Expression system for production of bioactive compounds, recombinant human adiponectin, in the silk glands of transgenic silkworms

신슬미,김봉윤,전형욱,Aeri Lee,이성원,성승현,박찬수,이종길,공현석,송영천,김경재 대한약학회 2014 Archives of Pharmacal Research Vol.37 No.5

Adiponectin is an adipocyte hormone involvedin glucose and lipid metabolism. The aim of this study was todevelop a human adiponectin expression system in transgenicsilkworm using a human adiponectin expression vector. The silk gland of the silkworm is a highly specializedorgan that has the wonderful ability to synthesize and secretesilk protein. To express human adiponectin in the silk glandof transgenic silkworm, targeting vectors pB-A3-adiponectin-IRES-RFP and pB-Ser1-adiponectin-IRES-RFPwere constructed and then introduced into the silkwormpupa. The transgenic silkworms were verified by PCR andthen generated. The level of adiponectin in the transgenicsilkworm was 6–10 ng/50 mg of freeze-dried powder, andwestern blotting using an antibody against human adiponectindemonstrated a specific band with a molecular weightof 30 kDa in the silkworm. These results showed that humanadiponectin introduced into the silkworm genome wasexpressed successfully on a large-scale.

식물추출물 형개와 황금의 모근생성 유도 및 재생 촉진 효과

신슬미,이은정,조영롱,조경혜 서울여자대학교 자연과학연구소 2008 자연과학연구논문집 Vol.20 No.-

Immunostimulatory Effects of Cordyceps militaris on Macrophages by the Enhanced Production of Cytokines through the Activation of NF-κB

신슬미,이성정,조경해,권정학,이성원,공현석,김경제 대한면역학회 2010 Immune Network Vol.10 No.2

Cordyceps militaris has been used in traditional medicine to treat numerous diseases and is reported to have antitumor and immunomodulatory activities in vitro and in vivo. The pharmacological and biochemical mechanisms of Cordyceps militaris extract (CME) on macrophages have not been clearly elucidated. In the present study, we tested the How CME induces the production of proinflammatory cytokines,transcription factor and the expression of co-stimulatory molecules was examined. Methods: We confirmed the levels of proinflammatory cytokines mRNA and protein of cytokines through RT-PCR and western blot analysis and followed by FACS analysis for the surface molecules. Results:CME increased the production of NO and proinflammatory cytokines, such as IL-1β, IL-6, TNF-α and PGE2, dose-dependently and induced protein levels of iNOS, COX-2, and proinflammatory cytokines in a concentration-dependent manner, as determined by western blot and RT-PCR analysis,respectively. The expression of co-stimulatory molecules such as ICAM-1, B7-1, and B7-2 was enhanced by CME. Furthermore, the activation of nuclear transcription factor NF-κB in macrophages was stimulated by CME. Conclusion:Based on these observations, CME increased proinflammatory cytokines through the activation of NF-κB, further suggesting that CME will provide potential use as an immune enhancing agent for treating immunological diseases.

동충하초의 NF-κB 신호를 통한 염증 반응 조절

신슬미,김경제,조경혜 서울여자대학교 자연과학연구소 2013 자연과학연구논문집 Vol.25 No.-

Cordyceps militaris (CM) has been used in traditional medicine to treat numerous diseases in vivo and in vitro. The molecular mechanism of CM pharmacological and biochemical actions on immune responses has not been clearly elucidated. In the present study, the water extract of CM (CME) and cordycepin (3’-deoxyadenosine) were prepared to examine the action mechanism of CM on immune response. Cordycepin, nucleoside analogue, is a metabolite of CM. This study demonstrated how CME or cordycepin itself regulates immune response by the production of pro-inflammatory cytokines ex-vivo. CME induced the expression of NO and pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in primary macrophages at mRNA and protein levels. On the other hand, cordycepin inhibited pro-inflammatory mediators in LPS-stimulated macrophages in a concentration of between 5 µg to 40 µg. The present results showed that CME induces immune response via NF-κB activation in macrophages meanwhile cordycepin inhibits inflammation via suppression of NF-κB activation in activated macrophages by LPS. These results demonstrate that Cordyceps militaris (CM) modulates immune responses, and it may potentially be useful immunomodulating agent for immune deficiency disorders.

