Lycium barbarum Polysaccharide Regulates Activation of RAW264.7 Macrophages Through MGL/TLR Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Lycium barbarum polysaccharides （LBP） in promoting the activation of RAW264.7 macrophages. MethodRAW264.7 macrophages were stimulated with LBP at different concentrations （50， 100， 200 mg·L-1）， and those stimulated with lipopolysaccharide （LPS） at 100 μg·L-1 and galactose （Gal） at 100 mg·L-1 as positive controls. After 24 h of LBP stimulation， the cell counting kit-8 （CCK-8） was used to detect the survival rate of RAW264.7 macrophages treated with LBP （0， 50， 100， 200， 400， 800 mg·L-1）. The levels of interleukin-6 （IL-6） and interleukin-12 （IL-12） in cell culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. The protein and mRNA expression of p38 mitogen-activated protein kinase （MAPK）， extracellular signal-regulated kinase （ERK）， c-Jun N-terminal kinase （JNK）， and nuclear factor κB （NF-κB） in Toll-like receptor 4 （TLR4）/Toll-like receptor 2 （TLR2）/macrophage galactose-type lectin （MGL） pathway of RAW264.7 macrophages was detected by Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） and Western blot. ResultCCK-8 results showed that compared with the results in the blank group， the survival rate of RAW264.7 macrophages decreased in the 400， 800 mg·L-1 LBP groups （P<0.05）. ELISA results showed that compared with the blank group， 50 mg·L-1 LBP could promote the secretion of IL-12 in RAW264.7 macrophages （P<0.01）. Compared with the blank group， 100 mg·L-1 LBP and 200 mg·L-1 LBP could promote the secretion of IL-6 in RAW264.7 macrophages （P<0.05， P<0.01）. Western blot results showed that compared with the blank group， the LBP groups （50， 100， 200 mg·L-1） enhanced protein expression levels of MAPK key molecules （p-p38 MAPK， p-ERK， p-NF-κB， and p-JNK） in TLR4， TLR2， and MGL pathways （P<0.05， P<0.01）. Compared with the model group， the 200 mg·L-1 LBP group could promote the expression level of p-NF-κB protein in RAW264.7 macrophages （P<0.01）. Real-time PCR results showed that compared with the blank group， the LBP groups （50， 100， and 200 mg·L-1） enhanced the mRNA expression levels of MAPK key molecules （p38 MAPK， ERK， NF-κB， and JNK） in TLR4 and TLR2 pathways （P<0.05， P<0.01）. Compared with the model group， the 50 and 200 mg·L-1 LBP groups could promote the mRNA expression levels of JNK and ERK2 in RAW264.7 macrophages （P<0.05， P<0.01）. ConclusionLBP can regulate the activation of RAW264.7 macrophages and participate in the immune response through the TLR2/TLR4/MGL pathway.

Effects of advanced glycation end products on osteoclasts at different stages of differentiation / 南方医科大学学报

OBJECTIVE@#To explore the effect of advanced glycation end products (AGEs) on osteoclasts at different stages of differentiation.@*METHODS@#Raw264.7 cells cultured were induced for osteoclastogenesis using RANKL, and the stages of differentiation of the osteoclasts were determined with TRAP staining. The cells were then randomly divided into control group, early-stage AGEs intervention group and late-stage AGEs intervention group. The viability of the cells after AGEs treatment was assessed using CCK-8 method. The cells were examined after the induction for osteoclastogenesis using TRAP staining, and the expression levels of RANK, NFATC-1, TRAF-6, TRAP and CTSK mRNAs were tested with RT-PCR; the expressions of CTSK and RANK proteins were detected using Western boltting.@*RESULTS@#We defined the initial 3 days of induction as the early stage of differentiation and the time beyond 3 days as the late stage of differentiation of Raw264.7 cells. Intervention with AGEs at 100 mg/L produced no significant effects on the viability of the cells, but AGEs suppressed the cell proliferation at a concentration exceeding 100 mg/L. The number of osteolasts in the early- and late-stage intervention groups was greater than that in the control group, but the cell count differed significantly only between the early-stage intervention group and control group ( < 0.05). The gene expressions of RANK, NFATC-1, TRAF-6, TRAP and CTSK all increased after the application of AGEs in both the early and late stages of differentiation, but the changes were significant only in the early-stage intervention group ( < 0.05). The changes in CTSK and RANK protein expressions were consistent with their mRNA expressions.@*CONCLUSIONS@#AGEs can affect the differentiation of osteoclasts differently when applied at different stages, and intervention with AGEs at the early stage produces stronger effect to promote osteoclast differentiation than its application at a late stage.

