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

Analysis of Intestinal Microbiome in Rheumatoid Arthritis (RA) Patients and Evaluation of RA Inhibitory Effect and Safety of Bifidobacterium bifidum ATT

정윤주 서울대학교 대학원 2019 국내박사

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation of the joints and extra-articular manifestations. Recent studies have shown that microorganisms affect RA pathogenesis. However, few studies have examined the microbial distribution of early RA patients, particularly female patients. In the present study, the gut microbiota profile and microbial functions in early RA female patients, including preclinical and clinically apparent RA cases, were assessed. Changes in microbiological diversity, composition, and function in each group were analyzed using quantitative insights into microbial ecology (QIIME) and phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt). The results revealed the dysbiosis due to decreased diversity in the early RA patients compared with healthy subjects. There were significant differences in the microbial distribution of various taxa from phylum to genus levels between healthy subjects and early RA patients. Phylum Bacteroidetes was enriched in early RA patients, while Actinobacteria, including the genus Collinsella, was enriched in healthy subjects. Functional analysis based on clusters of orthologous groups revealed that the genes related to the biosynthesis of menaquinone, known to be derived from gram-positive bacteria, were enriched in healthy subjects, while iron transport-related genes were enriched in early RA patients. Genes related to the biosynthesis of lipopolysaccharide (LPS), the gram-negative bacterial endotoxin, were enriched in clinically apparent RA patients. The obvious differences in microbial diversity, taxa, and associated functions of the gut microbiota between healthy subjects and early RA patients highlight the involvement of the gut microbiome in the early stages of RA. Rheumatoid factor (RF) is an autoantibody that is detected in approximately 80% of patients with rheumatoid arthritis. RF can be also detected in older individuals but the reason is unclear. There are a few RA patients who have a low level of RF in their blood but the reason for this is also unknown. Even though there are many unclear things about RF, several studies have presented a model for RA that immune complex, RF-human IgG-antigen, plays an important role in RA pathogenesis and development. The immune complex containing RF activates the complement after deposition in the joint, resulting in type III hypersensitivity reaction, which causes inflammation of the joint. Although there are a few studies on the association between RA and microbiome, there are no