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.

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.

Background : Ginseng (Panax ginseng) has been reported to exert an anti-inflammatory activity in a variety of inflammatory. However, inflammation-regulatory activity of wood-cultivated ginseng has not been thoroughly evaluated. In this study, we evaluated the anti-inflammatory effect of wood-cultivated ginseng and elucidated the potential mechanisms in LPS-stimulated RAW264.7 cells. Methods and Results : Inhibitory effects of the old wood-cultivated ginseng (WCG-O), young wood-cultivated ginseng (WCG-Y) and ginseng (G) on NO and PGE2 production were examined using the Griess assay and ELISA kit. Suppressive effects of WCG-O on inflammatory gene expression, transcriptional activation, and inflammation signaling events were investigated using Western blot analysis, RT-PCR analysis and luciferase activity reporter gene assay. WCG-O dose-dependently suppressed nitric oxide (NO) and Prostaglandin E2 (PGE2) production in LPS-stimulated RAW264.7 cells. In addition, WCG-O attenuated LPS-mediated overexpression of iNOS and COX-2. In addition, WCG-O blocked the expression of TNF-α and IL-1β in LPS-stimulated RAW264.7 cells. In elucidation of the potential mechanisms for anti-inflammatory effect, WCG-O inhibited the activation of IκK-α/β, the phosphorylation of IκB-α, and degradation of IκB-α, which results in the inhibition of p65 nuclear accumulation and NF-κB activation. In addition, WCG-O suppressed the activation of ERK1/2, p38 and JNK, which results in the inhibition of ATF2 nuclear accumulation. Conclusion : These results indicate that WCG-O may exert anti-inflammatory activity through the inhibiting NF-κB and MAPK signaling. From these findings, WCG-O has potential to be a candidate for the development of chemoprevention or therapeutic agents for the inflammatory diseases.

Background : Mistletoe has been used as the herbal medicine to treat hypertension, diabetes mellitus, inflammation, arthritis and viral infection. In this study, we evaluated the anti-inflammatory effect of extracts of branch from Taxillus yadoriki being parasitic in Neolitsea sericea (TY-NS-B) using in vitro model. Methods and Results : TY-NS-B significantly inhibited LPS-induced secretion of NO and PGE2 in RAW264.7 cells. TY-NS-B was also observed to inhibit LPS-mediated iNOS COX-2 expression. In addition, TY-NS-B attenuated production of inflammatory cytokines such as TNF-α and IL-1β induced by LPS. TY-NS-B blocked LPS-mediated inhibitor of IκB-α, and inhibited p65 translocation to the nucleus and NF-κB activation. Furthermore, TY-NS-B reduced the phosphorylation of MAPKs such as p38 and JNK, but not ERK1/2. In addition, TY-NS-B increased ATF3 expression and ATF3 knockdown by ATF3 siRNA attenuated TY-NS-B-mediated inhibition of pro-inflammatory mediator expression. Conclusion : Collectively, our results suggest that TY-NS-B exerts potential anti-inflammatory effects by suppressing NF-κB and MAPK signaling activation, and increasing ATF3 expression. These findings indicate that TY-NS-B could be further developed as an anti-inflammatory drug.

The initial segment (IS) in rodents is functionally and structurally distinct from other epididymal segments and plays an important role in sperm maturation. We previously showed that, in the mouse epididymis, basal cells (BCs) extend a narrow luminal-reaching projection only in the IS, while in all other regions, they mainly nestle at the base of the epithelium. We also found that BC projections are regulated by testicular luminal factors, and the present study was aimed at characterizing the signaling pathway involved in their formation and elongation. Previous studies reported that testicular luminal factors maintain the extracellular signal-regulated kinase (ERK) in a highly phosphorylated state in the IS. We report here that the BC projections, which we call axiopodia, periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement is controlled by MAPK/ERK signaling pathway. Our results suggest that ERK phosphorylation plays a key role in the formation and elongation of BC projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment.

진피 상층부 모세혈관은 젊은 사람에 비해 노인에서 그 수와 크기가 감소되어 있어 피부에서 모세혈관이 차지하는 비율이 나이에 따라 급격히 낮아진다. 연교는 물푸레나무과에 속하는 개나리 열매를 건조한 것으로 주로 염증성 또는 항균성 질환에 오랫동안 사용되어져온 약재로서 지금까지 피부 혈관신생과 관련된 효능은 보 고되지 않았다. 따라서 본 연구에서는 연교 추출물이 혈관신생과 관련된 인자들에 미치는 영향을 피부 각질형성 세포주를 이용하여 조사하고자 하였다. 우리는 먼저 연교 추출물이 혈관신생과 관련된 인자들의 발현에 어떤 영향을 주는지 알아보고자 각질형성세포에 연교 추출물을 처리하고 혈관신생과 관련된 55개 단백질의 발현을 분석하였다. 발현 변화를 보인 인자들 중 혈관내피세포 성장인자(VEGF, vascular endothelial growth factor) 는 강력한 혈관신생 촉진인자로서 연교 추출물에 의해 유의하게 발현이 증가되었다. 따라서 연교 추출물이 VEGF 생성에 미치는 영향에 대해 자세히 알아보고자 연교 추출물을 농도별로 세포에 처리하고 단백질 발현과 mRNA 발현 변화를 조사한 결과, 연교 추출물은 VEGF의 유전자 수준뿐만 아니라 단백질 수준의 발현을 2배 이상 농도 의존적으로 증가시켰다. 다음으로 연교 추출물에 의한 VEGF 발현 증가에 관여하는 신호전달 기전을 밝히고자 MAPK 활성을 살펴본 결과, 연교 추출물을 세포에 처리하면 5 min 내 p38 MAPK의 활성이 관찰되었 으며, 특이적 억제제 전처리를 통해 p38 MAPK 활성을 억제하면 연교 추출물을 처리하더라도 VEGF의 유전자 및 단백질 발현이 완전히 억제됨을 확인하였다. 이러한 결과로부터 연교는 피부 각질형성세포에 작용하여 p38 MAPK 활성을 통해 VEGF 생성을 유도함을 알 수 있었고, 피부에서 노화에 따른 표피아래 모세혈관 손상을 개선하는데 도움을 줄 수 있는 후보 물질로서 연교의 새로운 효능을 제안한다.

