15-hydroxyprostaglandin dehydrogenase is up-regulated by hydroxychloroquine in rheumatoid arthritis fibroblast-like synoviocytes

이수정 순천향대학교 대학원 2013 국내석사

15-hydroxyprostaglandin dehydrogenase (HPGD)는 prostaglandin E2 (PGE2) 이화작용의 비활성물질로 대사시키는 주요 효소로 알려져 있고, PGE2는 류마티스 관절염에서 주요 이화작용 요인으로 알려져 있다. 그러나 류마티스 관절염에서 HPGD 발현과 조절을 위한 연구가 거의 없다. 항류마티스약제는 관절 손상을 줄여주는 관절염 치료에 주요 약제로 이 연구는 류마티스 관절염 섬유아세포 유사 활막세포에서 정상 섬유아세포 유사 활막세포와 비교하고 HPGD의 발현을 확인한다. 그리고 류마티스 관절염 섬유아세포 유사 활막세포에서 HPGD의 발현으로 항류마티스약제 중 하나인 하이드록시클로로퀸 영향을 조사했다. 정상과 퇴행성관절염, 류마티스 관절염인 사람의 섬유아세포 유사 활막세포를 분리해서 HPGD의 단백질을 웨스턴 블로팅으로 확인했고 면역조직화학법으로 활막조직에서 HPGD의 발현을 확인했다. HPGD의 발현으로 항류마티스약제의 효과를 알아보기 위해 하이드록시클로로퀸을 세포에 처리했다. 활막에서 PGE2 방출은 효소 면역 검정법에 의해 분석했다. 면역조직 화학과 면역세포 화학 분석 및 웨스턴 블로팅 분석으로 HPGD의 발현이 정상과 퇴행성관절염 활막조직보다 류마티스 관절염에서 낮은 것을 확인했다. 그리고 류마티스 관절염 섬유아세포 유사 활막세포에서 HPGD의 발현은 항류마티스약제를 처리하면 증가했다. 항류마티스약제 중 하이드록시클로로퀸의 처리는 ERK와 SAPK/JNK, p38의 인산화에 영향이 있었다. 그리고 하이드록시클로로퀸 처리로 인한 PGE2 방출은 MAPK 경로 효소에서 특징적으로 나타났다. 따라서 HPGD의 감소는 류마티스 관절염과 같은 면역반응과 관련이 있고, 항류마티스약제는 HPGD발현 조절을 통한 류마티스 관절염의 발병과정에서 영향이 있을 것이다. 그 중 HPGD를 유도하는 하이드록시클로로퀸은 ERK 경로와 관련이 있을 것이다. Background: 15-hydroxyprostaglandin dehydrogenase (HPGD) is the key enzyme responsible for metabolic inactivation of prostaglandin E2 (PGE2) catabolism. PGE2 is one of the main catabolic factors involved in rheumatoid arthritis (RA). However, RA fibroblast-like synoviocytes (FLS) have so far not studied for HPGD expression and regulations. Disease modifying antirheumatic drugs (DMARDs) are most important drugs to treatment of arthritis that reduced effect of joint injury. The aim of present study was that evaluate the expression of HPGD in RA-FLS as well as compared with normal FLS. Also, we investigated the effect one of the most popular DMARDs, hydroxychloroquine on the expression of HPGD in RA-FLS. Methods: Human FLS was isolated from healthy individuals, osteoarthritis (OA) and RA patients. The protein level of HPGD was evaluated by western blotting. The expressions of HPGD in synovium were analyzed by immunohistochemistry. To investigate the effect of DMARDs in expression of HPGD, the cells were treated hydroxychloroquine (10 ?M). The release of PGE2 in synovium were analyzed by enzyme immunoassay. Results: In immunohistochemical, immunocytochmical and western blotting analysis, HPGD expression in human synovium is lower in RA synovium than normal and OA synovium. In RA-FLS, the expression of HPGD was increased by the treatments of DMARDs. Hydroxychloroquine treatment were affect phosphorylation of ERK, SAPK/JNK and p38. And PGE2 release with hydroxychloroquine treatment were significance mitogen-activated protein kinases pathway enzyme. Conclusion: Reduction of HPGD is associated with inflammatory responses, such as RA. DMARDs may have influence on the pathogenesis of RA through the regulation of HPGD expression. Hydroxychloroquine induced HPGD may be associated with the ERK pathway.

