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phosphorylated eif2α  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phosphorylated eif2α
    Cerebral I/R induces transient activation of ER stress transmembrane sensors, followed by apoptosis. (A) Schematic representation of the timeline of the experimental procedure. Mice were subjected to sham/MCAO for 1.5 hours followed by recovery/reperfusion. Brain tissues were collected at various time points during reperfusion for Western blotting, and brain infarction and neurological evaluation were performed after 24 hours recovery/reperfusion. (B) Triphenyl-tetrazolium chloride–stained cerebral coronal sections from representative brains, collected at 24 hours after reperfusion. The infarcted area is shown in white, and the normal area is shown in red. (C) Statistical analysis of the infarct volumes ( n = 5). **** P < 0.0001, I/R group vs . sham group, unpaired t- test. (D) Statistical analysis of the neurological scores 24 hours after reperfusion by Zea-Longa test ( n = 5). **** P < 0.0001, I/R group vs . sham group, Mann–Whitney U test. (E–J) The ischemic brain tissues were collected after 1.5 hours of MCAO (reperfusion 0 hours) or 1, 3, 6, 12, and 24 hours after reperfusion. The tissues from the sham group were collected at 1.5 hours after the sham surgery. (E) Representative western blot images of <t>p-eIF2α,</t> p-IRE1, ATF6, ATF4, and CHOP at different time points ( n = 3) after reperfusion. (F–J) Quantifications of the normalized levels of p-eIF2α/eIF2α (F), p-IRE1/IRE1 (G), ATF6/GAPDH (H), ATF4/GAPDH (I), and CHOP/GAPDH (J). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, I/R group vs . sham group, one-way analysis of variance followed by Dunnett’s multiple comparison test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; I/R: cerebral ischemia-reperfusion; IRE1: inositol requiring enzyme 1; MCAO: middle cerebral artery occlusion; p-: phosphorylated-; TTC: 2,3,5-triphenyl tetrazolium chloride.
    Phosphorylated Eif2α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphorylated eif2α/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1325 article reviews
    phosphorylated eif2α - by Bioz Stars, 2026-03
    98/100 stars

    Images

    1) Product Images from "Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress"

    Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

    Journal: Neural Regeneration Research

    doi: 10.4103/NRR.NRR-D-24-00044

    Cerebral I/R induces transient activation of ER stress transmembrane sensors, followed by apoptosis. (A) Schematic representation of the timeline of the experimental procedure. Mice were subjected to sham/MCAO for 1.5 hours followed by recovery/reperfusion. Brain tissues were collected at various time points during reperfusion for Western blotting, and brain infarction and neurological evaluation were performed after 24 hours recovery/reperfusion. (B) Triphenyl-tetrazolium chloride–stained cerebral coronal sections from representative brains, collected at 24 hours after reperfusion. The infarcted area is shown in white, and the normal area is shown in red. (C) Statistical analysis of the infarct volumes ( n = 5). **** P < 0.0001, I/R group vs . sham group, unpaired t- test. (D) Statistical analysis of the neurological scores 24 hours after reperfusion by Zea-Longa test ( n = 5). **** P < 0.0001, I/R group vs . sham group, Mann–Whitney U test. (E–J) The ischemic brain tissues were collected after 1.5 hours of MCAO (reperfusion 0 hours) or 1, 3, 6, 12, and 24 hours after reperfusion. The tissues from the sham group were collected at 1.5 hours after the sham surgery. (E) Representative western blot images of p-eIF2α, p-IRE1, ATF6, ATF4, and CHOP at different time points ( n = 3) after reperfusion. (F–J) Quantifications of the normalized levels of p-eIF2α/eIF2α (F), p-IRE1/IRE1 (G), ATF6/GAPDH (H), ATF4/GAPDH (I), and CHOP/GAPDH (J). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, I/R group vs . sham group, one-way analysis of variance followed by Dunnett’s multiple comparison test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; I/R: cerebral ischemia-reperfusion; IRE1: inositol requiring enzyme 1; MCAO: middle cerebral artery occlusion; p-: phosphorylated-; TTC: 2,3,5-triphenyl tetrazolium chloride.
    Figure Legend Snippet: Cerebral I/R induces transient activation of ER stress transmembrane sensors, followed by apoptosis. (A) Schematic representation of the timeline of the experimental procedure. Mice were subjected to sham/MCAO for 1.5 hours followed by recovery/reperfusion. Brain tissues were collected at various time points during reperfusion for Western blotting, and brain infarction and neurological evaluation were performed after 24 hours recovery/reperfusion. (B) Triphenyl-tetrazolium chloride–stained cerebral coronal sections from representative brains, collected at 24 hours after reperfusion. The infarcted area is shown in white, and the normal area is shown in red. (C) Statistical analysis of the infarct volumes ( n = 5). **** P < 0.0001, I/R group vs . sham group, unpaired t- test. (D) Statistical analysis of the neurological scores 24 hours after reperfusion by Zea-Longa test ( n = 5). **** P < 0.0001, I/R group vs . sham group, Mann–Whitney U test. (E–J) The ischemic brain tissues were collected after 1.5 hours of MCAO (reperfusion 0 hours) or 1, 3, 6, 12, and 24 hours after reperfusion. The tissues from the sham group were collected at 1.5 hours after the sham surgery. (E) Representative western blot images of p-eIF2α, p-IRE1, ATF6, ATF4, and CHOP at different time points ( n = 3) after reperfusion. (F–J) Quantifications of the normalized levels of p-eIF2α/eIF2α (F), p-IRE1/IRE1 (G), ATF6/GAPDH (H), ATF4/GAPDH (I), and CHOP/GAPDH (J). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, I/R group vs . sham group, one-way analysis of variance followed by Dunnett’s multiple comparison test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; I/R: cerebral ischemia-reperfusion; IRE1: inositol requiring enzyme 1; MCAO: middle cerebral artery occlusion; p-: phosphorylated-; TTC: 2,3,5-triphenyl tetrazolium chloride.