Background : Achyranthes japonica Nakai (AJ) is a perennial herb with a wide distribution in East Asia including Korea, China, and Japan, and it is mainly used as a medicinal plant. In Korea, AJ has been widely used to control pain and improve symptoms in OA patients. AJ contains several important phytochemicals such as saponins, inokosterone, ecdysterone, and oleanolic acid bisdesmoside. Methods and Results : The aim of this work was to investigate the antioxidant and anti-inflammatory activities of fermented and ethanol extracts of Achyranthes japonica Nakai (AJ). The extracts showed strong reductive power and nitrite scavenging, hydroxyl radical scavenging, superoxide radical scavenging, and DNA damage prevention activities. Treatment of RAW 264.7 macrophages with AJ inhibited lipopolysaccharide (LPS)-induced NO secretion and iNOS expression without affecting cell viability. AJ also inhibited cyclooxygenase 2 (COX-2) expression, leading to the suppression of COX-2-derived prostaglandin E2 production. These inhibitory effects of AJ were accompanied by reduced production of tumor necrosis factor-α and interleukins (IL)-1β, -6, and -10. Furthermore, AJ suppressed LPS-induced phosphorylation of extracellular signal regulated kinase, c-Jun N-terminal kinase, and p38. Moreover, AJ inhibited malondialdehyde production and myeloperoxidase activity in LPS-stimulated RAW 264.7 cells. Conclusion : The antioxidant activity of plants is closely related to their medicinal properties and is widely used as a parameter to determine the bioavailability of medicinal plants. The antioxidant and biological activities of AJ extracts might be due to the synergistic actions of multiple bioactive compounds. It can be concluded that AJ extracts are a potential source of biologically important drug candidates.

Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events including abiotic/biotic stress responses, innate immunity, hormone signaling and cell specificity in plants. The MAPK-mediated stress and ethylene signaling are recently known to be involved in nitogen-fixing symbiotic interactions; however, the biological role of MAPK for nodule development in legume plants is largely unknown. We here elucidated that MtMKK5-MtMPK3/6 cascade negatively regulate the nitrogen fixing nodule formation in Medicago truncatula. MtMKK5, an ortholog of SIMKK, overexpression significantly reduces the nodule formation in M. truncatula roots. MtMKK5 directly activates MtMPK3/6 by phosphorylation on the TEY motif within the activation loop in the cytoplasm, which might link to EFD as a negative regulator for nodule formation. EFD has a putative MAPK phosphorylation Thr residue and could be a target of the activated MtMPK3/6 in the nucleus. Consistently, a MAPK specific inhibitor U0126 enhances nodule formation and confers similar nodule phenotypes to the efd-1 mutant such as lower proliferation and differentiation to symbiotic tissues. Our works thus reveal a key negative signaling module mediated by MtMKK5-MtMPK3/6-EFD for symbiotic nitrogen fixing nodule organogenesis.

Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events including abiotic/biotic stress responses, innate immunity, hormone signaling and cell specificity in plants. The MAPK-mediated stress and ethylene signaling are recently known to be involved in nitogen-fixing symbiotic interactions; however, the biological role of MAPK for nodule development in legume plants is largely unknown. We here elucidated that MtMKK5-MtMPK3/6 cascade negatively regulate the nitrogen fixing nodule formation in Medicago truncatula. MtMKK5, an ortholog of SIMKK, overexpression significantly reduces the nodule formation in M. truncatula roots. MtMKK5 directly activates MtMPK3/6 by phosphorylation on the TEY motif within the activation loop in the cytoplasm, which might link to EFD as a negative regulator for nodule formation. EFD has a putative MAPK phosphorylation Thr residue and could be a target of the activated MtMPK3/6 in the nucleus. Consistently, a MAPK specific inhibitor U0126 enhances nodule formation and confers similar nodule phenotypes to the efd-1 mutant such as lower proliferation and differentiation to symbiotic tissues. Our works thus reveal a key negative signaling module mediated by MtMKK5-MtMPK3/6-EFD for symbiotic nitrogen fixing nodule organogenesis.