Complementary and Alternative Therapies for Inflammatory Diseases 2020View this Special Issue
Research Article | Open Access
Mi-Rae Shin, Jin A Lee, Min Ju Kim, Hae-Jin Park, Byeong Wook Park, Seung Bo Seo, Seong-Soo Roh, "Protective Effects of Phellinus linteus Mycelium on the Development of Osteoarthritis after Monosodium Iodoacetate Injection", Evidence-Based Complementary and Alternative Medicine, vol. 2020, Article ID 7240858, 12 pages, 2020. https://doi.org/10.1155/2020/7240858
Protective Effects of Phellinus linteus Mycelium on the Development of Osteoarthritis after Monosodium Iodoacetate Injection
Objective. The aim of this study was to identify the protective effects of Phellinus linteus mycelium (PLM) and its possible mechanisms in a model of monosodium iodoacetate- (MIA-) induced osteoarthritis (OA). Methods. Intra-articular injection of MIA was injected to 50 μL with 80 mg/mL using a 0.3 mL insulin syringe into the right knee joint. Changes in hindpaw weight-bearing distribution between the right (osteoarthritic) and left (contralateral control) legs were used as an index of joint discomfort. PLM (50, 100, and 200 mg/kg body weight) was orally administered once daily for 14 days from day 7 after MIA treatment. And then, various factors associated with inflammatory response and cartilage degeneration in cartilage tissues detected by western blotting. Results. PLM treatment showed a concentration-dependent elevation in change in hindpaw weight-bearing distribution (HWBD). PLM200 demonstrated the capacity to significantly increase HWBD, indicating that the change in weight-bearing distribution means the reduction of spontaneous pain. Our results indicate that PLM suppressed the inflammatory factors via NF-κB signaling pathway induced by p38 phosporlyation. Moreover, PLM200 exhibited a significant reduction of ROS produced by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. PLM100 and PLM200 inhibited the levels of matrix metalloproteinase (MMP)-1, one of proteinase that degrades extracellular matrix (ECM). Conclusions. Taken together, our results indicated that PLM has a strong chondroprotective effect through the suppression both ROS production and inflammation.
Osteoarthritis (OA) is nowadays one of the most prevalent chronic arthritis of human joint, and age and obesity are well known for its powerful risk factors [1, 2]. OA is characterized by cartilage degradation and inflammation and develops progressively over several years. Consequently, OA causes physical limitations, disability, mental stress, and socioeconomic burden . Therefore, both bodily pain and physical functioning by OA are lower than that for cardiovascular conditions, chronic respiratiory diseases, and gastrointestinal conditions . OA leads to much lower quality of life. Although OA was referred to as the noninflammatory arthritis historically, it is considered a low-grade inflammation disease [5, 6] triggered by factors like biomechanical stress associated with OA development . Therefore, anti-inflammatory drugs like nonsteroidal anti-inflammatory drugs (NSAIDs) were given to patients with symptomatic OA to remove OA symptoms [8, 9]. However, these drugs led the gastrointestinal and cardiovascular side-effects . Accordingly, new strategies about an effective and a safe herbal medicine for OA treatment is urgently needed.
Currently, many studies focused on describing the key factors responsible for the deepening of inflammatory processes . An analysis of the ever-increasing reports showed the important role of the cytokine network in the pathogenesis of OA . During the progression of OA, the degree of cytokine production can make changes to the duration and severity of OA . Such inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 are important factors participating in the pathogenesis of OA  and intimately related to cartilage matrix degradation. Matrix metalloproteinases (MMPs), proteolytic enzymes, are a family of zinc-dependent endoproteases and possess multiple roles in degradation of various proteins and tissue remodeling in the extracellular matrix (ECM). Moreover, MMPs promote cell migration, proliferation, and differentiation and could play a role in cell apoptosis, tissue repair, and angiogenesis . In addition, MMPs expression also increases during the inflammatory process. MMPs are secreted by inflammatory cells and promoted by inflammatory cytokines . Whereas, it generally counteracts inhibitory actions of endogenous TIMPs [17, 18]. Hence, the inhibition of inflammatory cytokines and MMPs may be a valid approach for OA treatment.
Medicinal fungi could produce diverse bioactive metabolites including anticancer drugs, antibiotics, and immunosuppressants [19–21]. Many medically meaningful metabolites had been reported from various edible fungi. Phellinus linteus is a famous medicinal mushroom that is widely used in Korea, Japan, China, and other Asian countries . The various components including polyphenols, pyrans, polysaccharides, and triterpenoids play a significant role in improving the health condition [23, 24]. Above all, Phellinus linteus mycelium (PLM) was used as medicines or healthy foods to treat several diseases such as infections, ulcer, cancer, and diabetes . In the previous reports, PLM showed wide spectrum of bioactivities, such as antioxidant, anti-inflammatory, cytotoxic, antiviral, and antidiabetic effects [26–28]. Especially, β-glucan isolated from mushroom is a natural polysaccharide and possess various biological effects such as immune-modulating properties, antitumor, anti-infection, and lowering blood cholesterol [29, 30].
