Journal: Frontiers in Pharmacology

Article Title: The TLR4-Active Morphine Metabolite Morphine-3-Glucuronide Does Not Elicit Macrophage Classical Activation In Vitro

doi: 10.3389/fphar.2016.00441

Figure Lengend Snippet: Effect of M3G co-administered with IFN-γ on expression of M1 markers in RAW264.7 cells. RAW264.7 cells (2 × 10 5 cells/well) were treated with 1 ng/ml IFN-γ alone or together with M3G (1, 5, 10, or 20 μM) for 12 h. Expression of iNOS, TNF-α, IL-6, and CD86 mRNA was determined by qRT-PCR. Results are shown relative to control (untreated) RAW264.7 cells. Mean ± SEM is shown, n = 4 independent experiments. ∗ p < 0.05, M3G + IFN-γ vs. IFN-γ alone, one-way ANOVA analysis with Dunnett’s multiple comparisons. Results are shown as mean ± SEM, n = 4–7 independent experiments.

Article Snippet: Amplification and quantification of cDNA was assessed, using TaqMan TM Fast Universal PCR Master Mix (Life Technologies, VIC, Australia) with AmpliTaq Gold TM DNA Polymerase and TaqMan TM Gene Expression Assays including human primers : IL-6 (Hs00985639-m1), IL-12 (Hs01011518-m1), IL-23 (Hs00900828-g1), TNFα (Hs01113624-g1), CXCL10 (Hs01124252-g1), and CXCL11(Hs04187682-g1) or mouse primers: iNOS (Mm00440502-m1), IL-6 (Mm00446190-m1), TNFα (Mm00443258-m1), CD86 (Mm00444543-m1) in a StepOnePlus 7500 real time PCR system (Applied Biosystems, Carlsbad, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Frontiers in Pharmacology

Article Title: The TLR4-Active Morphine Metabolite Morphine-3-Glucuronide Does Not Elicit Macrophage Classical Activation In Vitro

doi: 10.3389/fphar.2016.00441

Figure Lengend Snippet: Effect of M3G on expression of M1 markers in RAW264.7 cells. RAW264.7 cells (2 × 10 5 cells/well) were exposed to 0.001 or 0.01 ng/ml LPS, or M3G at the indicated concentrations (1, 5, 10, and 20 μM). The mRNA levels of iNOS, CD86, IL-6, or TNF-α were determined by qRT-PCR. Results are shown relative to control (untreated) RAW264.7 cells. Results are shown as mean ± SEM, n = 4–7 independent experiments. ∗∗ p < 0.01, LPS vs. control cells.

Article Snippet: Amplification and quantification of cDNA was assessed, using TaqMan TM Fast Universal PCR Master Mix (Life Technologies, VIC, Australia) with AmpliTaq Gold TM DNA Polymerase and TaqMan TM Gene Expression Assays including human primers : IL-6 (Hs00985639-m1), IL-12 (Hs01011518-m1), IL-23 (Hs00900828-g1), TNFα (Hs01113624-g1), CXCL10 (Hs01124252-g1), and CXCL11(Hs04187682-g1) or mouse primers: iNOS (Mm00440502-m1), IL-6 (Mm00446190-m1), TNFα (Mm00443258-m1), CD86 (Mm00444543-m1) in a StepOnePlus 7500 real time PCR system (Applied Biosystems, Carlsbad, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: Tailoring a traditional Chinese medicine prescription for complex diseases: A novel multi-targets-directed gradient weighting strategy

doi: 10.1016/j.jpha.2025.101199

Figure Lengend Snippet: Nao Yi Kang decoction (NYKD) inhibits glial cell activation and proliferation. (A, B) Immunofluorescent staining shows that NYKD inhibits astrocyte activation (green) and decreases the number of proliferating astrocytes: representative images of glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) double staining (A) and statistic diagram of the number of double positive cells of GFAP and PCNA. (C, D) Immunofluorescent staining shows that NYKD inhibits microglia activation (green) and decreases the number of proliferating microglia: representative images of ionized calcium-binding adaptor molecule 1 (Iba1) and PCNA double staining (C) and statistic diagram of the number of double positive cells of Iba1 and PCNA (D). (E, F) Immunofluorescent staining shows that NYKD decreased the number of M1 microglia: representative images of Iba1 and cluster of differentiation 86 (CD86) double staining (E) and statistic diagram of the number of double positive cells of Iba1 and CD86 (F). (G, H) Immunofluorescent staining shows that NYKD increases the number of M2 microglia: representative images of Iba1 and arginase 1 (Arg1) double staining (G) and statistic diagram of the number of double positive cells of Iba1 and Arg1 (H). Data are presented as mean ± standard deviation (SD) ( n = 5). ∗∗∗ P < 0.001 and ∗∗∗∗ P < 0.0001. ns: not significant. ICH: intracerebral hemorrhage; Medium: medium dose; DAPI: diamidino-phenyl-indole.