Pathogenic role of fibroblast like synoviocyte in the particle induced periprosthetic osteolysis

JAGGA SUPRIYA 한림대학교 대학원 2019 국내박사

Effects of CKD-M808, a novel histone deacetylase 6 inhibitor, on fibroblast-like synoviocytes and regulatory T cells in rheumatoid arthritis

손세희 서울대학교 대학원 2018 국내석사

Background: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease whose hallmark is joint destruction caused by synovial proliferation and leukocyte infiltration in synovial membrane. Histone deacetylase (HDAC) inhibitor was suggested as a promising therapeutic agent in cancer and autoimmune diseases. Recently, the toxicity of pan-HDAC inhibitor (pan-HDACi) has been reported in clinical trials. HDAC6 is a member of HDAC family, mainly targeting non-histone proteins. We examined the therapeutic effect of selective inhibitor of HDAC6 in RA. Objective: We investigated anti-inflammatory effects of a novel HDAC6 inhibitor, CKD-M808 (M808), on fibroblast-like synoviocytes (FLS), peripheral blood mononuclear cells (PBMCs) and regulatory T cells (Treg) in RA as well as adjuvant-induced arthritis (AIA) rat model. Methods: Cell viability was analyzed by cell-counting kit 8 (CCK-8). RA FLS were incubated with HDAC6 inhibitor, M808 and tubastatin A as a positive control, and then stimulated with IL-1β. The levels of metalloproteinase (MMP) -1, MMP-3, IL-6, CCL2, CXCL8, and CXCL10 were measured in culture supernatants of FLS by Magnetic Luminex Screening Assay multiplex kit (Luminex assay) or enzyme-linked immunosorbent assay (ELISA). Expression of HDAC6, α-tubulin, acetylated tubulin, cortactin, acetylated cortactin, ICAM-1, VCAM-1, and GAPDH in FLS were assessed by western blot. Wound healing assay and cell adhesion assay in FLS were performed. RA PBMCs were incubated with HDAC6 inhibitors, tubastatin A or M808, and then stimulated with LPS. TNF-α, IL-1β, IL-6, and IL-10 were measured in culture supernatants of PBMCs by Luminex assay and ELISA. CD4+CD25- T cells were isolated from RA PBMCs and induced to regulatory T cells. The markers of induced Treg (iTreg) were analyzed by flow cytometry. CD4+CD25- T cells (effector T cells, Teff) from healthy donor or murine splenocytes were stained with carboxyfluorescein succinimidyl ester (CFSE) or eFluor®670 and cocultured with iTreg from RA patients or Treg from murine splenocytes respectively. The proliferation of healthy Teff was analyzed by flow cytometry. Adjuvant-induced arthritis (AIA) was developed in Lewis rat. Clinical score of arthritis was measured after M808 treatment. Result: M808 decreased the production of MMP-1, MMP-3, IL-6, CCL2, CXCL8, and CXCL10 in RA FLS. M808 increased the acetylation of tubulin and cortactin and inhibited the expression of ICAM-1 and VCAM-1 in RA FLS. The migration of RA FLS and adhesion of U937 cells or Jurkat cells on RA FLS were declined with the treatment of M808. M808 reduced TNF-α and increased IL-10 production, but had negligible effects on IL-1β and IL-6 in RA PBMCs. The function of iTreg induced from RA cells under the presence of M808 significantly improved by suppressing the proliferation of Teff. M808 had enhanced suppressive function of Treg and improves the clinical score of arthritis in AIA rat model. Conclusion: A novel HDAC6 inhibitor, M808 exhibits the suppressive effects on inflammation by reducing the production of MMPs, pro-inflammatory cytokines, and chemokines, decreasing cell migration and adhesion of RA FLS, and improving the suppressive function of Treg derived from RA patients. M808 improved clinical score of arthritis in AIA animal model. M808 could be a potential therapeutic agent in RA.

