Anti-Inflammatory and Transepithelial Transport Activities of Rapeseed (Brassica napus) Napin-Derived Dipeptide Thr-Leu in Caco-2 and RAW264.7 Cocultures.

This study aimed to investigate the anti-inflammatory molecular activity of rapeseed napin-derived dipeptide Thr-Leu (TL) using Caco-2/RAW264.7 cell cocultures. This in vitro coculture intestinal inflammation model was used to assess the absorption, evolution, and anti-inflammatory effects of peptides. TL was absorbed by the intestinal epithelial cells with an apparent permeability of (2.48 ± 0.18) × 10-6 cm/s, primarily through the PepT1 pathway. TL treatment exerted anti-inflammatory and restorative effects on the impaired intestinal barrier function by enhancing the expression levels of occludin and ZO-1 in lipopolysaccharide (LPS)-induced Caco-2 cells. No significant change (P < 0.05) was detected in claudin-1 expression levels; however, the occludin expression levels were upregulated through the protein kinase C (PKC) signaling pathway. Compared with the LPS-induced group, TL (2.0 mM) reduced the levels of intracellular inflammation-related enzymes (iNOS: by 50.84%; COX-2: by 49.64%) on the coculture cell model. In addition, the interleukin (IL)-1ß, IL-6, and TNF-α levels in RAW264.7 cells were significantly (P < 0.05) downregulated following TL treatment (2.0 mM) due to the suppression of the phosphorylation of the JNK-independent pathway on the basolateral side of the coculture cell model. These findings highlight the potential use of TL in functional foods or nutraceuticals to prevent intestinal inflammation.

Adenophora Stricta Root Extract Alleviates Airway Inflammation in Mice with Ovalbumin-Induced Allergic Asthma.

Adenophora stricta Miq. (Campanulaceae family) is a traditional herb used for relieving cough and phlegm in East Asia. This study explored the effects of A. stricta root extract (AsE) in ovalbumin (OVA)-induced allergic asthma and lipopolysaccharide (LPS)-stimulated macrophages. Administration of 100-400 mg/kg AsE dose-dependently decreased pulmonary congestion and suppressed the reduction of alveolar surface area in mice with OVA-mediated allergic asthma. Histopathological analysis of lung tissue and cytological analysis of bronchioalveolar lavage fluid showed that AsE administration significantly attenuated inflammatory cell infiltration into the lungs. In addition, AsE also alleviated OVA-specific immunoglobulin E, interleukin (IL)-4, and IL-5 production, which are essential for OVA-dependent activation of T helper 2 lymphocytes. In Raw264.7 macrophage cells, AsE significantly blocked nitric oxide, tumor necrosis factor-α, IL-1ß, IL-6, and monocyte chemoattractant factor-1 production in response to LPS. Results from an immunoblot assay revealed that AsE inhibited the phosphorylation of c-jun N-terminal kinase, inhibitory-κB kinase α/ß, and p65 in LPS-stimulated cells. Furthermore, 2-furoic acid, 5-hydroxymethylfurfural, and vanillic acid 4-ß-D-glucopyranoside in AsE were shown to inhibit the production of proinflammatory mediators by LPS. Taken together, the present results suggest that A. stricta root will be a useful herb for relieving allergic asthma through managing airway inflammation.

Donepezil halts acetic acid-induced experimental colitis in rats and its associated cognitive impairment through regulating inflammatory/oxidative/apoptotic cascades: An add-on to its anti-dementia activity.

