This study was conducted to investigate the bioconversion of ginsenosides as well as anti-inflammatory activities of fresh ginseng Kkakdugi during fermentation. Fresh ginseng Kkakdugi reached proper ripeness, pH 4.30, and acidity 1.69% at 15oC after 10 days. Lactic acid bacteria grew until reaching 1.10×109 CFU/mL after 20 days of fermentation, and β- glucosidase activity increased from 1.154 to 1.885 units/g. The bioconversion of ginsenosides was confirmed based on increased content of Rg3, an aglycone, from 0.13 to 0.17 mg/g during fermentation through HPLC. Fresh ginseng Kkakdugi did not display cytotoxicity up to the concentrations of 80 μg/mL, regardless of ripening period. Nitrite production and expression of inflammation-related proteins, iNOS and COX-2, decreased in a dose-dependent manner regardless of ripening period. From these results, fresh ginseng Kkakdugi showed the bioconversion of ginsenosides to aglycone during the lactic acid fermentation as well as an anti-inflammatory effect through the reduction of NO production and iNOS and COX-2 expression

Aggregatibacter actinomycetemcomitans is an important pathogen in the development of localized aggressive periodontitis. Lipopolysaccharide (LPS) is a virulent factor of periodontal pathogens that contributes to alveolar bone loss and connective tissue degradation in periodontal disease. Our present study was designed to investigate the cytokine expression and signaling pathways regulated by A. actinomycetemcomitans LPS (Aa LPS). Cytokine gene expression profiling in RAW 264.7 cells was performed by microarray analyses. The cytokine mRNA and protein levels and related signaling pathways induced by Aa LPS were measured by RT-PCR, ELISA and western blotting. Microarray results showed that Aa LPS strongly induced the expression of NF-κB, NF-κB-related genes, inflammatory cytokines, TNF-α and IL-1β in RAW 264.7 cells. NF-κB inhibitor pretreatment significantly reduced the levels of TNF-α and IL-1β mRNA and protein. In addition, the Aa LPS-induced TNF-α and IL-1β expression was inhibited by p38/JNK MAP kinase inhibitor pretreatment. These results show that Aa LPS stimulates TNF-α and IL-1β expression through NF-κB and p38/JNK activation in RAW 264.7 cells, suggesting the essential role of this pathway in the pathogenesis of localized aggressive periodontitis.

This study examined the anti-osteoclastogenic effects of baicalin on receptor activator of NF-kB ligand (RANKL)- induced RAW264.7 cells. Baicalin is a flavonoid that is produced by Scutellaria baicalensis and is known to have multiple biological properties, including antibacterial, anti- inflammatory and analgesic effects. The effects of baicalin on osteoclasts were examined by measuring 1) cell via- bility; 2) the formation of tartrate-resistant acid phosphatase (TRAP) (+) multinucleated cells; 3) RANK/RANKL signa- ling pathways and 4) mRNA levels of osteoclast-associated genes. Baicalin inhibited the formation of RANKL-stimu- lated TRAP (+) multinucleated cells and also suppressed the RANKL-stimulated activation of p-38, ERK, cSrc and AKT signaling. Baicalin also inhibited the RANKL-stimu- lated degradation of IĸB in RAW264.7 cells. In addition, the RANKL-stimulated induction of NFATc1 transcription factors was found to be abrogated by this flavonoid. Baica- lin was further found to decrease the mRNA expression of osteoclast-associated genes, including carbonic anhydrase II, TRAP and cathepsin K in the RAW264.7 cells. Our data thus demonstrate that baicalin inhibits osteoclastogenesis by inhibiting the RANKL-induced activation of signaling molecules and transcription factors in osteoclast precursors.