멜라토닌이 섬유아세포 유사 활막세포에서 마이크로 RNA-146a와 인터루킨-6에 미치는 영향 : Melatonin-Stimulated microRNA-146a Increases Interleukin-6 in Human Fibroblast-Like Synoviocytes

노윤미 계명대학교 일반대학원 2012 국내석사

멜라토닌은 뇌의 송과선에서 생성되는 호르몬으로, 일주기 리듬에 관여하며, 항산화, 항노화, 증식억제, 면역조절 및 항염증 작용으로 잘 알려져 있으나, 관절질환에서 염증을 유발한다는 연구결과도 발표되어 있다. 이 연구는 관절질환에 작용하는 멜라토닌의 염증작용 기전을 마이크로 RNA와 연관하여 규명하고자 하였다. 퇴행성 관절염 환자 활막액을 1차 배양을 하여, 멜라토닌을 10 nM, 10 μM의 농도로 처리 하였고, 마이크로 RNA 표현은 실시간 중합효소 연쇄반응으로 측정, 인터루킨-6은 효소결합 면역흡착 분석법으로 측정, 세포자멸사는 다피 분석, 터넬 분석, 웨스턴 블롯을 실행하였다. 그리고 마이크로 RNA 유사제, 억제제를 형질 주입하여 발현정도를 측정하였으며, 마이크로 RNA-146a에 관련된 표적 유전자는 TargetscanHuman 6.0에서 예측하여 조절 기전의 3′비해석부위를 유전자 재조합한 후 마이크로 RNA-146a 유사제와 함께 형질주입 하여 발광효소 측정법으로 확인을 하였다. 또한 섬유아세포 유사 활막세포 뿐만 아니라 폐암 세포인 A549에서도 멜라토닌에 의한 마이크로 RNA-146a 발현을 확인하였다. 섬유아세포 유사 활막세포에 멜라토닌을 처리하였을 때 마이크로 RNA-146a 표현과 인터루킨-6 생성이 의미 있게 증가하였다. 또한, 마이크로 RNA-146a이 과발현 되면, 인터루킨-6 생성이 증가하며, 억제되면 인터루킨-6 생성이 감소되었다. 마이크로 RNA-146a 과발현은 세포자멸사에 영향이 없는데, 다피 분석, 터넬 분석, 케스페이지-3, p-53 발현 차이가 없었다. 마이크로 RNA-146a 유사제와 유전자 재조합된 CARD10을 형질주입하여 발광효소 측정 결과가 감소하였고, CARD10이 마이크로 RNA-146a에 관련된 표적 유전자임을 확인하였다. 활막세포와 달리 폐암세포주인 A549에 멜라토닌을 처리하고 마이크로 RNA-146a 발현을 확인한 결과 마이크로 RNA-146a 발현이 상당히 감소를 하였다. 따라서 본 연구결과 섬유아세포 유사 활막세포에서 멜라토닌의 자극을 주었을 때, RNA-146a의 발현을 증가시켜 인터루킨-6 생성을 증가시켰다. 멜라토닌은 다른 세포에서는 항염증작용을 유도 하지만, 섬유아세포 유사 활막세포에서 염증작용이 나타난다. 이 결과로 멜라토닌은 관절질환에서 RNA-146a를 통하여 항염증 작용을 나타내는 것으로 생각된다. Although the anti-inflammatory effect of melatonin is well recognized, the effect of melatonin on arthritic disease has been controversial. The aim of the current study was to investigate the role of melatonin in the regulation of microRNA-146a (miR-146a), Which is associated with arthritis disease. The expression of miR-146a in primary cultured fibroblast-like synoviocytes (FLS) was significantly increased by melatonin (10 nM) treatment. The effect of miR-146a on interleukin-6 (IL-6) productions in FLS transfected with miR-146a mimics and miR-146a inhibitors were determined. Overexpression of miR-146a in FLS stimulated IL-6 productions while knockdown of miR-146a attenuated IL-6 productions. Overexpression of miR-146a did not change the apoptosis level. The activity of luciferase containing 3′-untranslated region of caspase recruitment domain family member 10, Which encompasses the miR-146a binding site, was significantly decreased by luciferase reporter assay. Unlike FLS, the expression of miR-146a and IL-6 productions in human alveolar basal epithelial cells A549 treated with melatonin were significantly decreased. These results suggest that melatonin induces a pro-inflammatory effect in FLS, whereas melatonin induces an anti-inflammatory effect in other cells. This suggests that melatonin might exert a pro-inflammatory effect in arthritis via the regulation of miR-146a.

