Fig 1: MiR-23b-3p upregulation promoted the effects of obesity-induced IR of mice. a Body weight of mice after obesity-induced IR model construction, Acacetin injection and miR-23b-3p upregulation was measured. b Fat percent of mice was calculated after obesity-induced IR model construction, acacetin injection and miR-23b-3p upregulation. c-d Levels of fasting blood glucose (c) and fasting insulin (d) after obesity-induced IR model construction, acacetin injection and miR-23b-3p upregulation was determined. e-f AUC of mice for OGTT (e) and ITT (f) after obesity-induced IR model construction, acacetin injection and miR-23b-3p upregulation were quantified. g-j Levels of inflammatory cytokines CRP (g), IL-6 (h), TNF-α (i) and MCP1 (j) after obesity-induced IR model construction, acacetin injection and miR-23b-3p upregulation were detected with ELISA. k Relative expressions of IL-17 and Foxp3 after obesity-induced IR model construction, acacetin injection and miR-23b-3p upregulation were determined using qRT-PCR. GAPDH was used as internal control. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001, vs. Control; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, vs. Model; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. Acacetin+mimic control (MC). miR-23b-3p: MicroRNA-23b-3p; AUC: Area under the Curve; OGTT: Oral glucose tolerance test; ITT: Insulin tolerance test; CRP: C-reactive protein; IL-6: Interleukin-6; TNF-α: tumor necrosis factor-α; MCP1: monocyte chemoattractant protein 1; ELISA: enzyme-linked immunosorbent assay; Foxp3: Forkhead Box P3; qRT-PCR: quantitative real-time polymerase chain reaction
Fig 2: Robust and chronic visualization of blood plasma by in vivo transgene expression of Alb-mNG in hepatocytes(A) Schematic of approach for the in vivo experiments. AAV8-P3-Alb-mNG is administrated to mice via i.p. or i.v. injection (left). Alb-mNG expression was monitored by collecting a blood sample from the tail. Brain and liver tissues and blood were collected for morphological and biochemical examination (right).(B) Example of the fluorescence signals in blood samples collected on days 2 and 5 from a mouse that received i.p injection of AAV8-P3-Alb-mNG.(C) Plasma Alb-mNG fluorescence intensity over a time course of 8 weeks. Significant effect of time (one-way ANOVA: p < 0.05) (n = 6 mice).(D) Mouse liver images after 3 weeks of AAV8-P3-eGFP (positive control), PBS (negative control), and AAV8-P3-Alb-mNG i.p. injection. Scale bars, 500 μm.(E) mNG concentration in plasma samples (n = 3 mice). No significant effect of time (one-way ANOVA: p > 0.05)(F) Plasma albumin concentration using albumin ELISA in PBS- (gray, n = 2) and Alb-mNG-injected (green, n = 3) mice.(G) Plasma CRP levels for control or Alb-mNG i.p. injected mice during the 8 weeks of post-injection period (n = 6–12 mice).(H) Example images of liver (top panel) and brain slices (bottom panel) of control- (PBS) or Alb-mNG-expressing mice immunostained for macrophages (liver) or microglia (brain) by IBA1 (purple) and DAPI (yellow). Brain sections of lipopolysaccharide (LPS)-injected mice displayed reactive microglia morphology, while resting microglia are observed in the Alb-mNG mouse. Scale bars, 10 μm.Graphs show means ± SEM and individual values (except from C); ∗p < 0.05.
