Interleukin-1 alpha induces osteoclast activation and bone loss / 中国组织工程研究

BACKGROUND: Interleukin-1 is an important pro-inflammatory cytokine that has been documented in the regulation of bone inflammation and bone remodeling. A previous study has demonstrated that interleukin-1α can induce apoptosis while inhibiting osteoblast differentiation in MC3T3-E1 cells. OBJECTIVE: To investigate the role and mechanism of interleukin-1α on osteoclast activation and bone loss in mice. METHODS: (1) Cell test: RAW264.7 cells were either treated with interleukin-1α alone or with receptor activator of nuclear factor-κB ligand (RANKL) for 1 and 4 days. Cell viability was tested by cell counting kit-8 assay. The number of multinuclear osteoclasts was detected by tartrate resistant acid phosphatase assay. The mRNA and protein levels of osteoclast-specific genes and genes related to nuclear factor-κB pathway and Wnt/β-catenin pathway were tested by real-time fluorescence quantitative PCR, immunofluorescence staining or western blot. Bone marrow-derived macrophages were either treated with interleukin-1α alone or with RANKL and macrophage colony-stimulating factor for 7 days. The number of multinuclear osteoclasts was detected by tartrate resistant acid phosphatase assay. The protein levels of osteoclast-specific genes were tested by western blot. (2) Animal test: Twenty-four male C57BL/6J mice (6-8 weeks old) were assigned into two groups at random: control group and test group. Mice were subsequently treated with interleukin-1α solution or PBS by intraperitoneal injection twice a week for 5 weeks. Bone tissues from the femurs were performed with micro-computed tomography analysis and hematoxylin-eosin staining, tartrate resistant acid phosphatase, and immunofluorescence analysis. RESULTS AND CONCLUSION: Cell test: Interleukin-1α alone significantly increased RAW264.7 cell proliferation, but stimulated cell differentiation into osteoclasts in combination with RANKL (P < 0.05). Interleukin-1α significantly increased the expression of osteoclast-related markers and the number of tartrate resistant acid phosphatase-positive multinuclear cells in RAW264.7 cells and bone marrow-derived macrophages in the existence of RANKL or RANKL+macrophage colony-stimulating factor (both P < 0.05). Interleukin-1α was found to significantly enhance the nuclear factor-κB and Wnt/beta-catenin signaling in RAW264.7 cells (P < 0.05). Blocking of nuclear factor-κB or Wnt3 signaling not only reversed the activation of nuclear factor-κB and Wnt3 signaling but also weakened the enhanced expression of osteoclast-specific genes induced by interleukin-1α in RAW264.7 cells (P < 0.05). Animal test: interleukin-1α induced bone loss in mice while also upregulating the expression of osteoclast-specific markers, RANK, TRAF6 and p65, and Wnt3 in vivo (P < 0.05). The findings indicate that interleukin-1α can induce osteoclast activation and bone loss by promoting the nuclear factor-κB and Wnt signaling pathways.

Inhibitory Effect and Mechanism of Jingulian Extract on LPS-induced RAW264.7 Cell Inflammatory Response Based on PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

Objective:To explore the inhibitory effect and mechanism of Jingulian extract （JGL） on inflammation. Method:The following groups were set up in this study： a control group （10% fetal bovine serum）， a lipopolysaccharide （LPS） model group （0.5 mg·L^-1）， and JGL groups （10， 20， 40， 60， 80， 120， 160， 200， 250， 300 mg·L^-1 + 0.5 mg·L^-1LPS）. The RAW264.7 cells were cultured for 24 hours. Cell proliferation was detected by cell counting kit-8 （CCK-8） assay. Nitric oxide （NO） release was detected by Griess assay. The release of cytokines interleukin （IL）-1<italic>β</italic>， IL-6， IL-10， and tumor necrosis factor （TNF）-<italic>α</italic> was determined by enzyme linked immunosorbent assay （ELISA）. The expression of inducible nitric oxide synthase （iNOS） and intraprostaglandin peroxidase synthase 2 （PTGS2）/cyclooxygenase-2 （COX-2） was measured by real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） and the activation of key proteins in the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway by Western blot. Result:Compared with the control group， LPS （0.5 mg·L^-1）could promote the proliferation of RAW264.7 cells after stimulation for 24 hours （<italic>P</italic><0.01）. Compared with the model group， JGL had no significant effect on cell proliferation. Compared with the control group， LPS （0.5 mg·L^-1）increased the release of NO， IL-1<italic>β</italic>， IL-6， IL-10， and TNF-<italic>α</italic> （<italic>P</italic><0.01）. Compared with the model group， JGL （20-300 mg·L^-1）inhibited the release of NO in a dose-dependent manner after stimulation for 24 hours （<italic>P</italic><0.05） and reduced IL-1<italic>β</italic>， IL-6， and IL-10 （<italic>P</italic><0.05， <italic>P</italic><0.01）， but no obvious inhibition on the release of TNF-<italic>α</italic> was observed. LPS （0.5 mg·L^-1） could induce the expression of iNOS and PTGS2/COX-2 genes as compared with the control group （<italic>P</italic><0.05， <italic>P</italic><0.01）. JGL could down-regulate the mRNA expression of iNOS and PTGS2/COX-2 genes as compared with the model group （<italic>P</italic><0.05， <italic>P</italic><0.01）. LPS （0.5 mg·L^-1） could activate the PI3K/Akt pathway （<italic>P</italic><0.01） as compared with the control group， while JGL （10， 20， 40， and 80 mg·L^-1） decreased the expression of PI3K-p110， p-p85， and p-Akt （<italic>P</italic><0.01）， and inhibited the activation of PI3K/Akt pathway. Conclusion:JGL extract could significantly inhibit the inflammatory response and activation of the PI3K/Akt pathway induced by LPS in RAW264.7 cells. The anti-inflammatory effect was related to the inhibition of the PI3K/Akt pathway.