Cordycepin Suppresses Expression of Diabetes Regulating Genes by Inhibition of Lipopolysaccharide-induced Inflammation in Macrophages

신슬미,이성원,권정학,문선희,이성종,이종길,조경혜,하남주,김경제 대한면역학회 2009 Immune Network Vol.9 No.3

Background: It has been recently noticed that type 2 diabetes (T2D), one of the most common metabolic diseases, causes a chronic low-grade inflammation and activation of the innate immune system that are closely involved in the pathogenesis of T2D. Cordyceps militaris, a traditional medicinal mushroom, produces a component compound, cordycepin (3’-deoxyadenosine). Cordycepin has been known to have many pharmacological activities including immunological stimulating, anti-cancer, and anti-infection activities. The molecular mechanisms of cordycepin in T2D are not clear. In the present study, we tested the role of cordycepin on the anti-diabetic effect and anti-inflammatory cascades in LPS-stimulated RAW 264.7 cells. Methods: We confirmed the levels of diabetes regulating genes mRNA and protein of cytokines through RT-PCR and western blot analysis and followed by FACS analysis for the surface molecules. Results: Cordycepin inhibited the production of NO and pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in LPS-activated macrophages via suppressing protein expression of pro-inflammatory mediators. T2D regulating genes such as 11β-HSD1 and PPARγ were decreased as well as expression of co-stimulatory molecules such as ICAM-1 and B7-1/-2 were also decreased with the increment of its concentration. In accordance with suppressed pro-inflammatory cytokine production lead to inhibition of diabetic regulating genes in activated macrophages. Cordycepin suppressed NF-κB activation in LPS-activated macrophages. Conclusion: Based on these observations, cordycepin suppressed T2D regulating genes through the inactivation of NF-κB dependent inflammatory responses and suggesting that cordycepin will provide potential use as an immunomodulatory agent for treating immunological diseases.

Cordycepin Suppresses MHC-restricted Antigen Presentation and Leads to Down-regulation of Inflammatory Responses in Antigen Presenting Cells

신슬미,김경제,Seulah Kim,Bobae Hyun,Aeri Lee,Sungwon Lee,박찬수,Hyunseok KONG,송영천,이종길 한국생약학회 2013 Natural Product Sciences Vol.19 No.4

Cordyceps militaris, a traditional medicinal mushroom, produces a component compound, cordycepin (3'-deoxyadenosine). Cordycepin has many pharmacological activities including immunological stimulating, anti-cancer, and anti-infection activities. However, the therapeutic mechanism has not yet been elucidated. In this study, we examined the effects of cordycepin on the antigen-presenting function of antigen-presenting cells (APCs). Dendritic cells (DCs) were cultured in the presence of cordycepin and then allowed to phagocytose microspheres containing ovalbumin (OVA). After washing and fixing, the efficacy of OVA peptide presentation by DCs was evaluated using CD8 and CD4 T cells. Also, we confirmed the protein levels of proinflammatory cytokines through RT-PCR and Western blot analysis. Cordycepin decreased both MHC class I and class II-restricted presentation of OVA and suppressed the expression of both MHC molecules and the phagocytic activity toward exogenous OVA. The class II-restricted OVA presentation-regulating activity of cordycepin was also confirmed using mice that had been injected with cordycepin followed by soluble OVA. Furthermore, cordycepin suppressed the mRNA and protein levels of iNOS, COX-2, pro-inflammatory cytokines in a concentration-dependent manner. These results provide an understanding of the mechanism of the T cell response-regulating activity of cordycepin through the inhibition of MHC-restricted antigen presentation in relation to its actions on APCs.

