Obesity, a global health concern characterized by excessive fat accumulation, necessitates the discovery of anti-obesity compounds. Rottlerin, known for its anti-cancer effects as a mitochondrial uncoupler, has been a subject of interest. However, its impact on reducing intracellular lipid accumulation remains a gap in our understanding. This study aimed to fill this gap by dissecting the mechanism of rottlerin in 3T3-L1 adipocytes. We treated differentiated 3T3-L1 cells with 0-20 mM of rottlerin for 48 hours to assess its capability to induce lipid accumulation. Notably, we observed no cytotoxicity associated with the treatment of rottlerin up to 20 mM, indicating its safety at these concentrations. Lipid accumulation, measured by oil Red O, was downregulated dose-independently by rottlerin. We also found that key lipogenic enzymes, including SCD1 and DGAT1, were decreased. The transcription factor of lipogenic genes, SREBP1, was reduced by approximately 80% with rottlerin. LRP6, a crucial link between de novo lipogenesis mechanism reactions and Wnt signaling, was also degraded by around 70%. Interestingly, the downstream regulation of LRP6, b-catenin, and TCFL2 was diminished by rottlerin. Our data indicate that rottlerin alleviates adipocyte lipid accumulation by suppressing the LRP6/β-catenin/SREBP1c pathway. These findings underscore the potential of rottlerin as a safe nutraceutical for combating obesity.

Sprout products, such as broccoli, alfalfa, and cabbage, have positive health effects. Thus far, sprout foods have attracted attention owing to their good bioavailability. In particular, young broccoli sprouts exhibit anti-inflammatory, antioxidant, and anti-cancer effects. They contain 100 times more chemoprotective substances than adult broccoli. This study examined the anti-inflammatory effects of freeze-dried young sprout broccoli (FD-YB) in vitro using RAW264.7 macrophage cells. The FDYB powder antioxidant ability test showed that the radical-scavenging activity and superoxide dismutase enzyme activity increased in a dose-dependent manner. In addition, FD-YB was not cytotoxic to RAW264.7 cells, and nitric oxide production decreased after the FD-YB treatment of lipopolysaccharide-stimulated RAW264.7 cells in a dose-dependent manner. Furthermore, FD-YB significantly decreased the expression of inflammation-related proteins (Cyclooxygenase-2, Inducible nitric oxide synthase, and Prostaglandin E Synthase 2) and cytokines (Tumor necrosis factor- and Interleukin-6). In conclusion, FD-YB can be a potential nutraceutical for preventing and regulating excessive immune responses during inflammation.

본 연구는 국내산 가시오가피의 건강기능식품 소재로서 의 가능성을 확인하기 위해 산지별 채취한 가시오가피의 유효물질 함량 및 면역 증강 효과을 평가하였다. 태백, 철 원, 삼척, 강원도 농업기술원에서 수확한 가시오가피의 지 표성분인 eleutheroside B 및 eleutheroside E의 분석을 수 행하였으며, 면역 증강에 대한 효과를 관찰하기 위하여 MTT 세포독성 평가, NO 생성량과 cytokine 생성량을 측 정하였다. 지표성분 eleutheroside B의 함량은 채취 지역별 로 70% 에탄올 추출물에서는 2.96±0.11-6.24±0.05 mg/g로 태백에서 가장 높은 함량을 나타냈으며, 열수 추출물에서 는 1.11±0.05-2.11±0.03 mg/g로 태백에서 가장 높은 함량 을 나타냈다. Eleutheroside E 함량은 채취 지역별로 70% 에탄올 추출물에서는 4.93±0.20-10.79±0.03 mg/g을 나타냈으 며 철원에서 가장 높은 함량을 나타냈고, 열수 추출물에서 는 1.75±0.14-3.64±0.05 mg/g로 철원과 농업기술원에서 가장 높은 함량을 나타냈다. 또한, eleutheroside B 및 E 함량은 열수 추출물보다 70% 에탄올 추출물에서 더 높은 함량을 나타냈다. 채취 지역별 가시오가피의 70% 에탄올 추출물은 50-200 μg/mL 농도에서, 열수 추출물은 100-500 μg/mL 농도 에서 RAW 264.7 대식세포에 대한 세포독성을 나타내지 않 았으며, 대식세포의 활성화로 방출되는 NO 성성량을 측정 한 결과, 가시오가피 줄기 추출물에서 NO 생성량이 증가하 는 것을 확인하였으며, TNF-α, IL-6, IL-1β을 포함하는 cytokine의 방출을 측정한 결과 유의적인 증가를 나타냈다. 따라서 가시오가피 줄기는 면역 관련 질환의 개선을 위한 건강기능식품 소재로 활용 가능할 것으로 사료된다.

