Quantitative Study on Body Fats in Relation to the Levels of Adipokines and Cytokines

Lee, In Seok 경희대학교 2009 국내박사

Obesity can be characterized as a metabolic disorder and/or a state of chronic low-grade inflammation. This low-grade inflammation is associated with high blood levels of lipids, adipokines, and cytokines which contribute to the development of obesity. The purpose of this study was to investigate the blood levels of lipids, adipokines, and pro-inflammatory cytokines as prognostic indicators of obesity-related chronic diseases in normal, over-weight, and obese women. Sixty women were recruited and categorized according to their body mass index (BMI) into normal-weight (BMI <23 kg/m2), over-weight (BMI 23?25 kg/m2), and obese (BMI >25 kg/m2) groups. Anthropometric data and blood levels of lipids, adipokines, and pro-inflammatory cytokines were compared among the groups. The over-weight and obese subjects had significantly higher leptin levels than the normal subjects, whereas the normal subjects had significantly higher adiponectin levels than the over-weight and obese subjects (p < 0.05). The over-weight and obese subjects had a significantly higher concentration of TNF-? than the normal subjects (p < 0.05). The obese subjects had significantly higher concentrations of IFN-? than the normal and over-weight subjects (p < 0.05). The IL-1? and IL-6 plasma levels were not different among the three groups. In conclusion, this study confirmed that high levels of leptin and low levels of adiponectin were associated with the degree of obesity. Furthermore, high levels of TNF-? and IFN-? were associated with the degree of obesity. Adipose tissue mass (ATM) is an important source of adipokines. Increase in ATM contributes to chronic low-grade inflammation characterized by the high levels of pro-inflammatory cytokines. We investigated the effects of body fat reduction on blood levels of adipokines and pro-inflammatory cytokines in Korean women with BMIs >23 kg/m2. A total of 46 healthy women participated in the study. A registered dietician supervised the subjects as they conformed to a program of caloric restriction during a 12-week experimental period. Anthropometric assessments were carried out, and lipid, adipokine, and pro-inflammatory cytokine levels were analyzed at the beginning and the end of the experiment. Body weight and body fat mass decreased significantly in our subjects after 12 weeks of caloric restriction (p < 0.001). Triacylglycerol blood levels were significantly reduced in accordance with body fat reduction. However, the levels of other lipid parameters were unchanged. Adipokine, leptin, and visfatin levels were significantly decreased, while adiponectin increased significantly (p < 0.05). Levels of pro-inflammatory cytokines TNF-? and IFN-? were significantly reduced. These results indicate that body fat reduction via caloric restriction significantly affects the blood levels of adipokines and pro-inflammatory cytokines. High-fat diets induce an expansion of the adipose tissue (AT) that can be characterized by chronic low-grade inflammation. AT is an important source of adipokines and pro-inflammatory cytokines. The purpose of this study was to evaluate the effects of a shift from a high-fat diet to high-carbohydrate (CHO) diet on the blood levels of adipokines and pro-inflammation cytokines in mice fed a high-fat diet. Six-week-old male C57BL/6 mice were fed a high-fat diet (40% of the total calories) for 9 weeks to induce obesity, and then the diet was shifted to a high CHO diet (70% of the total calories) for 3 weeks. Body weight and organ weight as well as blood lipid levels were measured. The serum levels of adipokines and pro-inflammatory cytokines were analyzed. Shifting the diet from high fat to high CHO significantly decreased body weight, adipose tissues, and liver weight (p < 0.05). The lipid blood levels (TG, Total-chol, and LDL-chol) decreased. The leptin and resistin blood levels significantly decreased after the diet was shifted to a high-CHO diet (p < 0.05); however, the adiponectin concentrations did not change. The IL-6 and TNF-? serum levels were also significantly decreased by the high-CHO diet (p < 0.05). The IL-13 serum levels were significantly increased by the high-CHO diet (p < 0.05). Further, the serum levels of the TNF-? and supernatant IL-1? concentrations in mice fed a high-carbohydrate diet were significantly increased after the mice were shifted to a high-fat diet. On the other hand, the serum IL-4 and supernatant levels did not change. Conclusively, reduction of body weight and adipose tissues through shifts from a high-fat diet to a high-carbohydrate diet effectively improved low-grade inflammation states in mice fed a high-fat diet. Particularly, the reduction of body weight was associated with leptin, resistin, IL-6, and TNF-? levels.