Inhibition of bakuchiol and its derivatives in Psoraleae Fructus on nitric oxide production in lipopolysaccharide-activated RAW 264.7 cell lines / 中草药

Objective: To study bakuchiol and its derivatives of cyclohexane soluble part in 70% ethanol aqueous extract of Psoraleae Fructus and their inhibition on nitric oxide (NO) production in lipopolysaccharides (LPS)-activated murine macrophage RAW 264.7 cell lines. Methods: The compounds were separated and purified by silica gel column and high performance liquid chromatographies, and their structures were determined by spectroscopic data analyses. Using LPS-activated RAW 264.7 cell line models in vitro, all of the isolated compounds were evaluated for the inhibition against NO production. Results: Twelve compounds were obtained and identified as bakuchiol (1), 12,13-dihydro-12,13-epoxybakuchiol (2), Δ3,2-hydroxylbakuchiol (3), 12-oxobakuchiol (4), psoracorylifol B (5), psoracorylifol C (6), (12’S)-bisbakuchiol C (7), Δ1,3-bakuchiol (8), 13-methoxyisobakuchiol (9), bisbakuchiol B (10), bisbakuchiol A (11), and 12,13-dihydro-12,13-dihydroxybakuchiol (12), respectively. For the inhibition of NO production in the LPS-activated RAW 264.7 cell line model, a positive inhibitor, L-N6-(1-iminoethyl)-lysine (L-NIL), was used and showed the half maximal inhibitory concentration (IC50) value of (10.29 ± 1.10) μmol/L. The IC50 values of the assayed compounds 1, 3, 5, 10 and 11 were all more than 50 μmol/L, compounds 8, 9 and 12 were comparable to that of L-NIL, whereas the IC50 values of compounds 2, 4 and 7 were less than that of the positive inhibitor with statistically significance. Conclusion: Compound 4 is a new natural product. The results of the bioactivity assays indicated that compounds 2, 4, 7, 8, 9 and 12 are potential anti-inflammatory agents.

Study on anti-inflammatory active components and mechanism of Corydalis Bungeanae Herba / 中国中药杂志