previous studies that have revealed the relationship between gut microorganisms and RF, which is major autoantibody in RA etiology. Therefore, we investigated intestinal microflora according to RF level, one of the RA clinical indicators in the present work. As a result, though alpha and beta diversity were not different between groups divided by RF titer, specific taxa belonging to Actinobacteria phylum including Bifidobacterium genus showed less abundance relatively in RF negative groups than positive. Moreover, the least relative abundance was shown in patients with high titer of RF, over 60 IU/ml. Actinobacteria phylum and Bifidobacterium genus also showed a negative correlation with RF. In order to determine whether the reduction of the Bifidobacterium genus is the cause or the effects of the disease, we performed an experiment to analyze the effect of supplementation of heat-inactivated Bifidobacterium bifidum ATT, in RA model mice. The results showed that both the incidence and score of RA were significantly lower in Bifidobacterium fed group than the control. The production of autoantibody-like antibodies was also reduced, suggesting that the treatment with Bifidobacterium is effective for the suppression of RA. Since the supplementation of B. bifidum ATT was effective in preliminary RA in vivo study, it can be used as a food or pharmaceutical material. Prior to industrial use of the B. bifidum ATT as a commercial product, verification of safety is an important prerequisite, so the whole genome sequencing of B. bifidum ATT was conducted to acquire various information such as general genome information and function prediction as well as information related safety. In the genome of B. bifidum ATT, there were no virulence factor and genes involved in producing biogenic amines and phosphatidylserine, which plays an important role in platelet aggregation. Based on the microbiome analyses of patients with RA, preliminary in vivo study and safety assessment through whole genome sequencing, B. bifidum ATT showed the potential to be an effective source of a food material or drug substance for RA modulation with safety. Given further studies such as robust preclinical test and safety assurance through assays such as hemolytic activity and antibiotic resistance test would be implemented, B. bifidum ATT could be developed as a commercial product such as food ingredient or therapeutic agent in real life. 류마티스 관절염은 관절액을 생성하는 얇은 막인 관절 활막의 지속적인 염증반응이 특정인 만성 염증성 전신 질환이다. 염증의 파급으로 관절의 연골 손상, 골 미란, 관절의 파괴와 변형으로 통증을 유발하고 기능의 장애를 가져올 뿐만 아니라 폐섬유화증, 피부 궤양 등의 관절 외 증상도 함께 초래한다. 류마티스 관절염의 위험 요인으로는 환경적 요인과 유전적 요인이 있다. 류마티스 관절염의 주요 병인인 Th17 세포가 인체 공생 세균에 의해 유도된다는 연구결과를 비롯하여 최근 환경적 요인 중 미생물을 제시하는 연구들이 축적되고 있다. 차세대 유전체 분석법을 통해 직접 환자의 장내 균총을 분석하는 연구들도 다수 수행되었다. 