Fig 1: Diosmin-induced NEP expression depends on activate AhR. (A) N2a cells were treated with Dio (80 μM) with or without SR1 (2 μM) for 12 h. Whole cell lysates were collected and the NEP levels were determined. n = 3. (B) N2a cells were transfected with non-target control siRNA (NC) or siRNA targeting AhR (siAhR) for 48 h. The AhR levels were determined by western blotting. n = 3. (C) N2a cells were transfected with NC or siAhR for 48 h, followed by administration of vehicle or Dio (80 μM) for 12 h. NEP levels were then determined by western blotting. n = 3. APP/PS1 mice (8 months of age) received an intracerebroventricular injection with AAV9 carrying the shRNA targeting AhR (shAhR) or the shRNA targeting GFP (shGFP). Five days after injection, the mice were administrated with vehicle or Dio (40 mg/kg) for another 4 weeks. Total protein from the cortex and hippocampus tissues was prepared to determine the AhR levels by western blotting (D) and the NEP levels by western blotting (E) and ELISA (F), n = 3. (G) N2a cells were treated with Dio (80 μM) with or without SR1 (2 μM) for 12 h. n = 3. (H) APP/PS1 mice (8 months of age) were administrated with vehicle or Dio (40 mg/kg) for 4 weeks. Whole cell lysates of cells and animal tissues were collected. n = 3. The NEP enzyme activity in the samples was detected by a NEP Activity Assay Kit. Data were expressed as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. indicated group. Dio: diosmin; NC: nontarget control; NEP: Neprilysin; SR1: StemRegenin 1.
Fig 2: AhR transcriptionally upregulates NEP expression. (A-D) N2a cells were treated with Dio at the indicated concentrations for 6 h, and then the total mRNA was extracted (n = 3) (A); APP/PS1 mice (8 months of age) were administrated with vehicle or Dio (40 mg/kg) for 4 weeks. The WT littermates were used as control (B); N2a cells were treated with Dio (80 μM) with or without SR1 (2 μM) for 6 h (n = 3) (C); N2a cells were transfected with non-targeting siRNA (NC) or siRNA targeting AhR (siAhR) for 48 h, and then treated with Dio (80 μM) for another 6 h (n = 3) (D). The total RNA samples were extracted from the cell and tissue samples. The Nep mRNA levels were determined by qRT-PCR. n = 3. (E-G) HEK293T cells were co-transfected with NEP promoter-luciferase plasmid and WT AhR expression plasmid for 48 h. Whole cell lysates were collected and the AhR expression levels were determined by western blotting (E). After transfection, HEK293T cells were treated with Dio (80 μM), FICZ (250 nM), L-KN (3 μM) or I3C (10 μM) for another 18 h (n = 4) (F), or with Dio (80 μM) with or without SR1 (2 μM) for 18 h (n = 4) (G). Whole cell lysates were collected and the luciferase activity was determined. (H) HEK293T cells were co-transfected with WT AhR expression plasmid and WT or mutant (site1 or site2) NEP promoter-luciferase plasmid for 48 h. The cells were then treated with Dio (80 μM) for an additional 18 h, and then the luciferase activity was determined. n = 4. (I) ChIP-qPCR analysis of AhR binding to the NEP promoter in N2a cells. n = 3. Data were expressed as mean ± SD. ## p < 0.01 vs. WT treated with vehicle or N2a cell treated with DMSO, * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. indicated group. Dio: diosmin; I3C: indole-3-carbinol; L-KN: L-Kynurenine; Mut: mutant; SR1: StemRegenin 1; WT: wild type.
Fig 3: Activated AhR increases NEP expression in N2a cells and in the brain of APP/PS1 mice. (A and B) The cortex and hippocampus tissues from WT mice and APP/PS1 mice at the indicated age were collected, and homogenized, and the NEP levels were determined by western blot analysis (n = 3) (A) and ELISA (n = 6) (B). NEP expression was adjusted to β-actin, which was used as the loading control in western blot analysis (A) or normalized to the total protein in ELISA (B). (C) N2a cells were treated with DMSO or Dio (40 µM) for 6 h, the total mRNA was extracted and the mRNA levels of Cyp1a1, Cyp1b1 and Gst were determined by qRT-PCR, n = 3. (D) N2a cells were treated with DMSO, Dio (80 μM), FICZ (250 nM), I3C (10 μM) or L-KN (3 μM) for 24 h and the cell viability was measured by the CCK8 assay. n = 6. (E) N2a cells were treated with DMSO, Dio (40 μM), FICZ (250 nM), I3C (10 μM) or L-KN (3 μM) for 12 h. Whole cell extracts were examined by western blotting with the indicated antibodies. NEP expression was adjusted to that of β-actin, which was used as the loading control. n = 3. (F and G) N2a cells were treated with Dio at the indicated concentrations for 12 h (n = 3) (F) or with 80 μM Dio for the indicated time (n = 3) (G). The NEP protein levels were determined by western blotting. Eight-month-old APP/PS1 mice were treated with vehicle or Dio at the indicated concentration for 4 weeks. (H) The bodyweight was monitored during this period. At the end of the treatment, the mice were sacrificed and the cortex and hippocampus tissues were collected. The NEP protein levels were determined by western blot analysis (n = 3) (I) or ELISA (n = 4) (J). Data were expressed as mean ± SD. # p < 0.05; ## p < 0.01, ### p < 0.001 vs. indicated group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. indicated group. Dio: diosmin; Hippo: hippocampus; I3C: indole-3-carbinol; L-KN: L-Kynurenine;NEP: neprilysin;WT: wild type.
Fig 4: Activated AhR increases NEP expression in the brain of APP/PS1 mice. Eight-month-old APP/PS1 mice were treated with vehicle or Dio at the indicated concentration for 4 weeks. At the end of the treatment, the mice were sacrificed and the brain tissues were fixed and stained for NEP (green), NeuN (red) and DAPI (blue). NEP staining and quantification in cortex (A), and CA3 (B), CA1 (C) and DG (D) of the hippocampal regions. Scale bar: 20 μm. n = 6. Each point represents the mean intensity of NEP for 3-5 sections per mouse. Data were expressed as mean ± SD. ** p < 0.01, *** p < 0.001 vs. APP/PS1 group. CA: Cornus Ammonis; DG: dentate gyrus; Dio: diosmin; NEP: neprilysin;WT: wild type.
Fig 5: Diosmin ameliorates cognitive deficiency in APP/PS1 mice through the AhR-NEP pathway. (A) The experimental design of the mouse study. The APP/PS1 mice (8 months of age) were administrated vehicle or with Dio (40 mg/kg) for 4 weeks. On day 20 (d20), the mice were subjected to MWM assay. Quadrant IV was defined as the target quadrant. (B) Escape latency during spatial acquisition training. (C) Representative motion track during the spatial probe test. (D) Distance in the target quadrant, (E) time spent in the target quadrant, and (F) the number of platform crossings in the spatial probe test. (G) Swim speed during the spatial probe test. After the MWM assay was completed, the mice were subjected to the object recognition test. (H) The procedure for the object recognition test. The square frame represents the open field, the small square and circle represent the object. (I) Representative motion track and (J) the discrimination index of the object recognition test. n = 10. Data were expressed as mean ± SD. ## p < 0.01, ### p < 0.001 vs. WT mice treatment with vehicle, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. APP/PS1 mice treatment with vehicle. Dio: diosmin; MWM: Morris water maze; WT: wildtype.
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