Ulcerative colitis (UC) is a persistent inflammatory bowel disease (IBD) that is regarded as a risk factor for cognitive impairment. Donepezil (DON), a centrally acting acetylcholinesterase inhibitor (AChEI), is approved for the management of Alzheimer's disease (AD). We aimed to scrutinize the impact of DON on acetic acid (AA)-induced UC in rats and to evaluate its ability to attenuate inflammatory response, oxidative strain, and apoptosis in this model and its associated cognitive deficits. Rats were categorized into: normal, DON, AA, and AA + DON groups. DON (5 mg/kg/day) was administered orally for 14 days either alone or beginning with the day of UC induction. Colitis was evoked by a single transrectal injection of 1 ml of 4 % acetic acid. Results revealed that DON significantly improved the behavioral abnormalities with the mitigation of inflammation, apoptosis, and histopathological changes in the hippocampi of the colitis group. Moreover, DON significantly alleviated the macroscopic and microscopic changes associated with colitis. Interestingly, DON inhibited pro-inflammatory cytokines via suppression of AA-induced activation of nuclear factor kappa-B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1ß) in the colon, along with serum IL-1ß. DON inhibited colon lipid peroxidation, restored the antioxidants with a significant amelioration of the degree of neutrophil infiltration, and repressed colitis-induced matrix metalloproteinases-9 (MMP-9) production. Furthermore, DON decreased the Bax/Bcl-2 ratio and caspase-3 protein expressions. Eventually, in lipopolysaccharide (LPS)-treated RAW 264.7 macrophage cells, DON suppressed nitric oxide (NO) release, demonstrating the ability of DON to significantly curtail inflammation in immune cells. Taken together, DON ameliorated experimental colitis and its linked cognitive dysfunction, possibly via its antioxidant effect and modulation of pro-inflammatory cytokines and apoptosis. Thereby, DON could be a therapeutic nominee for UC and associated neurological disorders.

Mechanism of Buyang Huanwutang in Regulating Macrophage Cell Polarization Based on TLR4/NF-κB/NLRP3 Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Buyang Huanwutang in regulating macrophage polarization based on the Toll-like receptor 4 （TLR4） / nuclear factor-κB （NF-κB） / nucleotide-binding oligomerization domain-like receptor 3 （NLRP3） pathway. MethodRAW264.7 macrophages were intervened with lipopolysaccharide （LPS） of different concentrations （0， 1.25， 2.5， 5， 10， 20， 40， and 80 mg·L-1） for 24 hours. Cell Counting Kit-8 （CCK-8） assay was used to determine the cell viability of RAW264.7 macrophages. The optimal concentration was chosen to establish an in vitro inflammation model induced by LPS. Cells were divided into a blank group （20% blank serum）， a model group （20% blank serum + 10 mg·L-1 LPS）， a model control group （20% FBS + 10 mg·L-1 LPS）， low-， medium-， and high-dose （5%， 10%， and 20%） Buyang Huanwutang-containing serum groups， a high-dose （20%） Buyang Huanwutang combined with NLRP3 inhibitor MCC950 （50 μmol·L-1） group， a high-dose （20%） Buyang Huanwutang combined with reactive oxygen species （ROS） inhibitor NAC （10 μmol·L-1） group， and a high-dose （20%） Buyang Huanwutang combined with NF-κB inhibitor PDTC （10 μmol·L-1） group. Enzyme-linked immunosorbent assay （ELISA） was used to detect the expression of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， and tumor necrosis factor-α （TNF-α） in RAW264.7 macrophages. Flow cytometry was employed to measure ROS levels in macrophages. Western blot was used to determine the protein expression of M1-type macrophage-related factors inducible nitric oxide synthase （iNOS） and TNF-α， M2-type macrophage-related factors arginase-1 （Arg-1） and interleukin-10 （IL-10）， as well as the proteins in the TLR4/NF-κB/NLRP3 pathway. ResultCCK-8 results indicated that under 10 mg·L-1 LPS stimulation， RAW264.7 macrophages exhibited the highest cell viability （P<0.01）. Compared with the blank group， the model group showed significantly increased levels of IL-1β， IL-18， and TNF-α （P<0.05，P<0.01）， increased ROS expression （P<0.05，P<0.01）， increased protein expression of M1-type macrophage factors iNOS and TNF-α （P<0.01）， decreased protein expression of M2-type macrophage factors Arg-1 and IL-10 （P<0.05，P<0.01）， and upregulated expression levels of TLR4， myeloid differentiation factor 88 （MyD88）， phosphorylated inhibitor of NF-κB （p-IκB）/NF-κB inhibitor （IκB）， phosphorylated NF-κB （p-NF-κB） p65/NF-κB p65， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， and pro-Caspase-1 （P<0.05， P<0.01）. Compared with the model group， all Buyang Huanwutang-treated groups and inhibitor groups significantly reduced levels of IL-1β， IL-18， and TNF-α （P<0.01）， suppressed the expression of inflammatory factors in RAW264.7 macrophages， decreased cellular ROS expression levels （P<0.01）， downregulated M1-type macrophages iNOS and TNF-α protein expression （P<0.01）， upregulated M2-type macrophages Arg-1 and IL-10 protein expression （P<0.01）， and lowered protein expression levels of TLR4， MyD88， p-IκB/IκB， p-NF-κB p65/NF-κB p65， NLRP3， ASC， and pro-Caspase-1 （P<0.05， P<0.01）. ConclusionBuyang Huanwutang can improve macrophage inflammation， potentially by reducing macrophage ROS levels， inhibiting RAW264.7 macrophage polarization， and downregulating the protein expression levels of the TLR4/NF-κB/NLRP3 pathway.

