Hydrogen peroxide (H2O2) is originally an endogenous small molecule which is reduced into water in cells. In order to know the H2O2-induced oxidative stress in RAW 264.7 cells, first of all, the optimum concentration of exogenous H2O2 which show reactive cellular responses was determined as 40 μM by MTT assay, and followed by 40 μM H2O2 application in RAW 264.7 cells for 30 min, 1, or 2 hours. The expressional changes of essential proteins for cellular proliferation, epigenetic modification, inflammation, apoptosis, survival, and protection were assessed by immunoprecipitation high performance liquid chromatography (IP-HPLC) using 51 antisera. 40 μM H2O2 treatment down-regulated proliferation-related proteins, Ki-67, PCNA, CDK4, cyclin D2, cMyc, and PLK4, induced histone methylation/ deacetylation and DNA methylation by increasing levels of HDAC10 and DMAP1 and by decreasing levels of DNMT1 and KDM4D, activated inflammatory reaction by increasing levels of MCP-1, COX-2, CD68, LTA4H, CXCR4, and lysozyme, and dramatically up-regulated cellular apoptosis-, survival-, and protection-related proteins, AIF, PARP-1, caspase 9, c-caspase 9, pAKT1/2/3, SOD-1, HO-1, NF-kB, NRF2, and GSTO1 in RAW 264.7 cells. These observations suggest exogenous 40 μM H2O2-induced oxidative stresses which resulted global cellular responses including not only antioxidant, inflammation, and apoptosis but also proliferation and epigenetic modification. Particularly, 40 μM H2O2-induced apoptosis was mainly derived from PARP-1/AIF signaling leading parthanatos, and 40 μM H2O2-induced suppression of cMyc/MAX/MAD network was relevant to reduction of RAW 264.7 cell proliferation. Accordingly, H2O2 appears to affect RAW 264.7 macrophages in several ways eliciting not only oxidative stresses but also genome-wide DNA damage.

Suaeda asparagoides (Miq.) is a salt marsh plant, long been prescribed in traditional medicine for the treatment of hypertension and liver toxification in Asian countries. The powder of S. asparagoides was added at the ratio of 0, 5, and 10%, respectively, of grain-type Meju to manufacture Doenjang in brine according to the salt concentration (8 and 12%). After 24 weeks of fermentation, the Doenjang samples were determined to have an anti-inflammatory effect on RAW 264.7 cells. Evaluation of the anti-inflammatory effect of Doenjang added S. asparagoides powder extracted using 80% EtOH, was performed to study the inhibition of pro-inflammatory factors such as NF-κB (nuclar factor κB), NO (nitric oxide), TNF-α (tumor necrosis factor alpha), IL-6 (interleukin-6), iNOS (inducible nitric oxide synthase), and COX-2 (cyclooxygenase-2) in lipopolysaccharide (LPS)-induced RAW 264.7 cells. The results showed that the Doenjang extracts reduced the production of NO, IL-6, COX-2, and iNOS increased in the LPS-stimulated RAW cell without cytotoxicity. In the case of the NF-κB and TNF-α there was no significant difference between the control and samples. In conclusion, these results suggest that Doenjang added with the S. asparagoides powder acts as functional fermented food with anti-inflammation effect.

This study investigated the anti-inflammatory effects of processed (Beopje) curly dock (Rumex crispus L.) in LPS (lipopolysaccharide)-stimulated murine RAW 264.7 cells. The experimental group was classified into five groups : LPS no treatment, CD (curly dock), CD-B (CD processed through Beopje), LPS, LPS+CD-B (LPS+CD processed through Beopje) and LPS+CD (LPS+CD). Treatment of the Raw 264.7 cell lines using LPS led to a significant increase in NO production, pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β), and inflammation related genes (COX-2 and iNOS). Investigation of the inhibitory effects of CD and processed CD on NO production and expression of iNOS and COX-2 was done in LPS-induced RAW 264.7 cells. There was significant inhibition of NO production by LPS+CD and LPS+CD-B in a dose-dependent manner (p<0.05). Particularly, LPS+CD-B exhibited reduced mRNA expression of iNOS and COX-2 and NO production as compared to LPS+CD in Raw 264.7 cell lines (p<0.05). These results may explain some known biological activities of curly dock including the anti-inflammatory effects. CD-B in particular exhibited the highest anti-inflammatory effects of inhibiting production of NO, through the regulation of inflammatory related genes and pro-inflammatory cytokines. These results of Beopje processing might help decrease the anti-biological effects and increase several active substances of curly dock

