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: Chitosan treatment repairs damaged intestinal and blood–brain barriers in an MPTP-induced mouse model of PD. (A) Chitosan administration significantly increased ZO-1 and occludin expression levels, as detected by western blot. GAPDH was used as a loading control. (B) Chitosan treatment significantly increased the fluorescence intensity of ZO-1 (green, Alexa Fluor 488) and occludin (red, Alexa Fluor 594) in mouse colon tissue compared with the MPTP-induced PD group, and the fluorescence intensities of ZO-1 and occludin in PD group were lower than those in the control group. Scale bars: 10 μm. (C) Compared with MPTP-induced PD mice, chitosan treatment significantly decreased serum FITC-dextran levels, which are a measure of intestinal barrier integrity. (D) EB was used to monitor BBB permeability, and results were normalized to the control group. Chitosan treatment significantly reduced BBB damage. (E) EB measured by fluorescence microscopy imaging. Chitosan significantly restored BBB compared with MPTP-induced PD mice. Scale bars: 500 μm (upper) and 50 μm (lower). (F) EB of brain in mice measured by microplate reader. Chitosan treatment significantly decreased EB content compared with MPTP mice. All data are presented as the mean ± SD ( n = 3/group). * P < 0.05 (one-way analysis of variance followed by Tukey’s multiple comparisons test). BBB: Blood–brain barrier; DAPI: 4′,6-diamidino-2-phenylindole; EB: Evans blue; FITC-Dextran: Fluorescein isothiocyanate dextran; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson’s disease; 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: Expressing, Western Blot, Control, Fluorescence, Permeability, Microscopy, Imaging

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

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: Chitosan may reduce acetate levels, thereby activating the PPARD-AMPK signaling pathway, which promotes repair of the intestinal barrier and reduces neuroinflammation in an MPTP-induced mouse model of PD. (A, B) Western blot analysis of p-AMPK, AMPK, and PPARD levels in mouse colon tissue ( n = 3/group). Treatment with acetate significantly increased p-AMPK and PPARD expression. (C) Treatment with a PPARD antagonist significantly decreased mouse body weight ( n = 6/group). (D) There were no significant differences in fall latency among the groups in the rotarod test, which was used to assess motor dysfunction ( n = 6/group). (E–G) PPARD antagonist treatment significantly decreased PPARD, TH, ZO-1, and occludin expression, as determined by western blot ( n = 3/group). (H) Immunofluorescence staining for ZO-1 (green, Alexa Fluor 488) and occludin (red, Alexa Fluor 594) in mouse colon tissue ( n = 3/group). The PPARD antagonist treatment group exhibited markedly reduced ZO-1 and occludin mRNA expression levels in colon tissue. Scale bars: 10 μm. (I) QPCR was used to measure the mRNA levels of IL-1β, IL-6, IL-8, IL-10, TNF-α, and iNOS in mouse colon tissue ( n = 3/group). Treatment with the PPARD antagonist increased IL-6 and TNF-α mRNA levels, while IL-8 and iNOS levels were reduced. (J) ELISA was used to detect IL-1β, IL-6, IL-10, and TNF-α expression levels in mouse plasma ( n = 5/group). IL-1β, IL-6, and TNF-α expression levels were significantly increased in the PPARD antagonist treatment group. (K) QPCR was used to measure mRNA levels of IL-1β, IL-6, IL-8, IL-10, TNF-α, and iNOS in the SN ( n = 3/group). Treatment with the PPARD antagonist significantly increased the mRNA levels of IL-1β, IL-6, and IL-8. (L) Treatment with the PPARD antagonist reduced p-AMPK, but not AMPK, expression ( n = 3/group). GAPDH was used as the internal reference. 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, B) or unpaired t -test (C–L)). AMPK: Adenosine 5′-monophosphate-activated protein kinase; DAPI: 4′,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL-1β: interleukin-1 Beta; IL-6: interleukin-6; IL-8: interleukin-8; IL-10: interleukin-10; iNOS: inductible nitric oxide synthase; n.s.: not significant; NaA: sodium acetate; p-AMPK: phosphorylation adenosine 5′-monophosphate-activated protein kinase; PD: Parkinson’s disease; PPARD: peroxisome proliferator-activated receptor delta; 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: Western Blot, Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: CNS Neuroscience & Therapeutics