특히 초기 RA환자의 장내 균총 분석 결과 Prevotella copri라는 특정 세균이 건강한 사람 대비 상대적으로 풍부했으며, 이 Prevotella copri를 dextran sulfate sodium으로 염증을 유발한 쥐에 투여했을 때 염증이 더욱 심화되는 것 또한 밝혀졌다. 본 연구에서는 여성에게 호발하는 질환의 특성을 고려하여, 류마티스 관절염 초기 여성 환자 29명을 대상으로 채변 후 16S rRNA 유전자를 이용해 장내 균총을 분석하였다. 환자들은 자가항체는 검출되지만 관절 증상이 없는 환자(PC) 17명과, 자가항체가 검출되면서 관절 증상이 있는 명백한 류마티스 관절염 환자(ST) 12명의 두 그룹으로 다시 나뉘었다. 장내 균총에 미치는 치료제의 영향을 최소화하기위해 환자들은 모두 어떠한 치료도 받지않은 초기 환자들로만 구성되었다. 환자들의 장내 균총을 건강한 사람들의 그것과 비교한 결과, 다양성과 균총의 분포, 균의 기능에 있어서 모두 차이가 있었다. 다양성은 건강한 사람 대비 환자군에서 감소하였으며, 분포는 Actinobacteria phylum과 Collinsella genus가 건강한 사람 대비 환자군에서 감소한 것이 특징이었다. 뿐만 아니라 PICRUSt를 통한 미생물 기능 분석 결과는, 환자 군 중 명백히 증상이 있는 군 (ST)에서 그람 음성 세균 유래의 당지질(LPS) 생성에 관여하는 유전자의 발현이 증가한 것으로 나타났다. 따라서 본 연구는 초기 류마티스 관절염 환자군의 장내 균총은 건강한 사람과 확연히 다르며 특히 염증을 유발할 수 있는 당지질 관련 유전자가 증가한 것으로 보아, 미생물이 병인과 연관이 있음을 밝힐 수 있었다. 초기 류마티스 관절염 환자뿐만 아니라 진행된 환자를 포함하여 총 94명의 환자의 분변을 채변하여 두번째 연구를 진행하였다. 환자들은 자가항체인 Rheumatoid factor (RF) 정도에 따라 음성 그룹(RF ≤ 20 IU/ml)과 양성 그룹 (RF > 20 IU/m)으로 나뉘었다. 양성그룹은 다시 RF 검출양에 따라 저양성 (low positive, 20 < RF ≤ 60)와 고양성 (high prositive, RF > 60)그룹으로 나뉘었다. RF는 류마티스 관절염 환자의 80%에서 발견되는 자가항체로 질병의 진단 지표이다. 또한 인간 IgG 항체 – 미생물 유래 항원으로 이루어진 면역 복합체의 IgG Fc 부분에 결합하여 보체를 활성시켜 염증 반응으로 이어지는 면역반응을 촉발시키는 것으로도 알려져있다. RF 정도에 따른 장내 균총 분석 결과 균총의 다양성에는 차이가 없었으나 Actinobacteria phylum과 Bifidobacterium genus가 RF 양성군에서 음성군 대비 상대적으로 덜 풍부한 것으로 나타났고 특히 RF 고양성군에서 가장 덜 풍부하였다. 스피어만 상관분석결과 역시 Actinobacteria phylum 및 Bifidobacterium genus의 상대적 풍부도와 RF 정도는 통계적으로 유의미하게 음의 상관관계를 보였다. 따라서 이런 장내 균총 구성의 차이가 질병에 의한 것인지, 아니면 균총의 차이로 인해 질병이 시작되거나 증세가 심화되는지 알아보기 위해, 예비적인 동물실험을 수행하였다. 즉, 상대적으로 덜 풍부한 Bifidobacterium genus를 보충해주며 어떤 현상이 나타나는지에 대한 연구를 수행하였다. 동물 실험은 열로 불활성화 시킨 Bifidobacterium bifidum ATT 균을 type II collagen으로 관절염을 유발시킨 쥐에 투여하여 진행되었다. 그 결과 균을 급여하지 않은 쥐보다 균을 급여한 쥐에서 관절염의 발병이 늦었으며, 관절염 점수 또한 유의미하게 낮은 것을 관찰하였다. 류마티스환자의 자가 항체에 해당하는, type II collagen 특이적인 IgG 항체 생성 또한 균 급여군에서 유의미하게 적게 생성되었다. 따라서 RF 고양성군에서 상대적으로 적게 분포하고 있었던 Bifidobacterium을 보충해주면 질병이 조절되는 것을 알 수 있었고, 이를 바탕으로 B. bifidum ATT가 류마티스 관절염 환자들을 위한 건강기능 식품의 소재 또는 의약품의 소재가 될 수 있는 가능성을 확인하였다. 하지만 인간이 어떠한 물질을 식품이나 약으로 섭취할 때는 효능뿐만 아니라 안전성을 검증하는 것 또한 중요하다. 본 연구에서는 B. bifidum ATT균의 전체 유전자 서열을 해독 후 웹 기반 데이터베이스 프로그램을 이용하여 안전성을 검증하였다. B. bifidum ATT에는 플라스미드가 없었으므로 일차적으로 병원성이 없음을 검증하였다. 대표적인 유해균인 Listeria, S. aureus, E. coli, Enterococcus 균에 있는 병독성 유전자 또한 B. bifidum ATT의 유전자 내에 존재하지않음을 확인하였다. 뿐만 아니라 바이오제닉 아민인 histamine과 tysrosin의 생성에 필수적인 효소의 유전자와 혈액 응고에 관여하는 phosphatidyl serine 생성에 필요한 효소의 유전자도 존재하지 않음을 확인하였다. 결론적으로, 본 연구에서는 류마티스관절염 환자의 장내 균총 분석 결과, 예비적인 동물 실험과 전체 유전자 서열에 기반한 안전성 분석을 통해 B. bifidum ATT가 류마티스 관절염 조절에 유용한 식품 및 의약품 소재가 될 수 있음을 밝혔다. 철저한 전임상 실험과 용혈 활성, 항생제 저항성 등과 같은 실제 실험에 기반한 안전성 검증 등의 추가 연구가 수행된다면 B. bifidum ATT는 식품 원료나 치료제와 같은 상업적인 제품으로 개발될 수 있을 것이다.