The hypothesis of the current study was PLM supplementation could have chondroprotective effect via the inhibition of cartilage matrix degradatin on the articular cartilage. So, the efficacy and underlying mechanism of PLM were examined using the MIA-induced OA model.
2. Materials and Methods
Monosodium iodoacetate (MIA), phenyl methyl sulfonyl fluoride (PMSF), dithiothreitol (DTT), and diethylenetriaminepentaacetic acid (DTPA) were purchased from Sigma Aldrich Co., Ltd (St. Louis, MO, USA). The protease inhibitor mixture solution and ethylene diamine tetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). ECL western blotting detection reagents and pure nitrocellulose membranes were supplied by GE Healthcare (Piscataway, NJ, USA). 0.3 mL insulin syringe was obtained from BD Medical-Diabetes Care (Holdrege, USA). Besides, all other chemicals and reagents were purchased from Sigma Aldrich Co., Ltd. (St Louis, MO, USA). Primary antibody rabbit polyclonal antibodies against p47phox, p22phox, catalase, glutathione peroxidase-1/2 (GPx-1/2), phospho-p38 (p-p38), goat polyclonal antibodies against tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), mouse monoclonal antibodies against nuclear factor-kappa B p65 (NF-kBp65), cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), matrix metalloproteinase-1 (MMP-1), tissue inhibitor matrix metalloproteinase 1 (TIMP-1), histone, β-actin, and secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Moreover, rabbit polyclonal antireduced nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) was obtained from LifeSpan BioSciences (Seattle, WA, USA). The BCA protein assay kit is supplied by Thermo Fisher Scientific (Waltham, MA, USA).
2.2. Preparation of PLM
Phellinus linteus mycelium was obtained as a dried mycelium from Hankook Shinyak Corp. (Nonsan-si, Korea), and Green-lipped mussel powder was obtained from McFarlane Marketing (Aust) Pty Ltd. (Melbourne, Australia). Phellinus linteus mycelium was inoculated under optimal culture condition (at 30°C and pH 4-5) for 14 days on yeast malt extract glucose (YMG) agar. The extract condition was optimized for extraction temperature (100°C), extraction time (24 h), solvent amount (10 times purified water), and extraction frequency (1st). A 50 ton large-scale incubator was used to produce 7 g/L dry mycelium in 4 days after mass production of mushroom mycelium through the following manufacturing process (Table 1). The produced PLM analyzed the nutritional components in accordance with the method of food revolution at Korea Health Supplement Institute (Seongnam, Korea) . The composition of PLM is shown in Table 2.
2.3. β-Glucan Measurement of PLM
β-Glucan content in PLM was analysed using the K-YBGL kit (Megazyme, Ireland) as the mushroom and yeast β-glucan assay procedure. Extraction, laboratory analysis, and calculation were performed in accordance with the manufacturer’s instructions. β-glucan content was calculated as a percentage using the following equation:
Consequently, β-glucan content of PLM was 14.16 ± 6.27%.
2.4. Development of OA with MIA Injection and PLM Administration
Seven-week-old male Sprague Dawley rats weighing 200–250 g at the start of the experiment were purchased from DBL Co. (Eumseong, South Korea). The animals were housed three per cage in a room with controlled temperature conditions (23 ± 2°C), humidity (about 55 ± 5%), and lighting (12 h light/dark cycle) with free access to food and water. After adaptation (1 week), rats were randomly arranged in the descending order of weight and assigned into six groups of equal numbers (n = 7) without any statistical significance among the groups: Group 1: Nor group included the normal rats Group 2: Con group included the MIA control rats Group 3: GLM200 group included the green-lipped mussel 200 mg/kg-administered and MIA rats Group 4: PLM50 group included the PLM 50 mg/kg-administered and MIA rats Group 5: PLM100 group included the PLM 100 mg/kg-administered and MIA rats Group 6: PLM200 group included the PLM 200 mg/kg-administered and MIA rats
The normal and MIA control groups were administrated water using a stomach tube, while the other groups were orally administered green-lipped mussel 200 mg/kg or PLM 50, 100, and 200 mg/kg using a stomach tube for 2 weeks. OA was induced in SD rats; we employed the method of Wang with minor modifications . After anesthetization with injection of Zoletil mixture (Vibrac, France) 0.75 mg/kg intraperitoneally, rats were injected with MIA 80 mg/mL in a 50 μL volume using a 0.3 mL insulin syringe (31G needle) inserted through the patellar ligament into the intra-articular space of the right knee; normal rats were injected with an equivalent volume of saline . The rats in all groups were sacrificed after the end of experimental period. The rats were anesthetized using a mixture of Zoletil and xylazine and euthanized by isoflurane overdose. Right knee joint specimens were collected, and the muscles around the knee were joint quickly removed. The femoral cartilage of rat was removed through circular incision along the femoral edge of the articular capsule. In addition, the femur and tibial fibula were cut off along each bone surface. And then, the joint capsule was cut longitudinally and the synovial tissue was separated. The cartilage specimens were stored at −80°C until further study. The terminal blood samples were centrifuged at 3000 rpm for 20 min at 4°C and the serum was collected and stored at −80°C until analysis.
