Pathway In RAW264 Research Articles

Cytotoxicity and Autophagy Induced by Ivermectin via AMPK/mTOR Signaling Pathway in RAW264.7 Cells.

The widespread and excessive use of ivermectin (IVM) will not only cause serious environmental pollution, but will also affect metabolism of humans and other mammals that are exposed. IVM has the characteristics of being widely distributed and slowly metabolized, which will cause potential toxicity to the body. We focused on the metabolic pathway and mechanism of toxicity of IVM on RAW264.7 cells. Colony formation and LDH detection assay showed that IVM significantly inhibited the proliferation of and induced cytotoxicity in RAW264.7 cells. Intracellular biochemical analysis using Western blotting assay showed that LC3-B and Beclin-1 were upregulated and p62 was down-regulated. The combination of confocal fluorescence, calcein-AM/CoCl2, and fluorescence probe results showed that IVM could induce the opening of the mitochondrial membrane permeability transition pore, reduce mitochondrial content, and increase lysosome content. In addition, we focused on induction of IVM in the autophagy signal pathway. The Western blotting results showed that IVM increased expression of p-AMPK and decreased p-mTOR and p-S6K expression in protein levels, indicating that IVM activated the AMPK/mTOR signaling pathway. Therefore, IVM may inhibit cell proliferation by inducing cell cycle arrest and autophagy.

Synthetic Glabridin Derivatives Inhibit LPS-Induced Inflammation via MAPKs and NF-κB Pathways in RAW264.7 Macrophages

Glabridin is a polyphenolic compound with reported anti-inflammatory and anti-oxidative effects. In the previous study, we synthesized glabridin derivatives-HSG4112, (S)-HSG4112, and HGR4113-based on the structure-activity relationship study of glabridin to improve its biological efficacy and chemical stability. In the present study, we investigated the anti-inflammatory effects of the glabridin derivatives in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. We found that the synthetic glabridin derivatives significantly and dose-dependently suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2), and decreased the level of inducible nitric oxygen synthase (iNOS) and cyclooxygenase-2 (COX-2) and the expression of pro-inflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α). The synthetic glabridin derivatives inhibited the nuclear translocation of the NF-κB by inhibiting phosphorylation of the inhibitor of κB alpha (IκB-α), and distinctively inhibited the phosphorylation of ERK, JNK, and p38 MAPKs. In addition, the compounds increased the expression of antioxidant protein heme oxygenase (HO-1) by inducing nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) through ERK and p38 MAPKs. Taken together, these results indicate that the synthetic glabridin derivatives exert strong anti-inflammatory effects in LPS-stimulated macrophages through MAPKs and NF-κB pathways, and support their development as potential therapeutics against inflammatory diseases.

Cirsium japonicum var. maackii fermented with Pediococcus pentosaceus induces immunostimulatory activity in RAW 264.7 cells, splenocytes and CTX-immunosuppressed mice

Cirsium japonicum var. maackii (CJ), a perennial herb from the Compositae family, is a traditional antihypertensive, uretic antihepatitis, and antihemorrhagic medicine in Korea. However, CJ reported has minimal immune-enhancing effects. We investigated whether fermentation with Pediococcus pentosaceus (P. p) increases the immune efficacy of CJ through in vitro, ex vivo and in vivo. Results showed that CJ and P. p fermentation (PCJ) produced nitric oxide, induced immune-related cytokine secretion, and up-regulated the MAPK and NF-κB signaling pathways in RAW 264.7 cells. Furthermore, PCJ markedly stimulated proliferation and Th1 and Th2 response by inducing the production of cytokines in splenocytes. Oral administration of PCJ significantly alleviated cyclophosphamide (CTX)-induced immunosuppression in mice, as evidenced by increased immune-related cytokine production and improved gut microbiota. These results suggest that PCJ could have a positive impact on the development of functional food with the ability on immunomodulation and intestinal microenvironment improvement.

Boris knockout eliminates AOM/DSS-induced in situ colorectal cancer by suppressing DNA damage repair and inflammation.