Development of benzothiazole derivatives as PI3KC2γ inhibitor for rheumatoid arthritis

Yoo, Jaeho Sungkyunkwan university 2020 국내박사

Rheumatoid arthritis (RA) is a major disease of modern humans, and numerous studies have sought to find a therapeutic agent against RA. At present, the main treatment modalities of RA are disease-modifying anti-rheumatic drugs DMARD and biologics. To find out the new target for RA treatment, in the present study, I have suggested PI3K class2 gamma inhibitor. 6-(pyridin-4-yl)benzo[d]thiazol-2-amine was also evaluated in the RA tissue and in the RA animal model. An amino benzothiazole derivative was reported to inhibit PI3K kinase. Therefore, based on this evidence, in the present study, I demonstrated the relevance of amino benzothiazole to rheumatoid arthritis. To demonstrate the viability of MH7A (rheumatoid arthritis synovial cell) and raw 264.7 cell line, as well as the effect of rheumatoid arthritis model, 6-(pyridin-4-yl)benzo[d]thiazol-2-amine was evaluated RA tissue and in vivo model. Using a sample of tissue in the CIA model, I experimentally demonstrated that the target is effective in the rheumatoid arthritis by confirming the expression level of the target; the effect was also demonstrated in the rheumatoid arthritis model through the compound which is benzothiazole derivative. Amino benzothiazole derivatives are well known for their anti-inflammation effect. This compound was reported to have the effect of blocking TNF-α in MH7A cells; in vivo changes in physiological activity in cells were also demonstrated. The 6-(pyridin-4-yl)benzo[d]thiazol-2-amine compound was a PI3K class2 gamma inhibitor with druglikeness and inhibited by the cell. It was also effective in the model of CIA of rheumatoid arthritis disease. The present study demonstrates the 6-(pyridin-4-yl)benzo[d]thiazol-2-amine compound will be new RA treatment.

류마티스 관절염 환자에서 간질성 폐질환의 임상적 특징

김수경 대구가톨릭대학교 2008 국내석사

Background: Rheumatoid arthritis is a common systemic inflammatory disease affecting approximately 1-2% of the general population. Nearly 40% of patients with rheumatoid arthritis demonstrate some type of extra-articular manifestation involving skin, eye, heart, and lungs. A variety of pulmonary manifestations are associated with rheumatoid arthritis, and lung disease is the second most common cause of death after infection for patients with rheumatoid arthritis. We discussed clinical characteristics in patients with rheumatoid arthritis and with interstitial lung disease. Method: Thirty-two patients with rheumatoid arthritis and with interstitial lung disease and eighty-five patients with rheumatoid arthritis and without interstitial lung disease were included. Age, sex, disease duration, rheumatoid factor(RF), erythrocyte sedimentation rate(ESR), C-reactive protein(CRP) were measured in each group. Results: In high resolution computed tomography(HRCT) findings, usual interstitial pneumonia(UIP) was common in rheumatoid arthritis. Significant different variables between two groups were age, disease duration and ESR of clinical characteristics. Significant correlation was found between disease duration and interstitial pneumonia in rheumatoid arthritis, but longer disease duration was not predisposing factor for the interstitial pneumonia in rheumatoid arthritis in our study. Conclusion: It is suggested that longer disease duration may not be predisposing factor for the interstitial pneumonia in rheumatoid arthritis. Some data suggest a poor prognosis for rheumatoid arthritis associated interstitial pneumonia, further studies are required on the collagen vascular disease associated interstitial pneumonia.