2.5. Measurements of Hindpaw Weight-Bearing Distribution
Hindpaw weight-bearing distribution (HWBD) was assessed using an incapacitance meter that measures the distribution of weight bearing across each hindlimb. The balance in the weight-bearing capability of the hindpaws was disrupted after OA induction. A significant shift in weight from the arthritic site to the contralateral limb, i.e., a weight bearing deficit, was considered to be an index of pain. Pain was measured via the weight bearing of the paw load using an incapacitance tester (Linton Instrumentation, Norfolk, UK) . The hinpaw weight bearing distribution ratio (%) was calculated using the following equation:
2.6. Preparation of Cytosol and Nuclear Fractions
Protein extraction was performed according to the method of Komatsu with minor modifications . The cytosol fraction was homogenized with ice-cold lysis buffer A (250 mL) containing 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, and 1,250 μL protease inhibitor mixture solution. The homogenate was incubated at 4°C for 20 min. And then, 10% NP-40 was added and mixed well. After centrifugation (13,400 ×g for 2 min at 4°C) using Eppendorf 5415R (Hamburg, Germany), the supernatant liquid (cytosol fraction) was separated into a new e-tube. The left pellets were washed twice by buffer A, and the supernatant was discarded. Next, the pellets were suspended with lysis buffer C (20 mL) containing 50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 1% (v/v) glycerol, and 100 μL protease inhibitor mixture solution suspended and incubated at 4°C for 30 min. After centrifugation (13,400 ×g for 10 min at 4°C), the nuclear fraction was prepared to collect the supernatant. The protein concentration was determined using BCA protein assays by Thermo Fisher Scientific (Waltham, USA). Both cytosol and nuclear fractions were kept at −80°C before the analysis.
2.7. Immunoblotting Analysis
For the estimation of NF-κBp65 and histone (1 : 1000; Santa Cruz Biotechnology, Dallas, TX, USA), 10 μg of proteins from each nuclear fraction was electrophoresed through 8–10% sodium dodecylsulfate polyacrylamide gel (SDS-PAGE). Separated proteins were transferred to a nitrocellulose membrane, blocked with 5% (w/v) skim milk solution for 1 h, then incubated with primary antibodies to NF-κBp65 and histone, respectively, overnight at 4°C. After the blots were washed, they were incubated with anti-mouse IgG HRP-conjugated secondary antibody (1 : 3000; Santa Cruz Biotechnology) for 1.5 h at room temperature. In addition, 10–12 μg proteins of each cytosol fraction was eletrophoresed in 10–14% sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) for immunodetection of p-p38/COX-2/iNOS/TNF-α/IL-6/IL-1β/catalase/GPx-1/2/MMP-1/TIMP-1/β-actin (1 : 1000; Santa Cruz, USA). Each antigen-antibody complex was visualized using ECL western blotting detection reagents and detected by chemiluminescence with Sensi-Q 2000 Chemidoc (Lugen Sci Co., Ltd., Gyeonggi-do, Korea). Band densities were measured using ATTO Densitograph software (ATTO Corporation, Tokyo, Japan) and quantified as the ratio to histone or β-actin. The protein levels of the groups are expressed relative to those of the normal rat (represented as 1). We followed the methods of Mi-rae Shin et al. 2017 .
2.8. Statistical Analysis
Data are expressed as mean ± SEM. Statistical comparisons were assessed by one-way ANOVA followed by the least-significant differences (LSD) test (SPSS 22.0 for Windows, SPSS Inc., NY, USA). Values of were considered significant.
3.1. Change in Hindpaw Weight-Bearing Distribution
The MIA model is the most often used, being commonly chosen to evaluate the efficacy of pharmacological agents for pain management. Pain assessment assay commonly used is the incapacitance test, which measures the weight distribution between both hind limbs . The pain of knee joint leads to decrease of hindpaw weight-bearing distribution (HWBD), which has been proved in the previous study . The HWBD on 0, 1, and 2 weeks is measured in Figure 1. On day 0, the HWBD of MIA-treated groups was lower compared with that of the normal group (). After 2 weeks, the decreased HWBD was significantly increased at GLM200 and PLM200 groups (). The pain of knee joint could alleviate though PLM supplementation.
3.2. NOX4, p47phox, and p22phox in Cartilage Tissues
The NADPH oxidase family including NOX4, p47phox, and p22phox is one of the potent cellular sources in the overproduction of reactive oxygen species (ROS) . The expressions of NOX4, p47phox, and p22phox were examined, as shown in Figure 2. The treatment of MIA significantly led to upregulation of both NOX4 and p22phox in the cartilage tissue; however, PLM200 administration significantly suppressed the levels of all markers (NOX4; , p47phox; , and p22phox; ).