Caulerpa okamurae extract attenuates inflammatory interaction, regulates glucose metabolism and increases insulin sensitivity in 3T3-L1 adipocytes and RAW 264.7 macrophages / 中西医结合学报

OBJECTIVE@#To examine whether Caulerpa okamurae ethanolic extract (COE) could inhibit obesity-mediated inflammation, improve glucose metabolism and increase insulin sensitivity, using in vitro cell models of RAW 264.7 macrophages and 3T3-L1 adipocytes.@*METHODS@#We cocultured 3T3-L1 adipocytes in direct contact with lipopolysaccharide-stimulated RAW 264.7 macrophages and induced insulin resistance in 3T3-L1 adipocytes with tumor necrosis factor-α (TNF-α) in the presence or absence of 250 µg/mL of COE. We investigated various markers of inflammation, glucose regulation and insulin sensitivity in these models using Griess reagent to measure nitric oxide (NO) production, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose to measure glucose uptake, Western blot analysis to quantify protein expression and reverse transcriptase-polymerase chain reaction to evaluate mRNA expression.@*RESULTS@#We found that COE (250 µg/mL) significantly inhibited the lipopolysaccharide-induced inflammatory response in RAW 264.7 macrophages by downregulating NO production, nitric oxide synthase 2 expression and nuclear translocation of nuclear factor-κB. COE also showed similar anti-inflammatory activity in coculture, along with decreased TNF-α, interleukin-6 and monocyte chemoattractant protein mRNA expression. In addition, COE also improved glucose uptake in coculture by upregulating glucose transporter-4 (GLUT-4) and adiponectin and reducing serine phosphorylation of insulin receptor substrate-1 (IRS1). In the TNF-α-induced insulin resistance model of 3T3-L1 adipocytes, COE significantly improved both basal and insulin-stimulated glucose uptake, accompanied by phosphorylation of IRS1 at tyrosine 632, phospho-5' adenosine monophosphate-activated protein kinase α and glycogen synthase kinase-3β (Ser9) as well as upregulation of GLUT-4.@*CONCLUSION@#Together, these findings suggest that COE has potential to treat or prevent obesity-induced metabolic disorders.

Effects of Rehmanniae Extracts against Atherosclerosisin Mice and “IL-34”-Rho/Rock Pathway in Vitro / 中国药学杂志