Immunostimulatory Activity of Lactic Acid Bacteria Cell-Free Supernatants through the Activation of NF-κB and MAPK Signaling Pathways in RAW 264.7 Cells.

Lactic acid bacteria (LAB) can improve host health and has strong potential for use as a health functional food. Specific strains of LAB have been reported to exert immunostimulatory effects. The primary goal of this study was to evaluate the immunostimulatory activities of novel LAB strains isolated from humans and foods and to investigate the probiotic properties of these strains. Cell-free supernatants (CFS) obtained from selected LAB strains significantly increased phagocytosis and level of nitric oxide (NO) and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 in RAW264.7 macrophage cells. The protein expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, which are immunomodulators, was also upregulated by CFS treatment. CFS markedly induced the phosphorylation of nuclear factor-κB (NF-κB) and MAPKs (ERK, JNK, and p38). In addition, the safety of the LAB strains used in this study was demonstrated by hemolysis and antibiotic resistance tests. Their stability was confirmed under simulated gastrointestinal conditions. Taken together, these results indicate that the LAB strains selected in this study could be useful as probiotic candidates with immune-stimulating activity.

Anti-inflammatory effects of ß-ionone-curcumin hybrid derivatives against ulcerative colitis.

A series of ß-ionone-curcumin hybrid derivatives were designed and chosen to merge the biological characteristics of two parent molecules and to obtain a leading compound with higher biological activity. Through the initial screening, the structure activity relationship of their hybrid derivatives as inhibitors of nitric oxide (NO) production showed that meta-substituted derivatives exhibited the best inhibitory activity, among which 1h was the best one. In lipopolysaccharide-induced Raw264.7 macrophage cells, 1h showed anti-inflammatory activity by inhibiting the productions of NO and reactive oxygen species, the expressions of Interleukin-1ß and tumor necrosis factor-α, and the translocation of nuclear factor (NF)-κB from the cytosol to the nucleus. Furthermore, molecular docking simulation displayed that 1h could interact with cluster of differentiation 14 to inhibit the toll-like receptor 4/NF-κB signaling. In dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) of mice, 100 mg/kg of 1h could significantly reduce the colon length shortening and protect against colon injury, liver injury and oxidative stress in DSS-induced UC of mice. Besides, 1h was safety in vivo. In conclusion, 1h was the potential anti-inflammatory agent, and further investigations were underway in our laboratory.

In Vitro Anti-Inflammatory Activity of Three Peptides Derived from the Byproduct of Rice Processing.

