation of local and systemic inflammatory cascades

Abstract Due to the lack of effective treatments, osteoarthritis (OA) remains a challenge for clinicians. Quercetin, a bioflavonoid, has shown potent anti‐inflammatory effects. However, its effect on preventing OA progression and the underlying mechanisms are still unclear. In this study, Sprague–Dawley male rats were divided into five groups: control group, OA group (monosodium iodoacetate intra‐articular injection), and three quercetin‐treated groups. Quercetin‐treated groups were treated with intragastric quercetin once a day for 28 days. Gross observation and histopathological analysis showed cartilage degradation and matrix loss in the OA group. High‐dose quercetin‐group joints showed failure in OA progression. High‐dose quercetin inhibited the OA‐induced expression of MMP‐3, MMP‐13, ADAMTS4, and ADAMTS5 and promoted the OA‐reduced expression of aggrecan and collagen II. Levels of most inflammatory cytokines and growth factors tested in synovial fluid and serum were upregulated in the OA group and these increases were reversed by high‐dose quercetin. Similarly, subchondral trabecular bone was degraded in the OA group and this effect was reversed in the high‐dose quercetin group. Our findings indicate that quercetin has a protective effect against OA development and progression possibly via maintaining the inflammatory cascade homeostasis. Therefore, quercetin could be a potential therapeutic agent to prevent OA progression in risk groups.


| INTRODUC TI ON
Osteoarthritis (OA) is the most prevalent form of arthritis, which is the leading cause of disability among older adults and results in an inevitable economic and social burden on patients and families. 1,2 OA is generally characterized by progressive cartilage matrix destruction, subchondral bone sclerosis, and synovitis, resulting in pain, stiffness, and mobility loss. 3,4 Currently available therapeutic strategies for OA focus on relieving inflammation and pain. 5 In addition to joint replacement surgery, OA is commonly considered an incurable disease. Therefore, exploring the therapeutic agents that mitigate OA progression is critical for OA treatment.
The pathophysiology of OA is complex. Several factors are known to contribute to joint damage in OA, such as proinflammatory cytokines (e.g., interleukin-1 beta (IL-1β), tumour necrosis factor-alpha (TNFα) and IL-6), matrix metalloproteinases (MMPs), and aggrecan (a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)), which mediate extracellular matrix (ECM) degradation, as well as the inducible nitric oxide synthase-nitric oxide (NO) system. [6][7][8] Importantly, these factors interact closely with one another. For example, the production of MMP-13 and ADAMTS-5 can be efficiently induced by IL-1β in OA, resulting in cartilage breakdown. 9 Several dietary polyphenols such as quercetin, resveratrol, curcumin, epigallocatechin-3-gallate, rosmarinic acid, genistein, ginger, berries, silver fir, pine bark, and others have shown pain-relieving and anti-inflammatory effects and in OA models. 11 Quercetin, a flavonoid in many fruits, herbs, and vegetables, has shown anti-inflammatory, immunomodulatory, and antioxidative effects. [12][13][14][15] Quercetin has antiarthritic properties and joint protective effects in various inflammatory joint diseases. 16,17 Recent studies indicated that quercetin alleviates OA pathogenicity by inhibiting inflammation and apoptosis of chondrocytes, promoting macrophage polarization to the M2 phenotype. 18 The study also reported that quercetin inhibits endoplasmic reticulum stress-related cartilage degeneration via activating SIRT1/ AMPK signalling pathway and prevents the progression of OA in a rat model. 19 Quercetin suppresses OA progression and reduces the levels of pro-inflammatory cytokine IL-1β, IL-18, and TNFα in the OA rat model. 20 However, the anti-inflammatory activity of quercetin during OA development and progression is not fully understood.

| Chemicals and reagents
Quercetin and MIA were purchased from Sigma-Aldrich (St Louis, MO, USA). Quercetin was dissolved in normal saline. MIA was dissolved in water to make the stock solution and the working concentration was prepared by diluting the stock solution in PBS. A Bio-plex rat cytokine 22-plex assay kit (12005641) for TNFα, IL-1α,   IL-1β, IL-6, IL-7, IL-12p70, IL-18, IL-4, IL-5, IL-10, IL-2, IL-13, IL-17, IFN were purchased from Abcam. protective effect against OA development and progression possibly via maintaining the inflammatory cascade homeostasis. Therefore, quercetin could be a potential therapeutic agent to prevent OA progression in risk groups.