Article Snippet: The primary antibodies used were mouse anti-glial fibrillary acidic protein (GFAP) (1:1500, CSB-MA009369A0m; Cusabio Biotech Co., Ltd., Wuhan, China), mouse anti-proliferating cell nuclear antigen (PCNA) (1:1000, 2586; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba1) (1:500, 17198; Cell Signaling Technology), rabbit anti-PCNA (1:1000, 13110; Cell Signaling Technology), rabbit anti-cluster of differentiation 86 (CD86) (1:200, 19589; Cell Signaling Technology), and rabbit anti-arginase 1 (Arg1) (1:1000, 93668; Cell Signaling Technology).

Techniques: Activation Assay, Staining, Double Staining, Binding Assay, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: Osteopontin promotes dendritic cell maturation and function in response to HBV antigens

doi: 10.2147/DDDT.S81656

Figure Lengend Snippet: Blockage of OPN suppressed DC maturation and cytokine production after hepatitis B virus (HBV) antigenic stimulations in human PBMC. Notes: After administering OPN neutralizing antibody in vitro, the histograms ( A ) and expressions ( B ) of CD80, CD86, and HLA-DR in BDCA-1 + cells after HBV antigenic stimulations in human PBMCs are presented. Overlay plots are representative and show isotype control (shadow) and the expressions of the indicated molecules on DCs in the absence (green line) or presence (red line) of neutralizing antibody in each group. Levels of IL-12 ( C ), TNF-α, IFN-γ, and IL-4 ( D ) in human PBMC supernatant were measured after administering the OPN-neutralizing antibody in vitro as well as HBcAg and HBsAg stimulations. * P <0.05. Abbreviations: OPN, osteopontin; DCs, dendritic cells; HBV, hepatitis B virus; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; PBMC, peripheral blood mononuclear cells; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; Ab, antibody; IgG, Immunoglobulin gamma; HLA-DR, human leukocyte antigen-DR; BDCA-1, blood dendritic cell antigen 1.

Article Snippet: Expressions of surface molecules on DC were detected by flow cytometry using the following antibodies: PE-anti-mouse I-Ab/I-Ed (M5/114), PE-anti-mouse B7-1 (1G10), PE-anti-mouse B7-2 (Gl-1), FITC-anti-mouse CD11c (HL3), APC-anti-mouse TCRβ (H57-597), FITC-anti-human CD80 (L307.4), FITC-anti-human CD86 (2331 (FUN-1)), FITC-anti-human human leukocyte antigen DR (HLA-DR) (G46-6), PE-anti-human BDCA-1 (F10/21A3) and APC-anti-human CD3 (HIT3A) (all from BD Biosciences, San Diego, CA, USA).

Techniques: In Vitro

Journal: Drug Design, Development and Therapy

Article Title: Osteopontin promotes dendritic cell maturation and function in response to HBV antigens

doi: 10.2147/DDDT.S81656

Figure Lengend Snippet: OPN deficiency impaired DCs maturation and function after HBV antigenic stimulation in vitro. Notes: DCs derived from BM of the WT and OPN −/− mice were stimulated by HBV antigens. The histograms ( A ) and percentages ( B–D ) of CD80, CD86, and MHC-II molecules on the DC surface were detected by flow cytometry. Overlay plots are representative and show isotype control (shadow) and the expressions of the indicated molecules on DCs from WT mice (green line) or OPN −/− mice (red line) in each group. ( E ) Levels of IL-12 in the BMDC culture supernatants following different stimulations were measured by ELISA. * P <0.05; ** P <0.01. Abbreviations: OPN, osteopontin; DCs, dendritic cells; HBV, hepatitis B virus; BM, bone marrow; WT, wild-type; HBcAg, hepatitis B core antigen; HBsAg, surface antigen of the hepatitis B virus; MHC, major histocompatibility complex; IL, interleukin; BMDC, bone marrow-derived dendritic cell; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Expressions of surface molecules on DC were detected by flow cytometry using the following antibodies: PE-anti-mouse I-Ab/I-Ed (M5/114), PE-anti-mouse B7-1 (1G10), PE-anti-mouse B7-2 (Gl-1), FITC-anti-mouse CD11c (HL3), APC-anti-mouse TCRβ (H57-597), FITC-anti-human CD80 (L307.4), FITC-anti-human CD86 (2331 (FUN-1)), FITC-anti-human human leukocyte antigen DR (HLA-DR) (G46-6), PE-anti-human BDCA-1 (F10/21A3) and APC-anti-human CD3 (HIT3A) (all from BD Biosciences, San Diego, CA, USA).