Role of hypoxia in the inflammatory signaling pathway through JAK/STAT3/HIF-1α and the effect of hypoxia on Slug in rheumatoid arthritis fibroblast-like synoviocytes

김형진 성균관대학교 일반대학원 2016 국내박사

Objectives: To investigate the role of hypoxia on proinflammatory signaling pathways and the effect of hypoxia on slug expression in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) Method: RA FLS was cultured from synovial tissues of RA patients. The phosphorylation of ERK, JNK, and STAT3 proteins from RA FLS under hypoxic conditions were analyzed by Western blot. WP1066, an inhibitor of the JAK/STAT pathway, was used to confirm the signaling pathway in the regulation of HIF-1α. HIF-1α and Slug expressions in RA FLS under hypoxic conditions were analyzed using RT-PCR. Next, expressions of HIF-1α and Slug in RA synovial tissue were assessed by immunohistochemistry. The effect of TNF-α on Slug and HIF-1α expressions under normoxic conditions was assessed in RA FLS by using Western blot. Results: Under hypoxic conditions, the phosphorylation of STAT3, but not JNK and ERK proteins, was increased in RA FLS along with HIF-1α expression. Pre-treatment with WP1066 before induction of hypoxia inhibited the expression of p-STAT3 and HIF-1α expressions. Hypoxic conditions induced Slug mRNA expression from RA FLS. HIF-1α cDNA in RA FLS reproduced the Slug expression. Immunohistochemical staining showed that HIF-1α and Slug co-localized in the sublining area of RA synovium. Slug and HIF-1α expression were also enhanced when RA FLS was stimulated with TNF-α stimulation without hypoxia. Conclusion: In RA FLS, HIF-1α expression, induced by hypoxia, is regulated by JAK/STAT3 signaling pathway that leads to the expression of Slug. TNF-α alone can mimic the effect of hypoxia by inducing HIF-1α and Slug from RA FLS. This study suggests that hypoxia plays a pivotal role in the pathogenesis of RA and that TNF-α mimics hypoxia driven inflammation in RA FLS.