Corydalis Bungeanae Herba is often used to treat a variety of inflammatory diseases in traditional Chinese medicine. In order to determine its chemical material basis, the components of Corydalis Bungeanae Herba were isolated by automated purification system. Flavonoids and alkaloids were prepared, and all such components were identified by mass spectrometry. The effects of the components on the production of inflammatory mediators and pharmacological mechanisms in the lipopolysaccharide(LPS)-induced RAW264.7 cell inflammation model were examined. Mouse macrophages(RAW264.7) were first treated with LPS. The relationship between cell viability and LPS concentration was observed. Then, the effects of flavonoids components and alkaloid components with different administration concentrations on cell viability were detected to determine the maximum administration concentration. Secondly, 2.5, 5, 10 and 20 μg·mL~(-1) flavonoids components and alkaloid components were added respectively to observe the effects and mechanism of different concentrations of flavonoids components and alkaloid components on LPS-induced inflammation of RAW264.7 macrophages. Griess reagent assay was used to detect NO content in cell supernatant. The inflammatory cytokines(TNF-α, IL-1β and IL-6) in cell supernatant were determined by ELISA method. Western blot method was used to detect the intracellular nuclear factor(NF-κB) IκBα phosphorylation(p-IκBα), p65 phosphorylation(p-p65) and protein expression of TLR4, TLR2. The results showed that the alkaloid components inhibited the production of NO, TNF-α, IL-1β and IL-6 in a dose-dependent mannerin the concentration range of 2.5-20 μg·mL~(-1). In inflammation upstream pathways, the inhibitory effect of the alkaloid components on the TLR2 expression level was weaker than that of TLR4. In inflammation downstream, alkaloid components significantly inhibited phosphorylation of IκBα and p65 in a dose-dependent manner. These data suggested that the alkaloid components were the material basis components of Corydalis Bungeanae Herba, and its anti-inflammatory mechanism might be related to inhibiting the transmission of inflammatory signals in TLRs/NF-κB signaling pathways dominated by TLR4, interfering with the activation of inflammatory genes and inhibiting their over expression, and down-regulating the secretion level of inflammatory factors.

Anti-inflammatory study on 9,9'-O-di-(E）-feruloyl-meso-5,5'-dimethoxysecoisolariciresinol (LCA), an active ingredient in Litsea cubeba (Lour.) Pers / 药学实践杂志

Objective To evaluate the anti-inflammatory effects of LCA, which is the active component from Litsea cubeba (Lour.) Pers. Methods Pharmacodynamic evaluations of ear swelling and cotton ball granuloma models were used in animal experiments. In vitro experiment, lipopolysaccharide (LPS) stimulated monocyte macrophage RAW264.7 cells were used to evaluate the anti-inflammatory activity of LCA by detecting the secretions of nitric oxide (NO), tumor necrosis factor-α (TNF-α), the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein. Results In ear swelling experiment, the extent of ear swelling was significantly lower in the LCA treated group than the model group. The inhibition rate was greater in the high LCA concentration group than the positive drug group. In addition, LCA reduced the weight of cotton ball granuloma markedly. At the cell level, LCA significantly inhibited the secretions of NO, TNF-α and reduced the expressions of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. Conclusion LCA has significant anti-inflammatory effects in vitro and in vivo. The further studies are warranted for the development of anti-inflammatory drugs.

Intervention Effects of Berberine on Mice Macrophage Polarization Based on TLR 4/MyD88/NF-κB Signaling Pathway / 中国药房

OBJECTIVE：To study the effects of berberine on mic e macrophage polarization based on TLR 4-MyD88-NF-κB signaling pathway. METHODS ：Using mice RAW 264.7 macrophage as the object ，atorvastatin calcium as positive control ， inflammatory cell model was induced by lipopolysaccharide （LPS）；ELISA method was used to detect the contents of TNF-α，IL-6 and NF-κB in cell culture medium after treated with low，medium and high doses of berberine （5，10，20 μmol/L）for 24 h. The real-time fluorescence quantitative PCR was conducted to determine the mRNA expression of TLR 4 and MyD 88 in cells. Western blotting assay was used to detect the protein expression of TLR 4，MyD88，iNOS and CD 206 in cells. RESULTS ：Compared with blank control group ，the contents of TNF-α，IL-6 and NF-κB in cell culture medium，mRNA expression of TLR 4 and MyD 88， protein expression of TLR 4，MyD88 and iNOS in cells were increased significantly in LPS induction group （P＜0.05）. Compared with LPS induction group ，the contents of TNF-α and IL-6，mRNA and protein expression of TLR 4 and MyD 88 in atorvastatin calcium group ，berberine medium-dose and high-dose groupsas well as the content of NF-κ B and protein expression of iNOS in administration groups were decreased significantly ， while the content of NF-κB in berberine high-dose group was significantly lower than atorvastatin calcium group （P＜0.05）. The protein expressions of CD206 in atorvastatin calcium group and berberine high-dose group were increased significantly ，while the protein expression of CD 206 in berberine high-dose group was significantly higher than atorvastatin calcium group （P＜0.05）. CONCLUSIONS ：Different doses of berberine can intervene in mice macrophage polarization to different extents ，the mechanism of which may be associated with the regulation of TLR4/MyD88/NF-κB signaling pathway.