The Brother of Regulator of Imprinted Sites (BORIS, gene symbol CTCFL) has previously been shown to promote colorectal cancer cell proliferation, inhibit cancer cell apoptosis, and resist chemotherapy. However, it is unknown whether Boris plays a role in the progression of in situ colorectal cancer. Here Boris knockout (KO) mice were constructed. The function loss of the cloned Boris mutation that was retained in KO mice was verified by testing its activities in colorectal cell lines compared with the Boris wild-type gene. Boris knockout reduced the incidence and severity of azoxymethane/dextran sulfate-sodium (AOM/DSS)-induced colon cancer. The importance of Boris is emphasized in the progression of in situ colorectal cancer. Boris knockout significantly promoted the phosphorylation of γH2AX and the DNA damage in colorectal cancer tissues and suppressed Wnt and MAPK pathways that are responsible for the callback of DNA damage repair. This indicates the strong inhibition of colorectal cancer in Boris KO mice. By considering that the DSS-promoted inflammation contributes to tumorigenesis, Boris KO mice were also studied in DSS-induced colitis. Our data showed that Boris knockout alleviated DSS-induced colitis and that Boris knockdown inhibited the NF-κB signaling pathway in RAW264.7 cells. Therefore Boris knockout eliminates colorectal cancer generation by inhibiting DNA damage repair in cancer cells and relieving inflammation in macrophages. Our findings demonstrate the importance of Boris in the development of in situ colorectal cancer and provide evidence for the feasibility of colorectal cancer therapy on Boris.

Ginsenoside Rg1 attenuates dextran sodium sulfate-induced ulcerative colitis in mice

Ulceration colitis (UC) is a chronic and recurrent inflammatory disorder in the gastro-intestinal tract. The purpose of our study is to explore the potential mechanisms of ginsenoside Rg1 (GS Rg1) on dextran sulfate sodium (DSS)-induced colitis in mice and lipopolysaccharide (LPS)-induced RAW 264.7 cells. Acute colitis was induced in male C57BL/6 mice. In vitro model of LPS-induced RAW 264.7 cells to simulate enteritis model. The disease activity index (DAI), colon length, body weight and histopathological analysis were performed in vivo. Pro-inflammatory cytokines and markers for oxidative and anti-oxidative stress, MPO level were measured in vivo and in vitro. Nuclear erythroid 2-related factor 2 (Nrf2) and NF-κB p65 protein levels were analyzed using western blotting. Our results indicated that the UC models were established successfully by drinking DSS water. GS Rg1 significantly attenuated UC-related symptoms, including preventing weight loss, decreasing DAI scores, and increasing colon length. GS Rg1 ameliorated the DSS-induced oxidative stress. IL-1β, IL-6, and TNF-α levels were significantly increased in serum and cell supernatant effectively, while treatment with the GS Rg1 significantly reduced these factors. GS Rg1 reduced MPO content in the colon. GS Rg1 treatment increased SOD and decreased MDA levels in the serum, colon, and cell supernatant. GS Rg1 restored the Nrf-2/HO-1/NF-κB pathway in RAW 264.7 cells and UC mice, and these changes were blocked by Nrf-2 siRNA. Overall, GS Rg1 ameliorated inflammation and oxidative stress in colitis via Nrf-2/HO-1/NF-κB pathway. Thus, GS Rg1 could serve as a potential therapeutic agent for the treatment of UC.

Astragaloside IV Targets Macrophages to Alleviate Renal Ischemia-Reperfusion Injury via the Crosstalk between Hif-1α and NF-κB (p65)/Smad7 Pathways.

(1) Background: Astragaloside IV (AS-IV) is derived from Astragalus membranous (AM), which is used to treat kidney disease. Macrophages significantly affect the whole process of renal ischemia-reperfusion (I/R). The regulation of macrophage polarization in kidneys by AS-IV was the focus. (2) Methods: Renal tubular injury and fibrosis in mice were detected by Hematoxylin and Eosin staining and Masson Trichrome Staining, separately. An ELISA and quantitative real-time polymerase chain reaction were used to explore the cytokine and mRNA expression. Western blot was used to determine protein expression and siRNA technology was used to reveal the crosstalk of signal pathways in RAW 264.7 under hypoxia. (3) Results: In the early stages of I/R injury, AS-IV reduced renal damage and macrophage infiltration. M1-associated markers were decreased, while M2 biomarkers were increased. The NF-κB (p65)/Hif-1α pathway was suppressed by AS-IV in M1. Moreover, p65 dominated the expression of Hif-1α. In the late stages of I/R injury, renal fibrosis was alleviated, and M2 infiltration also decreased after AS-IV treatment. Hif-1α expression was reduced by AS-IV, while Smad7 expression was enhanced. Hif-1α interferes with the expression of Smad7 in M2. (4) Conclusions: AS-IV promoted the differentiation of M1 to M2, relieving the proinflammatory response to alleviate the kidney injury during the early stages. AS-IV attenuated M2 macrophage infiltration to prevent kidney fibrosis during the later stages.