Anti-inflammatory effect of leaves of Eriobotrya japonica

張榏峻 우석대학교 대학원 2009 국내석사

ABSTACT Chang, Ik Jun Dept. of Pharmacy Graduate school of Woosuk University (Directed by Prof. Shin, Tae Yong, Ph.D) Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, allergic rhinitis sinusitis, and rheumatoid arthritis. The discovery of drugs for the treatment of allergic disease is an important subject in human health. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-┒, interleukin (IL)-6, and IL-8 with immune regulatory properties. In the present study, we investigate the effect of leaves of Eriobotrya japonica Lindl. (Rosaceae) (LEJL) on the expression of pro-inflammatory cytokines and its possible mechanisms of action in human mast cells (HMC-1). LEJL dose-dependently inhibited phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 (PMACI)-induced gene expression and secretion of TNF-┒, IL-6, and IL-8. LEJL attenuated PMACI-induced activation of nuclear factor (NF)-┟B, and specifically blocked the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) but not that of c-jun N-terminal kinase. The inhibitory effect of LEJL on the pro-inflammatory cytokines was confirmed by using specific inhibitors for NF-┟B, p38 MAPK, and ERK. Our in vitro studies provide evidence that LEJL might contribute to the treatment of mast cell-derived allergic inflammatory diseases. 국문초록 비파엽의 항염증효과 Chang, Ik Jun Dept. of Pharmacy Graduate school of Woosuk University (Directed by Prof. Shin, Tae Yong, Ph.D) 비만세포 연계 알러지 염증 반응은 천식, 알레르기 비염, 부비강염, 류머티즘성 관절염과 같은 많은 질병과 관계가 있다. 알레르기 질병의 치료 약물 개발은 인류의 건강에 있어서 중요한 과제이다. 비만세포는 면역 조절 특성을 지닌 tumor necrosis factor (TNF)-┒, interleukin (IL)-6, and IL-8를 포함한 pro-inflammatory cytokine들의 생성과 합성을 유도한다. 이 논문에서는 인간 비만세포에서 pro-inflammatory cytokine들의 발현과 그 활성기전에 있어서 비파엽의 효과를 실험하였다. 비파엽은 phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 (PMACI) 유도 유전자 발현과 TNF-┒, IL-6, and IL-8의 분비를 농도의존적으로 저해하였다. 비파엽은 PMACI 유도 nuclear factor (NF)-┟B의 활성을 저해하였고, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 의 활성을 명확하게 차단하였다. 그러나 c-jun N-terminal kinase는 활성 차단이 나타나지 않았다. Pro-inflammatory cytokines에 대한 비파엽의 억제효과는 NF-┟B, p38 MAPK, 그리고 ERK의 각각의 약리적 저해제를 사용함으로써 확인하였다. 이러한 in vitro 연구는 비파엽이 비만세포 연계 알러지 염증반응 질병의 치료에 효과가 있다는 것을 증명한다.

Hypothermic Treatment Attenuates Liver Damage Following Asphyxial Cardiac Arrest In Rats Through Regulation Of Inflammation And Oxidative Stress