인간 섬유아세포 유사 활액막세포에서 IκBα 상위 억제제에 의한 IL-1β 매개 염증 반응 억제

권석현 전남대학교 대학원 2010 국내박사

IL-1β-NF-κB 축은 류마티스 관절염 병리 기전에 중요한 경로이고 염증성 활액막에서 전구 염증 매개체 생성의 중심 역할을 한다. 본 저자들은 섬유아세포 유사 활액막 세포(fibroblsat -like synoviocytes)가 IκB 상위 억제물의 과다 발현에 의해서 IL-1β 유도 독성으로부터 보호되는지 관찰하였다. 섬유아세포 유사 활액막 세포 감염시 아데노바이러스의한 돌연변이 Ad-IκBα (S32A, S36A)가 있는 경우 IL-1β 유도 핵 전위 방해와 NF-κB의 DNA 결합을 방해한다. 또한 Ad-IκBα (S32A, S36A)는 IL-1β 유도 염증 반응 즉, ENA-78과RANTES 그리고 MMP-1, MMP-3의 화학 반응 물질을 억제한다. 결과적으로 IL-1β 치료 후 섬유아세포 유사 활액막 세포의 세포분열을 증가는 Ad-IκBα (S32A, S36A)에 의해서 현저하게 감소된다. 그러나 IκBβ 돌연변이 Ad-IκBβ (S19A, S23A)는 IL-1β 독성을 방지하는데 효과가 없다. 이러한 결과는 IκBα 파괴 억제는 류마티스 관절염 환자에서 관절 파괴를 방지에 위한 중요한 표적이 된다. The IL‐1β‐NF‐κB axis is a key pathway in the pathogenesis of rheumatoid arthritis (RA) and is central in the production of proinflammatory mediators in the inflamed synovium. Therefore, The author examined whether fibroblast‐like synoviocytes (FLS) could be spared from IL‐1β‐induced toxicity by an overexpressing IκB super‐repressor. Infection of FLS with Ad‐IκBα (S32A, S36A), an adenovirus‐containing mutant IκBα, inhibited IL‐1β‐induced nuclear translocation and DNA binding of NF‐κB. In addition, Ad‐IκBα (S32A, S36A) prevented IL‐1β‐induced inflammatory responses; namely, the production of chemokines, such as ENA‐78 and RANTES, and activation of MMP‐1 and MMP‐3. Finally, increased cellular proliferation of FLS after IL‐1β treatment was significantly reduced by Ad‐IκBα (S32A, S36A). However, Ad‐IκBβ (S19A, S23A), the IκBβ mutant, was not effective in preventing IL‐1β toxicity. These results suggest that inhibition of IκBα degradation is a potential target for the prevention of joint destruction in patients with RA.

Effects of a novel spleen tyrosine kinase inhibitor, SKI-O-592, on rheumatoid arthritis fibroblast-like synoviocytes and collagen-induced arthritis