Piperlongumine (PL) is a natural product found in long pepper (Piper longum ). The pharmacological effects of PL are well known, and it has been used for pain, hepatoprotection, and asthma in Oriental medicine. No studies have examined the effects of PL on bone tissue or bone-related diseases, including osteoporosis. The current study investigated for the first time the inhibitory effects of PL on osteoclast differentiation, bone resorption, and osteoclastogenesis-related factors in RAW264.7 macrophages stimulated by the receptor activator for nuclear factor- κB ligand (RANKL). Cytotoxicity was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and osteoclast differentiation and bone resorption were confirmed by tartrate-resistant acid phosphatase (TRAP) staining and pit formation analysis. Osteoclast differentiation factors were confirmed by western blotting. PL exhibited toxicity in RAW264.7 macrophages, inhibiting osteoclast formation and bone resorption, in addition to inhibiting the expression of osteoclastogenesis-related factors, such as tumor necrosis factor receptor-associated factor 6 (TRAF6), c-Fos, and NFATc1, in RANKL-stimulated RAW264.7 macrophages. These findings suggest that PL is suitable for the treatment of osteoporosis, and it serves as a potential therapeutic agent for various bone diseases.

Previous studies demonstrated that rice bran oil has an anti-inflammatory effect. The current study investigated if the immune responses and energy metabolism phenotypes were affected by rice bran oil. Rice bran oil and palm oil, which served as an experimental control, were treated to RAW 264.7 macrophages respectively, followed by LPS stimulation so as to quantify the production of pro-inflammatory cytokines such as IL-6 and TNF-α. The expression of surface markers i.e. CD 80, CD 86 and MHC-classⅡ and mitochondrial metabolism phenotypes were also tested. The data exhibited rice bran oil suppressed inflammatory responses and mitochondrial metabolism were modulated by rice bran oil. These results warrant the necessity for the compositional and/or mechanistic studies to elucidate the details.

It is well-known that cultivated wild Panax ginseng has anti-inflammatory effect. However, a comparative study on cultivation period vs biofunctionality is currently lacking. In this study, 70% ethanol extracts of 3-years (yrs)-, 5-yrs-, or 7-yrs-old cultivated wild ginseng were evaluated for their inhibitory effects on RAW264.7 murine macrophages. Specifically, the production of pro-inflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor-alpha [TNF-α]), the expression of surface proteins (CD80, CD86, and MHC-II), and the phagocytic properties were investigated. RAW264.7 cells were induced by 500 ng/mL of lipopolysaccharide (LPS) and treated with 0.1, 1, and 10 ppm of samples. LPS-induced IL-6, TNF-α and surface proteins in all samples were downregulated in a dose-dependent manner. Both IL-6 and TNF-α were significantly reduced at 10 ppm of the 7-yrsold sample compared to 10 ppm of 3-yrs- and 5-yrs-old samples. CD80 and CD86 were also reduced at 10 ppm of all samples, and there was no difference among samples. The phagocytosis has no difference except in 10 ppm of 3 yr-old sample. The results suggest that cultivated wild ginseng extract has anti-inflammatory effect without decreasing phagocytosis.