생강은 다양한 생리활성이 확인됨에 따라 여러나라에서 전통의약재로서 폭넓게 사용되고 있다. 하지만 진저롤 및 그 유도체에서 유래되는 자극적인 향미로 인하여 식품소재로서의 사용에는 다소 어려움이 있는 실정이다. 본 연구에서는 생강을 Lactobacillus plantarum, Leuconostoc mesenteroides, Streptococcus thermophilus, 및 Lactobacillus acidophilus 등과 같은 유산균에 의한 발효를 통하여 생강의 관능특성을 증진시키려 하였다. 발효된 생강은 in vitro에서 전자공여능 및 cyclooxygenase-2 저해활성을 나타내었으며 또한 산화질소의 생성도 억제하였다. 특히 김치유래 유산균으로 발효한 생강추출물인 GLPe와 GLMe는 Raw 264.7 macrophages에서 prostaglandin-E2 의 생성을 60% 이상 저해하였으며 15-lipoxygenase에 대한 저해 활성도 나타내었다. 따라서 GLPe나 GLMe와 같은 발효생강 추출물은 섭취할 때 자극적인 향미로 인한 거부감이 적으며 염증이나 앨러지 등에 의해 야기되는 바람직하지 않은 증상을 일부 완화시킬 수 있는 건강식품소재로서 사용될 수 있음을 알 수 있었다.

Fraxinus rhynchophylla (Oleaceae), a deciduous tree, is known to have properties that include anti-inflammatory, convergence, febricide, antiblenophthalmia, antidiarrhea, antileukorrhea, and so forth. In addition, it has been used for traditional herbal medicine in East Asian countries, including Korea. In this study, we investigated the antioxidant and anti-inflammatory effects of Fraxinus rhynchophylla ethanol extract (FRE) in lipopolysaccharide (LPS)-induced murine macrophage Raw 264.7 cells with FRE pretreatment. We performed DPPH-assay, Western blot, and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). FRE showed 85% free radical scavenging activity at concentrations of 80 µg/ml. Results of this study also showed that FRE down-regulates Cox-2 and iNOS expression in mRNA and protein level. In conclusion, crude ethanol extract of Fraxinus rhynchophylla exhibited antioxidant and anti-inflammatory activities, and it may potentially provide a valuable source of natural herbal agent to inhibit inflammation.

Flavonoids have a range of biological activities, including anti-allergic, anti-inflammatory, anti-microbial, and anti-cancer activities, as demonstrated by in vitro studies. In this study, we investigated whether luteolin can be applied to suppression of lipopolysaccharide (LPS)-stimulated inflammatory responses in murine macrophages. Luteolin was found to reduce nitric oxide (NO) production in LPS-stimulated Raw 264.7 cells. In addition, expression of inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokine tumor necrotic factor-α (TNF-α) at the mRNA and protein levels were decreased. These inhibitory effects were found to be caused by the blockade of nuclear factor kappa-light- chain-enhancer of activated B cells (NF-κB) activation and phosphorylation of mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAP kinase. In addition, pre-treatment with luteolin resulted in reduced ganglioside expression levels and inhibited expression of GT1b in Raw 264.7 cells. On the basis of these observations, we suggest that luteolin has potential as an anti-inflammatory drug candidate, and ganglioside GT1b may play a role in the inflammatory process.

Veratric aicd is a phenolic compound, which is derived from medicinal mushrooms. In our study, veratric acid showed the effect on the reduction of nitric oxide (NO) production in LPS-stimulated RAW264.7 cells. The negative regulation of NO production in LPS-induced RAW264.7 cells were caused by the modulation of iNOS at mRNA and protein levels. The transcription factors for iNOS expression, including NF-κB, STAT-1, c-Jun, ATF-2, and Elk-1, were down-regulated by veratric acid. Because c-Jun, ATF-2, and Elk-1 is induced by p38, we determined that the effect of veratric acid on p38 expression, which was inactivated. Additionally, Akt and p110β, the catalytic subunit of PI3K, were inactivated by veratric acid. In the inhibition of p38 and PI3K, the inhibition of p38 did not modulate the expression of iNOS, the inhibition of PI3K, although, induced the synergic effect on the reduction of NO production. The results indicated veratric acid required p38 to regulate the expression of iNOS in LPS-stimulated RAW264.7 cells.