Inflammation is a contributing factor to the initiation and progression of many diseases, and some food-derived biofunctional peptides show high anti-inflammatory activity. In our previous study, we demonstrated that peptides derived from trypsin hydrolysis of rice protein show good immunological activity. In the present study, proteins of broken rice were extracted and identified by macroporous resin fractionation and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Subsequently, a bioinformatics prediction and in silico simulation approach was used to screen for peptides showing anti-inflammatory activity, including inhibition of the production of nitric oxide and proinflammatory cytokines (interleukin-1ß, interleukin-6, and tumor necrosis factor-α) by lipopolysaccharide-stimulated RAW264.7 mice macrophages. Three peptides (DNIQGITKPAIR, IAFKTNPNSMVSHIAGK, and IGVAMDYSASSKR) that demonstrated the highest binding affinity were synthesized, and their in vitro anti-inflammatory activity was investigated. This is the first study that integrates LC-MS/MS identification and bioinformatics prediction for reporting the anti-inflammatory activity of anti-inflammatory peptides derived from broken rice protein. The study findings revealed that the peptides derived from the byproduct of rice milling could be potentially used as natural anti-inflammatory alternativities.

Mycobacterium tuberculosis glycolipoprotein LprG inhibits inflammation through NF-κB signaling of ERK1/2 and JNK in LPS-induced murine macrophage cells.

Mycobacterium tuberoculosis (Mtb) is a contagious pathogen that causes human tuberculosis (TB). TB is a major global health threat that causes 9.6 million illnesses and 1.5 million deaths per year. Recent studies have suggested Mtb-secreted proteins as new candidates for therapeutic drugs and vaccines. LprG is a Mtb-secreted surface glycolipoprotein encoded by lprG (Rv1411c), which forms an operon with Rv1410c, where Rv1410c encodes P55, an efflux pump membrane protein. Various in vitro and in vivo studies have reported on the target-binding activity, cell envelope biosynthesis, and mycobacterial virulence of LprG. However, the anti-inflammatory effect of LprG in macrophages has not yet been investigated. In this study, we demonstrated that LprG can suppress lipopolysaccharide (LPS)-induced inflammation in a macrophage model. LprG inhibited LPS-stimulated nitric oxide (NO) production. LprG also suppressed expression of inducible cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) at the transcriptional and protein levels. In addition, LprG decreased mRNA expression of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α). Furthermore, LprG attenuated nuclear factor kappa-B (NF-κB) translocation and IκB phosphorylation. Moreover, LprG specifically inhibited phosphorylated kinases such as c-Jun N-terminal kinase (p-JNK) and extracellular signal-regulated kinase 1/2 (p-ERK1/2), but not p-p38. Taken together, these results suggest that LprG inhibits LPS-stimulated inflammation via downregulation of NO, COX-2, iNOS, and pro-inflammatory cytokines through the NF-κB, AP-1, and MAPK signaling pathways. The present study will aid in the development of anti-inflammatory medications using Mtb. The organism, which has long been regarded as a human pathogenic or human health-threating agent, can be utilized as a future medical resource.

Combination Treatment With Remdesivir and Ivermectin Exerts Highly Synergistic and Potent Antiviral Activity Against Murine Coronavirus Infection.

The recent COVID-19 pandemic has highlighted the urgency to develop effective antiviral therapies against the disease. Murine hepatitis virus (MHV) is a coronavirus that infects mice and shares some sequence identity to SARS-CoV-2. Both viruses belong to the Betacoronavirus genus, and MHV thus serves as a useful and safe surrogate model for SARS-CoV-2 infections. Clinical trials have indicated that remdesivir is a potentially promising antiviral drug against COVID-19. Using an in vitro model of MHV infection of RAW264.7 macrophages, the safety and efficacy of monotherapy of remdesivir, chloroquine, ivermectin, and doxycycline were investigated. Of the four drugs tested, remdesivir monotherapy exerted the strongest inhibition of live virus and viral RNA replication of about 2-log10 and 1-log10, respectively (at 6 µM). Ivermectin treatment showed the highest selectivity index. Combination drug therapy was also evaluated using remdesivir (6 µM) together with chloroquine (15 µM), ivermectin (2 µM) or doxycycline (15 µM) - above their IC50 values and at high macrophage cell viability of over 95%. The combination of remdesivir and ivermectin exhibited highly potent synergism by achieving significant reductions of about 7-log10 of live virus and 2.5-log10 of viral RNA in infected macrophages. This combination also resulted in the lowest cytokine levels of IL-6, TNF-α, and leukemia inhibitory factor. The next best synergistic combination was remdesivir with doxycycline, which decreased levels of live virus by ~3-log10 and viral RNA by ~1.5-log10. These results warrant further studies to explore the mechanisms of action of the combination therapy, as well as future in vivo experiments and clinical trials for the treatment of SARS-CoV-2 infection.