Protective effect of the cybister lateralimarginalia extract cl on renal function of rats with chronic nephritis induced by doxorubicin / 中国药学杂志

OBJECTIVE: To establish a model of chronic nephritis induced by doxorubicin and a model of RAW264.7 cell inflammation induced by lipopolysaccharide(LPS), and to study the protective effect of the Cybister lateralimarginalia extract CL on the renal function of rats with chronic nephritis induced by doxorubicin and its anti-inflammatory effect. METHODS: A rat model of chronic nephritis was established by a single intravenous injection of doxorubicin (6 mg·kg-1). After 2 weeks of modeling, normal control group and model control group were given normal saline while other groups were given intragastric administration of CL high and low dose, Shenyankangfu tablets and prednisone acetate respectively. The body weight of the rats was recorded every day. The 24 h urine was collected every weekend and the urine protein content was measured. After the last administration, the rats were dissected the next day, and the kidney and liver indexes were calculated. The levels of TP, ALB, TC, TG, BUN and Scr in serum were determined. The pathological changes of kidney tissues were observed. The lipopolysaccharide (LPS 10 μg·mL-1) was used to induce the RAW264.7 cell inflammation model. PBS was given to the normal control group and the model control group while CL low, medium and high dose were given to other groups respectively. The levels of TNF-α and iNOS in the supernatant of cells were detected by enzyme-linked immunosorbent assay(Elisa). RESULTS: Compared with the normal control group, the content of 24 h urine protein, the levels of TC, TG, BUN and Scr in serum, kidney index and liver index were significantly increased in the model control group (P<0.01 or P<0.05), but the body weight of rats, the levels of TP and ALB in serum were significantly decreased (P<0.01 or P<0.05). The secretion of pro-inflammatory factors TNF-α and iNOS were significantly increase in RAW264.7 cells with inflammatory induced by LPS (P<0.01). Compared with the model control group, the content of 24 h urine protein and the levels of TC, TG, BUN and Scr in serum were significantly decreased in the Shenyankangfu group, the prednisone acetate group and the CL group(P<0.01 or P<0.05), but the body weight of rats, the levels of TP and ALB in serum were significantly increased (P<0.01 or P<0.05); the pathological condition of renal tissue was improved to some extent. Each dose of CL could reduce the secretion of pro-inflammatory factors TNF-α and iNOS in RAW264.7 cells with inflammatory induced by LPS (P<0.01). CONCLUSION: The extract of Cybister lateralimarginalia has an obvious anti-inflammatory effect and has a good protective effect on chronic nephritis in rats.

Genistein regulates miR-21 to promote the apoptosis in LPS-activated RAW264.7 cells / 药学学报

The research is aimed to investigate the effect of genistein (GEN) on the apoptosis in lipopolysaccharide (LPS)-activated RAW264.7 cells and explore the pharmacological mechanism of GEN anti-atherosclerosis (AS). RAW264.7 cells were activated by LPS, the level of TNF-α and IL-6 mRNA were detected by qRT-PCR, the expression of COX-2 and iNOS were detected by Western blot. RAW264.7 cells were pretreated with GEN for 2 h, and then incubated with LPS for 24 h. After that, CCK8 kit was used for the cell viability, Annexin V-FITC/PI kit for the apoptosis of cell. qRT-PCR was used to detect the level of CHOP, caspase-3 and miR-21. Western blot was used to detect the expression of CHOP and caspase-3. Results showed that LPS (1 000 ng·mL-1) increased the expression of TNF-α, IL-6, COX-2 and iNOS in RAW264.7 cells compared with that in control group. GEN inhibited the cell activity and the level of miR-21, promoted the expression of CHOP and caspase-3 in LPS-activated RAW264.7 cells in a dose-dependent manner. miR-21 up inhibited the expression of CHOP and caspase-3 in LPS-activated RAW264.7 cells and this process was reversed by GEN treatment. miR-21 down promoted the expression of CHOP and caspase-3, which were further enhanced by GEN. These results indicate that GEN promotes the apoptosis of RAW264.7 cells activated by LPS through down regulating miR-21 and activating endoplasmic reticulum (ER) stress pathway.