K E Y W O R D S
inflammation, osteoarthritis, osteochondral degeneration, pro-inflammatory markers, quercetin Experiment Center. Visual observation of rats before the experiment showed no sign of illness or joint diseases. Rats were consuming food and water properly. Rats were housed under a controlled temperature (25 ± 2°C), a constant light cycle (12 h light/dark), and suitable humidity (45%-65%). All animals were allowed free access to food and water.
The experimental procedure was started 7 days after adaptive feeding. All animal procedures were approved by the Committee of Animal Experiments of Guangzhou University of Chinese Medicine.

| Induction of OA and experimental design
Rats were randomly divided into five groups (n = 6/group): control group, MIA (OA) group, and three quercetin-treated groups (25, 50, and 100 mg/kg, i.g., q.d.). The healthy control group and OA group were used to confirm the OA development after MIA injection. Three doses of quercetin were used to find out the optimal concentration of quercetin to prevent OA development. Pieces of the literature showed the beneficial effect of 10-100 mg/kg oral or peritoneal administration of quercetin on OA treatment in different animal models. 19,21 Based on these reports from the literature, this study chooses 25, 50, and 100 mg/kg quercetin concentrations. The animals were anaesthetised by isoflurane (2.5%), and the depth of anaesthesia was checked by a toe pinch. Rats were treated with a single intra-articular injection of 1 mg of MIA in the right knee. 22 Rats in the control group were administered an equivalent volume of PBS. Rats in the quercetin-treated groups received intragastric administration of quercetin from day 0 of MIA injection to day 27, once a day. The weight of the rats was measured every week. All rats were sacrificed on day 28, and the right knee cartilage tissues were collected. The joint surface morphology was macroscopically observed under the dissecting microscope.

| Synovial fluid and serum collection
Rats were euthanized with sodium pentobarbital (100 mg/kg, i.p.) and 500 μl of PBS was injected into the rat knee articular joint cavity.
The PBS was aspirated out after 5 times injections and withdrawal procedures. Then, the synovial fluid was centrifuged, and the supernatants were stored at −80 °C. Blood was collected from the abdominal aorta. After standing for 30 min at room temperature, the samples were centrifuged at 4500 RPM for 10 min and the serum was stored at −80°C.

| Micro-CT imaging
Micro-CT was used to assess tibial subchondral bone surface and secondary osteoporosis. The knee joints were collected, and the  All samples were thawed on ice, diluted 4-fold in serum, and diluted 2-fold in synovial fluid of the joint for the final assay. The final concentrations of cytokines were calculated by Bio-Plex Manager 6.1 software (Bio-Rad) based on standard curves.

| Statistical analysis
All data are presented as the mean ± standard deviation (SD). Data were analysed using one-way analysis of variance (anova). Relative indices were analysed using SPSS version 22.0 software (SPSS).
Data normal distribution was analysed by the Shapiro-Wilk test.
LSD (homogeneous variances) and Dunnett's C (unequal variances) were used to perform post hoc tests for multiple comparisons. The data were graphically presented using GraphPad Prism 7 (GraphPad Software Inc.). Differences were considered statistically significant at a p-value <0.05

| Quercetin prevented OA progression
The chemical structure of quercetin and the experimental design are listed in Figure 1A Furthermore, to evaluate the degree of cartilage degradation, we used the Mankin score system, which scores structural damage, cellular abnormalities, and matrix staining. 27 As presented in Figure 1E, the Mankin scores of the OA group were significantly higher than those of the control group, and the high-dose quercetin group exhibited significantly lower Mankin scores than the OA group. These results indicate that quercetin could protect articular OA-related cartilage damage.

| Quercetin retained the expression of aggrecan and collagen II in OA-progressing joints
Decreases in aggrecan and collagen II result in the compression or calcification of the ECM, followed by the acceleration of arthrosis. 28 To assess the effects of quercetin on MIA-treated rats, the protein levels of aggrecan and collagen II were evaluated. As shown in Figure 4C Data are presented as mean ± SD, n = 6. Significant differences between groups, ***p < 0.001.

