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-a (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-a: tumor necrosis factor-a; 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-1ß and IL-6; (d) liver mRNA expression of Crp, IL-1ß, 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).
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