류마티스 관절염 환자의 질병 활동도 지표로써 모발의 미세 원소측정

전재범 한양대학교 대학원 1994 국내석사

류마토이드 관절염 및 퇴행성 관절염에서 항 제II형 교원질 항체의 발현 양상에 관한 연구

홍정용 동국대학교 대학원 1998 국내박사

정형외과 영역에서 흔히 볼 수 있는 만성 관절염으로는 류마토이드 관절염, 퇴행성 관절염 등이 있으며 관절 연골의 파괴 과정을 이해하기 위해서는 교원질에 대한 이해가 중요하다. 교원질은 인체 결합 조직의 기본으로 13종류 이상이 알려져 있으며 그 중 제Ⅱ형 교원질이 연골 구성에 있어서 가장 중요하다. 교원질 유발 관절염(collagen-induced arthritis)은 1977년 Trentham들에 의하여 발표되었으며 쥐에 제Ⅱ형 교원질을 투여함으로써 발생된다. 이 관절염 모델은 임상적 및 조직 병리학적으로 류마토이드 관절염과 유사한 소견을 보이며 항 제Ⅱ형 교원질 항체가 증가된 소견을 보인다. 본 연구의 목적은 만성 관절염 환자에 있어 제Ⅱ형 교원질에 대한 IgG 및 IgM 항체의 시간적인 변화를 살펴보는 것이다. 연구 대상으로 정상군, 류마토이드 관절염군, 퇴행성 관절염군을 선택하였으며 혈액 채취를 시행하여 헐점을 분리한 후 효소 면역 측정을 시행하였다. 효소 면역 측정의 정밀도에서 변성된 인형 교원질을 제외한 다른 모든 측정값은 그 상관계수가 상당히 높아서 이 효소 면역 측정이 믿을 수 있는 수준인 것으로 판명되었으며, 변성된 인형 교원질의 경우에는 일정한 변성 패턴이 없어서 상관성이 결여된 것으로 추정하였다. 항체 역가의 시간적인 변화에서 류마토이드 관절염군, 퇴행성 관절염군에서 IgG 항체의 역가는 모든 군에서 시간적인 변화가 없었다. IgM 항체는 정상군에서는 시간적인 변화가 없었으나 관절염군에서는 시간적인 변화가 관찰되었는데, 항원의 종류에 따라 인형 교원질의 경우에는 류마토이드 관절염군 및 퇴행성 관절염군 모두에서 시간적인 변화를 확인할 수 있었으며, 무형 교원질의 경우에는 류마토이드 관절염에 국한되어서 시간적인 변화가 나타났다. 그러나 제Ⅱ형 계형 교원질의 경우는 이러한 시간적인 변화를 관찰할 수 없었다. 결론적으로 관절염군에서 IgG 항체는 시간적인 변화가 거의 없는데 반하여, IgM 항체는 시간적으로 증감하는 양상을 보여 추후 연구 과제로서 항체의 시간적 변화가 관절염의 악화 및 완화와 관련이 있는지를 알아보는 것이 중요할 것으로 생각한다. Collagen is the major structural protein in the human body, especially in connective tissues. Type II collagen is a main component of articular cartilage structure. Altered immunological conditions against type Ⅱ collagen may be closely related to the pathologic conditions of joint, especially arthritis. Since 1977, animal model for collagen-induced arthritis(CIA) has been developed. In those animals, high titers of anti-type Ⅱ collagen antibody were noticed. Pathologic findings were similar to rheumatoid arthritis, which suggested that rheumatoid arthritis might be one of the autoimmune diseases. There had been many reports about elevation of serum and synovial level of anti-type Ⅱ collagen antibody in rheumatoid arthritis patients. But majority of them did not discriminate the antibody titers according to the type of immunoglobulin(i.e. IgG, IgM). And the question whether the elevated antibody titers are cause or effect of the arthritis is still in controversy. In this study, the serum levels of anti-type Ⅱ collagen antibody were determined in three groups(control, degenerative arthritis and rheumatoid arthritis) with ELISA method. In each person, the serum levels of antibody IgG, IgM against human, bovine and chicken type Ⅱ collagens were determined individually. Since correlation coefficients of ELISA show high values except in the case of denatured human collagen, ELISA test is regarded confident. In terms of denatured human collagen, no constant denaturation pattern induces variable results. With regards to chronological changes, there is no change of IgG titer in all three groups. IgM titer of control group is not variable according to time change. But IgM titer of arthritis group changes from time to time. Chronological changes of antibody titers depend on the types of antigen. In case of human collagen, there are changes of antibody titer in degenerative arthritis and rheumatoid arthritis groups. And in case of bovine collagen, change of antibody titer is observed only in rheumatoid arthritis group. Conclusively, chronological change of IgG titer is not observed in arthritis group but IgM titer changes are observed in arthritis group. So it might be a topic of future research to evaluate the relationship between the chronological change of IgM antibody and disease aggravation.