Salvianolic Acid B Reduces the Inflammation of Fat Grafts by Inhibiting the NF-Kb Signalling Pathway in Macrophages.

Autologous fat grafting is a common method for soft tissue defect repair. However, the high absorption rate of transplanted fat is currently a bottleneck in the process. Excessive inflammation is one of the main reasons for poor fat transplantation. Salvianolic acid B (Sal-B) is a herbal medicine that shows promise for improving the effectiveness of fat transplantation. The aim of this study was to improve fat graft survival by injecting Sal-B into fat grafts locally. In vivo, 0.2 mL of Coleman fat was transplanted into nude mice along with Sal-B. The grafts were evaluated by histologic analysis at 2, 4, and 12 weeks posttransplantation and by microcomputed tomography at 4 weeks posttransplantation. In vitro ribonucleic acid sequencing, cell proliferation assays, anti-inflammatory activity assays, molecular docking studies, and kinase activity assays were performed in RAW264.7 cells to detect the potential mechanism. Sal-B significantly improved fat graft survival and attenuated adipose tissue fibrosis and inflammation. Sal-B also inhibited the polarization of M1 macrophages in fat grafts. In vitro, Sal-B inhibited the proliferation and activation of inflammatory pathways in RAW264.7 cells. In addition, Sal-B had an inhibitory effect on NF-κB (nuclear factor κ light polypeptide gene enhancer in B cells) signaling. This bioactivity of Sal-B may result from its selective binding to the kinase domain of the inhibitor of NF-κB kinase subunit β. Sal-B could serve as a promising agent for improving the effect of fat transplantation by inhibiting the polarization of M1 macrophages through NF-κB signaling.

The polysaccharide from Camellia oleifera fruit shell enhances immune responses via activating MAPKs and NF-κB signaling pathways in RAW264.7 macrophages.

Macrophage plays an important role in innate immune responses by secreting immune molecules and phagocytosis. Camellia oleifera fruit shell, accounting for approximately 60% weight of the single C. oleifera fruit, is rich in polysaccharides and has several biological activities such as anti-oxidation, lipid regulation and anticancer. However, the immunomodulatory activity of the polysaccharide from C. oleifera fruit shells (CPS) has not been reported. This study aimed to investigate the immunomodulatory activities and mechanisms of CPS in RAW264.7 macrophages. The Methyl Thiazolyl Tetrazolium (MTT) method was used to evaluate the effect of CPS on the cell viability of RAW264.7 macrophages, and cell morphology was pictured using microscope. The production of immune-related molecules, including nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor α (TNFα), interleukin (IL)-1β and IL-6, was detected by Griess assay and enzyme-linked immunosorbent assay (ELISA). The protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) and the phosphorylation level of mitogen-activated protein kinases (MAPKs) were analyzed through western blotting. The mRNA levels of related genes were tested using reverse transcription-polymerase chain reaction (RT-PCR). The nuclear translocation of nuclear factor-kappa B (NF-κB) was detected using immunofluorescence technology. The results indicated that CPS treatment stimulated the production of NO and PGE2 and significantly enhanced the protein expression of iNOS and COX2 with little effect on the cell morphology and viability. Also, the secretion and mRNA levels of TNFα were increased by the treatment of CPS. In addition, CPS treatment markedly upregulated the phosphorylation level of MAPKs including Extracellular Signal Regulated Kinase (ERK), P38, and c-Jun N-terminal Kinase (JNK) at different time points and caused the activation and nuclear translocation of NF-κB. Our data implied that CPS exerts immunomodulatory activities by activating MAPKs and NF-κB signaling pathways in RAW264.7 macrophages.

Ajania purpurea Extract Attenuates LPS-Induced Inflammation in RAW264.7 Cells and Peritonitis Mice.