3.3. p38-MAPK and NF-κBp65 in Cartilage Tissues
The previous study reported that p38-MAPK activity attenuates the transcriptional activity of the proinflammatory transcription factor, NF-κB [39, 40]. Activation of p38-MAPK affected the NF-κBp65 protein expression (Figure 3). The injection of MIA resulted in a significant increase of p38-MAPK compared with the normal group. In contrast, the treatment of all drugs significantly reduced the expression of p38-MAPK. Besides that, the NF-κBp65 expression in MIA control was elevated, whereas it was attenuated by GLM200, PLM100, and PLM200 administration ().
3.4. TNF-α, IL-6, IL-1β, COX-2, and iNOS in Cartilage Tissues
Activated NF-κB regulates the expression of inflammatory mediators and many cytokines . Figure 4 displays the effect of PLM on the expression of inflammation-related mediators such as COX-2, iNOS, and cytokines including TNF-α, IL-6, and IL-1β. The MIA treatment significantly augmented these protein expressions. On the other hand, PLM200 treatment significantly decreased all factors. Especially, the suppression effects of PLM200 was near the levels of the normal group (iNOS, IL-6, and IL-1β; , COX-2; , TNF-α; ).
3.5. MMP-1 and TIMP-1 in Cartilage Tissues
Cytokines upregulate metalloproteinase (MMPs) gene expression, and such prolytic enzymes degrade extracellular matrix, whereas TIMP-1 inhibits MMP-induced ECM degradation for the maintenance of homostasis . Our results in this study revealed that PLM200 significantly suppressed the expression of MMP-1 (), otherwise elevated TIMP-1 without a significance (Figure 5).
3.6. Catalase and Gpx-1/2 in Cartilage Tissues
The activity of ROS is balanced by enzymatic antioxidants such as catalase and GPx-1/2 . Antioxidant properties could reinforce the cellular antioxidant status. In our results, MIA control rats showed decreased expressions of catalase and GPx-1/2 compared with those of normal rats; however, PLM administration effectively upregulated these levels. Herein, antioxidant enzyme catalase significantly increased compared with MIA control rats by GLM200, PLM100, and PLM200 supplementation () (Figure 6).
Osteoarthritis (OA) is a chronic and degenerative joint disease characterized by intra-articular inflammation and cartilage degradation . Thus, inhibition of inflammation has been proposed as an effective strategy for improving the symptom or delaying the progression of OA. NSAIDs are the typical prescribed medications for treating disorders such as OA. But, the adverse effects about long-term uses of NSAIDs still existed . Phytochemicals can modulate inflammatory response and treat OA . Such nature-derived compounds have recently been paid attention to ideal drugs for OA due to their excellent anti-inflammatory activities and limited adverse effects [46, 47].
Phellinus linteus mycelium (PLM), a well-known medicinal mushroom, has been used in Asian countries for many centuries to prevent or treat diseases such as gastroenteric dysfunction, hemorrhage, diarrhea, rheumatoid arthritis, and cancers . Our findings revealed that PLM inhibited inflammatory responses in MIA-induced osteoarthritis rats. To the best of our knowledge, this is regarded the first report to demonstrate the chondroprotective effect of PLM- on MIA-induced OA. Above all, hindpaw weight-bearing distribution (HWBD) is measured. HWBD, as a measure of OA progression, was measured as the difference between MIA-injected and contralateral hind limbs. The groups with GML200 and PLM administration displayed a higher HWBD compared with that of the MIA control group. Especially, PLM200 showed a significant increase. The pain of cartilage leads to decrease of HWBD, which has been proved by several studies [49, 50]. These results demonstrate a balance and relief of cartilage discomfort in the PLM-treated group.
As mentioned earlier, the degradation of cartilage results from the imbalance between catabolic activity and the mechanical stresses, and the latter is primarily controlled by ROS and MMPs. Thus, our results can be explained that the antioxidant defense of PLM decreased the cartilage damage by inhibition of the formation of ROS and MMPs. Disruption of the cartilage homeostasis leads to cartilage destruction. ROS-induced damage is significantly greater in OA cartilage. ROS in the OA process is focused in particular on NADPH oxidase, which is heme-containing transmembrane proteins and especially includes Nox4, p47phox, and p22phox. Ultimately, NADPH oxidase catalyses the transfer of the electron from NADPH and generates superoxide anion (), giving rise to ROS [38, 51]. We measured protein expressions of NADPH oxidases associated with ROS production to investigate the role of PLM. Herein, excessive production of ROS damages nucleic acids, protein, lipids, and matrix components . Mitochondria are essential for cellular bioenergetics and regarded as the major cellular site for ROS production . Our present data indicate that treatment with PLM200 decreased NOX4, p47phox, and p22phox production in the cartilage. H2O2 is regarded as a representative form of ROS, and accumulation of H2O2 is linked closely to the progression of OA . In the previous study, H2O2-induced oxidative stress microenvironment and catalase and GPx-1/2 neutralize H2O2 to form H2O . After MIA injection, the antioxidant enzymes including catalase and GPx-1/2 were increased than those of the MIA control group. PLM treatment showed a significant upregulation of catalase, which alleviated the degradation of ECM and protected the cartilage from oxidative stress. As the result, this suggests that the antioxidant activity of PLM may contribute to reducing pain in the MIA rat model of OA.
