-Hee Jeong

Rebamipide Suppresses Collagen-Induced Arthritis Through Reciprocal Regulation of Th17/Treg Cell Differentiation and Heme Oxygenase 1 Induction

Rebamipide, a gastroprotective agent, has the ability to scavenge reactive oxygen radicals. Increased oxidative stress is implicated in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to investigate the impact of rebamipide on the development of arthritis and the pathophysiologic mechanisms by which rebamipide attenuates arthritis severity in a murine model of RA.

Eupatilin Exerts Antinociceptive and Chondroprotective Properties in a Rat Model of Osteoarthritis by Downregulating Oxidative Damage and Catabolic Activity in Chondrocytes.

Increases in oxidative stress are thought to be associated with the development of osteoarthritis (OA). Eupatilin, one of the major compounds present in artemisia species, was shown to have both anti-oxidative and anti-inflammatory properties. Here, we investigated the in vivo effects of eupatilin on pain severity and cartilage degradation in an experimental rat model of OA, along with the mechanisms of action underlying these effects. Experimental OA was induced via an intra-articular injection of monosodium iodoacetate (MIA), with oral administration of eupatilin initiated on the day of MIA injection. Pain was assessed by measuring the paw withdrawal latency and threshold. Cartilage destruction was analyzed macroscopically and histomorphologically. The effects of eupatilin on mRNA expression were investigated in interleukin-1β (IL-1β)-stimulated human OA chondrocytes. Eupatilin treatment exhibited clear antinociceptive effects, along with an attenuation of cartilage degradation in OA rats. Additionally, the number of osteoclasts present in the subchondral bone region was significantly decreased following eupatilin treatment. Eupatilin reduced the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), nitrotyrosine and inducible nitric oxide synthase (iNOS) in cartilage. mRNA levels of matrix metalloproteinase-3 (MMP-3), MMP13, and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) were reduced in IL-1β-stimulated human OA chondrocytes, while tissue inhibitor of metalloproteinases-1 (TIMP-1) was induced. Phosphorylated protein levels of the c-jun N-terminal kinase (JNK) was reduced by eupatilin. Taken together, these results suggest that eupatilin suppresses oxidative damage and reciprocally enhances extracellular matrix production in articular chondrocytes, making eupatilin a promising therapeutic option for the treatment of OA.

Ursodeoxycholic Acid Ameliorates Pain Severity and Cartilage Degeneration in Monosodium Iodoacetate-Induced Osteoarthritis in Rats

Osteoarthritis (OA) is a degenerative joint disease characterized by a progressive loss of cartilage. And, increased oxidative stress plays a relevant role in the pathogenesis of OA. Ursodeoxycholic acid (UDCA) is a used drug for liver diseases known for its free radical-scavenging property. The objectives of this study were to investigate the in vivo effects of UDCA on pain severity and cartilage degeneration using an experimental OA model and to explore its mode of actions. OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA) to the knee. Oral administration UDCA was initiated on the day of MIA injection. Limb nociception was assessed by measuring the paw withdrawal latency and threshold. Samples were analyzed macroscopically and histologically. Immunohistochemistry was used to investigate the expression of interleukin-1β (IL-1β), IL-6, nitrotyrosine and inducible nitric oxide synthase (iNOS) in knee joints. UDCA showed an antinociceptive property and attenuated cartilage degeneration. OA rats given oral UDCA significantly exhibited a decreased number of osteoclasts in subchondral bone legion compared with the vehicle-treated OA group. UDCA reduced the expression of IL-1β, IL-6, nitrotyrosine and iNOS in articular cartilage. UDCA treatment significantly attenuated the mRNA expression of matrix metalloproteinase-3 (MMP-3), -13, and ADAMTS5 in IL-1β-stimulated human OA chondrocytes. These results show the inhibitory effects of UDCA on pain production and cartilage degeneration in experimentally induced OA. The chondroprotective properties of UDCA were achieved by suppressing oxidative damage and inhibiting catabolic factors that are implicated in the pathogenesis of cartilage damage in OA.

Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice

Coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant synthesized in human body. This enzyme promotes immune system function and can be used as a dietary supplement. Rheumatoid arthritis (RA) is an autoimmune disease leading to chronic joint inflammation. RA results in severe destruction of cartilage and disability. This study aimed to investigate the effect of CoQ10 on inflammation and Th17 cell proliferation on an experimental rheumatoid arthritis (RA) mice model. CoQ10 or cotton seed oil as control was orally administrated once a day for seven weeks to mice with zymosan-induced arthritis (ZIA). Histological analysis of the joints was conducted using immunohistochemistry. Germinal center (GC) B cells, Th17 cells and Treg cells of the spleen tissue were examined by confocal microscopy staining. mRNA expression was measured by real-time PCR and protein levels were estimated by enzyme-linked immunosorbent assay (ELISA). Flow cytometric analysis (FACS) was used to evaluate Th17 cells and Treg cells. CoQ10 mitigated the severity of ZIA and decreased serum immunoglobulin concentrations. CoQ10 also reduced RANKL-induced osteoclastogenesis, inflammatory mediators and oxidant factors. Th17/Treg axis was reciprocally controlled by CoQ10 treatment. Moreover, CoQ10 treatment on normal mouse and human cells cultured in Th17 conditions decreased the number of Th17 cells and enhanced the number of Treg cells. CoQ10 alleviates arthritis in mice with ZIA declining inflammation, Th17 cells and osteoclast differentiation. These findings suggest that CoQ10 can be a potential therapeutic substance for RA.

Combination therapy with metformin and coenzyme Q10 in murine experimental autoimmune arthritis

Abstract Metformin (Met) and coenzyme Q10 (CoQ10) are reported to have therapeutic functions in several inflammatory diseases. These drugs have shown anti-inflammatory effects and have been utilized in mouse models of rheumatoid arthritis (RA). However, there is no evidence of the additive effect of Met and CoQ10 in RA. Although Met and CoQ10 may be involved in the improvement of mitochondrial dysfunction, limited information is available regarding whether this effect can improve mitochondrial dysfunction in RA in particular. In this study, we sought to determine whether Met and CoQ10 attenuate the severity of collagen-induced arthritis (CIA) and show an additive effect in a mouse model. The combination of Met and CoQ10 improved CIA, reducing joint inflammation, Th17 differentiation and IgG production. In contrast, the combination of Met and CoQ10 induced Treg differentiation. Osteoclastogenesis was reduced by the combination of Met and CoQ10. The protein expression of interleukin-1β, interleukin-6 and tumor necrosis factor-alpha in mice splenocytes exposed to lipopolysaccharide decreased after drug combination therapy. We also found that the expression of JC-1 and COX IV were enhanced by treatment with the combination of Met and CoQ10. Moreover, the combination of Met and CoQ10 promoted mitochondrial O2 consumption. These findings suggest that the combination of Met and CoQ10 reduced CIA severity, improving mitochondrial dysfunction compared to Met or CoQ10 alone. These results present a novel, significant preventive targets in RA and may enhance our understanding of its pathogenesis.

Augmented chondroprotective effect of coadministration of celecoxib and rebamipide in the monosodium iodoacetate rat model of osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease characterized by the progressive loss of articular cartilage and chronic pain. Although cyclooxygenase-2 (COX-2) inhibitors such as celecoxib are recommended to patients at high risk of gastrointestinal (GI) adverse events, COX-2 inhibitors do not completely prevent GI adverse events. Rebamipide, a gastroprotective agent, has anti-inflammatory properties and acts as an oxygen radical scavenger. The aim of this study was to investigate the in vivo effects of coadministration of rebamipide and celecoxib in an OA rat model. OA was induced by intra-articular injection of monosodium iodoacetate. Oral administration of rebamipide was initiated on the day of OA induction. In this study, rebamipide showed antinociceptive properties and attenuated cartilage degeneration. Rebamipide reduced the expression of matrix metalloproteinase 13, interleukin-1β, inducible nitric oxide synthase, and nitrotyrosine in OA cartilage. OA rats treated with celecoxib in combination with rebamipide demonstrated a higher pain threshold than those treated with monotherapy. Histological examination also showed that the joints from OA animals treated with combination therapy demonstrated less cartilage damage than those of animals treated with monotherapy. We showed that the potential benefit of combination therapy with celecoxib and rebamipide on pain and cartilage degeneration in OA.