Coffee (Coffea spp.) is one of the most important agricultural commodities, being widely consumed in the world. Various beneficial health effects of coffee have been extensively investigated, but data on habitual coffee consumption and its bio-physiological effect have not been clearly explained as well as it is not proved the cause and effect between drinking coffee and its bio-physiological reactions. We made the dialyzed coffee extract (DCE), which is absorbable through gastrointestinal tract, in order to elucidate the cellular effect of whole small coffee molecules. RAW 264.7 cells, a murine macrophage lineage, were directly treated with DCE, i.e., DCE-2.5 (equivalent to 2.5 cups of coffee a day), DCE-5, and DCE-10, for 12 hours, and their protein extracts were examined by immunoprecipitation high performance liquid chromatography (IP-HPLC). RAW 264.7 cells differently expressed the inflammation-related proteins depending on the doses of DCE. RAW 264.7 cells treated with DCE showed marked increase of cathepsin C, cathepsin G, CD20, CD28, CD31, CD68, indicating the activation of innate immunity. Particularly, the macrophage biomarkers, cathepsin G, cathepsin C, CD31, and CD68 were markedly increased after DCE-5 and DCE-10 treatments, and the lymphocyte biomarkers, CD20 and CD28 were consistently increased and became marked after DCE-10 treatment. On the other hand, RAW 264.7 cells treated with DCE showed consistent increase of IL-10, an anti-inflammatory factor, but gradual decreases of different pro-inflammatory proteins including TNFα, COX-2, lysozyme, MMP-2, and MMP-3. In particular, the cellular signaling of inflammation was gradually mitigated by the reduction of TNFα, COX-2, IL-12, and M-CSF, and also the matrix inflammatory reaction was reduced by marked deceases of MMP-2, MMP-3, and lysozyme. These anti-inflammatory expressions were consistently found until DCE-10 treatment. Therefore, it is presumed that DCE may have dynamic effects of innate immunity activation and pro-inflammation suppression on RAW264.7 cells simultaneously. These effects were consistently found in the highest dose of coffee, DCE-10 (equivalent to 10 cups of coffee a day in man), that might imply the small coffee molecules were accumulated in RAW 264.7 cells after DCE-10 treatment and produce synergistic cytokine effects for innate immunity activation and anti-inflammatory reaction concurrently.

7년 이상 장기간 배양으로 인한 고가의 산양삼은 항염증에 효과가 있다고 잘 알려져 있다. 하지만, 재배기간에 따른 항염증능의 비교 연구는 부족한 실정이다. 본 연구는, 3년, 5년, 7년 근령의 산양삼 추출물을 이용하여 RAW264.7 대식세포의 염증반응 모델에서의 항염증능을 비교분석하였다. 이를 위하여, 염증성 싸이토카인 (IL-6, TNF-α)의 분비, 표면 단백질(CD80, CD86, MHC-Ⅱ)의 발현 및 대식능을 분석하였다. 각 근령의 산양삼 추출물을 0.1, 1, 10ppm 농도로 RAW26.47 세포주에 처리한 후, LPS로 염증반응을 유도하였을 때, IL-6와TNF-α의 분비량이 모든 근령군에서 농도의 존적으로 감소함을 보였다. 하지만, 일반적으로 근령이 클수록 싸이토카인 분비 억제 효과가 높은 것으로 확인 되었다. 대식세포의 활성화마커인 표면 단백질의 발현양도 모든 실험군에서 농도의존적으로 감소 하는 경향을 나타내었다. 구체적으로는, CD80과 CD86의 발현은 3년삼, 5년삼, 7년삼의 10ppm에서 무처리 대조군과 비교하여 유의적으로 감소하였고, 근령간의 차이는 보이지 않았다. MHC-Ⅱ의 발현은 일정한 경향은 나타나지 않았고 3, 7년삼의 10ppm처리군에서 감소하였다. 대식능은 3년삼의 10ppm을 제외하고는 유의적 차이를 보이지 않았다. 이러한 결과는 산양삼 근령에 증가에 따라 항염증능이 증가하지만, 대식능에는 영향을 미치지 않는 것으로 평가되며 동물실험을 통하여 확증할 예정이다.