Inflammatory Effect of Light-Emitting Diodes Curing Light Irradiation on Raw264.7 Macrophage / 치위생과학회지

BACKGROUND: The light-emitting diode (LED) curing light used is presumed to be safe. However, the scientific basis for this is unclear, and the safety of LED curing light is still controversial. The purpose of this study was to investigate the effect of LED curing light irradiation according to the conditions applied for the polymerization of composite resins in dental clinic on the cell viability and inflammatory response in Raw264.7 macrophages and to confirm the stability of LED curing light. METHODS: Cell viability and cell morphology of Raw264.7 macrophages treated with 100 ng/ml of lipopolysaccharide (LPS) or/and LED curing light with a wavelength of 440~490 nm for 20 seconds were confirmed by methylthiazolydiphenyl-tetrazolium bromide assay and microscopic observation. The production of nitric oxide (NO) and prostaglandin E2 (PGE2) was confirmed by NO assay and PGE2 enzyme-linked immunosorbent assay kit. Expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in total RNA and protein was confirmed by reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: The LED curing light did not affect the viability and morphology of normal Raw264.7 cells but affected the cell viability and induced cytotoxicity in the inflammation-induced Raw264.7 cells by LPS. The irradiation of the LED curing light did not progress to the inflammatory state in the inflammation-induced Raw264.7 macrophage. However, LED curing light irradiation in normal Raw264.7 cells induced an increase in NO and PGE2 production and mRNA and protein expression of IL-1β and TNF-α, indicating that it is possible to induce the inflammatory state. CONCLUSION: The irradiation of LED curing light in RAW264.7 macrophage may induce an excessive inflammatory reaction and damage oral tissues. Therefore, it is necessary to limit the long-term irradiation which is inappropriate when applying LED curing light in a dental clinic.

Chuanliguspirolide, a new butylphthalide derivative from Chuanxiong Rhizoma and its inhibition on NO production in LPS-activated RAW264.7 and BV2 cell lines / 中草药

Objective: To study the chemical constituents of 95% ethanol aqueous extract of Chuanxiong Rhizoma and the bioactivities of inhibition on nitric oxide (NO) production in lipopolysaccharides (LPS)-activated murine macrophage RAW264. 7 and mouse microglia BV2 cell lines. Methods: The compounds were separated and purified by silica gel column and high performance liquid chromatographies, and their structures were determined by spectroscopic data analysis. Using LPS-activated RAW264. 7 and BV2 cell line models in vitro, all of the isolated compounds were evaluated for the inhibition against NO production. Results: Three butylphthalide derivatives were obtained and identified as Z-3', 8', 3'a, 7'a-tetrahydro-6, 3', 7, 7'a-diligustilide-8'-one (1), Z, Z'-3.3'a, 7. 7'a-diligustilide (2), and chuanliguspirolide (3), respectively. For the inhibition of NO production in the LPS-activated two cell lines, the half inhibitory concentration (IC50) values of compounds 1-3 and indomethacin as a positive control drug in RAW 264. 7 cell line model were (31.60 ± 2.62), (21.20 ± 0.61), (30.12 ± 2.90), and (54.62 ± 7.53) μmol/L, respectively, while IC50 values of compounds 1-3 and curcumin as a positive control drug in BV2 cell line model were (21.99 ± 4.40), (15.43 ± 1.34), (12.20 ± 3.40), and (10.58 ± 1. 41) μmol/L, respectively. Conclusion: Compound 3 named as chuanliguspirolide is a new one. The results of bioactivity assays indicated that compounds 1-3 are potential anti-inflammatory agents.

Active ingredients from Iris scariosa and Iris bloudowii with inhibitory activities against LPS-induced NO production in RAW264.7 macrophages / 中草药

Objective To study the anti-inflammatory constituents from the roots and rhizomes of Iris scariosa and I. bloudowii. Methods The chemical constituents were isolated and purified by column chromatography (silica gel, ODS, and sephadex LH-20), RP-preparative TLC, and semi-preparative HPLC. Their structures were identified by spectroscopic methods (MS, 1H-NMR, and 13C-NMR). The anti-inflammatory activities of the compounds were evaluated by the inhibition against NO production in lipopolysaeeharide (LPS) activated RAW 264.7 macrophages. Results Five compounds including tectoridin (1), iridin (2), nigricin 4’-O-β-D-glucoside (3), neomangiferin (4), and mangiferin (5) were isolated and identified from the roots and rhizomes of I. scariosa. Besides, seven compounds, nigricin4’-O-β-D-glucoside (3), nigricin (6), nigricanin (7), 5,5’-dihydroxy-3’,4’-dimethoxy-6,7- methylenedioxyisoflavone (8), nigricanin-4’-O-β-D-glucopyranoside (9), irifloside (10), and nigricin-4’-[O-β-D-glucopyranosyl- (1’’’→6″)-β-D-glucopyranoside] (11) were isolated and identified from the roots and rhizomes of I. bloudowii. Compounds 2, 3, 5, 6, 7, 9, 11, and previously reported chemical constituents of I. scariosa irigenin and irilone could inhibit NO production in RAW 264.7 macrophages to some extent. Conclusion Compounds 1-5 are isolated from I. scariosa and compounds 4 and 6-11 are isolated from I. bloudowii for the first time, respectively. The results of bioactivity assays indicated that compounds 2, 3, 5, 6, 7, 9, 11, and other two compounds irigenin and irilone isolated and purified from I. scariosa earlier are potential anti-inflammation agents.