The chicken combs extract alleviates pain and cartilage degradation in rat model osteoarthritis

Osteoarthritis (OA) is a degenerative and inflammatory disorder on particular joint inducing annihilation of articular cartilage. This study is aimed to investigate the therapeutic effect of extract of chicken combs (CCE) on pain severity and cartilage degeneration in an experimental model of rat OA. OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA) to the right knee. CCE, hyaluronic acid and celecoxib were administrated orally every day after MIA injection. Pain severity was estimated by evaluation of secondary tactile allodynia using the von Frey assessment test. The severity of cartilage degradation was examined by histological analysis and Mankin scoring system. Protein expression was observed by immunohistochemistry. Real-time polymerase chain reaction was used to measure mRNA level. CCE decreased secondary tactile allodynia revealed by a promoted pain withdrawal latency and pain withdrawal threshold. Cartilage destruction in the osteoarthritic joints was improved by CCE treatment. CCE also suppressed the expression of metalloproteinase (MMP)-3, -13, interleukin-1β, inducible nitric oxide synthase and nitrotyrosine increased in osteoarthritic joints. The mRNA level of MMP-1, 3, and -13 was down-regulated by CCE treatment. On the other hand, CCE treatment induced the gene expression of tissue inhibitor of metalloproteinase-1 and -3. CCE treatment demonstrates the therapeutic effect of pain relief and attenuates cartilage degeneration through the suppression of inflammatory mediators and metalloproteinases performing a pivotal function in OA pathogenesis.

Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the synovial joints. Although involvement of the fibroblast growth factor (FGF) signaling pathway has been suggested as an important modulator in RA development, no clear evidence has been provided. In this study, we found that synovial fluid basic FGF (bFGF) concentration was significantly higher in RA than in osteoarthritis (OA) patients. bFGF stimulates proliferation and migration of human fibroblast-like synoviocytes (FLSs) by activation of the bFGF-FGF receptor 3 (FGFR3)-ribosomal S6 kinase 2 (RSK2) signaling axis. Moreover, a molecular docking study revealed that kaempferol inhibited FGFR3 activity by binding to the active pocket of the FGFR3 kinase domain. Kaempferol forms hydrogen bonds with the FGFR3 backbone oxygen of Glu555 and Ala558 and the side chain of Lys508. Notably, the inhibition of bFGF-FGFR3–RSK2 signaling by kaempferol suppresses the proliferation and migration of RA FLSs and the release of activated T-cell-mediated inflammatory cytokines, such as IL-17, IL-21, and TNF-α. We further found that activated phospho-FGFR3 and -RSK2 were more highly observed in RA than in OA synovium. The hyperplastic lining and sublining lymphoid aggregate layers of RA synovium showed p-RSK2-expressing CD68+ macrophages with high frequency, while pRSK2-expressing CD4+ T-cells was observed at a lower frequency. Notably, kaempferol administration in collagen-induced arthritis mice relieved the frequency and severity of arthritis. Kaempferol reduced osteoclast differentiation in vitro and in vivo relative to the controls and was associated with the inhibition of osteoclast markers, such as tartrate-resistant acid phosphatase, integrin β3, and MMP9. Conclusively, our data suggest that bFGF-induced FGFR3–RSK2 signaling may play a critical role during the initiation and progression of RA in terms of FLS proliferation and enhanced osteoclastogenesis, and that kaempferol may be effective as a new treatment for RA.

Rebamipide ameliorates atherosclerosis by controlling lipid metabolism and inflammation