Techniques: In Vitro, Derivative Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Drug Design, Development and Therapy

Article Title: Osteopontin promotes dendritic cell maturation and function in response to HBV antigens

doi: 10.2147/DDDT.S81656

Figure Lengend Snippet: OPN produced by DCs plays an important role in response to HBV antigenic stimulation in vivo. Notes: At 24 hours after HepG2.2.15 cells supernatant direct injection into liver from the WT mice and OPN −/− mice ( A ) liver histology was assessed. Original magnification, 400×. ( B ) Leukocyte number and ( C ) expressions of CD80, CD86, and MHC-II molecules on DCs in liver from the WT mice and OPN −/− mice were measured. ( D ) Number of TCR + cells per gram of liver tissue were detected by an automated cell counter and flow cytometry. ( E ) Number of IFN-γ-producing T-cells and IL-4-producing T-cells were detected. HepG2 supernatant injection was used as control. n=5 per group. Adoptive transfer of CD11c + cells from the WT and OPN −/− mice as well as HepG2.2.15 supernatant direct injection into mice liver were performed. Then ( F ) the number of leukocyte per gram of liver tissue was counted in the OPN −/− mice at 0 and 24 hours; n=5 per group. ( G ) IFN-γ-producing cells in T-cells in liver from the OPN −/− mice were analyzed. n=5 per group. * P <0.05; ** P <0.01; *** P <0.001. Abbreviations: OPN, osteopontin; DCs, dendritic cells; HBV, hepatitis B virus; WT, wild-type; MHC, major histocompatibility complex; IL, interleukin; IFN, interferon; TCR, T-cell receptor; h, hour.

Article Snippet: Expressions of surface molecules on DC were detected by flow cytometry using the following antibodies: PE-anti-mouse I-Ab/I-Ed (M5/114), PE-anti-mouse B7-1 (1G10), PE-anti-mouse B7-2 (Gl-1), FITC-anti-mouse CD11c (HL3), APC-anti-mouse TCRβ (H57-597), FITC-anti-human CD80 (L307.4), FITC-anti-human CD86 (2331 (FUN-1)), FITC-anti-human human leukocyte antigen DR (HLA-DR) (G46-6), PE-anti-human BDCA-1 (F10/21A3) and APC-anti-human CD3 (HIT3A) (all from BD Biosciences, San Diego, CA, USA).

Techniques: Produced, In Vivo, Injection, Flow Cytometry, Adoptive Transfer Assay

Journal: Drug Design, Development and Therapy

Article Title: Osteopontin promotes dendritic cell maturation and function in response to HBV antigens

doi: 10.2147/DDDT.S81656

Figure Lengend Snippet: OPN administration significantly promoted the maturation of DCs from CHB patients after HBcAg stimulation. Notes: The expressions of CD80 ( A ), CD86 ( B ), and HLA-DR ( C ) in BDCA-1 + cells from the peripheral blood of CHB patients following HBcAg stimulation with or without exogenous OPN administration were measured by flow cytometry. CHB patients, n=20. ** P <0.01, *** P <0.001. ( D ) The percentages of IFN-γ-producing cell in CD3 + T-cells from the peripheral blood of CHB patients following HBcAg stimulation with or without exogenous OPN administration were measured by flow cytometry. Abbreviations: OPN, osteopontin; DCs, dendritic cells; CHB, chronic hepatitis B; HBV, hepatitis B virus; HBcAg, hepatitis B core antigen; IFN, interferon; HLA-DR, human leukocyte antigen-DR; BDCA-1, Blood Dendritic Cell Antigen 1.

Article Snippet: Expressions of surface molecules on DC were detected by flow cytometry using the following antibodies: PE-anti-mouse I-Ab/I-Ed (M5/114), PE-anti-mouse B7-1 (1G10), PE-anti-mouse B7-2 (Gl-1), FITC-anti-mouse CD11c (HL3), APC-anti-mouse TCRβ (H57-597), FITC-anti-human CD80 (L307.4), FITC-anti-human CD86 (2331 (FUN-1)), FITC-anti-human human leukocyte antigen DR (HLA-DR) (G46-6), PE-anti-human BDCA-1 (F10/21A3) and APC-anti-human CD3 (HIT3A) (all from BD Biosciences, San Diego, CA, USA).

Techniques: Flow Cytometry