Effects of H2 on osteoclast differentiation of RAW264.7 cells induced by RANKL and RANKL/TNF-α / 中国生化药物杂志

Objective To discuss the effect of H2 on osteoclast differentiation of RAW264.7 cells induced by RANKL and RANKL/TNF-α.MethodsRAW264.7 cells were treated with H2 in the presence of RANKL and RANKL/TNF-α.RAW264.7 cells viability was assessed by CCK-8.Test the Oxidative Stress of the induced RAW264.7.The number of TRAP-positive cells were counted under light microscopy.The levels of cathepsin K (CTK) and matrix metalloprotein-9(MMP-9) mRNA were analyzed by real-time PCR.ResultsH2 can not influence the RAW264.7 cell viability but can lower oxidative stress.The significant difference(P<0.05) indicated that H2 could significantly decrease the number of TRAP-positive MNCs.The significant difference among the 4 groups in CTK and MMP-9 genes (P<0.05) indicated that H2 could down-regulate their mRNA expression.ConclusionH2 can reduce the oxidative stress and inhibit differentiation of RAW264.7 cells into osteoclasts.

Water extract of Glycyrrhiza uralensis Fisch.enhanced the activation of RAW264.7 cells through TLR4 signaling pathway / 中华微生物学和免疫学杂志

Objective To investigate the effects of water extract of Glycyrrhiza uralensis Fisch.(GUWE) on the activation of RAW264.7 cells and the possible mechanism.Methods RAW264.7 cells were treated with GUWE containing different concentrations of polysaccharide (10, 50, 100, 500 μg/ml).Viability of these cells was analyzed by MTT assay.Phagocytic activity and surface molecules expressed on these cells were detected by flow cytometry.Levels of cytokines were analyzed by ELISA.Western blot assay was performed to analyze the activation of key molecules in TLR4 signaling pathway.Results GUWE at the concentration of 500 μg/ml significantly decreased the viability of RAW264.7 cells, but significantly increased the viability of RAW264.7 cells at concentrations of 50 μg/ml and 100 μg/ml.GUWE significantly enhanced the phagocytic activity of RAW264.7 cells as well as the expression of cytokines and costimulatory molecules in a dose-dependent manner.Further analysis indicated that the activation of RAW264.7 cells induced by GUWE was suppressed by TLR4 inhibitor.Moreover, GUWE enhanced the phosphorylation of NF-kB p65 and TLR4 downstream mitogen-activated protein kinases (MAPKs) including p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK).Conclusion This study indicates that GUWE promotes the activation of RAW264.7 cells through TLR4 signaling pathway.

Effects and mechanism of β-carotene on inflammatory factors in LPS-induced RAW264.7 cells / 中国免疫学杂志

Objective:To investigate the effects and mechanism of β-carotene on inflammatory factors (IL-1 β,IL-6,TNF-α) in LPS-induced RAW264.7 cells.Methods:Firstly,RAW264.7 cells of being induced by 4 (5 μg/ml)for 24 h were treated with different concentration of β-carotene (20,40,80,160 pmol/L)for 3 h.The cells viability was measured by MTIT,the mRNA relative expression of IL-1 β,IL-6,TNF-cα was detected by fluorescence quantitative PCR,the secretion capacity of IL-1 β,IL-6,TNF-α was detected by ELISA and the protein relative expression of NF-κB p65 protein was measured by Western blot.Secondly,RAW264.7 cells were induced by LPS(5 μg/ml) and different concentration of PDTC(1,5,10 μg/ml)for 24 h,NF-κB p65 protein was measured by Western blot and inflammatory factors were detected by fluorescence quantitative PCR and ELISA.Finally,compared the changes in the relative expression of inflammatory factors and NF-κB p65 protein between LPS+PDTC group and LPS+PDTC + β-carotene group.Results:Compared with the LPS-induced group,β-carotene could increase the cell viability of LPS-induced RAW264.7 cells and inhibied the relative expression of inflammatory factors and NF-κB p65 protein.Inhibited the relative expression of NF-κB p65 protein could reduce the relative expression of inflammatory factors.Compared with the LPS+PDTC group,LPS +PDTC + β-carotene group could inhibit the relative expression of inflammatory factors significantly (P＜0.05).But,there was little difference about the relative expression of NF-κB p65 protein between this two groups.Conclusion:β-carotene inhibits the relative expression of inflammatory factors(IL-1 β,IL-6,TNF-α) in LPS-induced RAW264.7 cells through inhibition of NF-κB p65 protein in NF-κB pathway,this pathway isn't unique.

