Journal: Neural Regeneration Research
Article Title: Chitosan alleviates symptoms of Parkinson’s disease by reducing acetate levels, which decreases inflammation and promotes repair of the intestinal barrier and blood–brain barrier
doi: 10.4103/NRR.NRR-D-23-01511
Figure Lengend Snippet: Acetate reverses chitosan-mediated repair of the intestinal barrier, increased inflammation in the colon, plasma, and SN, and promotes microglia activation in an MPTP-induced mouse model of PD. (A) Colon length ( n = 5/group). (B) ZO-1 and occludin expression, as assessed by western blot ( n = 3/group). All target proteins were normalized to the reference protein GAPDH. Compared with the chitosan group, acetate supplementation reduced ZO-1 and occludin expression levels. (C) Immunofluorescence staining for ZO-1 (green, Alexa Fluor 488) and occludin (red, Alexa Fluor 594) in mouse colon tissue ( n = 3/group). The immunofluorescence results were consistent with the western blot results. Scale bars: 10 μm. (D) The relative mRNA levels of IL-1β, IL-6, IL-8, IL-10, TNF-α, and iNOS in mouse colon tissue were measured by QPCR ( n = 3/group). Compared with the chitosan group, acetate supplementation resulted in an increase in IL-1β, IL-6, IL-10, TNF-α, and iNOS expression levels in the colon. The data shown in B-D were normalized to the control group. (E) The expression levels of inflammatory cytokines, including IL-1β, IL-6, IL-10, and TNF-α, in mouse plasma were measured by ELISA ( n = 5/group). Compared with the chitosan group, IL-1β and TNF-α levels were significantly increased in the plasma of the acetate group, while IL-10 expression was significantly decreased. (F) The mRNA levels of IL-1β, IL-6, IL-8, IL-10, TNF-α, and iNOS (normalized to the control group) in mouse SN tissue were determined via QPCR ( n = 3/group). Treatment with acetate enhanced TNF-α expression and decreased IL-6 and IL-10 expression in the SN. (G) Representative images of immunofluorescence staining for Iba1 (green, Alexa Fluor 488) and TH (red, Alexa Fluor 594) in the SN ( n = 3/group). The chitosan group exhibited fewer microglia than the MPTP group, while the chitosan + acetate group exhibited more microglia than the chitosan-only group. Scale bars: 50 μm. All data are presented as the mean ± SD. All experiments were repeated at least three times. * P < 0.05 (one-way analysis of variance followed by Tukey’s multiple comparisons test [A–C, G] or unpaired t -test [D–F]). DAPI: 4′,6-Diamidino-2-phenylindole; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Iba1: ionized calcium-binding adapter molecule 1; IL-1β: interleukin-1 beta; IL-6: interleukin-6; IL-8: interleukin-8; IL-10: interleukin-10; iNOS: inductible nitric oxide synthase; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; n.s.: no significance; NaA: sodium acetate; PD: Parkinson’s disease; QPCR: quantitative polymerase chain reaction; SN: substantia nigra; TH: tyrosine hydroxylase; TNF-α: tumor necrosis factor alpha; ZO-1: Zonula occludens-1.
Article Snippet: The primary antibodies used were as follows: rabbit anti-glyceraldehyde-3-phosphate dehydrogenase polyclonal antibody (GAPDH; 1:10,000, Proteintech, Wuhan, Hubei, China, Cat# 10494-1-AP, RRID: AB_2263076), rabbit anti-TH polyclonal antibody (1:5000, Proteintech, Cat# 25859-1-AP, RRID: AB_2716568), rabbit anti-zonula occludens-1 polyclonal antibody (ZO-1; 1:5000, Proteintech, Cat# 21773-1-AP, RRID: AB_10733242), rabbit anti-occludin polyclonal antibody (1:15,000, Proteintech, Cat# 27260-1-AP, RRID: AB_2880820), rabbit anti-AMPKα polyclonal antibody (1:1000, Cell Signaling Technology, Danvers, Massachusetts, USA, Cat# 2532, RRID: AB_330331), rabbit anti-phospho-AMPKα monoclonal antibody (1:1000, Cell Signaling Technology, Cat# 2535, RRID: AB_331250), and rabbit anti-PPARD polyclonal antibody (1:1000, Abcam, Cambridge, UK, Cat# ab23673, RRID: AB_2165902).
Techniques: Clinical Proteomics, Activation Assay, Expressing, Western Blot, Immunofluorescence, Staining, Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Real-time Polymerase Chain Reaction
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![Acetate reverses chitosan-mediated repair of the intestinal barrier, increased inflammation in the colon, plasma, and SN, and promotes microglia activation in an MPTP-induced mouse model of PD. (A) Colon length ( n = 5/group). (B) ZO-1 and occludin expression, as assessed by western blot ( n = 3/group). All target proteins were normalized to the reference protein GAPDH. Compared with the chitosan group, acetate supplementation reduced ZO-1 and occludin expression levels. (C) Immunofluorescence staining for ZO-1 (green, Alexa Fluor 488) and occludin (red, Alexa Fluor 594) in mouse colon tissue ( n = 3/group). The immunofluorescence results were consistent with the western blot results. Scale bars: 10 μm. (D) The relative mRNA levels of IL-1β, IL-6, IL-8, IL-10, TNF-α, and iNOS in mouse colon tissue were measured by QPCR ( n = 3/group). Compared with the chitosan group, acetate supplementation resulted in an increase in IL-1β, IL-6, IL-10, TNF-α, and iNOS expression levels in the colon. The data shown in B-D were normalized to the control group. (E) The expression levels of inflammatory cytokines, including IL-1β, IL-6, IL-10, and TNF-α, in mouse plasma were measured by ELISA ( n = 5/group). Compared with the chitosan group, IL-1β and TNF-α levels were significantly increased in the plasma of the acetate group, while IL-10 expression was significantly decreased. (F) The mRNA levels of IL-1β, IL-6, IL-8, IL-10, TNF-α, and iNOS (normalized to the control group) in mouse SN tissue were determined via QPCR ( n = 3/group). Treatment with acetate enhanced TNF-α expression and decreased IL-6 and IL-10 expression in the SN. (G) Representative images of immunofluorescence staining for Iba1 (green, Alexa Fluor 488) and TH (red, Alexa Fluor 594) in the SN ( n = 3/group). The chitosan group exhibited fewer microglia than the MPTP group, while the chitosan + acetate group exhibited more microglia than the chitosan-only group. Scale bars: 50 μm. All data are presented as the mean ± SD. All experiments were repeated at least three times. * P < 0.05 (one-way analysis of variance followed by Tukey’s multiple comparisons test [A–C, G] or unpaired t -test [D–F]). DAPI: 4′,6-Diamidino-2-phenylindole; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Iba1: ionized calcium-binding adapter molecule 1; IL-1β: interleukin-1 beta; IL-6: interleukin-6; IL-8: interleukin-8; IL-10: interleukin-10; iNOS: inductible nitric oxide synthase; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; n.s.: no significance; NaA: sodium acetate; PD: Parkinson’s disease; QPCR: quantitative polymerase chain reaction; SN: substantia nigra; TH: tyrosine hydroxylase; TNF-α: tumor necrosis factor alpha; ZO-1: Zonula occludens-1.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4542/pmc12094542/pmc12094542__NRR-21-377-g006.jpg)