The combination effects of boswellia serrata, grape seed and juniper berry extracts on the prevention of rheumatoid arthritis in mice

Park, Keon Woong 고려대학교 대학원 2015 국내석사

Boswellia serrata, a traditional herbal medicine in Asia and Africa, is used to treat a variety of inflammatory disorders, including inflammatory bowel disease, rheumatoid arthritis, and osteoarthritis. To develop an optimal combination of natural substances for the prevention of rheumatoid arthritis, we here investigated the combined effects of grape seed extract (GSE) and juniper berry extract (JBE) with Boswellia serrata extract (BSE). GSE, JBE, and BSE inhibited nitric oxide production in LPS-stimulated RAW264.7 cells, with BSE exhibiting the strongest inhibitory effect. Of the tested extracts, only GSE reduced prostaglandin E2 (PGE2) abundance in LPS-stimulated RAW264.7 cells. The optimal combination of BSE, GBE, and JBE at a ratio of 2:1:1 (w/w) was selected via in vitro assays using RAW264.7 cells and used to determine the preventive effects of the combination against rheumatoid arthritis in a mouse model of collagen-induced arthritis (CIA). Daily oral administration of the mixed extract formulation (100 or 200 mg/kg) and BSE (200 mg/kg) was initiated with the first collagen injection and continued for 7 weeks for arthritis induction. The mixed extract formulation significantly reduced the incidence of arthritis and serum IL-4 levels in CIA animals. Consistent with the observed biochemical alterations, histological observations revealed that the mixed extract formulation improved synovial hyperplasia and reduced inflammatory cell infiltration in a dose-dependent manner. Importantly, the observed preventive effects of the mixed extract formulation against rheumatoid arthritis were similar to those of BSE alone at the same dose (200 mg/kg). The results of this study suggest that an appropriate combination of GSE, JBE, and BSE could be used as a low-cost and effective alternative to BSE for the prevention of rheumatoid arthritis.

