Journal: British Journal of Pharmacology

Article Title: Artesunate interacts with the vitamin D receptor to reverse sepsis‐induced immunosuppression in a mouse model via enhancing autophagy

doi: 10.1111/bph.15158

Figure Lengend Snippet: Artesunate (AS) increases pro‐inflammatory cytokines release and bacterial clearance within LPS‐tolerant macrophages (n = 4). (a) LPS increased the release of TNF‐α (a1) and IL‐6 (a2) from peritoneal macrophages (PMs) in a dose‐dependent manner. (b) Schematic diagram of the establishment of the LPS‐tolerant macrophage model. (c) The level of TNF‐α (c1) and IL‐6 (c2) in LPS‐tolerant PMs (n = 5). (d) Effect of AS (5, 10 and 20 μg·ml−1) treatment on the level of TNF‐α (d1, d2) and IL‐6 (d3, d4) in LPS‐tolerant PMs and RAW264.7 cells (n = 5). (e) Effect of AS treatment (20 μg·ml−1) on the bacterial load in LPS‐tolerant RAW264.7 cells (n = 8). (f) Effect of AS (20 μg·ml−1) treatment on the mRNA level of TNF‐α (f1, f2) and IL‐6 (f3, f4) in LPS‐tolerant THP‐1 monocytes and THP‐1 derived macrophages (n = 5). (g) Effect of AS treatment (20 μg·ml−1) on the bacterial load in LPS‐tolerant THP‐1 derived macrophages (n = 8). One‐way ANOVA followed by Tukey's post hoc test; * P < 0.05

Article Snippet: Cell lines, culture and isolation of peritoneal macrophages from mice The murine macrophage‐like cell line RAW264.7 (CLS Cat#400319/p462_RAW‐2647, RRID:CVCL_0493) cells and human monocyte THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Derivative Assay

Journal: British Journal of Pharmacology

Article Title: Artesunate interacts with the vitamin D receptor to reverse sepsis‐induced immunosuppression in a mouse model via enhancing autophagy

doi: 10.1111/bph.15158

Figure Lengend Snippet: The vitamin D receptor is predicted to be an interactor candidate of artesunate (AS). (a) A total of 20 underlying signal molecules were selected via the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP). (b) Effect of AS on the relative mRNA levels of Vdr (n = 5). (c) Effect of AS on the protein levels of VDR (n = 5). (d) Effect of Vdr siRNA (d1) and Vdr‐KD lentiviral vector (d2) on TNF‐α levels in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). (e) Effect of Vdr‐OE lentiviral vector on TNF‐α level in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). (f1) Schematic diagram of the binding assay designed in our laboratory. (f2) Effect of VD3 on the binding of AS and VDR tracked by AS fluorophores (n = 5). AS with fluorophore 12‐(7‐oxycoumarinyl‐ethoxy) dihydroartemisinin was named AS I and AS with 12‐(‐1H‐benzo [de] isoquinoline‐1, 3(2H)‐dione‐2‐ethoxy) dihydroartemisinin was named AS II. (g) Effect of VD3 (100 nM) on TNF‐α levels in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). One‐way ANOVA followed by Tukey's post hoc test; ns, not significant; * P < 0.05

Article Snippet: Cell lines, culture and isolation of peritoneal macrophages from mice The murine macrophage‐like cell line RAW264.7 (CLS Cat#400319/p462_RAW‐2647, RRID:CVCL_0493) cells and human monocyte THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Plasmid Preparation, Binding Assay

Journal: British Journal of Pharmacology

Article Title: Artesunate interacts with the vitamin D receptor to reverse sepsis‐induced immunosuppression in a mouse model via enhancing autophagy

doi: 10.1111/bph.15158

Figure Lengend Snippet: Artesunate (AS) inhibits the nuclear translocation of VDR and modulates the transcription of its target gene Atg16l1. RAW264.7 cells were treated as described in the legend of Figure 2d. (a) Immunoblotting to observe the VDR level in the nuclear lysate. (b) Immunostaining to observe the nuclear translocation of VDR. VDR was probed using Alexa Fluor 555 (red). Representative images (bar = 5 μm) (b1). The karyoplasmic ratio of VDR was quantified from 100 cells (normalized to medium) (b2). (c) ChIP analysis for the binding of VDR to the Atg16l1 promoter. The protein–DNA complex was immunoprecipitated with anti‐VDR antibody or a negative control IgG. Representative agarose gels for the VDR‐binding region in the Atg16l1 promoter and Actb DNA in the input amplified using semiquantitative PCR. (d) The binding of VDR to the Atg16l1 promoter, normalized to Actb DNA in the input, analysed by qPCR (n = 5). (e) The protein level of ATG16L1 in LPS‐tolerant RAW264.7 cells treated with AS. (f) Change in ATG16L1 protein levels in LPS‐tolerant RAW264.7 cells (Vdr‐KD) treated with AS. (g) Change in ATG16L1 protein levels in Vdr‐OE LPS‐tolerant RAW264.7 cells treated with AS. One‐way ANOVA followed by Tukey's post hoc test; * P < 0.05