OBJECTIVE: To investigate the protective effects and mechanism of extracts of Rehmanniae against atherosclerosis by “IL-34”-Rho/Rock pathway in ApoE-/-mice. METHODS: ①Animal experiment 40 ApoE-/- mice were randomly divided into CTL group, Model group, Reh-L group (5 μg·g-1) and Ren-H group (10 μg·g-1), which were induced by low-density lipoprotein (LDL) for AS models. The serum levels of interleukin-34 (IL-34), LDL, mouse monocyte chemotactic protein 1 (MCP-1), mouse macrophage inflammatory protein 2(MIP-2) and endothelin-1 (ET-1) were detected by ELISA. AS index (AI) was calculated according to the serum lipids levels. ②Macrophage experiment RAW264.7 macrophages were cultured in vitro and divided into con group, ox-LDL group, IL-34 group and Reh group. IL-34-indeced foam cells formation by oil red O staining. The cholesterol ester levels and cholesterol efflux of RAW264.7 macrophages were detected. The immunofluorescence, qRT-PCR, Western blot analysis were used to detect RhoA and Rock1 expression. RESULTS: ①Animal experiment the serum levels of TC, TG, LDL-C, ET-1, IL-34, MCP-1, MIP-2 and AI, atherosclerotic lesion areas of the thoracic aorta of mice in Reh-H group were lower than Model group and Reh-L group, but with higher HDL-C level (P<0.05). Compared with the CTL group, the RhoA and Rock1 levels of aorta tissues in Model group were significantly increased by qRT-PCR and Western blot (P<0.05). But the RhoA and Rock1 levels of aorta tissues in Reh-H group were lower than Model group or Reh-L group (P<0.05). ②Macrophage experiment the cholesterol ester level and cholesterol efflux, RhoA and Rock1 expressions of RAW264.7 macrophages induced by ox-LDL and IL-34 were more than con group and ox-LDL group (P<0.05). Compared with IL-34 group, the cholesterol ester level and cholesterol efflux, RhoA and Rock1 expressions of RAW264.7 macrophages in Reh group were significantly decreased (P<0.05). CONCLUSION: The extracts of Rehmanniae can protectagainst atherosclerosis in ApoE-/- AS mice models induced by LDL through inhibiting “IL-34”-Rho/Rock pathway.

Anti-inflammatory effect of corynoline isolated from Corydalis bungeana Turcz / 中国药科大学学报

To isolate corynoline from Corydalis bungeana Turcz.and study its anti-inflammatory mechanism via TLRs/NF-κB signal pathway.Corynoline was extracted by 80％ ethanol and purified by silicagel column chromatography.The structure and purity of corynoline was determined by UPLC,MS,1H NMR and 13C NMR.In the course of experiment,the cytotoxicity of corynoline was evaluated by MTT assay.And the inflammation model was established by RAW264.7 macrophages induced by lipopolysaccharide(LPS),which was intervened by coryno line.The expression levels of TLR4,TLR2 and nuclear transcription factor-κB(NF-κB) signaling pathways related proteinsin RAW264.7 macrophages were detected by Western blot.Furthermore,the expressionof NF-κB p65 mRNA and nuclear p65 were determined by the real-time fluorescence quantitative PCR(RT-qPCR) and Western blot.Results showed that 5-40 μmol/L corynoline reduced the expression level of TLR4 and TLR2,and inhibited the phosphorylation level of IκBα and the phosphorylation and nuclear translocation of p65 at gene and protein levelin a dose-dependent manner in LPS-induced RAW264.7 cells.This study indicated the protective effect of corynoline on LPS-induced RAW264.7 macrophages may be related with the inhibition of TLRs/NF-κB inflammatory signaling pathway.

Anti-inflammatory and anti-arthritic effects of Guge Fengtong Formula: in vitro and in vivo studies / 中国天然药物

Rheumatoid arthritis (RA) is the most common inflammatory arthritis and a major cause of disability. Presently, the clinical therapeutic medicines for inflammatory and arthritic diseases are unsatisfactory due to severe adverse effects or ineffectiveness. The Guge Fengtong formula (GGFT), containing the standardized extracts of Dioscoreae Nipponicae Rhizoma, Spatholobi Caulis, and Zingiberis Rhizoma, has long been used for RA treatment by Chinese doctorsin China. However, the detailed anti-inflammatory and anti-arthritic activity of GGFT has not been reported so far. In the present work, we aimed to evaluate the anti-inflammatory and anti-arthritic effects of GGFT using three in vivo animal models, and tried to uncover its preliminarythe underlying mechanism of action mechanism in RAW 264.7 macrophages. The obtained results indicated that GGFT significantly attenuated ear edema, decreased carrageenan-induced paw edema, reduced the arthritis score, and reversed the weight loss of the complete Freund's adjuvant (CFA)CFA-injected rats. Additionally, marked decrease of in synovial inflammatory infiltration and synovial lining hyperplasia in the joints and decline of inflammatory factors (TNF-α and IL-1β) in the serum were observed in the GGFT-treated rats. In lipopolysaccharide-activated RAW264.7 macrophages, GGFT reduced the production of NO, PGE2, and IL-6, and inhibited the expression of iNOS, COX-2, and NF-κB expression. Our results demonstrated that GGFT possessed considerable anti-inflammatory activity and have had potential therapeutic effects on adjuvant induced arthritis in rats, which provided providing experimental evidences for its traditional application in the treatment of RA and other inflammatory diseases.