    Techniques Used: Activation Assay, Western Blot, Staining, MANN-WHITNEY, Comparison, Binding Assay

    OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.
    Figure Legend Snippet: OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

    Techniques Used: Activation Assay, Cell Culture, Control, Western Blot, MTT Assay, Comparison, Binding Assay, In Vitro

    Neuroserpin inhibits ER stress–mediated signaling transduction induced by OGD/R. (A) Schematic representation of the timing of the experimental procedures. Neuroserpin (NSP; 20 ng/mL) was added to cortical neurons (7 DIV) 4 hours before OGD and during OGD, followed by reperfusion. Cell lysates were collected at the indicated time points of reperfusion for the examination of ER stress signaling molecules. (B) Representative western blots showing phosphorylated and total levels of ER stress sensors and apoptosis-related proteins. (C) Representative western blots showing levels of puromycin. (D–K) Quantitative analysis of the normalized p-PERK/PERK (D, n = 3), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 5), CHOP (J, n = 5), and cleaved-caspase-3 (K, n = 4). GAPDH and α-tubulin were used as loading controls. ** P < 0.01, *** P < 0.001, OGD/R vs . control; # P < 0.05, ## P < 0.01, OGD/R + NSP vs . OGD/R, unpaired t -test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; i.c.v.: intracerebroventricular; NSP: neuroserpin; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase.
    Figure Legend Snippet: Neuroserpin inhibits ER stress–mediated signaling transduction induced by OGD/R. (A) Schematic representation of the timing of the experimental procedures. Neuroserpin (NSP; 20 ng/mL) was added to cortical neurons (7 DIV) 4 hours before OGD and during OGD, followed by reperfusion. Cell lysates were collected at the indicated time points of reperfusion for the examination of ER stress signaling molecules. (B) Representative western blots showing phosphorylated and total levels of ER stress sensors and apoptosis-related proteins. (C) Representative western blots showing levels of puromycin. (D–K) Quantitative analysis of the normalized p-PERK/PERK (D, n = 3), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 5), CHOP (J, n = 5), and cleaved-caspase-3 (K, n = 4). GAPDH and α-tubulin were used as loading controls. ** P < 0.01, *** P < 0.001, OGD/R vs . control; # P < 0.05, ## P < 0.01, OGD/R + NSP vs . OGD/R, unpaired t -test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; i.c.v.: intracerebroventricular; NSP: neuroserpin; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase.

    Techniques Used: Transduction, Western Blot, Control, Binding Assay, In Vitro



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    OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of <t>p-PERK/PERK</t> (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: <t>phosphorylated-;</t> PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.
    Anti Dna Pk Pser2056, supplied by Boster Bio, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of <t>p-PERK/PERK</t> (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: <t>phosphorylated-;</t> PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.
    Rabbit Monoclonal Antibody Hy P80276, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of <t>p-PERK/PERK</t> (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: <t>phosphorylated-;</t> PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.
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    Image Search Results


    Cerebral I/R induces transient activation of ER stress transmembrane sensors, followed by apoptosis. (A) Schematic representation of the timeline of the experimental procedure. Mice were subjected to sham/MCAO for 1.5 hours followed by recovery/reperfusion. Brain tissues were collected at various time points during reperfusion for Western blotting, and brain infarction and neurological evaluation were performed after 24 hours recovery/reperfusion. (B) Triphenyl-tetrazolium chloride–stained cerebral coronal sections from representative brains, collected at 24 hours after reperfusion. The infarcted area is shown in white, and the normal area is shown in red. (C) Statistical analysis of the infarct volumes ( n = 5). **** P < 0.0001, I/R group vs . sham group, unpaired t- test. (D) Statistical analysis of the neurological scores 24 hours after reperfusion by Zea-Longa test ( n = 5). **** P < 0.0001, I/R group vs . sham group, Mann–Whitney U test. (E–J) The ischemic brain tissues were collected after 1.5 hours of MCAO (reperfusion 0 hours) or 1, 3, 6, 12, and 24 hours after reperfusion. The tissues from the sham group were collected at 1.5 hours after the sham surgery. (E) Representative western blot images of p-eIF2α, p-IRE1, ATF6, ATF4, and CHOP at different time points ( n = 3) after reperfusion. (F–J) Quantifications of the normalized levels of p-eIF2α/eIF2α (F), p-IRE1/IRE1 (G), ATF6/GAPDH (H), ATF4/GAPDH (I), and CHOP/GAPDH (J). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, I/R group vs . sham group, one-way analysis of variance followed by Dunnett’s multiple comparison test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; I/R: cerebral ischemia-reperfusion; IRE1: inositol requiring enzyme 1; MCAO: middle cerebral artery occlusion; p-: phosphorylated-; TTC: 2,3,5-triphenyl tetrazolium chloride.