류마티스관절염 마우스모델에서 CFHR5의 관절염 완화 효과

김경미 계명대학교 대학원 2024 국내석사

Rheumatoid arthritis is a chronic inflammatory arthritis that is related to the immune complement response. Complement factor H-related 5 (CFHR5) exhibits anti-inflammatory effects by binding to complement C3, an inflammatory response protein. It is not known how CFHR5 affects arthritis-based autoimmunity. This study aimed to confirm the effect of CFHR5 in collagen-induced arthritis (CIA), a rheumatoid arthritis mouse model. CIA mice were induced by injecting type 2 collagen twice into DBA/1J mice. After 23 d, the mice were divided into three groups, and until day 42, the positive control group, methotrexate, was intraperitoneally administered twice a week, the low dose of 0.5 mg/kg group and the high dose 1.5 mg/kg group were intraperitoneally administered CFHR5 once a week. Clinical severity score and arthritis index were measured every other day. After sacrificing the mouse on day 42, foot tissue was removed and H&E and Toluidine blue staining were performed. Compared to the negative control group, both low-dose and high-dose CFHR5 treatment resulted in statistically significant reductions in the clinical arthritis index (Low-dose CFHR5 group, P <0.01; High-dose CFHR5 1.5 mg/kg, P <0.001). The arthritis index also significantly decreased compared to the negative control group (Low-dose CFHR5 group, P <0.05; High-dose CFHR5, P <0.001). Four parameters were assessed in both H&E and Toluidine blue stained sections: synovial hyperplasia, pannus formation, cartilage destruction, and bone erosion. The Toluidine blue staining evaluation criteria were specific to this stain. In all three standard parameters (Matrix staining, Surface regularity, Cartilage thickness), the positive control group, low dose, and high dose CFHR5 demonstrated a significant reduction compared to the negative control group. In this study, the administration of CFHR5 attenuated the activity of arthritis tissue inflammation and joint damage severity in a CIA mouse model, suggesting that CFHR5 has an anti-inflammatory effect in rheumatoid arthritis, and detailed mechanistic studies are needed. 류마티스관절염은 만성 염증성 관절염으로, 면역 보체 반응과 관련이 있는 것으로 보고된다. Complement factor H-related 5 (CFHR5)는 염증 반응 단백질인 보체 C3에 결합하여 항염증 효과를 나타내는 것으로 알려져 있다. CFHR5가 관절염을 기반으로 한 자가면역에 어떤 영향을 미치는지는 밝혀진 바가 없다. 본 연구는 류마티스관절염 마우스 모델인 Collagen Induce Arthritis(CIA)에서 CFHR5의 효과를 확인하고자 했다. DBA/1J 마우스에 제2형 콜라겐을 2회 주입하여 CIA 마우스 유도하였다. 23일 후 마우스를 3개의 그룹으로 나누어 42일까지 양성 대조군 methotrexate는 주 2회, 저용량 0.5 mg/kg, 고용량 1.5 mg/kg 군은 CFHR5 주 1회 복강 내 투여하였다. 격일로 임상 중증도 점수와 관절염 지수를 측정하였다. 42일 째 마우스를 희생시킨 후 발 조직을 적출하여 H&E과 Toluidine blue 염색을 진행하였다. 저용량, 고용량 CFHR5의 치료 효능을 음성 대조군과 비교한 결과 임상 관절염 지수 및 관절염 발생 빈도는 음성 대조군과 비교하였을 때 통계적으로 유의미하게 감소하였다. H&E 염색과 Toluidine blue 염색 결과 H&E 염색 평가 기준인 4개의 매개변수 (synovial hyperplasia, Pannus formation, Cartilage destruction, Bone erosion)와 Toluidine blue 염색 평가 기준인 3개의 매개변수(Matrix staining, Surface regularity, Cartilage thickness)에서 모두 양성 대조군, 저용량, 고용량 CFHR5는 음성 대조군과 비교하였을 때 통계적으로 유의한 감소 소견을 보였다. 본 연구에서는 CIA 마우스 모델에서 CFHR5의 투여는 관절염 조직 염증의 활동성 및 관절 손상의 중증도를 약화시켰다. 이는 류마티스관절염에 CFHR5가 항염증 효과가 있음을 시사하며, 자세한 기전 연구가 필요하다.

Global Metabolite Profiling of Synovial Fluids for the Discovery of Potential Biomarkers of Rheumatoid Arthritis

XUAN JINHUA 고려대학교 대학원 2012 국내석사

Rheumatoid arthritis (RA) is an autoimmune disease that causes inflammation in multiple joints. There is still no cure for RA and the diagnosis remains difficult. A global metabolite profiling was performed to identify distinctive metabolic patterns of RA using synovial fluids by comparing RA with osteoarthritis (OA). Twenty nine synovial fluid samples were analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOF MS), a total of 63 metabolites were positively annotated. Firstly, the differences in metabolite signatures were visualized using a hierarchical clustering analysis and a principle component analysis. Secondly, partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis OPLS-DA) models were established, of which both good capabilities of discrimination. Potential biomarkers were selected based on the variable importance for projection (VIP) values higher than 1. Furthermore, using Welch’s t-test with P < 0.01, a set of 17 metabolites were identified as dominant discriminatory biomarkers that contributed to the differentiation between the two groups such as RA and OA. Several amino acids were generally found to be down-regulated in RA, which implied altered metabolic changes with disease perturbation. Finally, the use of synovial metabolite profiling in diagnosis was investigated with 100% specificity and sensitivity. This study demonstrated that a metabolomics-based approach is an effective tool for investigating synovial biomarkers and can potentially facilitate diagnosis of diseases.