The present study was investigated the antibacterial effect of several sodium salts and the sodium salt mixture composed with sodium chlorate, sodium azide and sodium cyanide on Salmonella gallinarum (S. gallinarum) infection in murine derived macrophage RAW 264.7 cells. In the infection assay of S. gallinarum in macrophage cells pretreated with 15 mM sodium chlorate, 0.3 mM sodium cyanide, 0.3 mM sodium azide and the sodium salt mixture (15 mM sodium chlorate, 0.3 mM sodium cyanide, 0.3 mM sodium azide), respectively, the numbers of S. gallinarum in all treated-groups tended to decrease in the process of time after treatment, but the group treated with sodium cyanide was no significant difference compared with control. After 24 hours of treatment, the number of S. gallinarum in sodium azide (p<0.05), sodium chlorate (p<0.001) and the sodium salt mixture (p<0.001) treated-group was significantly decreased compared with control, and that in the sodium salt mixture treated-group was decreased the higher than all groups. The results of this study demonstrated that the sodium salt mixture composed with sodium chlorate, sodium azide and sodium cyanide, has the antimicrobial activity for S. gallinarum and may be beneficial on the control of intracellular pathogens.

Macrophages can recognize antigens and microorganisms, and then initiate an appropriate defense. However, there has been a lack of comprehensive information regarding the genes that are modulated by commensal yeasts, including Saccharomyces cerevisiae or Saccharomyces exiguus. In addition, it is not clear to what extent the beneficial yeasts modulate the immune response against microbes and/or microbial toxins. Using DNA microarray, which contains approximately 25,000 genes, we studied interactions between host cells and yeast/bacterial toxin (LPS) by analyzing the transcriptional response of macrophages stimulated by Saccharomyces exiguus and/or Lipopolysaccharides. Thirty three genes were identified to be modulated by more than two folds between groups of macrophage cells. Pathway analysis provided insight into the mutual interactions. Of particular interest was the responses elicited by fungus in murine macrophage cells, including modulation of immunity/defense, cellular signal transduction, cell proliferation/differentiation, and transport. This finding indicates that the yeast induces immune response pathways as well as those associated with cell proliferation and transport. Among the 33 genes identified from the DNA microarray screening, eight genes were further checked by RT-PCR analysis using gene specific primers. Compared to those of negative control, sequential treatment with the yeast strain followed by LPS apparently induced expression of Tnfaip3, IL7R, and CD86, while it inhibited expression of Cxcl10 and CD83. In conclusion, this study identified the genes that are up-regulated by Saccharomyces exiguus. A further study is needed in order to determine whether these genes are modulated at the protein level, and also for their roles in control of immune responses.

Salmonellosis is the commonest zoonosis worldwide that generally causes enterocolitis and foodborne poisoning which represents a considerable public health burden. Salmonella spp. are potential enteric pathogens and intracellularly replicates in host cells resulting in chronic infections. The medical treatments for salmonellosis have been difficult yet and had a serious problem including the increasing emergence of antibiotic resistance. The present report was designated to investigate the antibacterial effects of Saururus chinensis Baill ethanol extract (SCEE) on pure culture and infection with Salmonella enterica serovar Typhimurium (S. typhimurium) in murine derived macrophage RAW 264.7 cells. In determination of antibacterial activity of SCEE against S. typhimurium, bacterial viability was markedly decreased compared to the control. Also, SCEE significantly induced morphological change (p<0.05) of RAW 264.7 cells. In infection assay of S. typhimurium in RAW 264.7 cells pretreated with 100㎍/㎖ of SCEE, which is a non-cytotoxic concentration, bacterial uptake ability of macrophage was increased corresponding with morphological change, whereas bacterial survival rates within macrophage were markedly reduced compared with untreated control. Furthermore, nitric oxide (NO) production in SCEE-treated cells was slightly increased until 2 h but showed a tendency of decrease after 4 h until 24 h post infection compared with untreated control with S. typhimurium infection. Taken together, these findings demonstrated that SCEE has the antibacterial activity for S. typhimurium and the protective effects against S. typhimurium infection through activating murine macrophage independent on NO, suggesting that SCEE may be beneficial on the disease caused by intracellularly replicating pathogens as a safe alternatives of conventional chemotherapies.