Owing to its diverse range of bioactive compounds, Ganoderma lucidumhas garnered significant research attention for health promotion and disease prevention. Ganodermanondiol, which has a triterpenoid structure, is one of the major active compounds of G. lucidum. In the present study, the anti-inflammatory effects of ganodermanondiol were investigated to evaluate its usefulness as a functional ingredient. Ganodermanondiol (0.5–2 g/mL) significantly inhibited the production of nitric oxide (NO), the expression of the cytokines tumor necrosis factor (TNF)??and interleukin 6 (IL-6), and the expression of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in lipopolysaccharide-induced RAW 264.7 (murine macrophage) cells. Ganodermanondiol (0.5–2 g/mL) also inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) signal molecules, including p38 and c-Jun N-terminal protein kinase (JNK) in RAW 264.7 cells. Ganodermanondiol significantly inhibited the essential factors involved in the inflammatory responses of RAW 264.7 cells and would, therefore, serve as a potential prophylactic and therapeutic agent for immune-related diseases.

This research investigated the immunoenhancing effect through the intracellular MAPKs and NF-B signaling pathways in macrophages activated by crude polysaccharides (YBP) of barley sprouts. YBP extracted from barley sprouts is composed of xylose (25.8%), arabinose (24.1%), galactose (23.4%), and galacturonic acid (11.7%). YBP did not affect the cytotoxicity and showed superior secretion of nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)- by RAW264.7 cells. Also, YBP dose dependently increased IL-6, TNF-, and inducible nitric oxide synthase (iNOS) mRNA gene expression. In the western blot, YBP strongly induced the phosphorylation of the p38, JNK, ERK, and IB pathways in RAW 264.7 cells. In the anti-pattern recognition receptor (anti-PRRs) assay, the effect of YBP on NO secretion strongly decreased toll-like receptor (TLR) 4 and Dectin1 antibodies, whereas IL-6 and TNF- secretion by YBP mainly decreased SR and CD14. Therefore, we concluded that YBPinduced NO, IL-6, and TNF- were secreted via the MAPKs, while NF-B pathways through TLR4, Dectin1, SR, and CD14 receptors existed in a macrophage surface and were involved in the immunoenhancing effect.

This study aims to evaluate the anti-inflammatory effect of Auricularia auricula-judae ethanol extract (AEE) in LPS-induced RAW264.7 cells and dextran sulfate sodium (DSS)-induced acute colitis mice. The highest levels of total polyphenols and DPPH radical scavenging activity were observed in AEE. Also, NO production was reduced in a dose-dependent manner in RAW264.7 cells stimulated by lipopolysaccharide (LPS). AEE was suspended in distilled water and administration per oral at different doses of 100 and 300 mg/kg body weight every day with 3% DSS in drinking water for 7 days. The sample AEE 100 and 300 mg/kg significantly increased body weight, colon length and decreased disease activity index (DAI) score. Histological features showed that 100 and 300 mg/kg of AEE suppressed edema, mucosal damage and the loss of crypts induced by DSS. The serum levels of TNF- and IL-6 were measured in acute colitis mice using ELISA kits. Levels of TNF- and IL-6 in serum were significantly decreased by topical application of AEE. Therefore, AEE increases antioxidant and anti-inflammatory activity, and it is thought that it can effectively help prevent colitis.

Macrophages secrete various cytokines and inflammatory mediators, resulting in playing critical roles in inflammation and immunity. In this study, we investigated anti-inflammatory and immune enhancing properties of PB203, which is a water-soluble extract powder from the fruit of Actinidia polygama, in macrophages. A. polygama is a medicinal plant traditionally known to treat abdominal pain, stroke and rheumatoid arthritis. However, the molecular mechanism for the immune modulation of PB203 is still unclear. Therefore, we assessed the effects of PB203 on the lipopolysaccharide (LPS)-induced inflammation and immune activation, and elucidated its action mechanism in mouse macrophage, RAW264.7 cells. PB203 significantly suppressed not only the levels of nitric oxide (NO), prostaglandin E2, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), but also the mRNA expression of inducible NO synthase, cyclooxygenase-2, TNF-α and IL-1β in LPS-stimulated RAW264.7 cells. We also found that these anti-inflammatory activities of PB203 were mediated through the inhibition of toll-like receptor 4 and nuclear factor kappa B (NF-κB) induced by LPS. On the other hand, in normal macrophages, PB203 dose-dependently elevated the gene expression of immunomodulators including granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1 and TNF-α in a statistically significant manner. The expression of IL-10, IL-1β, IL-6, and interferon-γ were also remarkably upregulated by the treatment of 500 μg/mL PB203. In addition, PB203-mediated production of NO and TNF-α was attenuated by NF-κB inhibition in RAW264.7 cells. Interestingly, PB203 promoted the production of nuclear factor erythroid-2-related factor 2, resulting in the increased level of heme oxygenase-1, which is a representative antioxidant enzyme, in both LPS-stimulated and normal RAW264.7 cells. Taken all together, these results suggest that PB203 may have great potential as the candidate of anti-inflammatory agent for improving inflammatory diseases or immune enhancing agent for preventing infectious diseases. Keywords: Actinidia polygama extract (PB203); macrophages; immunomodulator; nuclear factor kappa B (NF-κB); heme oxygenase-1 (HO-1)