Effects of Rab7 gene silencing on cytokine and MAPK signal pathway activated by R848 in macrophage / 中国免疫学杂志

Objective:To investigate the effect of Rab7 on cytokine induced by TLR7 (Toll like receptor-7) R848 activated in Raw264.7,and discusses the influence of Rab7 on MAPK signal transduction.Methods: TLR7 downstream cytokines such as TNF-α,IL-6,IFN-α,IFN-β and IP-10 activated by R848 were detected through Q-PCR in Rab7 silenced mouse macrophages,and then analysis of phosphorylation of MAPK determined with Western blot showed the effect of Rab7 on signal transduction of MAPK.Results: Rab7 inhibit production of cytokine activated by TLR7,and also,Rab7 had an inhibitory effect on MAPK signal pathway.Conclusion: The experimental results further illustrate that the Rab7 is the TLR7 signal transduction pathway negative regulatory factor,and to participate in MAPK signaling pathway.

Regulation Effects of Isoquercitrin on Inflammatory in LPS-induced RAW264.7 Cell / 医药导报

Objective To investigate the influence of isoquercitrin on the inflammatory factors in LPS-induced RAW264.7 cells.Methods MTT method was used to detect inhibition ratio of RAW264.7 cells induced by isoquercitrin.The level of TNF-α in culture medium was measured by ELISA.Nitric oxide (NO) was detected by Nitrate Assay Kit.Western blotting was used to investigate the influence on the productions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).Results The half inhibitory concentration (IC50) of isoquercitrin was 65.73 μmol·L-1.LPS had no inhibitory effect on the cells.Compared with LPS group,the level of TNF-α was decreased to 74.80% and 60.57% in isoquercitrin (20,10 μmol·L-1) groups in a dose-dependent manner.The results measured by Nitrate Assay Kit revealed that isoquercitrin (20,10 μmol·L-1) could suppress production of NO,the level of NO decreased to 79.34% and 68.81%(P<0.05).The Western blotting results showed that isoquercitrin (20,15,10 μmol·L-1) inhibited the productions of iNOS and COX-2 (P<0.05).Conclusion Isoquercitrin has anti-inflammatory effects by inhibiting the productions of TNF-α,NO,iNOS and COX-2,and the most effective dose for the inhibition is 10 μmol·L-1.

Comparison of anti-inflammatory activity of ethanol extract of coptis chinensis Franch.and Berberine hydrochloride in vitro / 中国免疫学杂志

Objective:To study the anti-inflammatory effect of the ethanol extract of Coptis chinensis Franch.in vitro.Methods: An inflammatory cell model was established by stimulating the mouse peritoneal macrophages in vitro with lipopolysaccharide(LPS).LPS stimulated of RAW264.7 cells for a long time after administration of intervention.Effect of ethanol extract of Coptis chinensis Franch.on RAW264.7 cell growth activity was analyzed by MTT assay.The production of tumor necrosis factor-α(TNF-α),IL-1β,IL-6,NO,prostaglandin E2(PGE2) was determined by ELISA.The expression of mRNA of TNF-α,induced nitric oxide synthase(iNos) and HO-1 was detected by real-time fluorescent quantitative PCR(RT-PCR).Results: The ethanol extract of Coptis chinensis had no inhibition effect on the scope of RAW 264.7 cells the scope of 5-80 mg/L.Each treatment group concentration of interleukin-6 (IL-6),interleukin-1β (IL-1β),TNF-α,NO,prostaglandin E2 (PGE2) content of LPS stimulation model group were significantly (P<0.01),and content was not related to concentration.Real-time quantitative (RT-PCR) showed,the concentration of each treatment group were significantly lower iNos,HO-1 and TNF-α mRNA expression (P<0.05,P<0.05,P<0.01),and content was not related to concentration.Conclusion: Coptis chinensis Franch.ethanol extract has anti-inflammatory effects in vitro,the mechanism may be related to inhibition of TNF-α,NO and other inflammatory factors and the impact of the activation of arachidonic acid (AA) metabolism.