Porphyromonas gingivalis lipopolysaccharide (Pg LPS) is an important virulence factor in chronic periodontitis. The aim of this study was to compare the expression of inflammatory cytokine genes in Escherichia coli LPS (Ec LPS) and Pg LPS-stimulated mouse macrophage RAW 264.7 cells. Cells were treated with Ec LPS and Pg LPS for 18 hours, and the cytokine gene expression profile was assessed using microarrays and confirmed by real-time PCR. Microarray analysis showed that both types of LPS induced a significant increase in the expression of IL-17β, IL-2, Ccl4, Cxcl2 and TNFα compared with the control. However, LT-b was up-regulated by Pg LPS but not by Ec LPS. Real-time PCR analysis of these genes showed similar results for LT-b, Ccl4, Cxcl2, and TNF- but found that IL-17β and IL-2 were upregulated by Pg LPS but not by Ec LPS. These data indicate that Pg LPS stimulates the transcription of IL-17β, IL-2, Ccl4, Cxcl2, LT-b, and TNFα, all of which may be involved in the pathogenesis of chronic periodontitis.

The attachment and adhesion of RAW 264.7 and MC3T3-E1 cells to titanium (Ti) discs with various degrees of roughness was investigated. The attachment, adhesion, and proliferation of these cells were evaluated after 4 hr, 24 hr and 7 day incubations. Both RAW 264.7 and MC3T3-E1 cells showed a time-dependant correlation between attachment and adhesion on the surface of the titanium discs. Both types of cells tended to have higher survival rate on these discs as the surface roughness increased. The percentage of adherent inflammatory RAW 264.7 cells was greater than MC3T3-E1 cells at 24 hr, but this was reversed at 7 days in culture. The morphology of osteoblastic MC3T3-E1 cells at 24 hr, determined using a surface emission microscope (SEM), appeared flattened and spread out while inflammatory RAW 264.7 cells were predominantly spherical in shape. The adhesion of both cell types on the titanium discs was dependant on the levels of fibronectin adsorbed on the disc surface, indicating that serum constituents modulate the efficient adhesion of these cells. Our data indicate that the cellular response to the titanium surface is dependent on the types of cells, surface roughness and serum constituents.

Nitric oxide(NO) is synthesized via the oxidation of L-arginine by a family of nitric oxide synthases(NOS), which are either constitutive(cNOS) or inducible(iNOS). The induction of iNOS in tissues can lead to the sustained production of high concentrations of NO which may exert pro-inflammatory effects including vasodilation, edema, cyototoxicity, and its activity can be mediated by various pro-inflammatory cytokine, including interferonγ(INF- γ), tumor necrosis factor, IL-1 and IL-6. The enzyme, iNOS, became a new target for pharmacologcal research with the aim to find new substances for the treatment of chronic inflammatory disorders. Murine macrophages produce large amounts of NO when activated with TFN-γ plus LPS. Murine macrophage-like cell line, RAW 264.7, is a suitable cell model to perform in vitro studies regarding the iNOS system. Artemisin feddei Lev. et Vnt.(Compositae) is a perennial herb growing in Korea. The aerial parts have been used in foik medicine as antiinflammatory, antipyretic, choleretic and diuretic agent. Sesquiterpenelactones were isolated from this plant. In the course of screening for NO inhibitory activity from medicnial plants, the aqueous extract of this plant was found to have a significant activity.