Park Yoonsoo 강원대학교 대학원 2021 국내박사

Hypothermic treatment is known to protect against cardiac arrest (CA) including improving survival rate. However, little study has evaluated CA-induced liver damages and effects of hypothermia on the damages. This study, therefore, was designed to determine possible protective effects of hypothermia on the liver after asphyxial CA (ACA). Rats were subjected to 5-minute ACA followed by return of spontaneous circulation (ROSC). Body temperature was controlled at 37 ± 0.5℃ (normothermia group) or 33 ± 0.5℃ (hypothermia group) for 4 h after ROSC. Livers were examined at 6 h, 12 h, 24 h and 2 days, respectively, after ROSC. Histopathological examination was performed by hematoxylin and eosin staining, and changes of pro-inflammatory cytokines (tumor necrosis factor (TNF-α) and interleukin (IL)-2) and anti-inflammatory cytokines (IL-4 and IL-13) expressions were investigated by immunohistochemistry. For oxidative stress and antioxidant effects, superoxide anion radical production was evaluated using dihydroethidium fluorescence straining, and endogenous antioxidants (superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2)) were examined using immunohistochemistry. In histopathology examination, sinusoidal dilatation and vacuolization were shown after ACA, however, the CA-induced structural alterations were prevented by hypothermia. In immunohistochemical examination, expressions of pro-inflammatory cytokines were reduced in the hypothermia group compared with those in the normothermia group, and expressions of anti-inflammatory cytokines have been increased in the hypothermia group compared with the normothermia group. In conclusion, hypothermic treatment for 4 h following ACA in rats inhibited increases of pro-inflammatory cytokines and stimulated anti-inflammatory cytokines expression. Increased numbers of mast cells in the normothermia group with ACA were significantly reduced in the hypothermia group with ACA. Gradual increase in superoxide anion radical production was found with time in the normothermia group with ACA, but the production was significantly suppressed in the hypothermia group with ACA. Immunoreactivities of SOD1 and SOD2 were increased in the hypothermia group with ACA compared to those in the normothermia group. This study suggests that hypothermic treatment after ACA events has an ability to reduce liver damages through regulation of inflammation, oxidative stress and expressions of antioxidant enzymes. □ Keywords Antioxidants, Anti-inflammatory cytokines, Asphyxial cardiac arrest, Liver, Oxidative stress, Therapeutic hypothermia, Pro-inflammatory cytokines 치료적 저체온 요법은 생존율 개선을 포함하여 심정지로 인한 손상을 보호하는 것으로 알려져 있다. 하지만, 치료적 저체온 요법 적용함으로써 심정지에 따른 간 손상 완화 기전에 관한 연구는 미비한 실정이다. 이에 따라, 본 연구에서는 심정지 랫드 모델에서 치료적 저체온 요법을 적용하여 간 손상이 완화되는 것을 확인하였다. 본 연구에서는 랫드를 이용하여 5분 동안 질식성 심정지 유발 후, 자발적 순환 회복 (return of spontaneous circulation, RoSC)을 유도하는 방법으로 심정지 모델을 정립하였다. 치료적 저체온 요법은 RoSC 직후 4시간 동안 실험동물의 체온을 33 ± 0.5℃로 유지함으로써 처치하였고, 간 조직의 조직학적 분석은 RoSC 후 각각 6시간, 12시간, 1일 및 2일에 수행하였다. 조직 병리학적 분석을 위해 hematoxylin and eosin 염색하였고, 전염증성 사이토카인 (tumor necrosis factor α, TNF- α; interleukin 1β, IL-1 β) 및 항염증성 사이토카인 (interleukin 4, IL-4; interleukin 13, IL-13)을 관찰하기위하여 면역조직 화학 염색을 수행하였다. 또한, 산화적 스트레스 및 항산화 효소의 변화를 확인하기 위하여 dihydroethidium (DHE) 조직 형광 염색 및 초과산화물 불균등화 효소 (superoxide dismutases, SODs)에 대한 면역조직 화학 염색을 수행하였다. 심정지 유발후에 조직 병리학적인 변화로 간 조직의 혈관 팽창 및 공포 형성이 관찰되었고, 이들은 치료적 저체온 처치로 완화되는 것이 확인되었다. 더 나아가, 정상 체온 군에 비하여, 저체온 처치를 한 실험동물의 간 조직에서 심정지 유발에 따른 전염증성 사이토카인의 면역반응성이 유의하게 감소하였으며, 항염증성 사이토카인의 면역반응성은 유의하게 증가한 것을 확인하였다. 또한, 정상 체온 군에 비하여, 저체온 처치를 한 실험동물의 간 조직에서 심정지 유발에 따른 초과산화물 음이온 생성이 유의하게 감소하고, 내인성 항산화 효소의 면역반응성은 유의하게 증가한 것을 확인하였다. 본 연구 결과를 토대로, 치료적 저체온 요법 적용은 염증성 사이토카인의 조절, 산화적 스트레스 완화 및 항산화 효소 증진을 통하여 심정지로 인한 간 손상을 완화할 수 있는 치료적 전략으로 사용될 수 있음을 시사한다. □ 핵심주제어 항산화, 항염증성 사이토카인, 질식성 심정지, 간, 산화적 스트레스, 치료적 저체온 요법, 전염증성 사이토카인

Changes in analgesic efficacy of morphine over time following nerve injury in a rat model of peripheral neuropathy