The Role of CABIN1 and EC-SOD in Rheumatoid Arthritis : 류마티스 관절염을 유도한 형질전환동물에서의 CABIN1과 EC-SOD 유전자의 기능학적 분석

이준구 경북대학교 대학원 2014 국내박사

Calcineurin (CN) is a calcium- and calmodulin-dependent serine/threonine phosphatase. In immune cells, CN controls the activity of a wide range of transcription factors, including NF-AT, NF-kB, c-fos, and Elk-1. CN plays an important role in synoviocyte activation and arthritis progression in vivo and this function is tightly linked to dysregulated intracellular Ca2+ store and Ca2+ response triggered by pro-inflammatory cytokines. In the present study, transgenic mice expressing human Cabin1 (hCabin1) were generated, driven by type II collagen promoter and the efficiency of these mice was investigated by experimental arthritis. These transgenic mice successfully expressed hCabin1 in joint tissue as well as other organs like liver, heart and brain. The over-expression of hCabin1 reduced the disease severity during collagen-induced arthritis. In fibroblast-like synoviocytes (FLSs) from hCabin1 transgenic mice, the productions of these cytokines including, IL-2, IL-4, and IFN-γ were decreased and MMPs was also depressed in transgenic mice FLS. In addition, these effects were only found in the joint tissue, which is a major inflammation site. These findings will provide a better knowledge of the pathogenic mechanisms of rheumatoid arthritis and a potential animal model of the chronic inflammatory conditions, including atherosclerosis and transplantation. Calcineurin-binding protein 1 (Cabin1) regulates calcineurin phosphatase activity as well as the activation, apoptosis, and inflammatory responses of fibroblast-like synoviocytes (FLSs), which actively participate in the chronic inflammatory responses in rheumatoid arthritis (RA). However, the mechanism of action of Cabin1 in FLS apoptosis is not clear. The aim of this study was to define the regulatory role of Cabin1 in FLSs of mice with collagen-induced arthritis (CIA). Transgenic mice overexpressing human Cabin1 in joint tissues, under the control of a type II collagen promoter, were generated. hCabin1 expression in joints and FLSs was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of cytokines, matrix metalloproteinases (MMPs), and apoptosis-related genes in FLSs was determined by enzyme-linked immunosorbent assay, gelatin zymography, and RT-PCR, respectively. Joints were histologically examined after H&E and TRAP staining. hCabin1-transgenic CIA mice had less severe arthritis than wild-type CIA mice, based on hind paw thickness and histology. This was accompanied by significantly enhanced apoptosis in transgenic mice, evidenced by significantly more TUNEL-positive cells in synovial tissues. The expression of inflammatory cytokines and MMPs was reduced, and the transgenic CIA mice exhibited decreased Akt activation and increased expression of p53, caspase-3, caspase-9, and Bax. hCabin1 plays a critical role in promoting apoptosis of FLSs and in attenuating inflammation and the destruction of cartilage and bone in RA. These findings help elucidate the pathogenic mechanisms of RA and suggest that Cabin1 is a potential target for RA treatment. Oxidative stress such as reactive oxygen species (ROS) within the inflamed joint have been indicated as being involved as inflammatory mediators in the induction of arthritis. Correlations between extracellular-superoxidedimutase (EC-SOD) and inflammatory arthritis have been shown in several animal models of RA. However, there is a question whether the over-expression of EC-SOD on arthritic joint also could suppress the progression of disease or not. In the present study, the effect on the synovial tissue of experimental arthritis was investigated using EC-SOD over-expression model. The over-expression of EC-SOD in joint tissue was confirmed by RT-PCR and immunohistochmistry. The degree of the inflammation in EC-SOD transgenic mice was suppressed in the collagen-induced arthritis model. In a cytokine assay, the production of proinflammatory cytokines such as, IL-1β, TNFα, and metalloproteinases (MMPs) was decreased in fibroblast-like synoviocyte (FLS) but not in peripheral blood. Histological examination also showed repressed symptoms in EC-SOD transgenic mice. In conclusion, these data suggest that the over-expression of EC-SOD in FLS contributes to the activation of FLS and protection from joint destruction by depressing the production of the proinflammatory cytokines and MMPs. These results provide EC-SOD transgenic mice with a useful animal model for inflammatory arthritis research.