Macrophages have important roles in the progression of inflammation. Ajania purpurea Shih. is a member of the Ajania Poljakor family that grows in Tibet (China). Extracts from plants in this genus have anti-bacterial and anti-inflammatory properties. However, there are few reports on the activity and mechanism of Ajania purpurea. Here, we confirmed the anti-inflammatory effect of Ajania purpurea Shih. ethanol extract (EAPS) by examining the levels of inflammatory factors in a mouse model of peritonitis and RAW264.7 cells. The main components of EAPS detected by LC-MS analysis included piperine and chlorogenic acid. In particular, in lipopolysaccharide (LPS)-induced RAW264.7 cells, EAPS inhibited the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-induced RAW264.7 cells, lowered the levels of nitric oxide (NO) and prostaglandin E2 (PGE2), as well as the release of inflammatory factors such as tumor necrosis factor-alpha (TNF-α) and pro-inflammatory cytokines such as interleukin (IL)-1β and IL-6. In addition, Western blot analysis and immunofluorescence staining verified that EAPS inhibited the activity of the nuclear factor-kappaB (NF-κB) pathway by reducing the nuclear translocation of the p65 subunit. Furthermore, in a mouse model of peritonitis, EAPS inhibited the release of inflammatory factors, as well as the recruitment of immune cells including neutrophils and macrophages. These findings indicated that EAPS suppressed LPS-induced inflammation via inhibiting the NF-κB pathway in RAW264.7 cells and mice with peritonitis. Thus, EAPS may be a viable therapeutic method for the treatment of inflammation and related disorders.

3ʹ,4ʹ-Dihydroxyflavone mitigates inflammatory responses by inhibiting LPS and TLR4/MD2 interaction

BackgroundWe previously reported the potential inhibitory activity of 3ʹ,4ʹ-dihydroxyflavone (DHF) on nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated macrophages. PurposeWe investigated the underlying molecular mechanisms of DHF in LPS-activated macrophages and evaluated its effect on LPS-induced septic shock in mice. MethodsTo explore the anti-inflammatory effect of DHF, nitrite, PGE2, and cytokines were measured in vitro and in vivo experiments. In addition, to verify the molecular signaling pathway, quantitative real time-PCR, luciferase assay, nuclear extraction, electrophoretic mobility shift assay, immunocytochemistry, immunoprecipitation, molecular docking analysis, and myeloid differentiation 2 (MD2)-LPS binding assay were conducted. ResultsDHF suppressed the LPS-induced expression of proinflammatory mediators through nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor 3 (IRF3) inactivation pathways in RAW 264.7 macrophages. Importantly, molecular docking analysis and in vitro binding assays showed that DHF interacts with the hydrophobic pocket of MD2 and then interferes with the interaction between LPS and toll-like receptor 4 (TLR4). DHF inhibited LPS-induced oxidative stress by upregulating nuclear factor erythroid 2-related factor 2 (Nrf2). Treatment of LPS-induced endotoxemia mice with DHF reduced the expression levels of pro-inflammatory mediators via the inactivation of NF-κB, AP-1, and signal transducer and activator of transcription 1 (STAT1) in the lung tissue, thus increasing the survival rate. ConclusionTaken together, our data first time revealed the underlying mechanism of the DHF-dependent anti-inflammatory effect by preventing LPS from binding to the TLR4/MD2 complex. Therefore, DHF may be a possible anti-inflammatory agent for the treatment of LPS-mediated inflammatory diseases.

Regulatory effect of isovitexin on MAPK/NF-κB signal in mice with acute ulcerative colitis

The aim of this study was to investigate the anti-inflammatory effects and mechanism of isovitexin on ulcerative colitis mice and RAW264.7 cells. The results showed that isovitexin had strong antioxidant and anti-inflammatory effects, and could restore intestinal barrier integrity (p < 0.01). In addition, isovitexin inhibited the expression of MyD88, TLR4 and NF-κB p65 proteins. At the same time, isovitexin can inhibit the activation of MAPK/NF-κB signaling pathway in RAW264.7 cells. In conclusion, isovitexin has a protective effect on UC mice, and its improvement mechanism of UC might be related to MAPK/NF-κB signaling 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 action of ark shell (Scapharca subcrenata) protein hydrolysate in LPS-stimulated RAW264.7 murine macrophages.