임은정 고려대학교 대학원 2010 국내박사

Morphine has been considered as a potent analgesic, but its analgesic efficacy in neuropathic pain is highly controversial. Moreover, because of the severe adverse consequence, its use in clinical practices is strictly limited. Pain due to peripheral nerve injury is a dynamic and progressive process and its underlying mechanisms alter over time following nerve injury. Thus, the time course following nerve injury may be an important factor determining the efficacy of analgesics. In the present study, we examined whether analgesic efficacy of morphine against neuropathic pain changed over time following peripheral nerve injury. If so, we investigated whether this alteration is related to pro-inflammatory responses of spinal glia, which has been recently considered as a contributor to morphine tolerance, using the rat tail model of peripheral neuropathy. Morphine (1 and 2 mg/kg, i.p.) was excellent for the relief of mechanical allodynia at the early time (2 weeks following nerve injury), whereas the effect was significantly reduced at the late time (16 weeks following nerve injury). In addition, morphine efficacy was gradually reduced over time after nerve injury. RT-PCR results indicated that mRNA for TLR2 and TLR4, Iba-1 (a marker for microglia) and GFAP (a marker for astrocyte), and pro-inflammatory cytokines, such as IL-1b, IL-6 and TNF-a were significantly increased in the spinal cord 2 weeks after nerve injury. Of pro-inflammatory cytokine transcripts, IL-1b and IL-6 were kept highly expressed up to 16 weeks following nerve injury. At the late time, IL-6 but not IL-1b immunoreactivity (IR) was observed to be co-localized with OX-42, a marker for microglia, in the spinal cord. To address the involvement of pro-inflammatory responses of spinal microglia in the progressive reduction of morphine efficacy, we examined whether inhibition of glia or pro-inflammatory cytokines could restore the reduction of morphine efficacy at the late time. While single treatment of morphine (1 mg/kg, i.p.) did not attenuate mechanical allodynia at the late time, the co-administration of morphine AV411 (2.5 and 25 mg/rat, i.t.) as a glia-specific phosphodiesterase inhibitor, IL-6 neutralizing antibody (0.001 and 0.01 mg/rat, i.t.) or IL-1 receptor antagonist (25 mg/rat, i.t.) significantly suppressed mechanical allodynia. The present results suggest that morphine efficacy for the relief of neuropathic pain was reduced over time following nerve injury and this alteration is involved, in part, in pro-inflammatory responses of spinal microglia to nerve injury. 모르핀은 아주 유능한 진통제로 간주되어 왔지만, 신경병증성 통증에 대한 모르핀의 진통 효과 여부는 논란의 대상이 되어 왔다. 최근 들어, 신경병증성 통증에 대한 모르핀의 진통효과 감소가 신경손상 후에 유발되는 신경병증성 통증의 기전이 시간경과에 따라 다양하게 변하기 때문일 수 있다는 가능성이 제시되었다. 이에, 본 연구에서는 신경병증성 통증 중 기계적 이질통에 대한 모르핀의 효과가 신경 손상 후 시간이 지나감에 따라 변하는지와 그와 관련된 기전을 알아보았다. 꼬리 신경을 손상시키는 신경병증성 통증 모델을 이용하여, 신경 손상 후 초기(2주)와 후기(16주)에 모르핀 (1, 2 mg/kg) 을 처치하였다. 그 결과 신경 손상 후 초기에는 모르핀의 진통효과가 탁월하였으나 후기에는 효과가 현저하게 감소하였으며, 신경손상 후 11주와 15주에 모르핀을 처치한 결과와 종합해보면 시간이 지남에 점진적으로 감소하는 것을 알 수 있었다. 이 결과가 단순히 나이가 증가하여 나타난 결과인 가를 알아보기 위해, 후기(16주) 군의 수술시점인 6주보다 14주 더 자란 20주령 쥐를 대상으로 한 실험에서, 신경손상 후 2주에 모르핀을 준 효과가 초기(2주) 군의 모르핀 효과와 유사한 것으로 보아 나이에 따른 효과가 아니라는 것을 알 수 있었다. 모르핀 진통효과의 감소가 모르핀 수용체 감소와 관련되는지 알아본 결과, 신경 손상 후 초기에 감소되었던 모르핀 수용체가 오히려 복원된 것으로 보아 적어도 수용체의 양적인 변화와는 관련이 없음을 알 수 있었다. 최근 들어 신경병증성 통증 유발과 모르핀 내성이 신경계 세포와 관련이 있을 가능성이 제시된다는 점에 착안하여 신경교세포의 활성을 조사한 결과, 신경 손상 후 초기에 현저하게 증가되었던 신경계 세포의 형태학적이나 양적인 변화가 후기에 복원되는 것으로 보아, 신경 손상 후 감소된 모르핀 효과가 적어도 신경계 세포의 형태적 변화와는 직접적인 관련이 없음을 알 수 있었다. 그러나 신경 손상 후 초기(2주)에 척수 내에 증가되었던 IL-1b, IL-6이 다른 염증성 사이토카인과는 달리 신경 손상 후 후기(16주)에까지 지속적으로 증가되어 있었다. 또한 In vivo에서 이중 면역반응 실험 결과에서는 신경 손상 후 후기에 증가된 염증성 사이토카인 중 IL-6이 척수네 미세신경교 세포에서 발현되었으며, 사이토카인을 발현하는 세포 근원은 in vitro 실험 결과와도 일치하였다. 그러므로 말초 신경 손상 후 후기에 미세 신경교 세포에서 발현된 IL-6이 모르핀의 진통효과 억제와 관련이 있을 것으로 추측하였다. 이 결과를 바탕으로 신경교 세포나 염증성 사이토카인의 억제가 저하된 모르핀의 진통효과를 복원시키는지 알아본 결과, AV411, IL-6 neutralizing antibody, IL-1 receptor antagonist 모두 후기(16주)에 저하되었던 모르핀의 진통효과를 유의하게 복원하였다. 결론적으로 신경병증성 통증에 대한 모르핀의 진통효과는 신경 손상 후 시간 경과에 따라 점차적으로 감소되는 것을 확인하였다. 이 변화는 신경 손상에 의한 척수의 신경교 세포와 염증성 사이토카인 활성 특히, IL-6의 활성과 적어도 부분적으로 관련이 있음을 알 수 있었다. 향후 척수의 신경교 세포나 염증성 사이토카인에 대한 조절이 모르핀을 포함한 진통제의 민감성을 높일 수 있는 기전이 될 것이라 판단되어 좀 더 다양한 많은 연구가 진행되어야 할 것이라고 생각한다.