Article Title: Ligustrazine Alleviates Blood–Brain Barrier Damage by Downregulating Expression of miR ‐297c‐5p

doi: 10.1111/cns.70367

Figure Lengend Snippet: LSZ increased levels of TJs in b. End3cells treated with OGD. (A) Representative WB analysis of occludin and ZO‐1. (B) Levels of occludin and ZO‐1 in b. End3 cells treated with OGD for 3 h. (C) Levels of occludin in b. End3 cells treated with OGD for 3 h and the effects of LSZ in ELISA. (D) Representative WB analysis of occludin and ZO‐1. (E) Levels of occludin and ZO‐1 in cells with 3‐h OGD and LSZ treatment. Data are presented as means ± SEM, n = 6 in each group. ** p < 0.01, *** p < 0.001 versus sham group; # p < 0.05, ## p < 0.01 versus OGD group.

Article Snippet: The membranes were then probed with antibodies for polyclonal rabbit antimouse occludin (1:1000; 27260‐1‐AP; Proteintech), polyclonal rabbit anti‐mouse ZO‐1 (1:1000; 21773‐1‐AP; Proteintech), polyclonal rabbit anti‐mouse caveolin‐1 (1:1000; ab2910; Abcam), polyclonal rabbit anti‐mouse caveolin‐2 (1:1000; AF5409; Affinity), polyclonal rabbit anti‐mouse caveolin‐3 (1:1000; ab2912; Abcam), and anti‐β‐actin (1:10000; A2228; Sigma‐Aldrich) served as the loading control.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Ligustrazine Alleviates Blood–Brain Barrier Damage by Downregulating Expression of miR ‐297c‐5p

doi: 10.1111/cns.70367

Figure Lengend Snippet: MiR‐297c‐5p transfection regulated the levels of occludin in b. End3 cells. (A) Transfection efficiency was detected under confocal microscope. The pseudo red color was postprocessing of FAM color. (B) Representative of WB analysis of occludin, caveolin‐1, caveolin‐2, and caveolin‐3 in different treatments. (C) MiR‐297c‐5p agomir decreased levels of occludin, but miR‐297c‐5p antagomir increased it. MiR‐297c‐5p agomir increased levels of caveolin‐1 (D), caveolin‐2 (E), and caveolin‐3 (F), but miR‐297c‐5p antagomir decreased them. Data are presented as means ± SEM, n = 6 experiments in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 versus sham group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus miR‐297c‐5p agomir/antagomir group.

Article Snippet: The membranes were then probed with antibodies for polyclonal rabbit antimouse occludin (1:1000; 27260‐1‐AP; Proteintech), polyclonal rabbit anti‐mouse ZO‐1 (1:1000; 21773‐1‐AP; Proteintech), polyclonal rabbit anti‐mouse caveolin‐1 (1:1000; ab2910; Abcam), polyclonal rabbit anti‐mouse caveolin‐2 (1:1000; AF5409; Affinity), polyclonal rabbit anti‐mouse caveolin‐3 (1:1000; ab2912; Abcam), and anti‐β‐actin (1:10000; A2228; Sigma‐Aldrich) served as the loading control.

Techniques: Transfection, Microscopy

Journal: CNS Neuroscience & Therapeutics

Article Title: Ligustrazine Alleviates Blood–Brain Barrier Damage by Downregulating Expression of miR ‐297c‐5p

doi: 10.1111/cns.70367

Figure Lengend Snippet: MiR‐297c‐5p involved in regulating occludin in vivo. (A) The virus expression site in the hippocampus was detected by GFP expression on AAV under a confocal microscope. (B) Representative of WB analysis of occludin. (C) AAV‐miR‐297c‐5p significantly decreased the expression of occludin. Data are presented as means ± SEM; n = 6 in each group. ** p < 0.01 versus Scramble group.