Effect of P2X7 R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7 / 中国病理生理杂志

AIM: To establish a cell line of stable silencing of P2X7 receptor (P2X7R) expression through short hairpin RNA ( shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line.METHODS:Stable silencing of P2X7 R gene in the RAW264.7 cells was achieved by re-combinant shRNA plasmid targeting murine P2X7 R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X7 R silencing in G418 resistant cells was verified by immunofluorescent micros-copy and real-time PCR, respectively.The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation as-say.The cell cycle distribution and apoptosis were evaluated by flow cytometry.RESULTS:The expression of P2X7 R at mRNA and protein levels was down-regulated by 80% in shP2X7 R group compared with negative control ( NC) plasmid transfection.In addition, P2X7 R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05).Compared with NC cells, P2X7R silencing resulted in an increase in the phagocytosis of the cells ( P<0.05) .CONCLUSION:A cell line RAW264.7 of stable silencing of P2X7 R expression was successfully es-tablished.P2X7 R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macro-phages.

Antioxidant activity and anti-inflammatory activity of ethanol extract and fractions of Doenjang in LPS-stimulated RAW 264.7 macrophages

BACKGROUND/OBJECTIVES: Fermentation can increase functional compounds in fermented soybean products, thereby improving antioxidant and/or anti-inflammatory activities. We investigated the changes in the contents of phenolics and isoflavones, antioxidant activity and anti-inflammatory activity of Doenjang during fermentation and aging. MATERIALS/METHODS: Doenjang was made by inoculating Aspergillus oryzae and Bacillus licheniformis in soybeans, fermenting and aging for 1, 3, 6, 8, and 12 months (D1, D3, D6, D8, and D12). Doenjang was extracted using ethanol, and sequentially fractioned by hexane, dichloromethane (DM), ethylacetate (EA), n-butanol, and water. The contents of total phenolics, flavonoids and isoflavones, 2,2-diphenyl-1 picryl hydrazyl (DPPH) radical scavenging activity, and ferric reducing antioxidant power (FRAP) were measured. Anti-inflammatory effects in terms of nitric oxide (NO), prostaglandin (PG) E2 and pro-inflammatory cytokine production and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions were also measured using LPS-treated RAW 264.7 macrophages. RESULTS: Total phenolic and flavonoid contents showed a gradual increase during fermentation and 6 months of aging and were sustained thereafter. DPPH radical scavenging activity and FRAP were increased by fermentation. FRAP was further increased by aging, but DPPH radical scavenging activity was not. Total isoflavone and glycoside contents decreased during fermentation and the aging process, while aglycone content and its proportion increased up to 3 or 6 months of aging and then showed a slow decrease. DM and EA fractions of Doenjang showed much higher total phenolic and flavonoid contents, and DPPH radical scavenging activity than the others. At 100 microg/mL, DM and EA fractions of D12 showed strongly suppressed NO production to 55.6% and 52.5% of control, respectively, and PGE2 production to 25.0% and 28.3% of control with inhibition of iNOS or COX-2 protein expression in macrophages. CONCLUSIONS: Twelve month-aged Doenjang has potent antioxidant and anti-inflammatory activities with high levels of phenolics and isoflavone aglycones, and can be used as a beneficial food for human health.