실험적 포도막염에서 pro-inflammatory cytokine의 역할에 관한 연구

박종문 충남대학교 대학원 1998 국내박사

Cytokines produced during the effector phases of natural and specific immunity serve to mediate or regulate immune as well as inflammatory responses. In natural immunity, endotoxin directly stimulates mononuclear phagocytes to secrete cytokines which often regulate the synthesis of cytokines from other cells. To evaluate the roles of pro-inflammatory cytokines including TNF, IL-1, IL-6, and IL-8 in experimental uveitis, numbers of infilterating inflammatory cells, IL-6 bioassay, mRNA analysis for various cytokines and IL-8 were investigated. In this study, there was no significant difference in the number of infiltering cells in TNF-R mice compared to congenic controls, but significant difference in the number of infiltering cells was found in IL-1R^-^ and TNF-R^-^ /il-1R^-^ mice. IL-6 level in aqueous humor was reduced in IL-1R^-^ and TNF-R^-^ /il-1R^-^ mice. mRNAs of TNF-α, IL-1α, IL-1Ra, IL-6, and iNOS were detected in all experimental mice after LPS or IL-1α injection. IL-1α and IL-1Rα mRNA were detected in TNF-R^-^ mice without injection of LPS. mRNA expression of IL-1β, IL-10, IFN-γ and CD8 was not detected in all experimental mice with or without LPS injection but IL-10 mRNA was detected in all experimental mice after IL-1α injection. Anti-IL-8 reduced the number of infilterating cells, but the difference was not statisticaly significant. Anti-IL-8 reduced free IL-8 in viterous humor, but total IL-8 level was not reduced with anti-IL-8. These results suggest that IL-1 is appears to have a more important role than in LPS-induced uveitis, IL-6 may not be indicator of inflammation progress, and IL-8 showes to be minor contributor.