Role of Cordycepin and Adenosine on the Phenotypic Switch of Macrophages via Induced Anti-inflammatory Cytokines

신슬미,문선희,박윤희,권정학,이승정,이종길,조경혜,김경제 대한면역학회 2009 Immune Network Vol.9 No.6

Background: Chronic low grade inflammation is closely linked to type II diabetes, obesity, and atherosclerosis. Macrophages play a key role in the regulation of pro- or anti- inflammatory actions at the lesion sites of disease. Components of cordyceps militaris, cordycepin and adenosine, have been used for the modulation of inflammatory diseases. The effects of cordycepin in the modulation of macrophages have yet to be be elucidated. We investigated the effects of cordycepin and adenosine on the morphological changes of macrophages under the inflammatory condition of LPS and an anti-inflammatory condition involving high concentrations of adenosine. Methods: We confirmed the mRNA levels of the M1/M2 cytokine genes through RT-PCR and morphological change. Results: LPS-activated macrophages returned to their inactivated original shape, i.e., they looked like naïve macrophages, through the treatment with high concentrations of cordycepin (40 μg/ml). LPS and adenosine activated macrophages also returned to their original inactivated shapes after cordycepin treatment; however, at relatively higher levels of cordycepin than adenosine. This change did not occur with relatively low concentrations of cordycepin. Adenosine down-regulated the gene expression of M1 cytokines (IL-1β, TNF-α) and chemokines (CX3CR1, RANTES), such as cordycepin. Additionally, M2 cytokines (IL-10, IL-1ra, TGF-β) were up-regulated by both cordycepin and adenosine. Conclusion: Based on these observations, both cordycepin and adenosine regulated the phenotypic switch on macrophages and suggested that cordycepin and adenosine may potentially be used as immunomodulatory agents in the treatment of inflammatory disease. Background: Chronic low grade inflammation is closely linked to type II diabetes, obesity, and atherosclerosis. Macrophages play a key role in the regulation of pro- or anti- inflammatory actions at the lesion sites of disease. Components of cordyceps militaris, cordycepin and adenosine, have been used for the modulation of inflammatory diseases. The effects of cordycepin in the modulation of macrophages have yet to be be elucidated. We investigated the effects of cordycepin and adenosine on the morphological changes of macrophages under the inflammatory condition of LPS and an anti-inflammatory condition involving high concentrations of adenosine. Methods: We confirmed the mRNA levels of the M1/M2 cytokine genes through RT-PCR and morphological change. Results: LPS-activated macrophages returned to their inactivated original shape, i.e., they looked like naïve macrophages, through the treatment with high concentrations of cordycepin (40 μg/ml). LPS and adenosine activated macrophages also returned to their original inactivated shapes after cordycepin treatment; however, at relatively higher levels of cordycepin than adenosine. This change did not occur with relatively low concentrations of cordycepin. Adenosine down-regulated the gene expression of M1 cytokines (IL-1β, TNF-α) and chemokines (CX3CR1, RANTES), such as cordycepin. Additionally, M2 cytokines (IL-10, IL-1ra, TGF-β) were up-regulated by both cordycepin and adenosine. Conclusion: Based on these observations, both cordycepin and adenosine regulated the phenotypic switch on macrophages and suggested that cordycepin and adenosine may potentially be used as immunomodulatory agents in the treatment of inflammatory disease.