염증은 신체 특정 조직의 감염 및 손상에 관한 생체 반응이며, 매개하는 주요 대상은 면역세포이다. 염증은 급성과 만성 염증으로 나뉘며 신체 조직의 감염 및 손상부위의 규모에 따라 구분 할 수 있다. 염증의 범위가 크게 발현되거나 급성염증 형태로 진행되지 않을 때 만성 염증으로 진행되며 대표적인 만성 염증 질환인 장 질환(Inflammatory bowel disease)의 일종인 크론병 (Crohn’s disease)이나 관절질환인 류머티스성 관절염(Rheumatoid arthritis)으로 나타난다. 낮은 수준이기는 하나 비만 역시 염증성 질환으로 분류할 수 있다. 연리초속 식물이 고래에 신장염을 치료하는 민간처방으로 주로 사용됐기에 이에 착안하여 털연리초(Lathyrus palustris)를 이용하여 세포독성과 항염증 활성 효과를 평가하였다. 대식세포인 RAW 264.7 세포에서 염증 유발 인자인 lipopolysaccharide (LPS)로 자극 후 NO와 PGE2 같은 염증 매개 물질들의 억제 효과를 확인하였다. 털연리초 에탄올 추출물을 처리한 후 염증 매개 물질의 저해율(%)을 측정했을 때 NO 및 PGE2 생성을 농도 의존적으로 현저하게 억제하는 농도는 40 ㎍/mL이었으며 특이적으로 PGE2 발현을 74% 이상 강력히 억제함을 확인하였다. 따라서 본 연구결과는 털연리초의 에탄올 추출물이 유의성 있는 항염증 효과를 나타내었고 이러한 생리활성 효과는 예방의학적 소재로서의 가능성을 충분히 제시할 수 있기에 염증 질환의 예방 및 비만 억제를 위한 기능성 건강식품의 개발로 이어질 것으로 기대된다. 또한 염증 과 관련된 사이토카인 물질인 IL-4, IL-13 및 염증 지표 단백질 인 iNOS, COX-2의 억제 메커니즘과 항염증 활성을 나타내는 핵심 성분의 추가적인 연구가 차후 필요할 것으로 판단된다.

Recently, as a natural substance has been emphasized interest in research to enhance the immune function. Green lettuce (Lactuca sativa L.) is a popular vegetable used fresh and it contains various phytochemicals and antioxidant compounds, and has been reported to have various physiological activities such as antibacterial, antioxidant, antitumor and anti-mutagenic. However, only a few studies have investigated on the mechanism of action of immune-enhancing activity of lettuce. Therefore, in this study, the immunomodulatory activities and potential mechanism of action of Green lettuce extracts (GLE) were evaluated in the murine macrophage cell line RAW264.7. GLE significantly increased NO levels by RAW264.7 cells, as well as expressions of immunomodulators such as iNOS, COX-2, IL-1β, IL-6, IL-12, TNF-α and MCP-1. Although GLE activated ERK1/2, p38, JNK and NF-κB, GLE-mediated expressions of immunomodulators was dependent on p38, JNK and NF-κB. In addition, TLR4 inhibition blocked GLE-mediated expressions of immunomodulators and activation of p38, JNK and NF-κB. Taken together, these results demonstrated that TLR4-MAPK/NF-κB signalling pathways participated in GLE-induced macrophage activation and GLE could be developed as a potential immunomodulating functional food.

Quercus mongolica (QM), which belongs to fagaceae, is one of the oak native to Korea. We evaluated the antiinflammatory effect of branches extracted with 70% ethanol of QM (QM-B) and elucidated the potential signaling pathway in LPS-induced RAW264.7 cells. The QM-B showed anti-inflammatory activity through inhibition of NO production. The QM-B dose-dependently suppressed NO production by inhibiting iNOS, COX-2 and IL-6 expression in LPS-induced RAW264.7 cells. The QM-B inhibited the degradation and phosphorylation of IκB-α and NF-κB activation. The QM-B suppressed the phosphorylation of p38 and ERK1/2. Also, the QM-B increased HO-1 expression. These results suggested that QM-B may utilize anti-inflammatory activity by suppressing NF-κB and MAPK signaling pathway and inducing HO-1 expression indicated that the QM-B can be used as a natural anti-inflammatory drugs.

Zanthoxylum piperitum D.C. (ZP) peels has been used as a natural spice and herb medicine for hypertension reduction, for strokes, and for its anti-bacterial and anti-oxidant activity. However, the anti-inflammatory mechanisms employed by ZP have yet to be completely understood. In this study, we elucidate the anti-inflammatory mechanism of ZP in lipopolysaccharide (LPS)-induced RAW264.7 cells. We evaluated the effects of ZP in LPS-induced levels of inflammatory cytokines, prostaglandin E2 (PGE2), and caspase-1 using ELISA. The expression levels of inflammatoryrelated genes, including cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS), were assayed by Western blot analysis. We elucidated the effect of ZP on nuclear factor (NF)-κB activation by means of a luciferase activity assay. The findings of this study demonstrated that ZP inhibited the production of inflammatory cytokine and PGE2 and inhibited the increased levels of COX-2 and iNOS caused by LPS. Additionally, we showed that the anti-inflammatory effect of ZP arises by suppressing the activation of NF-κB and caspase-1 in LPS- induced RAW264.7 cells. These results provide novel insights into the pharmacological actions of ZP as a potential candidate for development of new drugs to treat inflammatory diseases.