Article Snippet: Cell lines, culture and isolation of peritoneal macrophages from mice The murine macrophage‐like cell line RAW264.7 (CLS Cat#400319/p462_RAW‐2647, RRID:CVCL_0493) cells and human monocyte THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Translocation Assay, Western Blot, Immunostaining, Binding Assay, Immunoprecipitation, Negative Control, Amplification

Journal: British Journal of Pharmacology

Article Title: Artesunate interacts with the vitamin D receptor to reverse sepsis‐induced immunosuppression in a mouse model via enhancing autophagy

doi: 10.1111/bph.15158

Figure Lengend Snippet: Artesunate's (AS) effect is autophagy‐dependent through VDR in vitro. (a) Effect of Atg16l1 siRNA on TNF‐α (a1) and IL‐6 (a2) levels in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). (b) Effect of bafilomycin (Baf) (10 ng·ml−1) on TNF‐α (b1) and IL‐6 (b1) levels in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). (c) Effect of 3‐MA (5 mM) on TNF‐α (c1) and IL‐6 (c2) levels in LPS‐tolerant RAW264.7 cells treated with AS (n = 5). (d) Effect of 3‐MA, Ly294002 (10 μM), or Baf on the bacterial clearance in LPS‐tolerant RAW264.7 cells treated with AS (n = 8). (e1) LPS increased the protein levels of LC3B‐I, LC3B‐II and ATG5 in a dose‐dependent manner in RAW264.7 cells. (e2) The protein levels of LC3B‐I, LC3B‐II and ATG5 over time in RAW264.7 cells treated with LPS (100 ng·ml−1). The level of expression peaked at 1 h. (f) Representative image of immunofluorescence staining of LC3B in LPS‐tolerant RAW264.7 cells treated with AS (bar = 2 μm). (f1) Relative fluorescent puncta indicating LC3B aggregation were quantified from 100 cells; the number in the medium group was normalized as 1 (f2). (g) The protein levels of LC3B‐II, ATG16L1 and ATG5 in LPS‐tolerant RAW264.7 cells treated with AS. (h) Changes in LC3B‐II, ATG16L1 and ATG5 protein levels in LPS‐tolerant RAW264.7 cells (Vdr‐KD) treated with AS. (i) Changes in LC3B‐II, ATG16L1 and ATG5 protein levels in LPS‐tolerant RAW264.7 cells (Vdr‐OE) treated with AS. One‐way ANOVA followed by Tukey's post hoc test; ns, not significant; * P < 0.05

Article Snippet: Cell lines, culture and isolation of peritoneal macrophages from mice The murine macrophage‐like cell line RAW264.7 (CLS Cat#400319/p462_RAW‐2647, RRID:CVCL_0493) cells and human monocyte THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vitro, Expressing, Immunofluorescence, Staining

Journal: British Journal of Pharmacology

Article Title: Artesunate interacts with the vitamin D receptor to reverse sepsis‐induced immunosuppression in a mouse model via enhancing autophagy

doi: 10.1111/bph.15158

Figure Lengend Snippet: Artesunate (AS) inhibits the physical interaction between VDR and NF‐κB p65 in LPS‐tolerant macrophages. RAW264.7 cells were treated as described in the legend of Figure 2d. (a) The cytoplasm (a1) and nuclear (a2) lysate were used for an IP experiment using anti‐VDR antibodies and the associated NF‐κB p65 (p65) was detected by immunoblotting (IB). (b) Immunostaining to observe the co‐localization of p65 and VDR. p65 was probed using Alexa Fluor 488 (green). VDR was probed using Alexa Fluor 555 (red). Representative images are shown (bar = 5 μm) (b1). The co‐localization of VDR and p65 (b2) and the karyoplasmic ratio of p65 (b3) was quantified from 100 cells (normalized to medium). (c) The p65 level in the nuclear lysate was detected using elisa and WB. (d) Change in the p65 level in Vdr‐KD (d1) or Vdr‐OE (d2) LPS‐tolerant RAW264.7 cells treated with AS. (e) Change in the TNF‐α level in p65‐KD (e1) or p65‐OE (e2) LPS‐tolerant RAW264.7 cells treated with AS (n = 5). One‐way ANOVA followed by Tukey's post hoc test; ns, not significant; * P < 0.05