Potential anti-inflammatory effects of ark shell (Scapharca subcrenata) protein hydrolysates were investigated. Ark shell protein hydrolysates were prepared using Alcalase® and pepsin and were designated ASAH and ASPH, respectively. The nitric oxide (NO) inhibitory activity of ASAH and ASPH was determined in lipopolysaccharides (LPS)-stimulated RAW264.7 murine macrophages, and the results showed that ASAH inhibited better NO inhibitory activity than ASPH. ASAH suppressed inflammatory mediator, a prostaglandin E2, secretion of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), and production of reactive oxygen species (ROS) dose dependently. It inhibited the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and simulated heme oxygenase-1 (HO-1) protein expression. However, the pharmacological approach revealed that pretreatment with zinc protoporphyrin ІX (ZnPP), an inhibitor of HO-1, reversed the anti-inflammatory effect of ASAH. Moreover, ASAH upregulated phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK1/2, and p38 MAPK. To find out the role of MAPKs phosphorylation, MAPKs inhibitors were used, and the results showed that ASAH-mediated HO-1 protein expression and Nrf2 nuclear translocation were abolished. Taken all together, this study revealed that ASAH has a potential anti-inflammatory activity through regulation of the MAPK-dependent HO-1/Nrf2 pathway. PRACTICAL APPLICATIONS: Food-derived marine bioactive peptides, due to their pivotal role in biological activities, are gaining much attention recently. However, the anti-inflammatory activities of ark shell protein hydrolysates still remain to be investigated. This study investigated that ASAH shows potential anti-inflammatory activities through regulation of the MAPK-dependent HO-1/Nrf2 pathway in RAW264.7 murine macrophages. These findings indicated that ASAH may be used as a dietary supplement, functional food, and medicinal drug for the management of inflammation and inflammation-associated diseases.

LncRNA H19 via miR-29a-3p is involved in lung inflammation and pulmonary fibrosis induced by neodymium oxide

The occupational and environmental health safety of rare earths has attracted considerable attention. In China, the rare earth neodymium oxide (Nd2O3) is extensively refined and utilized. However, the mechanisms of Nd2O3-induced lung injury are elusive. In the present study, we found that exposure of mice to Nd2O3 caused an inflammatory reaction and fibrosis in lung tissues, which was in relation to the Nd2O3-induced higher levels of the lncRNA H19 (H19), tumor necrosis factor receptor 1 (TNFRSF1A), p-p65, and p-IKKβ and lower levels of miR-29a-3p. Further, in mouse monocyte macrophage leukemia cells (RAW264.7), Nd2O3 induced an inflammatory reaction, increases of H19 and TNFRSF1A levels, decreases of miR-29a-3p levels, and activation of the nuclear factor (NF)-κB signaling pathway. Further, we established that miR-29a-3p regulates TNFRSF1A expression. Up-regulation of miR-29a-3p and down-regulation of H19 blocked the Nd2O3-induced secretion of TNF-α, MIP-1α, and IL-6; the increases of TNFRSF1A levels; and activation of the NF-κB signaling pathway in RAW264.7 cells. Further, in Nd2O3-treated RAW26.4 cells, H19 inhibited the expression of miR-29a-3p, which targets TNFRSF1A, and activated the NF-κB signaling pathway to enhance the expression of TNF-α, MIP-1α, and IL-6. Moreover, for mice, up-regulation of miR-29a-3p reversed lung tissue inflammation, pulmonary fibrosis, and activation of the NF-κB signaling pathway induced by Nd2O3. In sum, the present investigation shows that H19 via miR-29a-3p is involved in lung inflammation and pulmonary fibrosis induced by Nd2O3, which is a mechanism for the Nd2O3-induced lung inflammatory response and pulmonary fibrosis. This information is useful for development of a biomarker of Nd2O3-induced lung injury.