Dietary Aloe QDM Complex Reduces Obesity-Induced Insulin Resistance and Adipogenesis in Obese Mice Fed a High-Fat Diet

신슬미,이종길,김경제,김설아,오희은,공현석,신은주,도선길,조태형,박영인 대한면역학회 2012 Immune Network Vol.12 No.3

Obesity-induced disorders contribute to the development of metabolic diseases such as insulin resistance, fatty liver diseases,and type 2 diabetes (T2D). In this study, we evaluated whether the Aloe QDM complex could improve metabolic disorders related to blood glucose levels and insulin resistance. Male C57BL/6 obese mice fed a high-fat diet for 54 days received a supplement of Aloe QDM complex or pioglitazone (PGZ) or metformin (Met) and were compared with unsupplemented controls (high-fat diet; HFD) or mice fed a regular diet (RD). RT-PCR and western blot analysis were used to quantify the expression of obesity-induced inflammation. Dietary Aloe QDM complex lowered body weight,fasting blood glucose, plasma insulin, and leptin levels, and markedly reduced the impairment of glucose tolerance in obese mice. Also, Aloe QDM complex significantly enhanced plasma adiponectin levels and insulin sensitivity via AMPK activity in muscles. At the same time, Aloe QDM decreased the mRNA and protein of PPARγ/LXRα and scavenger receptors in white adipose tissue (WAT). Dietary Aloe QDM complex reduces obesity-induced glucose tolerance not only by suppressing PPARγ/LXRα but also by enhancing AMPK activity in the WAT and muscles, both of which are important peripheral tissues affecting insulin resistance. The Aloe QDM complex could be used as a nutritional intervention against T2D.

Role of Salvia miltiorrhiza for Modulation of Th2-derived Cytokines in the Resolution of Inflammation

문선희,김경제,신슬미,김설아,오희은,한신하,이승정 대한면역학회 2011 Immune Network Vol.11 No.5

Background: Salvia miltiorrhiza (SM) has been used to treat inflammatory diseases including edema and arthritis; however,the anti-inflammatory mechanism of SM action remains unresolved. Methods: The effects of an ethanol extract of SM (ESM) on pro-inflammatory cytokines such as TNF-α,IL-1β, IL-6, and NO, and on anti-inflammatory cytokines including IL-4, IL-10, TGF-β, and IL-1Ra have been studied in an attempt to elucidate the anti-inflammatory mechanism in murine macrophages. Results: ESM inhibited the production of pro-inflammatory cytokines via down-regulation of gene and protein expression whereas it increased the anti-inflammatory cytokines. Furthermore, ESM inhibited the expression of the chemokines, RANTES and CX3CL1, as well as of inflammatory mediators such as TLR-4 and 11β-HSD1. Conclusion: These results indicated that the regulatory effects of ESM may be mediated though the suppression of pro-inflammatory cytokines as well as the induction of anti-inflammatory cytokines. Consequently, we speculate that ESM has therapeutic potential for inflammation-associated disorders.

Immunomodulatory Effects of Dioscoreae Rhizome Against Inflammation through Suppressed Production of Cytokines Via Inhibition of the NF-κB Pathway

김설아,이승정,신슬미,현보배,공현석,한신하,Aeri Lee,김경제 대한면역학회 2012 Immune Network Vol.12 No.5

Dioscoreae Rhizome (DR) has been used in traditional medicine to treat numerous diseases and is reported to have anti-diabetes and anti-tumor activities. To identify a bioactive traditional medicine with anti-inflammatory activity of a water extract of DR (EDR), we determined the mRNA and protein levels of proinflammatory cytokines in macrophages through RT-PCR and western blot analysis and performed a FACS analysis for measuring surface molecules. EDR dose-dependently decreased the production of NO and pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α, and PGE_2, as well as mRNA levels of iNOS, COX-2, and pro-inflammatory cytokines, as determined by western blot and RT-PCR analysis, respectively. The expression of co-stimulatory molecules such as B7-1 and B7-2 was also reduced by EDR. Furthermore, activation of the nuclear transcription factor, NF-κB, but not that of IL-4 and IL-10, in macrophages was inhibited by EDR. These results show that EDR decreased pro-inflammatory cytokines via inhibition of NF-κB-dependent inflammatory protein level, suggesting that EDR could be a useful immunomodulatory agent for treating immunological diseases.