F I G U R E 2
Quercetin inhibited levels of inflammatory mediators and growth factors in the synovial fluid of OA-progressing joints. The expressions of pro-and anti-inflammatory cytokines, chemokines and growth factors in the synovial fluid were analysed. The values are presented as the mean ± SD, n = 6. Significant difference compared to OA group, *p < 0.05, **p < 0.01, and ***p < 0.001.

F I G U R E 3
Quercetin inhibited levels of inflammatory mediators and growth factors in the serum of OA-progressing rats. The expressions of pro-and anti-inflammatory cytokines, chemokines and growth factors in the serum of each group were analysed. The values are presented as the mean ± SD, n = 6. Significant difference compared to OA group, *p < 0.05, **p < 0.01, and ***p < 0.001.

| Quercetin prevented subchondral bone damage and bone loss in OA-progressing joints
The OA group showed significant subchondral bone injury accompanied by massive osteophyte formation compared with the control group ( Figure 5A The values are presented as the mean ± SD, n = 6. Significant difference compared to OA group, *p < 0.05, **p < 0.01, and ***p < 0.001. that quercetin treatment can effectively protect subchondral bone damage in OA-progressing joints.

| DISCUSS ION
OA is the most prevalent arthritic disease that affects the joints. OA is an age-related degenerative disease with a complicated pathology involving inflammation and ECM degradation. The majority of available therapies focus on relieving symptoms, but there are challenges to slowing the progression of the disease. Therefore, it is important to find new therapeutic agents to mitigate the development and progression of OA in vulnerable patients. 29 Quercetin has been demonstrated to exert anti-inflammatory activity in multiple disease models. [30][31][32] However, the anti-inflammatory activity of quercetin during OA development and progression is not fully understood. is still a debate about which aggrecanase plays a major role in OA mainly due to discrepancies in results from murine OA models and human OA. 53,54 In murine OA models, ADAMTS5 is the major aggrecanase. 55 In addition, ADAMTS4 is overexpressed in human OA but ADAMTS5 expression remains similar in synovial fibroblasts of both healthy donors and OA patients. 54 Proinflammatory mediators, such as IL-1β or TNFα, induce the expression of ADAMTS4 and ADAMTS5. 56  Aggrecan is a high-molecular-weight proteoglycan that has a pivotal role in cartilage structure and the joints' functions. Aggrecan is one of the main components of the cartilage ECM, which also protects collagen from degradation. The loss of ECM aggrecans in the early stages of OA is one of the critical events in the development of the disease. 57 The major structure of the ECM is composed of aggrecan and collagen type II, which preserve the normal physiological functions of cartilage. Loss of aggrecan and collagen type II contributes to the acceleration of OA progression. Thus, inhibition of the degradation of aggrecan and collagen type II may represent a novel treatment for OA. 58 Our results indicate a novel role of quercetin in preserving cartilage integrity by rescuing type II collagen and aggrecan from MIA-induced degradation.
Quercetin, a single extract natural product, has anti-inflammatory properties and has shown potential to treat various inflammatory diseases. 59 Quercetin is a senolytic drug that induces apoptosis of senescent cells. 60 Senescent cells are associated with inflammation and tissue destruction. Therefore, the anti-inflammatory and mitigation of OA-induced tissue degeneration effects of quercetin could be associated with its senolytic property. Previous studies had reported the role of quercetin in the treatment of fully developed OA. 18,19,21,61 In this study, we investigated the effect of quercetin on the prevention of OA progression and its role in the homeostasis of OA pathogenicity-related inflammatory cascades. This is the first study to evaluate the synovial and serum levels of 22 cytokines, chemokines, chemokine receptors, and growth factors in OA rats and quercetin-treated OA-progressing rats. Three doses of quercetin were used to evaluate the dose-dependent protective effect against OA development. The limitation of this study is the lack of a mechanism clarifying how quercetin prevents OA progression.
However, the OA joint is a complex microenvironment with the in-