Ruxolitinib-loaded black phosphorus nanosheets actively target proximal tubule cells to ameliorate nephrotic syndrome by reducing inflammation and oxidative stress

Nephrotic syndrome (NS) is one of the most common pediatric kidney diseases characterized by massive proteinuria and generalized edema that accounts for considerable mortality. Although the etiology of NS is not yet fully understood, altered mitochondrial metabolism, higher levels of oxidative stress, and chronic inflammation are considered the main causes of NS. Thus, reducing the level of reactive oxygen species (ROS) and inhibiting inflammation may effectively improve NS. In this study, the small molecule compound ruxolitinib phosphate salt (RP) was identified and we found it could improve NS by inhibiting cellular inflammation and reducing ROS production to exert a cytoprotective effect, but it also had some side effects. A novel two-dimensional material, black phosphorus nanosheets (BPNSs), was used to efficiently load RP and proximal tubule cells (PTCs) targeting peptide G3-C12 through electrostatic interaction. The modification of G3-C12 promoted cellular uptake of BPNSs, and the endocytosis of BPNSs@G3-C12 was primarily via micropinocytosis. RP@BPNSs@G3-C12 actively targeted PTCs and released RP in a pH-responsive manner in the acidic microenvironment of inflammation. It inhibited LPS-triggered activation of the NF-κB and JNK signaling pathways in RAW264.7 cells, and reduced the production of inflammatory factors such as NO, TNF-α, and IL-6. RP@BPNSs@G3-C12 further exhibited cytoprotective effect against doxorubicin damage, oxidative stress and apoptosis. The anti-apoptotic effect may be mainly through the Caspase 3 signaling pathway. Moreover, RP@BPNSs@G3-C12 treatment could also reduce renal inflammation, improve renal injury, apoptosis and oxidative stress in vivo, thereby improve renal functions of NS patients.

Piperine Derived from Piper nigrum L. Inhibits LPS-Induced Inflammatory through the MAPK and NF-κB Signalling Pathways in RAW264.7 Cells.

Piperine, an important natural product, has a good anti-inflammatory effect. However, few researchers have studied its mechanism in these pathways. The objective of this research was to evaluate the molecular mechanism underlying the anti-inflammatory responses of piperine in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The purification and characterization of piperine from Piper nigrum L. were determined by HPLC, UPLC-Q-TOF-MS and 1H NMR. Then, the anti-inflammatory activity was evaluated by a reagent test kit, ELISA kits, RT-PCR and Western blot experiments. The results suggested that piperine (90.65 ± 0.46% purity) at a concentration of 10–20 mg/L attenuated the production of NO and ROS, downregulated the protein and mRNA expression levels of TNF-α, IL-1β and IL-6, and upregulated the protein and mRNA transcription levels of IL-10. Meanwhile, the Western blot results indicated that piperine could inhibit the phosphorylation levels of the ERK, JNK, p38 and p65 proteins. Our findings suggest that piperine is a potential anti-inflammatory substance, whose molecular mechanism may be to regulate the key factors of the NF-κB and MAPK signalling pathways.

Anti-inflammatory and antioxidant activities of methanol extract of Piper betle Linn. (Piper betle L.) leaves and stems by inhibiting NF-κB/MAPK/Nrf2 signaling pathways in RAW 264.7 macrophages

Oxidative stress and chronic inflammation are closely linked to various diseases. However, previous studies have demonstrated that plant extracts could prevent and alleviate these adverse outcomes. Piper betle Linn. (Piper betle L.) is a cosmopolitan plant that belongs to the Piperaceae family, whose leaves are edible and possess several health benefits. This study sought to characterize the anti-inflammatory and antioxidant effects of a methanol extract of Piper betle L. leaves and stems (MPBLLS). MPBLLS was found to have a dose-dependent radical scavenging effect, as demonstrated by the 2,2-diphenyl-1-picrylhydrazyl assay. Additionally, MPBLLS inhibited the lipopolysaccharide (LPS)-stimulated production of nitric oxide and prostaglandin E2 by reducing the expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 macrophages without affecting cell viability. Furthermore, our findings suggested that the inhibitory effects of MPBLLS on pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and interleukin-6 were due to the inhibition of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in LPS-treated RAW 264.7 macrophages. MPBLLS and hydroxychavicol, a major constituent of MPBLLS, suppressed LPS-induced translocation of NF-κB p65 from cytoplasm to nucleus. Interestingly, MPBLLS increased nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels and transcription levels of Nrf2 target genes in a dose-dependent manner. Collectively, our findings suggest that MPBLLS could serve as a basis for the development of novel orally-administered therapies due to its inhibitory effects on oxidative and inflammatory stress. Data AvailabilityThe data presented in this study are available on request from the corresponding author.