Aralia cordata (A. cordata), which belongs to Araliaceae, is a perennial herb widely distributed in East Asia. We evaluated the anti-inflammatory effect of stems (AC-S), roots (AC-R) and leaves (AC-L) extracted with 100% methanol of A. cordata and elucidated the potential signaling pathway in LPS-stimulated RAW264.7 cells. The AC-L showed a strong anti-inflammatory activity through inhibition of NO production. AC-L dose-dependently inhibited NO production by suppressing iNOS, COX-2 and IL-β expression in LPS-stimulated RAW264.7 cells. AC-L inhibited the degradation and phosphorylation of IκB-α, which donated to the inhibition of p65 nuclear accumulation and NF-κB activation. Furthermore, AC-L suppressed the phosphorylation of ERK1/2 and p38. These results suggested that AC-L may utilize anti-inflammatory activity by blocking NF-κB and MAPK signaling pathway and indicated that the AC-L can be used as a natural anti-inflammatory drugs.

Honey used as conventional medicine has various pharmacological properties. In the honey and anti-inflammatory effect, Gelam honey and Manuka honey has been reported to exert anti-inflammatory activity. However, the anti-inflammatory effect and potential mechanisms of acacia honey (AH) are not well understood. In this study, we investigated anti-inflammatory activity and mechanism of action of AH in LPS-stimulated RAW264.7 cells. AH attenuated NO production through inhibition of iNOS expression in LPS-stimulated RAW264.7 cells. AH also decreased the expressions of IL-1β, IL-6 and TNF-α as pro-inflammatory cytokines, and MCP-1 expression as a pro-inflammatory chemokine. In the elucidation of the molecular mechanisms, AH decreased LPS-mediated IκB-α degradation and subsequent nuclear accumulation of p65, which resulted in the inhibition of NF-κB activation in RAW264.7 cells. AH dose-dependently suppressed LPS-mediated phosphorylation of ERK1/2 and p38 in RAW264.7 cells. In addition, AH significantly inhibited ATF2 phosphorylation and nuclear accumulation of ATF2 in LPS-stimulated RAW264.7 cells. These results suggest that AH has an anti-inflammatory effect, inhibiting the production of pro-inflammatory mediators such as NO, iNOS, TNF-α, IL-6, IL-1β and MCP-1 via interruption of the NF-κB and MAPK/ATF2 signaling pathways.

Mushrooms have been widely cultivated and consumed as foods and herbal medicines owing to their various biological properties. However, few studies have evaluated the anti-inflammatory effects of mushrooms. Here, we investigated the effects of mushroom extracts (MEs) on lipopolysaccharide (LPS)-induced inflammation in macrophages (RAW264.7 cells). First, we extracted MEs with either water or ethanol. Using LPS-treated RAW264.7 cells, we measured cell proliferation and NO production. Gene expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 (IL-6), and IL-1β was assessed by RT-PCR, and protein abundance of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) and phosphorylation of p65 were determined by immunoblotting. MEs prepared using both water and ethanol inhibited LPS-induced inflammation in RAW264.7 cells. Nitric oxide (NO) levels induced by LPS were reduced by treatment with MEs. Isaria japonica Yasuda water extracts and Umbilicaria esculenta (Miyoshi) Minks ethanol extracts significantly decreased the mRNA expression of inflammation-related cytokine genes including TNF-α, IL-6, and IL-1β. Similarly, the protein abundance of iNOS and COX-2 was also decreased. The phosphorylation of p65, a subunit of nuclear factor-κB was at least partly suppressed by MEs. This study suggests that mushrooms could be included in the diet to prevent and treat macrophage-related chronic immune diseases.