Article Snippet: Cell lines, culture and isolation of peritoneal macrophages from mice The murine macrophage‐like cell line RAW264.7 (CLS Cat#400319/p462_RAW‐2647, RRID:CVCL_0493) cells and human monocyte THP‐1 (CLS Cat# 300356/p804_THP‐1, RRID:CVCL_0006) cells were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: Formulation of Gamma-Oryzanol Encapsulated Nanoparticles and Their Modulation Effects on Inducible Nitric Oxide Synthase and Nitric Oxide in LPS-Stimulated RAW 264.7 Macrophages

doi: 10.3390/pharmaceutics18030365

Figure Lengend Snippet: Cell viability of RAW 264.7 macrophages treated with ORZ-NPs, blank NPs, or ORZ powder at 0–200 μg mL −1 for 48 h, assessed by MTT assay. Cell viability is presented as the percentage of viable cells relative to the untreated, LPS-stimulated control (set as 100% viability). Data are expressed as the mean ± SD of three independent experiments ( n = 3). Statistical significance: * p < 0.05 and *** p < 0.001. Abbreviations: LPS, lipopolysaccharide; mL, milliliter; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; n , number; NPs, nanoparticles; ORZ, gamma-oryzanol; ORZ-NPs, gamma oryzanol encapsulated nanoparticles; SD, standard deviation; μg; microgram.

Article Snippet: RAW 264.7 macrophage-like cells were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: MTT Assay, Control, Standard Deviation

Journal: Pharmaceutics

Article Title: Formulation of Gamma-Oryzanol Encapsulated Nanoparticles and Their Modulation Effects on Inducible Nitric Oxide Synthase and Nitric Oxide in LPS-Stimulated RAW 264.7 Macrophages

doi: 10.3390/pharmaceutics18030365

Figure Lengend Snippet: Inhibitory effects of the ORZ-NPs on iNOS protein expression levels and NO production in LPS-stimulated RAW 264.7 macrophages. Cells were pre-treated with blank NPs, ORZ-NPs, or ORZ (0–50 µg mL −1 ) for 1 h, followed by LPS stimulation (1 µg mL −1 ) for 24 h. Representative Western blot showing iNOS protein expression levels, with β-actin as a loading control ( A ). Quantification of iNOS protein expressed as fold change relative to the untreated, LPS-treated control. iNOS was normalized to β-actin and derived from three independent experiments ( B ). NO production measured by Griess assay, presented as percentage of the untreated, LPS-stimulated control ( C ). Data are expressed as mean ± SD ( n = 3). Statistical significance: * p < 0.05, ** p < 0.01, and *** p < 0.001. Abbreviations: h, hour; iNOS, inducible nitric oxide synthase; kDa, kilodalton; LPS, lipopolysaccharide; mL, milliliter; n , number; NO, nitric oxide; NPs, nanoparticles; ORZ, gamma-oryzanol; ORZ-NPs, gamma oryzanol encapsulated nanoparticles; SD, standard deviation; μg, microgram.

Article Snippet: RAW 264.7 macrophage-like cells were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Expressing, Western Blot, Control, Derivative Assay, Griess Assay, Standard Deviation

Journal: Pharmaceutics

Article Title: Formulation of Gamma-Oryzanol Encapsulated Nanoparticles and Their Modulation Effects on Inducible Nitric Oxide Synthase and Nitric Oxide in LPS-Stimulated RAW 264.7 Macrophages

doi: 10.3390/pharmaceutics18030365

Figure Lengend Snippet: Effect of the ORZ-NPs on the secretion of pro-inflammatory cytokines, including TNF-α ( A ) and IL-6 ( B ) in LPS-stimulated RAW 264.7 macrophages. Cells were pre-treated with blank-NPs, ORZ-NPs, or ORZ powder (0–50 µg mL −1 ) for 1 h, followed by LPS stimulation (1 µg mL −1 ) for 24 h. Cytokine levels were measured by ELISA assay and are presented as percentages relative to the untreated, LPS-stimulated control. Data are shown as mean ± SD of three independent experiments ( n = 3). Statistical significance: *** p < 0.001. Abbreviations: ELISA, enzyme-linked immunosorbent assay; h, hour; IL-6, interleukin-6; LPS, lipopolysaccharide; mL, milliliter; n , number; NPs, nanoparticles; ORZ, gamma-oryzanol; ORZ-NPs, gamma oryzanol encapsulated nanoparticles; SD, standard deviation; TNF-α, tumor necrosis factor-alpha; μg, microgram.

Article Snippet: RAW 264.7 macrophage-like cells were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Standard Deviation