Fig 3: L-DOPA treatment in EAU Gnat1rd17 mice decreases retinal infiltration of immune cells. Absolute numbers of neuroretina-infiltrating CD4+, CD8+, and CD11b+ cells in EAU Gnat1rd17 mice treated with vehicle or L-DOPA given (a) i.p. or as (b) eye drops (day 14 after immunization) were measured by flow cytometry. The data shown are the mean ± SEM of 6–8 neuroretinas from 6–8 mice per group and are representative of two experiments. * p 0.05 and *** p 0.001 by unpaired t-test for comparisons between groups receiving i.p. injections of vehicle and L-DOPA, respectively, and paired t-test for comparisons between groups of vehicle-treated contralateral eyes and L-DOPA-treated ipsilateral eyes in the same animals. (c) Absolute numbers of CD3+ T cells, naïve CD4+ T cells (CD4+CD44low), activated CD4+ T cells (CD4+CD44high), CD11b+ (CD11bhighCD11clow, macrophages, and neutrophils), and CD11c+ (CD11chighMHC-IIhigh, dendritic cells) in spleens of EAU Gnat1rd17 mice treated with i.p. injections of vehicle or L-DOPA. Flow cytometry was used to analyze the data on day 14 after immunization, and the results are shown as the means ± SEM of 4 mice in each group (unpair t-test). Shown is one representative experiment of two independently performed experiments. (d) qRT-PCR analysis of Crp mRNA levels in the liver of EAU Gnat1rd17 mice treated with i.p. injections of vehicle or L-DOPA. Data were collected on day 14 following immunization and are presented as the mean ± SEM of 4 mice in each group (unpair t-test).
Fig 4: EAU in Gnat1rd17 mice results in an increased accumulation of immune cells in the neuroretina accompanied by local overexpression of inflammatory mediators. (a) Quantification of total CD4+, CD8+, and CD11b+ cell infiltrates in the neuroretina by flow cytometry in EAU WT and Gnat1rd17 mice on day 14 after immunization. Data are given as the mean ± SEM of 10 neuroretinas per group. Differences between groups: ** p 0.01 by unpaired t-test. (b) Flow cytometry analysis of splenocytes collected from EAU WT and Gnat1rd17 mice on day 14 after immunization shows no statistically significant difference between the groups for total numbers of CD3+ T cells, naïve CD4+ T cells (CD4+CD44low), activated CD4+ T cells (CD4+CD44high), CD11b+ (CD11bhighCD11clow, macrophages, and neutrophils), or CD11c+ (CD11chighMHC-IIhigh, dendritic cells). Data are shown as mean ± SEM of 5 mice in each group (unpair t-test). (c) Spleen mRNA expression of IL-1β and IL-6; (d) liver mRNA expression of Crp, IL-1β, and IL-6; and (e) serum CRP levels show no statistically significant difference between WT and Gnat1rd17 mice on day 14 after immunization. Data shown are the mean ± SEM of 5 mice in each group (unpaired t-test). (f) mRNA levels of cytokine, chemokine, and neuroinflammatory/immune cell marker genes in the neuroretinas of EAU WT and Gnat1rd17 mice on day 14 after immunization. The data are presented as the mean ± SEM of 8–10 neuroretinas from 4–5 mice per group, with one representative of two experiments shown. Significant differences between groups: * p 0.05, ** p 0.01, and *** p 0.001 (unpaired t-test).
Fig 5: SAA1 decreased but CRP was not significantly changed in 600 µL BALF collected from intranasal S. pneumoniae infection mice. (A) Detection of S. pneumoniae in both blood and BALF 24 h post-PBS- or S. pneumoniae D39-nasal administration. n = 18. All individual data points are non-negative. Data are presented as mean ± SEM. (B) CRP in BALF displayed no significant difference between non-infection (PBS-intranasal administration, n = 6) and infection (S. pneumoniae D39-nasal administration, n = 6) groups. Student’s t-test was used for statistical analysis (p = 0.28, no significant difference). The experiment was independently repeated three times with similar trends observed. The data presented are from one representative experiment. (C) SAA1 in BALF decreased in infection (S. pneumoniae D39-nasal administration, n = 6) group in comparison to non-infection (PBS-intranasal administration, n = 6) group. Student’s t-test was used for statistical analysis (p < 0.05, showed significant difference). The experiment was independently repeated three times with similar trends observed. The data presented are from one representative experiment.
Supplier Page from Thermo Fisher Scientific for Mouse CRP ELISA Kit