In vivo and in vitro anti-inflammatory activity of Cryptostegia grandiflora Roxb. ex R. Br. leaves

BACKGROUND: Despite Cryptostegia grandiflora Roxb. ex R. Br. (Apocynaceae) leaves are widely used in folk Caribbean Colombian medicine for their anti-inflammatory effects, there are no studies that support this traditional use. Therefore, this work aimed to evaluate the effect of the total extract and primary fractions obtained from Cryptostegia grandiflora leaves, using in vivo and in vitromodels of inflammation, and further get new insights on the mechanisms involved in this activity. RESULTS: Ethanolic extract of Cryptostegia grandiflora leaves, and its corresponding ether and dichloromethane fractions, significantly reduced inflammation and myeloperoxidase activity (MPO) in ear tissue of mice treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Histological analysis revealed a reduction of edema and leukocyte infiltration. Complementarily, we demonstrated that extract and fractions reduced nitric oxide (NO•) and prostaglandin E2 (PGE2) production in LPS-stimulated RAW 264.7 macrophages, as well as scavenging activity on DPPH and ABTS radicals. CONCLUSIONS: Our results demonstrated for the first time the anti-inflammatory activity of Cryptostegia grandiflora leaves, supporting its traditional use. This activity was related to inhibition of MPO activity, and PGE2 and NO• production. These mechanisms and its antioxidant activity could contribute, at least in part, to the anti-inflammatory effect showed by this plant.

Efecto de Rhizophora mangle L sobre la producción de anión superóxido en macrófagos murinos RAW 264, 7 / Effect of Rhizophora mangle L on the superoxide anion production in RAW 264, 7 murine macrophages

Introducción: el extracto acuoso de la corteza de Rhizophora mangle L. (mangle rojo) posee varias propiedades farmacológicas: en el tratamiento de la mastitis bovina, la curación de heridas,las infecciones uterinas y las úlceras gastroduodenales; debido a sus propiedades antiséptica, cicatrizante, antiinflamatoria y antioxidante. Sin embargo, no se han completado los estudios de la actividad antioxidante a todos los niveles de complejidad para dilucidar los mecanismos de acción involucrados en este efecto farmacológico. Objetivo: determinar el efecto del extracto acuoso de Rhizophora mangle y su fracción polifenólica sobre la producción de anión superóxido en una línea celular de macrófagos murinos RAW 264,7, estimulados con forbol 12-myristato 13-acetato o lipopolisacárido. Métodos: la evaluación de la actividad antioxidante del extracto de Rhizophora mangle y su fracción polifenólica sobre la producción de anión superóxido en macrófagos RAW 264.7, activados con lipopolisacárido o forbol 12-myristato 13-acetato, se determinó mediante el método de reducción del ferricitocromo c. Resultados: el extracto de Rhizophora mangle y su fracción polifenólica, inhibieron la producción de anión superóxido en macrófagos RAW 264,7, activados con ambos tipos de agentes de forma dependiente de la concentración de taninos. La comparación de las líneas de regresión reveló diferencias significativas (p< 0,05), resultando este efecto superior en la fracción para ambos tipos de agentes, y en presencia de lipopolisacárido para ambas muestras. Conclusiones: el extracto acuoso de Rhizophora mangle mostró actividad antioxidante a nivel celular, evidenciada por la reducción del estrés oxidativo en macrófagos mediante la inhibición de la producción de anión superóxido. A su vez se demostró que los compuestos polifenólicos presentes en el extracto fueron los principales responsables de los efectos antioxidantes observados en este estudio