김선욱 서울대학교 대학원 2020 국내석사

배경: 류마티스 관절염 (RA)은 만성적인 염증성 자가면역 질환으로서, 백혈구의 침윤 및 활막 조직의 활성화를 동반하며, 골격 및 연골 조직의 파괴를 특징으로 한다. 활막 세포는 류마티스 관절염의 발병 기전에 중요한 역할을 하며, 활막 세포의 침습성은 관절 조직의 손상과 관련이 있다. 비장 티로신 키나제 (SYK)는 비 수용체 티로신 키나제로서, 면역 수용체의 신호전달에 중요한 역할을 함이 보고된 바 있다. 따라서, 본 연구는 새로운 SYK 억제제인 SKI-O-592 의 효과를 류마티스 관절염 환자 유래 활막 세포의 침습성과 단핵구의 염증 및 콜라겐 유도 관절염 마우스 모델에서 확인하는 것을 목적으로 하고 있다. 실험방법: 활막세포는 류마티스 관절염으로 진단받은 환자의 활막 조직으로부터 분리하였다. 활막세포는 SYK 의 억제제인 SKI-O-592의 처리 1시간 후에 TNF-α로 48시간 자극되었다. 자극 후, 세포 생존능은 cell-counting kit 8 에 의해 분석되었다. 활막세포 상층액의 IL-6, IL-8, CXCL10, MMP-3 의 농도 및 단핵구 세포 상층액의 TNF-α 농도는 효소면역법으로 측정하였다. 활막세포의 이동능을 평가하기 위해 상처 치유 분석과 Transwell 체계를 이용한 이동, 침습능을 분석 하였다. 세포 부착능은 활막세포와 calcein-AM 으로 표지한 THP-1 세포를 공배양하여 평가하였다. 활막세포에서 VCAM-1, ICAM-1, α-튜불린, β-액틴 및 인산화 된 SYK, JNK, p38, ERK, c-Jun, MKK4 의 단백질 발현은 웨스턴 블롯으로 평가하였다. DBA/1J 마우스에서 콜라겐 유도 관절염을 유발하고, SKI-O-592를 투여한 후, 관절염의 임상 점수와 조직학적 점수를 평가하였다. 실험결과: SKI-O-592 는 세포독성 없이 류마티스 관절염 환자의 활막세포에서 CXCL10 과 같은 케모카인의 농도를 감소시켰다. 손실된 부분 및 다공성 막을 통과하여 이동, 침습한 활막세포의 수는 SKI-O-592 투여 군에서 감소하였다. TNF-α의 자극 15분 후 JNK 와 p38 의 인산화는 SKI-O-592 투여에 의해 감소하며, 이는 농도 의존적 으로 감소하였다. SKI-O-592는 세포 생존능에 영향을 미치지않고 IgG 자극 하에서 THP-1 세포의 TNF-α 분비를 감소시키는 효과를 보였다. 또한, 콜라겐 유도 관절염 동물 모델에서 약물 투여 후 관절염의 임상점수와 조직학적 점수의 감소를 관찰하였다. 결론: 새로운 비장 티로신 키나제 억제제인 SKI-O-592 는 류마티스 관절염 환자로부터 유래된 활막세포들의 침습성을 감소시키고, JNK와 p38을 포함하는 MAPK 인자들의 인산화를 억제하였다. 또한, 단핵구 세포에서 항 염증 효과를 가지며, 동물 모델 에서도 임상 점수와 관절 파괴의 개선을 통한 치료 효능을 보였다. 따라서 SKI-O-592는 류마티스 관절염의 잠재적인 치료 제제가 될 수 있다고 사료된다. Background/Purpose: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by bone and cartilage destruction with leukocyte infiltration and activation at synovial tissue. The fibroblast-like synoviocytes (FLS) have a central role in disease pathogenesis and their invasiveness correlates with articular damage in RA patients. Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase known to have a crucial role in immune receptor signaling. This study aims to evaluate the inhibitory effect of a novel small-molecule SYK inhibitor, SKI-O-592, on the invasiveness of RA FLS and inflammation of monocytes in vitro and mouse collagen-induced arthritis (CIA) model in vivo. Methods: FLS were isolated from synovial tissues of RA patients. FLS were treated with SKI-O-592 for 1 hr and then stimulated with tumor necrosis factor-alpha (TNF-α) for 48 hr. After stimulation, cell viability was measured using cell counting kit-8 (CCK-8) assay. The levels of IL-6, IL-8, CXCL10, MMP-3, and TNF-α were measured in culture supernatant of RA FLS and THP-1 cells by ELISA. Wound healing assay and transwell migration, invasion assay using RA FLS was performed to evaluate cell migration ability. The adhesion ability of FLS was evaluated by the co-culture of calcein-AM labeled THP-1 cells. And the expression of VCAM-1, ICAM-1, α-tubulin, β-actin, total and phosphorylated SYK, c-Jun N-terminal kinase (JNK), p38, ERK, phosphorylated c-Jun, mitogen-activated protein kinase kinase 4 (MKK4) was determined by Western blotting. CIA was developed in DBA/1J mice. The clinical score of arthritis and the histological score was evaluated after treatment with SKI-O-592. Results: SKI-O-592 reduced the secretion of chemokine, CXCL10 in RA FLS. The number of migrated cells to wounded region and through pore membrane was decreased by SKI-O-592. Phosphorylation of JNK and p38 was reduced by SKI-O-592 after TNF-α stimulation. SKI-O-592 decreased secretion of TNF-α levels dose-dependently in THP-1 cells with IgG stimulation. The viability and proliferation of FLS and THP-1 were not affected by SKI-O-592. In the CIA model, clinical score of arthritis and histological score was decreased following SKI-O-592 treatment. Conclusion: SKI-O-592 inhibited the invasiveness of RA FLS and had an anti-inflammatory effect in monocytes. SKI-O-592 had a therapeutic effect in the mouse CIA model by improving clinical and histological scores with ameliorating joint destruction. In conclusion, a novel SYK inhibitor, SKI-O-592, may provide a new therapeutic option for RA patients.