Atherosclerosis is a chronic inflammatory disease caused by the accumulation of excess lipid in the aorta and the severity is regulated by T lymphocytes subsets. Rebamipide has therapeutic activity in collagen induced arthritis (CIA) by controlling the balance between T helper (Th) 17 and regulatory T (Treg) cells. In this study, we aimed to determine whether rebamipide reduces the development of atherosclerosis. To investigate the therapeutic effect of rebamipide, ApoE-KO mice fed a western diet were administered rebamipide orally for 8 weeks. Mice were sacrificed followed by the evaluation of plaque formation in the aorta or immunohistochemistry for IL-17 and Foxp3. Serum was also prepared to determine the pro-inflammatory cytokine levels. The ability of rebamipide to regulate lipid metabolism or inflammation was confirmed ex vivo. Results The oral administration of rebamipide decreased plaque formation in atherosclerotic lesions as well as the markers of metabolic disorder in ApoE-deficient mice with atherosclerosis. Pro-inflammatory cytokines were also suppressed by rebamapide. In addition, the population of Th17 was decreased, whereas Treg was increased in the spleen of rebamipide-treated ApoE deficient mice. Rebamipide also ameliorated the severity of obese arthritis and has the capability to reduce the development of atherosclerosis by controlling the balance between Th17 and Treg cells. Thus, rebamipide could be a therapeutic agent to improve the progression of inflammation in metabolic diseases.

THU0070 Dual Targeting of Kaempferol on the Kinase Activities of FGFR3 and RSK2 Suppresses Rheumatoid Arthritis in Vitro and in Vivo

Background Rheumatoid arthritis (RA) is a chronic inflammatory joint disease. Because increased angiogenesis combined with proliferation of fibroblast-like synoviocytes (FLSs) plays a critical role in RA pathogenesis, signaling factors and growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) in the RA synovium have been considered as important treatment targets. FGF expression is increased in RA synovial fluid (SF), as compared to osteoarthritis (OA) SF. Kaempferol is a flavonoid present in abundant amounts in edible plants. We previously discovered that kaempferol bound to the active pocket of the RSK2 N-terminal kinase domain, inhibiting RSK2 N-terminal kinase activity. Objectives This study was undertaken to investigate the activation of fibroblast growth factor receptor 3 (FGFR3) and FGFR3-RSK2 signaling pathways play key roles in the development of RA. Methods All crystal structures of the kinase domains of the receptor tyrosine kinases were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). RSK2+/+ and RSK2-/- mouse embryonic fibroblasts (MEFs) and JB6 JB6 Cl41 mouse skin epidermal cells were used in in vitro study. Early passages (P3) of the human FLSs were cultured with DMEM. Collagen-induced arthritis (CIA) was induced in DBA/1J mice by immunization with type II collagen (CII). Kaempferol (2 mg/kg) dissolved in 10% DMSO was administered through intraperitoneal injection three times a week after induction of arthritis. Histologic analysis of the joints was performed using Safranin O, H&E staining. The frequencies of interleukin-17-producing CD4+ Th17 cells and CD4+CD25+Foxp3+ Treg cells were analyzed by flow cytometry. Results We found that kaempferol targeted the active pocket of FGFR3 kinase domain by forming hydrogen bonds with R groups of Lys508, Glu555, and Ala558. We further confirmed that when kaempferol binds to FGFR, FGFR kinase activity was inhibited in vitro and ex vivo. Higher FGF content was found in the SF of RA patients compared to that of OA patients. We demonstrated that the activation of FGFR3 and RSK2 by bFGF stimulation is directly affected by synovial fibroblast proliferation and cell migration in the tissues of RA patients through activation of the inflammatory response and macrophage- and T-cell-mediated immune responses. Notably, we found that oral administration of kaempferol suppressed arthritis incidence and score, proteoglycan decomposition, and the Th17 lineage differentiation of naïve T cells in a CIA mouse model. We also found that inhibition of FGFR3 and RSK2 by kaempferol suppressed the populations of CD4+/IL-17+ and CD4+/SRC+ cells, which inhibited STAT3 phosphorylation at Ser727 and Tyr705. Moreover, we found that kaempferol inhibited osteoclastogenesis by stimulating M-CSF and RANKL stimulation ex vivo and in vitro. Conclusions The activation of the FGFR3 and RSK2 signaling pathways induced by FGF play a key role in RA development in humans, and dual targeting of kaempferol on the FGFR3 and RSK2 suppressed CIA in vitro and in vivo. References Cho, Y.Y., Yao, K., Pugliese, A., Malakhova, M.L., Bode, A.M., and Dong, Z. 2009. A regulatory mechanism for RSK2 NH(2)-terminal kinase activity. Cancer Res 69:4398-4406. Disclosure of Interest None declared