Anti-Oxidant and Anti-Inflammatory Substance Generated Newly in Paeoniae Radix Alba Extract Fermented with Plant-Derived Lactobacillus brevis 174A.

Fermentation of medicinal herbs can be a significant technique to obtain bioactive compounds. Paeoniae Radix (PR) used in the present study is a well-known herbal medicine that exhibits anti-inflammatory and immunomodulatory activity. The aim of this study is to explore the possibility that a bioactive compound is newly generated in PR extract by fermentation with a plant-derived lactic acid bacteria Lactobacillus brevis 174A. We determined the anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The PR extract fermented with Lactobacillus brevis 174A markedly increased the total phenolic content, decreased intracellular ROS levels, inhibited the release of nitric oxide (NO). It also suppressed inflammatory cytokines IL-6, TNF-ɑ, while simultaneously downregulating the gene expressions of iNOS, IL-6, TNF-ɑ, and IL-1ß compared to the unfermented PR extract. Furthermore, the bioactive compound newly generated from the fermentation was identified as pyrogallol. It inhibits the inflammatory responses in a dose-dependent manner suggesting that fermentation of the herbal extract used as a medium together with the plant-derived lactic acid bacterial strain may be a practical strategy to produce medicines and supplements for healthcare.

Metabolic Conversion and Removal of Manganese Ferrite Nanoparticles in RAW264.7 Cells and Induced Alteration of Metal Transporter Gene Expression.

BACKGROUND: Manganese Ferrite Nanoparticles (Mn-IONPs) are widely used in biomedical field and their cytotoxicity has been initially explored, but the mechanism remains obscure. The nano-bio interactions are believed to be crucial for cytotoxicity mechanism, while little data have been acquired. METHODS: Mn-IONPs were synthesized by thermal decomposition of acetylacetonate precursor. After physicochemical characterization, we analyzed the metabolic conversion and removal of Mn-IONPs in RAW264.7 cells by Prussian blue staining, TEM, HRTEM and elemental quantitative analysis, followed by gene expression evaluation using quantitative RT-PCR. RESULTS: Mn-IONPs were successfully synthesized. Both the uptake and cytotoxicity of Mn-IONPs on RAW264.7 cells were time- and dose-dependent. After internalized, Mn-IONPs were passed to daughter cells with passages on. Meanwhile, Mn-IONPs were exocytosed and digested to metal ions and further excreted out, resulted in the labeling rate and ions contents decreased gradually. As ion influx related genes, the expressions of ZIP14, IRP2, FtH and DMT1 were suppressed within 24 hours but overexpressed to a plateau at the 48th hour in a dose-dependent manner. At the 72nd hour, ZIP14 and DMT1 mRNA levels decreased toward normal, while IRP2 and FtH kept up-regulated. As efflux related genes, FPN, SLC30A10 and Hamp2 genes were up-regulated within 24-72 hours; SPCA1 was suppressed at the 24th and 72nd hour, while overexpressed at the 48th hour. All the efflux related genes' mRNA had a dose-dependent increasing manner at the corresponding time points. CONCLUSION: Mn-IONPs showed time- and dose-dependent cytotoxicity and cell labeling rate in RAW264.7 cells. Accompanying with the intracellular catabolic breakdown and exocytosis of Mn-IONPs, RAW264.7 cells also secreted and re-uptook manganese and iron ions to maintain intracellular homeostasis in the succeeding passages. And the metabolic conversion of Mn-IONPs in RAW264.7 cells can affect the expression of ZIP14, DMT1, FPN, SLC30A10, IRP2, FtH, Hamp2 and SPCA1 genes.