Suppressive effects of acetone extract from the stem bark of three Acacia species on nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells

Objective To compare the inhibitory effects of acetone extracts from the stem bark of three Acacia species (Acacia dealbata, Acacia ferruginea and Acacia leucophloea) on nitric oxide production. Methods The lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells were used to investigate the regulatory effect of acetone extracts of three Acacia stem barks on nitric oxide production and the expression of inducible nitric oxide synthase, cyclooxygenase-2 and tumor necrosis factor-α. Further, the phenolic profile of acetone extracts from the Acacia barks was determined by liquid chromatography-mass spectrometry/mass spectrometry analysis. Results All the three extracts significantly decreased LPS-induced NO production as well as the expression of inducible nitric oxide synthase, cyclooxygenase-2 and tumor necrosis factor-α in a concentration dependent manner (25, 50 and 75 μg/mL). In the liquid chromatography-mass spectrometry/mass spectrometry analysis, acetone extract of Acacia ferruginea bark revealed the presence of 12 different phenolic components including quercetin, catechin, ellagic acid and rosmanol. However, Acacia dealbata and Acacia leucophloea barks each contained 6 different phenolic components. Conclusions The acetone extracts of three Acacia species effectively inhibited the NO production in LPS-stimulated RAW 264.7 cells and the presence of different phenolic components in the bark extracts might be responsible for reducing the NO level in cells.

The regulatory effect of cardamonin on TLR4/MyD88/NF-κB/iNOS pathway / 中国药理学通报

Aim Toassesstheregulatoryeffectsofcar-damonin (CDN ) on toll-like receptor (TLR )-4/MyD88/NF-κB/iNOS signaling pathway in lipopolysac-charide (LPS )-stimulated RAW264. 7 macrophage cells.Methods LPS-stimulatedRAW264.7cells were divided into three groups:vehicle-treated group, LPS-treated group and LPS +CDN-treated group.Cell viability was assessed by CCK-8 assay.The concentra-tion of nitric oxide (NO)in cell culture medium was measured by Griess reagent.The mRNA levels of iN-OS,COX-2,MCP-1 ,TNF-α,IL-6 and IL-1βwere de-termined by reverse transcription real-time quantitative PCR(RT-qPCR).The protein levels of inducible nitric oxide synthase(iNOS),TLR4,myeloid differentiation factor 88(MyD88),nuclear factor κB(NF-κB)phos-phorylated (p )-p65 ,inhibitor κBα(IκBα),and p-IκBαweredeterminedbyWesternblot.Results 1~50 μmol·L-1 CDN had no cytotoxicity in RAW264. 7 cells.However,CDN inhibited the LPS-induced secre-tion of nitric oxide(NO)and mRNA expressions of iN-OS,COX-2,MCP-1 ,TNF-α,IL-6 and IL-1βin a dose-dependent manner.Moreover,50 μmol · L-1 CDN inhibited the LPS-induced up-regulation of iNOS, TLR4,MyD88,NF-κB p-p65,p-IκBαand down-reg-ulationofIκBα.Conclusion Cardamonininhibitsthe production of NO via a mechanism associated with the inhibition of TLR4/MyD88/NF-κB/iNOS pathway.

Suppressive effects of acetone extract from the stem bark of three Acacia species on nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells

Objective: To compare the inhibitory effects of acetone extracts from the stem bark of three Acacia species (Acacia dealbata, Acacia ferruginea and Acacia leucophloea) on nitric oxide production. Methods: The lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells were used to investigate the regulatory effect of acetone extracts of three Acacia stem barks on nitric oxide production and the expression of inducible nitric oxide synthase, cyclooxygenase-2 and tumor necrosis factor-α. Further, the phenolic profile of acetone extracts from the Acacia barks was determined by liquid chromatography-mass spec-trometry/mass spectrometry analysis. Results: All the three extracts significantly decreased LPS-induced NO production as well as the expression of inducible nitric oxide synthase, cyclooxygenase-2 and tumor necrosis factor-α in a concentration dependent manner (25, 50 and 75 μg/mL). In the liquid chromatography-mass spectrometry/mass spectrometry analysis, acetone extract of Acacia ferruginea bark revealed the presence of 12 different phenolic components including quercetin, catechin, ellagic acid and rosmanol. However, Acacia dealbata and Acacia leucophloea barks each contained 6 different phenolic components. Conclusions: The acetone extracts of three Acacia species effectively inhibited the NO production in LPS-stimulated RAW 264.7 cells and the presence of different phenolic components in the bark extracts might be responsible for reducing the NO level in cells.