In addition, we examined the expression of matrix metalloproteinase (MMP)-1 and TIMP-1. The extracellular matrix is composed of proteoglycans (aggrecans) that retain water and of collagen II (95%). MMPs are a class of proteolytic enzymes involved in the occurrence, development, and progression of OA that promote the degradation of cartilage matrix components such as collagen II, which is a crucial factor in balancing the synthesis and degradation of the ECM of articular cartilage [16, 53]. Among MMPs, MMP-1 is a soluble proteinase known as collagenase-1 and degrades the extracellular matrix of cartilage and plays a main role in the occurrence, development, and progression of OA . Meanwhile, activated MMP-1 is mainly regulated by tissue inhibitor of metalloproteinase (TIMP)-1 as the chief MMP regulator. Moreover, TIMP-1 is produced by connective tissue and resists against collagenase, gelatinase, and stromelysin [54, 55]. In this study, MIA control expressed an increased MMP-1 expression, and it also suggested that MMP-1 is associated with destruction of cartilage. However, PLM supplementation significantly blocked the MMP-1 upregulation of the MIA control group. These results suggest that the anticatabolic effect of PLM may be due to the inhibition of MMP-1.
Mitogen-activated protein kinases (MAPKs), which are a family of serine/threonine kinases, integrate and process various extracellular signals or cytokines . The p38 MAPK corresponds to an integral component of the proinflammatory signaling pathway about external stress signals and is associated with inflammation, cell differentiation, cell growth, and cell death [56, 57]. Several studies have shown that blocking the p38 activity attenuates the transcriptional activity of the proinflammatory transcription factor NF-κB without altering its DNA-binding activity. The NF-κB signaling pathway is involved in the regulation of inflammatory mediators and cytokines in the progression of OA . NF-κB is a transcription factor localized to the cytoplasm and is rendered inactive by a constitutive interaction with the inhibitory protein IκB . NF-κBp65 is separated from IκB and translocates to the nucleus to regulate the expression of inflammatory mediators and cytokines . Many studies have demonstrated inflammatory cytokines such as IL-1β, TNF-α, and IL-6 play vital roles in OA pathogenesis [37, 51], which is intimately related to degradation of the extracellular matrix (ECM). Besides, COX is a key proinflammatory enzyme that converts arachidonic acid into prostaglandins. The excessive iNOS induces the overproduction of NO. NO generates ONOO− through reacting with O2-, and this can accelerate OA . Inhibition of NF-κB activation is associated with the downregulation of COX-2 expression and synthesis. Moreover, higher levels of these cytokines in the initiation and progression of articular cartilage destruction have been shown to correlate with pain and physical function of patients with OA . Our results suggest that suppression of NF-κB activation may underlie the mechanism by which PLM inhibits the inflammatory response in cartilage tissues. That is, the elevated expressions of COX-2 and iNOS were markedly attenuated by both PLM100 and PLM200 treatments, and three cytokines such as IL-1β, TNF-α, and IL-6 significantly decreased by PLM200.
Taken together, it suggests that PLM could inhibit excessive production of ROS and elevate anti-oxidant effect through catalase upregulation. Moreover, PLM could lead to NF-κB inactivation through inhibition of p38 MAPK and it will suppress sequentially both inflammation factors and components related to degradation of cartilage matrix (Figure 7). As the result, PLM treatment may be the potential therapeutic candidate in patients with osteoarthritis.
The data used to support the findings of this study are included within the article.
All animal procedures were approved by the Animal Research Ethics Committee of the Daegu Haany University (Permit Number: DHU2019-095).
Conflicts of Interest
The authors declare that they have no conflicts of interest.
J.A. Lee and M.J. Kim performed the experiments and were responsible for data acquisition. B.W. Park and S.B. Seo made PLM through a manufacturing process of mass production. M.R. Shin performed data analysis and drafted the article. H.J. Park and S.S. Roh were responsible for the conception and design of the study. All authors have participated actively in carrying out and improving the study, and all have approved the submission of this article.
This research was supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) through the Encouragement Program for the Industries of Economic Cooperation Region (No. P0006194), and the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIT) (No .2018R1A5A2025272).
- D. Pereira, E. Ramos, and J. Branco, “Osteoarthritis,” Acta Médica Portuguesa, vol. 28, no. 1, pp. 99–106, 2015.
- H. P. French, R. Galvin, N. F. Horgan, and R. A. Kenny, “Prevalence and burden of osteoarthritis amongst older people in Ireland: findings from the Irish longitudinal study on ageing (TILDA),” The European Journal of Public Health, vol. 26, no. 1, pp. 192–198, 2016.
- M. J. Elders, “The increasing impact of arthritis on public health,” The Journal of Rheumatology Supplement, vol. 60, pp. 6–8, 2000.