전염증성 사이토카인을 억제하는 구강 유산균 Weissella cibaria CMU의 분자기전 연구

김민정 원광대학교 일반대학원 2020 국내석사

In this study examined the pro-inflammatory cytokines inhibitory effects of Weissella cibaria CMU (W. cibaria CMU), which is a probiotic strain isolated from saliva in healthy children. Aggregatibacter actinomycetemcomitans (A. a) was treated in RAW264.7 cells which were macrophagocytes to induce inflammation, and the effect of W. cibaria CMU on the produced inflammation medium was confirmed. RAW264.7 cells were treated with W. cibaria CMU with a multiplicity of infection (MOI) 0.1, 1, 10, 100 and 1000 to perform MTS assay there by measuring the cell survival rate. RAW264.7 cells were treated with W. cibaria CMU in MOI 0.1, 1 and 10 to measure nitric oxide (NO) production and then induced with A. a to perform NO assay. Expression of inflammatory cytokines such as Interleukin (IL)-1β and IL-6 has been performed with reverse transcription-polymerase chain reaction (RT-PCR) and real-time polymerase chain reaction (real-time PCR) to measure the amount of mRNA. The iNOS protein expression, phosphorylation of IKKα/β and IκBα and the translocation NF-κB p65 into the nucleus were confirmed by Western blot. As a result of MTS assay performed to examine the cytotoxicity of W. cibaria CMU to RAW264.7 cells, it was confirmed that there was no significant change in cell survival rate in MOI 0.1, 1, 10, 100 and 1000. NO assay confirmed dose-dependently suppressed NO production in A. a-induced RAW264.7 cells. As a result of measuring the mRNA amount of IL-1β and IL-6 by RT-PCR and real-time PCR experiments, it was confirmed that the expression amount of inflammatory cytokine in the A. a-induced RAW264.7 cells were reduced in a dose-dependent manner. As a result of Western blot, it was confirmed that protein expression amount of iNOS, p-IKKα/β, p-IκBα and NF-κB p65 (in the nucleus) also suppressed in a dose-dependent manner. Based on such a result, the anti-inflammatory effects of W. cibaria CMU was confirmed in RAW264.7 cells in which the inflammatory reaction was induced to A. a in this experiment. Based on this, it is determined that W. cibaria CMU can prevent chronic inflammatory diseases such as periodontitis. 본 연구에서는 프로바이오틱스 균주인 건강한 어린이의 타액에서 분리한 Weissella cibaria CMU (W. cibaria CMU)의 염증성 사이토카인 억제 효과를 알아보고자 하였다. 대식세포인 RAW264.7 세포에 Aggregatibacter actinomycetemcomitans (A. a)를 처리하여 염증을 유도하였고, 생성된 염증 매개체에 대해 W. cibaria CMU가 미치는 영향을 확인하고자 하였다. RAW264.7 세포에 W. cibaria CMU를 multiplicity of infection (MOI) 0.1, 1, 10, 100, 1000으로 처리하여 MTS assay를 진행하였고 이를 통해 세포 생존율을 측정하였다. Nitric oxide (NO) 생성량을 측정하기 위해 RAW264.7 세포에 W. cibaria CMU를 MOI 0.1, 1, 10으로 처리한 후 A. a로 자극하여 NO assay를 진행하였다. Interleukin (IL)-1β와 IL-6와 같은 염증성 사이토카인의 발현은 reverse transcription-polymerase chain reaction (RT-PCR)과 real-time polymerase chain reaction (real-time PCR)을 시행하여 mRNA 양을 측정하였다. Western blot을 통해 iNOS 단백질 발현, IKKα/β 및 IκBα의 인산화와 핵 내로의 NF-κB p65 이동을 확인하여 W. cibaria CMU의 분자기전을 연구하였다. RAW264.7 세포에 대한 W. cibaria CMU의 세포 독성을 알아보기 위해 진행한 MTS assay 결과 MOI 0.1, 1, 10, 100, 1000에서 세포 생존율은 유의한 변화가 없음을 확인하였다. 또한 NO assay를 통해 A. a 유도된 RAW264.7 세포에서 NO 생성량이 용량 의존적으로 억제됨을 확인하였다. RT-PCR, real-time PCR 실험을 통해 IL-1β와 IL-6의 mRNA 양을 측정한 결과 A. a 유도된 RAW264.7 세포에서 염증성 사이토카인의 발현양이 용량 의존적으로 감소하였음을 확인할 수 있었다. Western blot 결과 iNOS, p-IKKα/β, p-IκBα, 핵 내의 NF-κB p65의 단백질 발현량 또한 용량 의존적으로 억제됨을 확인하였다. 이러한 결과를 통해 본 실험에서 A. a로 염증반응이 유도된 RAW264.7 세포에서의 W. cibaria CMU 항염증 효과를 확인하였고, 이를 토대로 W. cibaria CMU는 치주염과 같은 만성 염증질환을 예방할 수 있는 것으로 판단된다.