In vitro anti-inflammatory potential of marine macromolecules cross-linked bio-composite scaffold on LPS stimulated RAW 264.7 macrophage cells for cartilage tissue engineering applications.

Biomaterials serve as an integral component of tissue engineering. They are designed to provide architectural framework of native extracellular matrix so as to encourage cell growth and eventual tissue regeneration. Naturally occurring biopolymers as scaffolds offer options for cartilage tissue engineering due to anti-inflammatory, biocompatibility, biodegradability, low toxicity of degradation by-products and plasticity in processing into a variety of material formats. Here we studied in vitro anti-inflammatory potential of marine macromolecules cross-linked bio-composite scaffold composed of hydroxyapatite, alginate, chitosan and fucoidan named as HACF on LPS stimulated RAW 264.7 macrophage cells. The effects of HACF on the viability of RAW264.7 cells, nitrite level, intracellular ROS as well as the mRNA levels of NF-κB, iNOS, COX-2, TNF-α, IL-1ß and IL-6 were examined in LPS induced RAW264.7 macrophage cells. The results revealed that HACF hydrogel scaffold exerts anti-inflammatory effect by inhibiting the production of ROS, suppress NF-kB translocation to the nucleus and thereby inhibiting the production of inflammatory mediators. Hence, our results confirm that HACF has a strong anti-oxidant capacity to inhibit inflammation associated gene expression by suppressing NF-kB signaling pathway. It clearly reveals the anti-oxidant and anti-inflammatory effect of HACF hydrogel scaffold on LPS induced RAW 264.7 cells.

Structural properties and in vitro and in vivo immunomodulatory activity of an arabinofuranan from the fruits of Akebia quinata.

In our continuous searching for natural active polysaccharides with immunomodulatory activity, an arabinofuranan (AQP70-3) was isolated and purified from the fruits of Akebia quinata (Houtt.) Decne. by using ion-exchange chromatography and gel permeation chromatography for the first time. AQP70-3 contained both α-l-Araf and ß-l-Araf, and the absolute molecular weight was 1.06 × 104 g/mol. The backbone of AQP70-3 comprised →5)-α-l-Araf-(1→, →3,5)-α-l-Araf-(1→, and →2,5)-α-l-Araf-(1→, with branches of →1)-ß-l-Arafand →3)-α-l-Araf-(1→ residues. Biological assay suggested that AQP70-3 can stimulate phagocytic activity and promote the levels of nitric oxide (NO), interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α) of RAW264.7 cells. Furthermore, AQP70-3 was found to increase the production of reactive oxygen species (ROS) and NO in zebrafish embryo model.

Isolation and characterization of ent-pimarane diterpenoids from Sigesbeckia pubescens.

Five new ent-pimarane diterpenoids ent-16-nor-2-oxopimar-8(14)-ene-15,19-dial (1), ent-16-nor-2α,19-dihydroxypimar-8-en-15-al (2), 3-O-acetyldarutigenol (3), 19-O-acetylkirenol (4), ent-16-nor-3ß,15-dihydroxypimar-8(14)-ene (5) were isolated and characterized from the ethanol extract of Sigesbeckia pubescens. Their structures were elucidated on the basis of spectroscopic analysis. The absolute configuration of C-15 in compounds 3 and 4 was assigned using Snatzke's method. All these compounds were assessed for their anti-inflammatory potential by measuring the inhibitory effects on NO production in LPS-induced RAW 264.7 macrophage cells and compound 4 showed significantly inhibitory activity with IC50 value of 5.9 µM.