Inhibitory effect of berberine on pannus formation through modulation of MIF in adjuvant-induced polyarthritis rats

김성훈 경희대학교 대학원 2012 국내석사

류마티스 관절염은 다발성 관절의 염증 및 파괴를 특징으로 하는 만성염증성 자가면역 질환으로 병인은 정확하게 밝혀지지 않았다. 하지만 환경적, 유전적인 병인에 의해 복합적으로 발생하는 면역반응 결과 생산되는 싸이토카인 같은 매개물질이 염증세포의 비정상적인 증가를 초래하여 활막염으로 인한 활막세포의 과도한 증식과 신생혈관의 생성, 지속적인 염증 반응을 일으키는 것을 특징으로 한다. 대식세포 유주 억제 인자 (Macrophage migration inhibitory factor, MIF)는 TNF-α 또는 IFN-γ와 같은 염증성의 자극에 반응 하는대식세포에 의해 유도 될 수 있으며, 류마티스 관절염 환자의 활막세포에서유래된 MIF는 단핵구에 의해 TNF-α를 분비시키고 다시 활막세포를 활성시킨다. 또한 류마티스 관절염 환자의 활막세포에서 MIF는 MMP-1과 MMP3의발현을 전사인자수준에서 증가시킨다. 류마티스 관절염의 병인에 관련된 중요한 요소로써 MIF를 통한 활막세포의 비대증에 관한연구는 자가면역질환의병인 이해에 중요하다. 그러나 류마티스 관절염의 활막세포를 대상으로 한 연구는 부족한 실정이다. 본 연구에서 동물모델을 만들기 위해 adjuvant induced arthritis 동물 모델을 이용하였다. AIA로 모델은 10일 후부터 증상이 유도되게 되는데 증상과 동시에 berberine(1, 10 or 20mg/kg, I.P) 치료를 병행하여 10-30일 동안 매일 치료하였다. 유도된 다발성 관절염 모델에 대한 평가는 몸무게, 관절염지표, 발목의비중을 통하여 형태학적인 변화를 외형적으로 관찰하여 효과를 보고 조직학적인 염색을 하여 다시 확인 하였다. 또한 발목관절에서의 염증성 싸이토카인 IL-1β,TNF-α, IFN-γ의 발현 정도를 확인하였다. 세포실험에 있어서 류마티스 관절염 환자의 활막세포를 통해 berberine을 처리하였을 경우의 세포의 증식과 침투성을 관찰하였으며 IL-1β으로 활성화 된 활막세포에서의 MMP-1, MMP-3, MMP-13의 발현 정도를 관찰하였고 berberine약물을 처리한 AIA모델과 활막세포에서의 MIF의 발현 정도를 관찰하였다. 결록적으로 berberine치료는 동물모델에서의 외형적, 조직학적,염증성 싸이토카인 등의 치료효과를 보았고 세포실험에서의 MMP1, MMP3, MMP-13의 감소, 활막세포의 증식, 침투성을 치료한다는 것을 증명하였다. 또한 MIF인자를 동물 모델실험과 세포실험에서 모두 치료효과를 증명하였다. 위 실험을 통하여 berberine은 류마티스 관절염환자들에게 좋은 치료방법이 될 것이라고 사료된다. The objective of this study was to investigate the inhibitory effect of berberine, a major component of Coptidis Rhizoma, on pannus formation in knee joints of the adjuvant-induced polyarthritis (AIA) rats, which is an animal model of human rheumatoid arthritis (RA), through the modulation of macrophage migration inhibition factor (MIF). Adjuvant-induced polyarthritis was induced by intradermal injection of a mixed suspension of dried Mycobacterium tuberculosis and incomplete Freund's adjuvant to the Lewis rats. The rats were daily given berberine (1, 10 or 20mg/kg, i.p.) from day 10 to 30 after arthritis induction. Berberine significantly alleviated arthritic symptoms, evaluated by body weight, ankle joint volume and arthritis index affected limbs of the AIA rats, which was also confirmed by histochemical observations of knee joint sections obtained on day 30. The increases in the mRNA expression of MIF and pro-inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ in the knee synovium of the AIA rats were significantly inhibited by berberine treatment. In vitro inhibitory effects of berberine on proliferation and invasiveness of IL-1β-stimulated human fibroblast-like synoviocytes obtained from the rheumatoid arthritis patients were also verified using BrdU cell proliferation assay and Transwell invasion assay, respectively. The increased secretions of MIF and MMPs in IL-1β-stimulated fibroblast-like synoviocytes and subsequent inhibition of those secretions by berberine were also identified in vitro. In vivo pannus formation and its invasion into cartilage were histologically observed in knee joints of the AIA rats on day 30 after arthritis induction. The berberine treatment evidently suppressed the pannus expansion and invasion in addition to the inhibition of inflammatory responses such as thickness of synovium and inflammatory cell infiltration into synovium. In summary, beberine significantly inhibited proliferation, invasiveness and inflammation response of IL-1β-stimulated human rheumatoid fibroblast like-synoviocyte through the modulation of MIF-mediated cell signaling. It also suppressed arthritic symptoms and pannus formation in knee joints of the AIA rats, suggesting its strong potential as a therapeutic agent for treating rheumatoid arthritis.