Anti-inflammatory effects of the compounds, isolated from Sanguisorba officinalis L. on Toll-like receptor 9-stimulated dendritic cells

Tultul, Mst Shanta Islam 제주대학교 2020 국내석사

Anti-inflammatory Effects of Methyl 4-(β-Dglucopyranosyloxy)-3-hydroxy-5-methoxybenzoate Isolated from Sanguisorba officinalis

Rahman, Mohammad Saydur 제주대학교 2020 국내박사

Anti-inflammatory Effects of Fermented Lotus Root and Linoleic Acid in Lipopolysaccharide-induced RAW 264.7 Cells

김성민 가천대학교 일반대학원 2020 국내석사

Lotus root, the edible rhizome of Nelumbo nucifera, is a popular traditional herbal medicine in East Asia. It has been known to several beneficial effects, including anti-oxidative effects and anti-inflammatory effects. However, the mechanisms underlying the anti-inflammatory effects of lotus root are still largely unknown. Furthermore, several recent studies have reported that physiological functions of plant-based foods were further enhanced through fermentation. Thus, the aim of this study is to examine the mechanisms underlying the anti-inflammatory effects of fermented lotus root (FLR) and its major constituent linoleic acid (LA) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. First, cytotoxicity assay was carried out to investigate whether FLR and LA affect cell viability. When RAW 264.7 cells were treated with FLR or LA, there was no significant cytotoxicity, and cell proliferation increased by LPS was reduced. Nitric oxide (NO) production induced by LPS was decreased due to treatment with FLR in a dose-dependent manner, and treatment with LA significantly decreased NO production at the highest concentration. In addition to that, increased mRNA expression of immune genes including nitric oxide synthase 2 (Nos2), prostaglandin-endoperoxide synthase 2 (Ptgs2), tumor necrosis factor-alpha (Tnf-α), interleukin-1 beta (Il1b), and interleukin-6 (Il6) was reduced when cells were treated with FLR or LA. Phosphorylation of nuclear factor-kappa B (NF-κB) and inhibitor of kappa B alpha (IκBα) was the highest when cells were induced by LPS for 1 h, and treatment with FLR or LA inhibited phosphorylation of NF-κB and IκBα. Besides, nuclear translocation of NF-κB p65 was blocked by treatment with FLR or LA. These results suggest that FLR and LA attenuate pro-inflammatory response through inhibition of NF-κB activation in LPS-induced RAW 264.7 cells. Nelumbo nucifera의 식용 뿌리줄기인 연근은 동아시아에서 전통적인 한약재로서 사용되어왔다. 연근은 항산화 효과 및 항염증 효과를 비롯하여 몇 가지 유익한 효능을 가지는 것으로 알려져 있으나, 연근이 가지는 항염증 효과의 근본적인 메커니즘은 잘 알려져 있지 않다. 또한, 최근 여러 연구들에 의해 식물유래 식품들이 가지는 생리활성 기능이 발효를 통해 더욱 향상되었음이 밝혀졌다. 따라서, 이 연구의 목적은 lipopolysaccharide (LPS)로 유도된 RAW 264.7 세포에서 발효연근 (FLR)과 그 주요 성분인 리놀레산 (LA)의 항염증 효과와 함께 근본적인 메커니즘을 평가하는 것이다. 먼저 FLR과 LA가 세포 생존율에 미치는 영향을 조사하기 위해 세포독성시험을 수행하였다. RAW 264.7 세포가 FLR과 LA로 처리되었을 때 유의미한 세포 독성은 나타나지 않았으며 LPS에 의해 증가된 세포 증식은 감소되었다. LPS에 의해 유도된 nitric oxide (NO) 생성은 FLR 처리에 의해 농도의존적으로 감소하였으며, LA는 가장 고농도 (100 μM)에서만 유의미하게 NO 생성을 감소시켰다. 또한 면역관련 유전자인 nitric oxide synthase 2 (Nos2), prostaglandin-endoperoxide synthase 2 (Ptgs2), tumor necrosis factor-alpha (Tnf-α), interleukin-1 beta (Il1b) 및 interleukin 6 (Il6)의 mRNA 발현 증가는 FLR 또는 LA 처리에 의해 감소되었다. Nuclear factor-kappa B (NF-κB)와 inhibitor of κB alpha (IκBα)의 인산화 수준은 세포가 LPS에 의해 1시간동안 처리되었을 때 가장 높았으며, NF-κB와 IκBα의 인산화는 FLR 또는 LA 처리에 의해 억제되었다. FLR 또는 LA 처리는 NF-κB p65의 핵 전위 또한 억제하였다. 이러한 결과는 LPS로 유도된 RAW 264.7 세포에서 FLR과 LA가 NF-κB 활성화의 억제를 통해 전염증성 반응을 약화시킨다는 것을 시사한다.