The purpose of this study was to analyze the anti-inflammatory effect of Chung-Dae Indigo Pulverata Levis, indigo naturalis) produced during indigo dyeing. As a result of in vitro cytotoxicity experiments using RAW 264.7 cell, Chung-Dae extract did not inhibit cell proliferation in Raw 264.7 cells in the range of 1~32 μg/mL. NO production was significantly reduced when Chung-Dae extracts were treated at concentrations of 2, 8, and 32 μg/mL (p<0.05). The pro-inflammatory cytokines TNF-α, IL-6, IL-1β and IFN-γ significantly decreased when the Chung-Dae extract was treated at concentrations of 2, 8, and 32 μg/mL compared to the LPS group, and similarly, the TNFα and IL-6 mRNA levels also decreased. Additionally, the mRNA level of COX-2 was also suppressed. At the protein expression level, the expression of TNF-α, IL-6, iNOS and COX-2 were observed with LPS and Chung-Dae extract significantly decreased compared to the group treated with only LPS (p<0.05). From the above results, it shows that Chung-Dae extract, a plant-derived compound, inhibits the inflammatory response induced by LPS in RAW 264.7 cells. and in particular, regulates the inflammatory response by inhibiting the expression of pro-inflammatory cytokines and inflammation-related enzymes.

This study aims to investigate the effects of exogenous succinic acid (SCA) on Brucella (B.) abortus infection in macrophage RAW 264.7 cells and ICR mice. Firstly, the in vitro experiment was conducted by MTT cytotoxicity and bacterial internalization assay to evaluate the uptake of B. abortus into macrophage cells. Two non-cytotoxic concentrations of SCA demonstrated attenuated invasion of Brucella into macrophages at 30 and 45 min post- infection (pi). Secondly, ICR mice were treated with SCA and infected with B. abortus. On day-14 pi, spleen and blood serum were collected to evaluate the bacterial burden and total spleen weight as well as the production of cytokine/chemokine, respectively. The results showed that SCA treatment promoted bacterial growth and reduced the total spleen weight in mice. Furthermore, SCA treatment increased the level of IL-10 cytokine in the sera, while dampening the production of MCP-1 chemokine compared to the control. The results of bacterial load in spleen and spleen weight together with cytokine/chemokine production profile in the sera indicated that SCA induced the host anti-inflammatory response which is beneficial for the survival of Brucella. Therefore, these findings suggest that SCA contributed to host immunity against Brucella infection and the emerging potential topic-immunometabolism should be invested for further investigations.

본 연구는 침출차 원료로 레드비트를 활용하기 위해서 세포독성, 항염증 및 항산화 등의 활성을 조사하고자 설계되었다. 항산화 효능은 DPPH 및 ABTS 라디컬 소거 활성을 분석하여 평가하였다. RAW 264.7 세포를 통해 MTT 분석으로 세포 생존율을 평가하고 LPS로 유도하여 활성산소종과 염증 매개체(일산화질소, TNF-α, IL-6 등)의 생성량을 확인하였다. 그 결과, DPPH 및 ABTS 라디컬 소거 활성은 농도 의존적으로 증가하였으며, 모든 농도에서 세포독성이 없음을 확인하였다. 또한, LPS로 유도한 RAW 264.7 세포에서 활성산소종(ROS)과 일산화질소(NO), IL-6, TNF-α 생성량을 유의적으로 감소시켰다. 따라서 이러한 결과는 레드비트가 안전한 항산화 및 항염증 효과를 가진 침출차의 원료로서 높은 잠재력을 가지고 있음을 시사한다.

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.

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.