Anti-rheumatoid Arthritis Effect of Lactic Acid Bacteria-fermented Mycelium in RAW264.7 Macrophage and MH7A Human Rheumatoid Fibroblast-like Synoviocyte

Youngjun Soh 고려대학교 대학원 2024 국내석사

Regulation of the interaction between synovial cells and B cells by BAFF-mediated VCAM-1 expression

윤성식 세종대학교 대학원 2020 국내석사

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that leads to expansion of synovium and joint destruction. Fibroblast-like synoviocytes (FLS) also play a key role in the pathogenesis of RA by producing inflammatory cytokines that contribute to cartilage destruction. RA-FLS develop a unique aggressive phenotype that increases invasiveness into the extracellular matrix and further exacerbates joint damage. Tumor necrosis factor- α (TNF- α) is a pro-inflammatory cytokines that triggers the expression of inflammatory molecules, including other cytokine and cell adhesion molecules. TNF- α induces the expression of B cell activating factor(BAFF) which plays a role in maturation and maintenance of B cells. TNF- α also induces vascular cell adhesion molecule-1(VCAM-1) which helps regulate inflammation -associated vascular adhesion and the transendothelial migration of leukocytes, such as macrophages, T cells and B cells. The pathogenesis of RA is complex and en-compasses many cell types, including T cells, B cells, and macrophages which is believed to play a significant role in the process of RA. While interactions between T cells and B cells and between T cells and FLSs have been deeply studied, but interactions between B cells and FLSs are not. So we thought that cell-cell contact between B cells and FLSs may be significant in the patho-physiology of RA, and the interactions between TNF- α induced BAFF and VCAM-1 is related with the cell-cell interactions. Here, we investigated whether BAFF and VCAM-1 have relationship with B lymphocytes and FLSs interactions using WiL2-NS and MH7A synovial cells. We used RT-PCR, Western blot analysis, transcriptional activity assay, transfection using siRNA, MTT assay to measure the interactions between two cells especially cell bindings. Taken together, we found that TNF- α-induced the upregulation of BAFF and VCAM-1 increased the interaction between MH7A cells and WiL2-NS. Thus, the pathological mechanism of progression of RA could be related with the BAFF-mediated interaction of synovial cells and B lymphocytes.