Effect of Kudinoside D on ox-LDL-induced lipid accumulation and LPS-induced inflammation / 中国药理学通报

Aim To investigate the effects of Kudino- side D on lipid accumulation induced by oxidized low density lipoprotein ( ox-LDL) and inflammation induced by lipopolysaccharide ( LPS ) in RAW264.7 cells.Methods Foam cells were established by incubating the RAW264.7 cells with ox-LDL.The concentration of lipid droplets in the cells was observed by oil red staining, and the level of total cholesterol (TC) in cells was measured by enzyme method.The gene and protein expressions of scavenger receptors CD36 and SR-A1, ATP binding cassette transporters A1 and Gl ( ABCA1 and ABCGI) were detected by RT-qPCR and Western blot, respectively.The expressions of inter- leukin-6 (IL-6), interleukin-1 (3 (IL-ip), monocyte chemoattractant protein-1 (MCP-1 ) and tumor necrosis factor-a (TNF-a) were detected by ELISA and RT-qPCR.The protein expressions of mTOR and p-mTOR were detected by Western blot.Results Compared with model group, the high dose of Kudinoside D decreased the content of TC and down-regulated the gene and protein expression of SR-A induced by ox-LDL.Meanwhile Kudinoside D also decreased the levels of IL-ip and MCP-1 and down-regulated the protein expression of p-mTOR induced by LPS.Conclusions Kudinoside D may reduce the intracellular TC content by down-regulating the gene and protein expression of SR-A1.Kudinoside D may play an anti-inflammatory role through mTOR pathway.

Regucalcin enhances adipocyte differentiation and attenuates inflammation in 3T3-L1 cells.

Dysregulation of adipocyte differentiation and dysfunction play key roles in the pathogenesis of obesity and associated disorders such as diabetes and metabolic syndrome, and as such, a better understanding of the molecular mechanism of adipogenesis may help to elucidate the pathological condition of obesity and its associated disorders. Regucalcin (RGN) plays multiple regulatory roles in intracellular Ca2+ signaling pathways in mammalian cells. Here, we report that overexpression of RGN enhances lipid accumulation in 3T3-L1 adipocyte cells after adipogenic stimulation, accompanied by upregulation of adipocyte differentiation marker proteins. In contrast, genetic disruption of RGN inhibited adipogenic stimulation-induced differentiation of 3T3-L1 cells. Furthermore, RGN overexpression in differentiated 3T3-L1 adipocytes blocked inflammatory crosstalk between 3T3-L1 adipocytes and RAW264.7 macrophages in a transwell coculture system. Knockdown of RGN expression in cocultured 3T3-L1 adipocytes enhanced their susceptibility to RAW264.7 macrophage-mediated inflammation. These results suggest that RGN is required for 3T3-L1 adipocyte differentiation and that it exerts anti-inflammatory activity against 3T3-L1 adipocyte inflammation after coculture with RAW264.7 macrophages. Thus, RGN may be a novel regulator of adipocyte differentiation and act as a suppressor of inflammation in macrophage-infiltrated adipocyte tissue.

Seven new chemical constituents from the roots of Gentiana macrophylla pall.

Gentimilegenins A, B (1, 2), (6R, 8R)-6-hydroxy swerimuslactone A (3), (6R, 8S)-6-hydroxy swerimuslactone A (4), 4-hydroxy roburic acid methyl ester (5), (±) 3'-hydroxy gentioxepine (6), N-heptacosanoyl anthranilic acid (7a), N-nonacosanoyl anthranilic acid (7b), together with 40 known compounds were isolated from the roots of Gentiana macrophylla Pall. Their structures were elucidated on the basis of comprehensive analysis of HRESIMS, IR, 1D-, 2D-NMR and X-ray diffraction. The anti-inflammatory effects of selected compounds were also evaluated through the detection of their inhibitory effects on NO production in LPS-induced RAW264.7 macrophage cells.

Mycobacterium tuberculosis-Secreted Protein, ESAT-6, Inhibits Lipopolysaccharide-Induced MMP-9 Expression and Inflammation Through NF-κB and MAPK Signaling in RAW 264.7 Macrophage Cells.