- J. Y. Reginster, “The prevalence and burden of arthritis,” Rheumatology, vol. 41, no. suppl_1, pp. 3–6, 2002.
- T. M. Griffin and C. R. Scanzello, “Innate inflammation and synovial macrophages in osteoarthritis pathophysiology,” Clinical and Experimental Rheumatology, vol. 37, no. Suppl 120, no. 5, pp. 57–63, 2019.
- M. Millerand, F. Berenbaum, and C. Jacques, “Danger signals and inflammaging in osteoarthritis,” Clinical and Experimental Rheumatology, vol. 37, no. Suppl 120, no. 5, pp. 48–56, 2019.
- Y. Y. Chow and K. Y. Chin, “The role of inflammation in the pathogenesis of osteoarthritis,” Mediators of inflammation, vol. 2020, Article ID 8293921, p. 16, 2020.
- J. W. Bijlsma, F. Berenbaum, and F. P. Lafeber, “Osteoarthritis: an update with relevance for clinical practice,” The Lancet, vol. 377, no. 9783, pp. 2115–2126, 2011.
- W. Zhang, R. W. Moskowitz, G. Nuki et al., “OARSI recommendations for the management of hip and knee osteoarthritis, part II: OARSI evidence-based, expert consensus guidelines,” Osteoarthritis and Cartilage, vol. 16, no. 2, pp. 137–162, 2008.
- L. S. Lohmander, D. McKeith, O. Svensson et al., “A randomised, placebo controlled, comparative trial of the gastrointestinal safety and efficacy of AZD3582 versus naproxen in osteoarthritis,” Annals of the Rheumatic Diseases, vol. 64, no. 3, pp. 449–456, 2005.
- B. J. E. de Lange-Brokaar, A. Ioan-Facsinay, G. J. V. M. van Osch et al., “Synovial inflammation, immune cells and their cytokines in osteoarthritis: a review,” Osteoarthritis and Cartilage, vol. 20, no. 12, pp. 1484–1499, 2012.
- C. T. Vangsness Jr., W. S. Burke, S. J. Narvy et al., “Human knee synovial fluid cytokines correlated with grade of knee osteoarthritis—a pilot study,” Bulletin of the NYU Hospital for Joint Diseases, vol. 69, no. 2, pp. 122–127, 2011.
- P. Wojdasiewicz, Ł. A. Poniatowski, and D. Szukiewicz, “The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis,” Mediators of Inflammation, vol. 2014, Article ID 561459, p. 19, 2014.
- J. C. Fernandes, J. Martel-Pelletier, and J. P. Pelletier, “The role of cytokines in osteoarthritis pathophysiology,” Biorheology, vol. 39, no. 1-2, pp. 237–246, 2002.
- W. Zheng, H. Zhang, Y. Jin et al., “Butein inhibits IL-1β-induced inflammatory response in human osteoarthritis chondrocytes and slows the progression of osteoarthritis in mice,” International Immunopharmacology, vol. 42, pp. 1–10, 2017.
- G. Q. Zeng, A. B. Chen, W. Li, J. H. Song, and C. Y. Gao, “High MMP-1, MMP-2, and MMP-9 protein levels in osteoarthritis,” Genetics and Molecular Research, vol. 14, no. 4, pp. 14811–14822, 2015.
- W. Bode, C. Fernandez-Catalan, F. Grams et al., “Insights into MMP-TIMP interactions,” Annals of the New York Academy of Sciences, vol. 878, pp. 73–91, 1999.
- H. Nagase, R. Visse, and G. Murphy, “Structure and function of matrix metalloproteinases and TIMPs,” Cardiovascular Research, vol. 69, pp. 562–573, 2006.
- R. Nicoletti, M. L. Ciavatta, E. Buommino, and M. A. Tufano, “Antitumor extrolites produced by Penicillium species,” International journal of analytical, pharmaceutical and biomedical sciences, vol. 2, pp. 1–23, 2008.
- T. B. Ng and H. X. Wang, “Pharmacological actions of cordyceps, a prized folk medicine,” The Journal of Pharmacy And Pharmacology, vol. 57, no. 12, pp. 1509–1520, 2005.
- J. Y. Han, J. Im, J. N. Choi et al., “Induction of IL-8 expression by Cordyceps militaris grown on germinated soybeans through lipid rafts formation and signaling pathways via ERK and JNK in A549 cells,” Journal of Ethnopharmacology, vol. 127, no. 1, pp. 55–61, 2010.
- T. Zhu, S. H. Kim, and C. Y. Chen, “A medicinal mushroom: Phellinus linteus,” Current Medicinal Chemistry, vol. 15, no. 13, pp. 1330–1335, 2008.
- W. Chen, H. Tan, Q. Liu et al., “A review: the bioactivities and pharmacological applications of Phellinus linteus,” Molecules, vol. 24, no. 10, p. 1888, 2019.
