phospho ampkα antibodies  (Sino Biological)

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 alleviates motor dysfunction and improves DA neuron survival in an MPTP-induced mouse model of PD. (A) Experimental timeline of behavioral tests and sample collection from the different treatment groups, including control, NaA alone, MPTP-induced PD model, MPTP + NaA, chitosan treatment, MPTP + chitosan + PPARD antagonist, and SCFA treatment. (B) Experimental timeline of cell treatment with acetate, an AMPK agonist, and a PPARD agonist. (C) Chitosan significantly increased mouse body weight ( n = 7/group). (D) In the rotarod test, fall latency was increased after chitosan treatment ( n = 7/group). (E) Chitosan administration significantly increased TH expression, as determined by western blot assay. GAPDH was used as loading control ( n = 3/group). (F) Chitosan treatment significantly increased the number of TH-positive dopaminergic neurons (red, Alexa Fluor 594), as determined by immunofluorescence staining ( n = 3/group). Scale bars: 100 μm. (G) UHPLC-MS/MS was used to detect DA, DOPAC, and HVA levels in striatum tissue ( n = 4/group). Treatment with chitosan significantly upregulated the levels of DA, DOPAC/DA, and (DOPAC + HVA)/DA, but there was no significant change in HVA/DA levels. All data are presented as the mean ± SD. All experiments were repeated at least three times. * P < 0.05 (two-way analysis of variance followed by Tukey’s multiple comparisons test (C) or one-way analysis of variance followed by Tukey’s multiple comparisons test (D–G). AMPK: Adenosine 5′-monophosphate-activated protein kinase; DA: dopamine; DOPAC: 3,4-dihydroxyphenylacetic acid; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HVA: homovanillic acid; ig: intragastrical administration; ip: intraperitoneal administration; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; n.s.: no significance; NaA: sodium acetate; PD: Parkinson’s disease; PPARD: peroxisome proliferator-activated receptor delta; SCFA: short-chain fatty acid; SN: substantia nigra; TH: tyrosine hydroxylase.

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: Control, Expressing, Western Blot, Immunofluorescence, Staining, Tandem Mass Spectroscopy

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: The PPAR and AMPK signaling pathways are enriched in genes whose expression is altered by acetate supplementation, as determined by RNA sequencing of colon tissue from a mouse model of PD. (A) Venn diagram of DEGs among three datasets. (B) KEGG pathway enrichment analysis showed that these DEGs were enriched in the PPAR and AMPK signaling pathways, which are associated with inflammation. (C) Heatmap analysis of 183 common DEGs between MPTP vs . MPTP + Chitosan and MPTP + Chitosan vs . MPTP + Chitosan + NaA ( n = 2/group). (D) qPCR verification analysis of the mRNA levels (normalized to the control group) of the PPAR and AMPK signaling pathway–related genes whose expression was altered in mouse colon tissue ( n = 3/group). All data are presented as the mean ± SD. All experiments were repeated three times. * P < 0.05 (one-way analysis of variance followed by Tukey’s multiple comparisons test). AMPK: Adenosine 5′-monophosphate-activated protein kinase; DEGs: differentially expressed genes; FABP5: fatty acid-binding protein 5; FASN: fatty acid synthase; KEGG: Kyoto Encyclopedia of Genes and Genomes; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; n.s.: not significant; NaA: sodium acetate; PD: Parkinson’s disease; PPAR: peroxisome proliferators-activated receptor; PPARD: peroxisome proliferator-activated receptor delta; qPCR: quantitative polymerase chain reaction; SCD1: stearoyl-coenzyme A desaturase 1; SCD4: stearoyl-coenzyme A desaturase 4.

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: Protein-Protein interactions, Expressing, RNA Sequencing, Control, 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: Acetate may relieve inflammation by activating the PPARD/AMPK signaling pathway in Caco-2 cells. (A, B) Western blot analysis of PPARD, p-AMPK, and AMPK expression in Caco-2 cells treated with a PPARD agonist or left untreated. Compared with the acetate group, PPARD and p-AMPK expression levels were significantly increased in the group treated with acetate and the PPARD agonist. (C) qPCR was used to detect the mRNA level of IL-1β, IL-6, IL-8, IL-10, TNF-α, and iNOS in Caco-2 cells treated with a PPARD agonist or left untreated. Compared with the acetate group, IL-1β and TNF-α were down-regulated, and iNOS was up-regulated, in cells treated with the PPARD agonist. (D, E) Western blot analysis of AMPK, p-AMPK, and PPARD expression levels in Caco-2 cells treated with an AMPK agonist or left untreated. p-AMPK expression was significantly lower in the group treated with acetate and an AMPK agonist than in the acetate-only group. PPARD expression was not altered by treatment with the AMPK agonist. (F) qPCR was used to detect the mRNA levels of IL-1β, IL-6, IL-8, IL-10, TNF-α, and iNOS in Caco-2 cells treated with the AMPK agonist or left untreated. Treatment with the AMPK agonist reduced IL-1β, iNOS, IL-6, and TNF-α expression levels compared with treatment with acetate alone. GAPDH was used as loading control in the western blot assays. All data (normalized by control group) are presented as the mean ± SD ( n = 3/group). All experiments were repeated at least three times. * P < 0.05 (one-way analysis of variance followed by Tukey’s multiple comparisons test). AMPK: Adenosine 5′-monophosphate-activated protein kinase; 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.: no significance; p-AMPK: phosphorylation adenosine 5’-monophosphate-activated protein kinase; PPARD: peroxisome proliferator-activated receptor delta; qPCR: quantitative polymerase chain reaction; TNF-α: tumor necrosis factor alpha.

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, Control, Phospho-proteomics, 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