-20pt?>Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes contagious tuberculosis (TB). Recently, Mtb-secreted proteins have been considered virulence factors and candidates for drugs and vaccines. Among these proteins, 6-kDa early secreted antigenic target (ESAT-6) is known to be able to induce component of matrix metalloproteinase-9 (MMP-9) in epithelial cells, leading to recruitment of macrophages. However, detailed function of ESAT-6 during macrophage recruitment to inflammatory sites remains unknown. Thus, the objective of the present study was to elucidate such function of EAST-6 and mechanism(s) involved. In the present study, we have found that recombinant ESAT-6 purified in the form of ESAT-6 double-connected structure (2E6D) could inhibit lipopolysaccharide (LPS)-induced potential of cell migration and inflammation in murine macrophage cells. Interestingly, 2E6D suppressed LPS-induced MMP-9 expression at both protein and mRNA levels as well as its enzyme activity. Levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes as known upregulators of MMP-9 were significantly decreased when 2E6D has been treated. In addition, nitric oxide (NO) as a second messenger was also significantly decreased by treatment with the purified 2E6D. Furthermore, 2E6D inhibited LPS-induced phosphorylation of IκB and translocation of NF-κB. Moreover, 2E6D suppressed phosphorylation of MAPK signaling proteins. Taken together, these results suggest that ESAT-6 can suppress LPS-induced MMP-9 and inflammation by downregulating COX-2, iNOS, and NO through NF-κB and MAPK signaling.

Anti-Inflammatory Activity of Some Characteristic Constituents from the Vine Stems of Spatholobus suberectus.

The dried vine stems of Spatholobus suberectus are commonly used in traditional Chinese medicine for treating gynecological and cardiovascular diseases. In this study, five new compounds named spasuberol A (2), homovanillyl-4-oxo-nonanoate (5), spasuberol C (6), spasuberoside A (14), and spasuberoside B (15), together with ten known compounds (1, 3, 4, 7-13), were isolated from the dried vine stems of S. suberectus. Their chemical structures were analyzed using spectroscopic assays. This is the first study interpreting the detailed structural information of 4. The anti-inflammatory activity of these compounds was evaluated by reducing nitric oxide overproduction in RAW264.7 macrophages stimulated by lipopolysaccharide. Compounds 1 and 8-10 showed strong inhibitory activity with half maximal inhibitory concentration (IC50) values of 5.69, 16.34, 16.87, and 6.78 µM, respectively, exhibiting higher activity than the positive drug l-N6-(1-iminoethyl)-lysine (l-NIL) with an IC50 value of 19.08 µM. The IC50 values of inhibitory activity of compounds 2 and 4-6 were 46.26, 40.05, 45.87, and 28.29 µM respectively, which were lower than l-NIL, but better than that of positive drug indomethacin with an IC50 value of 55.44 µM. Quantitative real-time polymerase chain reaction analysis revealed that assayed compounds with good anti-inflammatory activity, such as 1, 6, 9, and 10 at different concentrations, can reduce the messenger RNA (mRNA) expression of some pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2). The anti-inflammatory activity and the possible mechanism of the compounds mentioned in this paper were studied preliminarily.

Investigation on activation in RAW264.7 macrophage cells and protection in cyclophosphamide-treated mice of Pseudostellaria heterophylla protein hydrolysate.

Our previous study has demonstrated that Pseudostellaria heterophylla protein hydrolysate (PPH) has immunomodulatory activity on murine spleen lymphocytes. The aim of this study was to investigate the excitation of PPH in RAW264.7 macrophage cells and the protective effect in cyclophosphamide (CTX)-treated mice. The results showed PPH of 50 µg/mL could stimulate macrophages resulting in significant promotions of nitric oxide (NO) production, endocytosis and reactive oxygen species formation. Meanwhile, enzyme-linked immunosorbent assay (ELISA) revealed that the levels of tumor necrosis factor-α and interleukin-10 were significantly upregulated by PPH. Furthermore, 50 mg/kg per day PPH restored the T lymphocyte proliferation and natural killer cell activity, and increased NO production and pinocytosis of peritoneal macrophages in CTX-treated mice. These findings indicate PPH plays a crucial role in RAW264.7 macrophage cells activation and in the protection against immunosuppression in CTX-treated mice and could be used as a potential immunostimulant agent.