류마티스 관절염을 억제하는 물질의 탐색 및 기전 연구

강민경 경남대학교 대학원 2018 국내석사

Rheumatoid arthritis is an autoimmune disease that causes chronic inflammatory in the joints and causes fatal disorders in the joints. There are various inflammatory factors in the joint of patients with rheumatoid arthritis. The pro-inflammatory cytokines TNF-α, IL-6 and Nitric Oxide (NO) are the major causes and play an important role in the immune system. However, if it is overproduced, it will cause an inflammatory reaction. Thus, it is important to regulate TNF-α, IL-6 and NO. In this study, we examined the effect of inhibiting inflammatory by using 628 kinds of natural products in the RAW 264.7 cells induced by LPS. As a result of screening, Cassia angustifolia Vahl was selected. Cassia angustifolia Vahl is widely used as laxatives. It is toxic to humans and can be used as a small amount of drug, and can not be used as a raw material for food. Therefore, several biological experiments were conducted to confirm the inhibitory effect of Cassia angustifolia Vahl methanol extract (CAM) on rheumatoid factor. The inflammatory response of RAW 264.7 cells was induced by LPS and the inhibitory effect of inflammatory factors was confirmed by MTT reduction assay, NO production assay, Morphological assay, ELISA assay. As a result, inhibition of rheumatoid arthritis related factors of Cassia angustifolia Vahl in LPS induced RAW 264.7 cells was confirmed. Thus, Cassia angustifolia Vahl was given to the possibility to a natural drug material. 류마티스 관절염은 자가면역질환 중 하나이며 관절에 만성적인 염증이 생겨 치명적인 장애를 일으키는 질환이다. 류마티스 관절염 환자의 관절 안에는 여러 가지 염증인자들이 생성되어 있다. 주로 pro-inflammatory cytokines인 TNF-α, IL-6와 NO가 주원인인데, 이들은 일반적으로 면역작용에서 중요한 역할을 하지만 과잉으로 생산되면 염증반응을 일으키게 된다. 따라서 류마티스 관절염은 염증으로부터 이어지며 과잉 생산되는 염증 인자들을 조절하는 것이 중요하다. 본 연구에서는 국내·외에서 자생하는 628종의 천연물자원을 대상으로 염증반응이 유도된 RAW 264.7 세포에 염증억제 효과를 가지는 물질을 탐색하기 위하여 screening을 실시하였고, 그 결과 센나엽을 선별할 수 있었다. 센나엽은 통변효과가 뛰어나 주로 완하제로 사용하고 있으며 사람에게 독성이 있어 소량의 약물로는 사용할 수 있으나 식품의 원료로는 사용하지 못하는 생약으로 분류되어 있다. 따라서 센나엽 methanol 추출물(CAM)의 류마티스 관련 인자 억제 효과를 확인하기 위해 여러 생물학적 실험을 실시하였다. RAW 264.7 세포의 염증반응은 LPS로부터 유도하였고, MTT reduction assay, NO production assay, Morphological assay, ELISA assay를 통하여 염증 인자 억제 효과를 확인하였다. 따라서 본 연구는 LPS로부터 염증반응이 유도된 RAW 264.7 세포에서 센나엽의 류마티스 관절염 관련 인자의 억제 효과를 확인하여 류마티스 관절염 치료제 개발의 가능성을 제시하고자 하는 바이다.