Article Snippet: The membranes were then probed with antibodies for polyclonal rabbit antimouse occludin (1:1000; 27260‐1‐AP; Proteintech), polyclonal rabbit anti‐mouse ZO‐1 (1:1000; 21773‐1‐AP; Proteintech), polyclonal rabbit anti‐mouse caveolin‐1 (1:1000; ab2910; Abcam), polyclonal rabbit anti‐mouse caveolin‐2 (1:1000; AF5409; Affinity), polyclonal rabbit anti‐mouse caveolin‐3 (1:1000; ab2912; Abcam), and anti‐β‐actin (1:10000; A2228; Sigma‐Aldrich) served as the loading control.

Techniques: In Vivo, Virus, Expressing, Microscopy

Journal: CNS Neuroscience & Therapeutics

Article Title: Ligustrazine Alleviates Blood–Brain Barrier Damage by Downregulating Expression of miR ‐297c‐5p

doi: 10.1111/cns.70367

Figure Lengend Snippet: MiR‐297c‐5p directly targeted occludin by dual‐luciferase activity assay. (A) Alignment of binding sequences for miR‐297c‐5p in the 3′‐UTRs of Ocln mRNA. (B) The intensity of interaction between miR‐297c‐5p and 3′‐UTRs of Ocln mRNA was detected by luciferase reporter assay after 48 h incubation. Data are presented as means ± SEM; n = 3 in each group. *** p < 0.001 versus mimics NC group.

Article Snippet: The membranes were then probed with antibodies for polyclonal rabbit antimouse occludin (1:1000; 27260‐1‐AP; Proteintech), polyclonal rabbit anti‐mouse ZO‐1 (1:1000; 21773‐1‐AP; Proteintech), polyclonal rabbit anti‐mouse caveolin‐1 (1:1000; ab2910; Abcam), polyclonal rabbit anti‐mouse caveolin‐2 (1:1000; AF5409; Affinity), polyclonal rabbit anti‐mouse caveolin‐3 (1:1000; ab2912; Abcam), and anti‐β‐actin (1:10000; A2228; Sigma‐Aldrich) served as the loading control.

Techniques: Luciferase, Activity Assay, Binding Assay, Reporter Assay, Incubation

Journal: CNS Neuroscience & Therapeutics

Article Title: Ligustrazine Alleviates Blood–Brain Barrier Damage by Downregulating Expression of miR ‐297c‐5p

doi: 10.1111/cns.70367

Figure Lengend Snippet: The verification of miR‐297c‐5p targeting occludin in MCAO model. (A) Neurological deficit was analyzed in MCAO mice after pretreatment of RNA oligo and LSZ. (B) Representative of WB analysis of occludin. (C) Level of occludin expression was presented as means ± SEM, n = 3 in each group. ** p < 0.01, *** p < 0.001 versus sham group, # p < 0.05, ## p < 0.01 versus MCAO group, $ p < 0.05 versus scramble group (Scram: Scramble, Ago:Agomir, Anta:Antagomir).

Article Snippet: The membranes were then probed with antibodies for polyclonal rabbit antimouse occludin (1:1000; 27260‐1‐AP; Proteintech), polyclonal rabbit anti‐mouse ZO‐1 (1:1000; 21773‐1‐AP; Proteintech), polyclonal rabbit anti‐mouse caveolin‐1 (1:1000; ab2910; Abcam), polyclonal rabbit anti‐mouse caveolin‐2 (1:1000; AF5409; Affinity), polyclonal rabbit anti‐mouse caveolin‐3 (1:1000; ab2912; Abcam), and anti‐β‐actin (1:10000; A2228; Sigma‐Aldrich) served as the loading control.

Techniques: Expressing