Discovery of Pyrrolo[2,3-d]pyrimidine derivatives as potent and selective colony stimulating factor 1 receptor kinase inhibitors

Colony stimulating factor 1 receptor kinase (CSF1R) plays an integral role in tumor-associated macrophage repolarization and has emerged as a novel therapeutic target for cancer immunotherapy. Most of the current CSF1R kinase inhibitors lack selectivity between CSF1R kinase and other type III growth factor receptor members. Herein, we report a potent and selective CSF1R inhibitor 18h, which displays an IC50 value of 5.14 nM against CSF1R and achieves selectivity over other type III receptor tyrosine kinases (>38-fold). 18h inhibits the phosphorylation of CSF1R and its downstream signaling pathway in RAW264.7, THP-1, and M–NFS–60 cells. Treatment with this compound leads to alteration of the macrophage polarization in RAW264.7 macrophages in a dose-dependent manner. In vivo, 18h demonstrates acceptable pharmacokinetic profiles and suppresses the tumor growth in a mouse xenograft model inoculated with M–NFS–60 cells.

Effects of Sparassis latifolia neutral polysaccharide on immune activity via TLR4-mediated MyD88-dependent and independent signaling pathways in RAW264.7 macrophages.

BackgroundSparassis latifolia (S. latifolia) is a precious edible fungus with multiple biological activities. To date, no study has been investigated the underlying molecular mechanism of immunoregulation caused by the neutral polysaccharide of S. latifolia.Materials and methodsTo investigate immunomodulatory mechanism of S. latifolia neutral polysaccharide (SLNP), SLNP was obtained from S. latifolia and its structure, immune receptors and regulation mechanism were studied.ResultsS. latifolia neutral polysaccharide consisted of arabinose, galactose, glucose, xylose, and mannose with a molar ratio of 6:12:63:10:5. SLNP was a pyran polysaccharide with a relative molecular weight of 3.2 × 105 Da. SLNP promoted the proliferation of RAW264.7, which further induced the secretions of nitric oxide, TNF-α, IL-6, and IFN-β, and upregulated the immune receptor TLR4 expression. Moreover, SLNP increased remarkably the levels of TRAF6, IRF3, JNK, ERK, p38, and p38 mRNA and protein mediated by TLR4.ConclusionS. latifolia neutral polysaccharide regulated the immune function of RAW264.7 through MyD88-dependent and -independent signaling pathways mediated by TLR4 receptor, which suggests that SLNP is a new immunomodulator.

Taxifolin attenuates inflammation via suppressing MAPK signal pathway in vitro and in silico analysis

ObjectiveTaxifolin is a natural flavonoid compound that can be isolated from onions, grapes, oranges and grapefruit. It also acts as a medicine food homology with extraordinary antioxidant and anti-inflammatory activity. This study aims to explain the protective effects and potential mechanisms of taxifolin against inflammatory reaction. MethodsLevels of interleukin (IL)-6, IL-1β and intracellular reactive oxygen species (ROS) were assessed in different time after the treatment of taxifolin in RAW264.7 cells induced by lipopolysaccharide (LPS). Subsequently, the mRNA and protein levels of inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α and the phosphorylation expression levels of the MAPK signal pathway were also evaluated. A silico analysis was used to explain the binding situation for the investigation of taxifolin and MAPK signal pathway. And then MAPK inhibitors were used to reveal the expression level of iNOS, VEGF, COX-2 and TNF-α in RAW264.7 cells. ResultsIt was demonstrated that cell inflammatory damage induced by LPS was significantly alleviated after the treatment of taxifolin. Then, the mRNA and protein levels of iNOS, VEGF, COX-2 and TNF-α were reduced and the phosphorylation expression levels of the MAPK signal pathway were down-regulated remarkably as well. In silico analysis, taxifolin could form a relatively stable combination with MAPK signal pathway. MAPK inhibitors showed increasing or decreasing effect in the mRNA levels of iNOS, VEGF, COX-2 and TNF-α, which suggesting that taxifolin down-regulated iNOS, VEGF, COX-2 and TNF-α expressions were not entirely through the MAPK pathway. ConclusionThis finding demonstrated that taxifolin improved the inflammatory responses that partly involved in the phosphorylation expression level of MAPK signal pathway in RAW264.7 cells exposed to acute stress.