- K. Kojima, T. Ohno, M. Inoue, H. Mizukami, and A. Nagatsu, “Phellifuropyranone a: a new furopyranone compound isolated from fruit bodies of wild Phellinus linteus,” Chemical & Pharmaceutical Bulletin, vol. 56, no. 2, pp. 173–175, 2018.
- S. C. Huang, P. W. Wang, P. C. Kuo et al., “Hepatoprotective principles and other chemical constituents from the mycelium of Phellinus linteus,” Molecules, vol. 23, no. 7, p. 1705, 2018.
- H. J. Park, “Anti-allergic and anti-inflammatory activity of Phellinus linteus grown on Panax ginseng,” Food Science and Biotechnology, vol. 26, no. 2, pp. 467–472, 2017.
- T. Nakamura, S. Matsugo, Y. Uzuka, S. Matsuo, and H. Kawagishi, “Fractionation and anti-tumor activity of the mycelia of liquid-cultured Phellinus linteus,” Bioscience, Biotechnology, and Biochemistry, vol. 68, no. 4, pp. 868–872, 2004.
- S.-H. Kim, Y.-S. Song, S.-K. Kim, B.-C. Kim, C.-J. Lim, and E.-H. Park, “Anti-inflammatory and related pharmacological activities of the n-BuOH subfraction of mushroom Phellinus linteus,” Journal of Ethnopharmacology, vol. 93, no. 1, pp. 141–146, 2004.
- D. H. Lee and H. W. Kim, “Innate immunity induced by fungal β-glucans via dectin-1 signaling pathway,” International Journal of Medicinal Mushrooms, vol. 16, no. 1, pp. 1–16, 2014.
- W. Chanput, M. Reitsma, L. Kleinjans, J. J. Mes, H. F. J. Savelkoul, and H. J. Wichers, “β-Glucans are involved in immune-modulation of THP-1 macrophages,” Molecular Nutrition & Food Research, vol. 56, no. 5, pp. 822–833, 2012.
- Korea food & drug administration, “Food Code,” 2019, http://www.foodsafetykorea.go.kr/foodcode/01_02.jsp?idx=263.
- Z.-M. Wang, Y.-C. Chen, and D.-P. Wang, “Resveratrol, a natural antioxidant, protects monosodium iodoacetate-induced osteoarthritic pain in rats,” Biomedicine & Pharmacotherapy, vol. 83, pp. 763–770, 2016.
- K. Y. Chin, S. K. Wong, F. Z. Japar Sidik et al., “The effects of annatto tocotrienol supplementation on cartilage and subchondral bone in an animal model of osteoarthritis induced by monosodium iodoacetate,” International Journal of Environmental Research and Public Health, vol. 16, no. 16, p. 2897, 2019.
- Y. M. Lee, E. Son, S. H. Kim, O. S. Kim, and D. S. Kim, “Anti-inflammatory and anti-osteoarthritis effect of Mollugo pentaphylla extract,” Pharmaceutical Biology, vol. 57, no. 1, pp. 74–81, 2019.
- S. Komatsu, “Extraction of nuclear protein,” Methods in Molecular Biology, vol. 355, pp. 73–77, 2007.
- M. R. Shin, K. J. Kim, S. H. Kim et al., “Comparative evaluation between sulfasalazine alone and in combination with herbal medicine on DSS-induced ulcerative colitis mice,” BioMed Research International, vol. 2017, Article ID 6742652, p. 10, 2017.
- J. de Sousa valente, “The pharmacology of pain associated with the monoiodoacetate model of osteoarthritis,” Frontiers in Pharmacology, vol. 10, no. 974, 2019.
- S. Drevet, G. Gavazzi, L. Grange, C. Dupuy, and B. Lardy, “Reactive oxygen species and NADPH oxidase 4 involvement in osteoarthritis,” Experimental Gerontology, vol. 111, pp. 107–117, 2018.
- R. N. Saha, M. Jana, and K. Pahan, “MAPK p38 regulates transcriptional activity of NF-κB in primary human astrocytes via acetylation of p65,” The Journal of Immunology, vol. 179, no. 10, pp. 7101–7109, 2007.
- K. K. Brown, S. A. Heitmeyer, E. B. Hookfin et al., “P38 MAP kinase inhibitors as potential therapeutics for the treatment of joint degeneration and pain associated with osteoarthritis,” Journal of Inflammation, vol. 5, no. 1, p. 22, 2008.
- E. Endrinaldi, E. Darwin, N. Zubir, and G. Revilla, “The effect of mesenchymal stem cell wharton’s jelly on matrix metalloproteinase-1 and interleukin-4 levels in osteoarthritis rat model,” Open Access Macedonian Journal of Medical Sciences, vol. 7, no. 4, pp. 529–535, 2019.
- A. Hosseinzadeh, K. Bahrampour Juybari, M. J. Fatemi et al., “Protective effect of ginger (zingiber officinale roscoe) extract against oxidative stress and mitochondrial apoptosis induced by interleukin-1β in cultured chondrocytes,” Cells Tissues Organs, vol. 204, no. 5-6, pp. 241–250, 2017.