Introduction: the aqueous extract of Rhizophora mangle L (red mangrove) bark has some pharmacological properties, that is, the treatment of bovine mastitis, wound healing, uterine infections and stomach ulcers, due to its antiseptic, healing, antiinflammatory and antioxidant properties. However, the studies of antioxidant activity at all complexity levels have not been completed in order to elucidate the mechanisms of action involved in this pharmacological effect. Objective: to determine the effect of Rhizophora mangle aqueous extract and its polyphenolic fraction on superoxide anion production in a cellular line of RAW 264,7 murine macrophages, stimulated with phorbol 12-myristate 13-acetate or lipopolysaccharide. Methods: the evaluation of the antioxidant activity of Rhizophora mangle extract and its polyphenolic fraction on superoxide anion production in RAW 264.7 macrophages activated with lipopolysaccharide or phorbol 12-myristate 13-acetate was made using the C ferricytochrome reduction method. Results: Rhizophora mangle extract and its polyphenolic fraction inhibit the superoxide anion production in RAW 264.7 macrophages activated with both types of agents depending on tannin concentration. The comparison of the regression lines showed significant differences (p< 0.05), resulting this effect higher in the fraction for both types of agents, and the presence of lipopolysaccharide for both samples. Conclusions: Rhizophora mangle aqueous extract showed antioxidant activity at cellular level, evidenced by reduction of oxidative stress in macrophages, through the inhibition of superoxide anion production. At the same time, it was shown that polyphenolic compounds, present in the extract, were the main responsible for the antioxidant effects observed in this study

A comparison of anti-inflammatory activities of green tea and grapefruit seed extract with those of microencapsulated extracts

We compared the effects of grapefruit seed extract (GFSE), green tea extract (GT) and their microencapsulated extract on anti-inflammatory activities in murine RAW 264.7 macrophages cell line. In order to protect the bioactive compounds in the extracts, they were microencapsulated with maltodextrin and H2O. Nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha), inducible nitric oxide synthase (iNOS) protein expression and thiobarbiturate reactive substances (TBARS) were analyzed in LPS activated RAW 264.7 macrophages. The green tea extract at the range of 100-600 microg/mL inhibited NO, PGE2 production and iNOS protein expression without cytotoxicity in a dose-dependent manner. Grapefruit seed extract had strong inhibitory effects on NO and PGE production and iNOS protein expression at the range of 5-20 microg/mL without cytotoxicity. Microencapsulation of green tea extract had further inhibitory effects on NO and PGE2 production and on iNOS protein expression, whereas microencapsulated GFSE did not show any further inhibitory effects on these parameters. Taken together, our results suggest that GSFE might be a promising candidate for preventing inflammation related diseases, such as cardiovascular disease, cancer or diabetes, and the microencapsulation of green tea extract could improve its bioactivity.

The Chloroform Fraction of Carpinus tschonoskii Leaves Inhibits the Production of Inflammatory Mediators in HaCaT Keratinocytes and RAW264.7 Macrophages

Inflammation is the immune system's response to infection and injury-related disorders, and is related to pro-inflammatory factors (NO, PGE2, cytokines, etc.) produced by inflammatory cells. Atopic dermatitis (AD) is a representative inflammatory skin disease that is characterized by increasing serum levels of inflammatory chemokines, including macrophage-derived chemokine (MDC). Carpinus tschonoskii is a member of the genus Carpinus. We investigated the anti-inflammatory activity of C. tschonoskii by studying the effects of various solvent fractions prepared from its leaves on inflammatory mediators in HaCaT and RAW264.7 cells. We found that the chloroform fraction of C. tschonoskii inhibited MDC at both the protein and mRNA levels in HaCaT cells, acting via the inhibition of STAT1 in the IFN-gamma signaling pathway. In addition, the chloroform fraction significantly suppressed the expression of inflammatory factors induced by lipopolysaccharide stimulation, except COX-2 and TNF-alpha. These results suggest that the chloroform fraction of C. tschonoskii leaves may include a component with potential anti-inflammatory activity.