Fig 1: DRD5 receptor is highly expressed in colonic macrophages.A RT-qPCR analysis of gene expression of DRD1, DRD2, DRD3, DRD4, and DRD5 in IECs (CD45-Epcam+) and LP cells (CD45+Epcam-) isolated from WT mice (n = 3 mice per group). B RT-qPCR analysis of gene expression of DRD1, DRD4, and DRD5 in T cells, B cells, NK cells, DC cells, monocytes, neutrophils, macrophages, and ILCs cells isolated from the colonic LP of WT mice (n = 3 mice per group). C Immunofluorescent labeling of TH (green), Cx3cr1(red), and DAPI (blue) in colon sections from Cx3cr1 reporter mice. The close proximity of macrophages with dopaminergic neurons is indicated by arrow. Scale bar, 30 µm. D Immunofluorescent labeling of DRD5 (green), F4/80 (red), and DAPI (blue) in colon sections from WT mice. The merging of DRD5 with F4/80+ in macrophages indicated by the arrowhead. Scale bar, 30 µm. E Immunofluorescent labeling of DRD5 (green), Arg1(red), and DAPI (blue) in colon sections from WT mice. The merging of DRD5 with Arg1+ in macrophages indicated by the arrowhead. Scale bar, 30 µm. F Immunofluorescent labeling of DRD5 (green), Inos (red), and DAPI (blue) in colon sections from WT mice. The merging of DRD5 with Inos+ in macrophages indicated by the arrowhead. Scale bar, 30 µm. Data are pooled from three independent experiments (A, B). Error bars show means ± SEM. ***p < 0.001. Two-tailed unpaired student’s t-test.
Fig 2: Mechanism of AS-IV was tested after transfected with miR-203 inhibitor in LPS-induced inflammatory damage. (A) The level of miR-203 was tested through qRT-PCR. (B) Cell viability was measured through CCK-8 assay. (C) Apoptosis was measured through PI and FITC-conjugate Annexin V staining. (D) The related protein expressions of apoptotic process were tested through western blot. (E-F) The levels of TNF-α, IL-6 and iNOS were detected through western blot analysis. (G) Concentration of TNF-α and IL-6 were tested using ELISA. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the indicated group, #p < 0.05 and ##p < 0.01 compared to LPS group.
Fig 3: Immunoflorescence staining results of iNOS (96 h). A) Lamella of gill tissue (arrow). Control group. IF. 20 μm. B) Weak positive reaction in lamellar cells of gill tissue (arrows). Low dose toxic group. IF. 20 μm. C) Positivity in lamellar cells of gill tissue (arrow). High dose toxic group. IF. 20 μm. D) Liver tissue, vena centralis (arrow). Control group. IF. 20 μm. E) Positive reactions in hepatocytes (arrows). Low dose toxic group. IF. 20 μm. F) Positive reactions in hepatocytes (arrows). High dose toxic group. IF. 20 μm. G) Weak positive reactions in neurons (arrows). Control group. IF. 20 μm. H) Positivity in neurons (arrows). Low dose toxic group. IF. 20 μm. I) Positivity in neurons (arrows). High dose toxic group. IF. 20 μm.
Fig 4: Assessments of inflammatory cell infiltration in tumors harvested at late stages.Tissue sections from tumor xenografts harvested at end-point of study, i.e. 41–61 days post-implantation of cells, were immune-labelled for iNOS (M1-macrophages), Arg1 (M2-macrophages) or MPO (neutrophils). Intratumoral and peritumoral scores in left and right panels, respectively. Middle panel; representative images from each staining from randomly selected tumors.
Fig 5: METP NPs reverse the proinflammatory phenotype in BMDMs under LPS stimulation in vitro. (A) Expression levels of the CD80 and CD86 macrophage surface markers according to flow cytometry analysis. (B) Secretion levels of the TNF-α and IL-1β proinflammatory cytokines according to ELISA (pg/mL). (C and D) Protein expression level of the iNOS and ARG1 macrophage markers according to Western blot analysis. (E) The gene transcription levels of Nos2, TNF-α, IL-1β, IL-6, Cxcl9 and actin were detected by qRT-PCR in all four groups. The concentration of MET NPs is shown in µg/mL, and the concentration of DS16570511 is shown in µM. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
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