- S. Vila, “Inflammation in osteoarthritis,” Puerto Rico Health Sciences Journal, vol. 36, no. 3, pp. 123–129, 2017.
- L. S. Simon, “Nonsteroidal anti-inflammatory drugs and their risk: a story still in development,” Arthritis research & therapy, vol. 15, no. Suppl 3, p. S1, 2013.
- K. S. Kim, D. H. Oh, H. M. Choi et al., “Pyrrolidine dithiocarbamate, a NF-kappaB inhibitor, upregulates MMP-1 and MMP-13 in IL-1beta-stimulated rheumatoid arthritis fibroblast-like synoviocytes,” European Journal of Pharmacology, vol. 613, no. 1–3, pp. 167–175, 2009.
- Y. Luo, P. Shang, and D. LiLuteolin, “A flavonoid that has multiple cardio-protective effects and its molecular mechanisms,” Frontiers in Pharmacology, vol. 8, p. 692, 2017.
- J. Fei, B. Liang, C. Jiang, H. Ni, and L. Wang, “Luteolin inhibits IL-1β-induced inﬂ in rat chondrocytes and attenuates osteoarthritis progression in a rat model,” Biomedicine & pharmacotherapy, vol. 109, pp. 1586–1592, 2019.
- H. H. Su, Y. C. Chu, J. M. Liao et al., “Phellinus linteus mycelium alleviates myocardial ischemia-reperfusion injury through autophagic regulation,” Frontiers in Pharmacology, vol. 8, p. 175, 2017.
- N. Schuelert and J. J. McDougall, “Grading of monosodium iodoacetate-induced osteoarthritis reveals a concentration-dependent sensitization of nociceptors in the knee joint of the rat,” Neuroscience Letters, vol. 465, no. 2, pp. 184–188, 2009.
- Y. Yang, Y. Wang, Y. Kong et al., “Carnosine prevents type 2 diabetes-induced osteoarthritis through the ROS/NF-κB pathway,” Frontiers in Pharmacology, vol. 9, p. 598, 2018.
- F. Rousset, F. Hazane-Puch, C. Pinosa et al., “IL-1beta mediates MMP secretion and IL-1beta neosynthesis via upregulation of p22phox and NOX4 activity in human articular chondrocytes,” Osteoarthritis and Cartilage, vol. 23, no. 11, pp. 1972–1980, 2015.
- C. Y. Chuang, G. Degendorfer, and M. J. Davies, “Oxidation and modification of extracellular matrix and its role in disease,” Free Radical Research, vol. 48, no. 9, pp. 970–989, 2014.
- B. C. Kieseier, C. Schneider, J. M. Clements et al., “Expression of specific matrix metalloproteinases in inflammatory myopathies,” Brain, vol. 124, no. 2, pp. 341–351, 2001.
- S. McDonnell, M. Morgan, and C. Lynch, “Role of matrix metalloproteinases in normal and disease processes,” Biochemical Society Transactions, vol. 27, no. 4, pp. 734–740, 1999.
- W. G. Gho, Y. Choi, K.-H. Park, and J.-K. Huh, “Expression of collagenases (matrix metalloproteinase-1, 8, 13) and tissue inhibitor of metalloproteinase-1 of retrodiscal tissue in temporomandibular joint disorder patients,” Journal of the Korean Association of Oral and Maxillofacial Surgeons, vol. 44, no. 3, pp. 120–127, 2018.
- S. Kumar, J. Boehm, and J. C. Lee, “p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases,” Nature Reviews Drug Discovery, vol. 2, no. 9, pp. 717–726, 2003.
- J. M. Kyriakis and J. Avruch, “Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation,” Physiological Reviews, vol. 81, no. 2, pp. 807–869, 2001.
- M. C. de Andrés, A. Takahashi, and R. O. C. Oreffo, “Demethylation of an NF-κB enhancer element orchestrates iNOS induction in osteoarthritis and is associated with altered chondrocyte cell cycle,” Osteoarthritis and Cartilage, vol. 24, no. 11, pp. 1951–1960, 2016.
- T. Pan, R. Chen, D. Wu et al., “Alpha-mangostin suppresses interleukin-1β-induced apoptosis in rat chondrocytes by inhibiting the NF-κB signaling pathway and delays the progression of osteoarthritis in a rat model,” International Immunopharmacology, vol. 52, pp. 156–162, 2017.
- M. Ostojic, V. Soljic, K. Vukojevic, and T. Dapic, “Immunohistochemical characterization of early and advanced knee osteoarthritis by NF-κB and iNOS expression,” Journal of Orthopaedic Research, vol. 35, no. 9, pp. 1990–1997, 2017.
- K. B. Marcu, M. Otero, E. Olivotto, R. Maria Borzi, and M. B. Goldring, “NF-κB signaling: multiple angles to target OA,” Current Drug Targets, vol. 11, no. 5, pp. 599–613, 2010.
Copyright © 2020 Mi-Rae Shin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.