    Journal: Neural Regeneration Research

    Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

    doi: 10.4103/NRR.NRR-D-24-00044

    Figure Lengend Snippet: Cerebral I/R induces transient activation of ER stress transmembrane sensors, followed by apoptosis. (A) Schematic representation of the timeline of the experimental procedure. Mice were subjected to sham/MCAO for 1.5 hours followed by recovery/reperfusion. Brain tissues were collected at various time points during reperfusion for Western blotting, and brain infarction and neurological evaluation were performed after 24 hours recovery/reperfusion. (B) Triphenyl-tetrazolium chloride–stained cerebral coronal sections from representative brains, collected at 24 hours after reperfusion. The infarcted area is shown in white, and the normal area is shown in red. (C) Statistical analysis of the infarct volumes ( n = 5). **** P < 0.0001, I/R group vs . sham group, unpaired t- test. (D) Statistical analysis of the neurological scores 24 hours after reperfusion by Zea-Longa test ( n = 5). **** P < 0.0001, I/R group vs . sham group, Mann–Whitney U test. (E–J) The ischemic brain tissues were collected after 1.5 hours of MCAO (reperfusion 0 hours) or 1, 3, 6, 12, and 24 hours after reperfusion. The tissues from the sham group were collected at 1.5 hours after the sham surgery. (E) Representative western blot images of p-eIF2α, p-IRE1, ATF6, ATF4, and CHOP at different time points ( n = 3) after reperfusion. (F–J) Quantifications of the normalized levels of p-eIF2α/eIF2α (F), p-IRE1/IRE1 (G), ATF6/GAPDH (H), ATF4/GAPDH (I), and CHOP/GAPDH (J). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, I/R group vs . sham group, one-way analysis of variance followed by Dunnett’s multiple comparison test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; I/R: cerebral ischemia-reperfusion; IRE1: inositol requiring enzyme 1; MCAO: middle cerebral artery occlusion; p-: phosphorylated-; TTC: 2,3,5-triphenyl tetrazolium chloride.

    Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

    Techniques: Activation Assay, Western Blot, Staining, MANN-WHITNEY, Comparison, Binding Assay

    OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

    Journal: Neural Regeneration Research

    Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

    doi: 10.4103/NRR.NRR-D-24-00044

    Figure Lengend Snippet: OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

    Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

    Techniques: Activation Assay, Cell Culture, Control, Western Blot, MTT Assay, Comparison, Binding Assay, In Vitro

    Neuroserpin inhibits ER stress–mediated signaling transduction induced by OGD/R. (A) Schematic representation of the timing of the experimental procedures. Neuroserpin (NSP; 20 ng/mL) was added to cortical neurons (7 DIV) 4 hours before OGD and during OGD, followed by reperfusion. Cell lysates were collected at the indicated time points of reperfusion for the examination of ER stress signaling molecules. (B) Representative western blots showing phosphorylated and total levels of ER stress sensors and apoptosis-related proteins. (C) Representative western blots showing levels of puromycin. (D–K) Quantitative analysis of the normalized p-PERK/PERK (D, n = 3), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 5), CHOP (J, n = 5), and cleaved-caspase-3 (K, n = 4). GAPDH and α-tubulin were used as loading controls. ** P < 0.01, *** P < 0.001, OGD/R vs . control; # P < 0.05, ## P < 0.01, OGD/R + NSP vs . OGD/R, unpaired t -test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; i.c.v.: intracerebroventricular; NSP: neuroserpin; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase.

    Journal: Neural Regeneration Research

    Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

    doi: 10.4103/NRR.NRR-D-24-00044

    Figure Lengend Snippet: Neuroserpin inhibits ER stress–mediated signaling transduction induced by OGD/R. (A) Schematic representation of the timing of the experimental procedures. Neuroserpin (NSP; 20 ng/mL) was added to cortical neurons (7 DIV) 4 hours before OGD and during OGD, followed by reperfusion. Cell lysates were collected at the indicated time points of reperfusion for the examination of ER stress signaling molecules. (B) Representative western blots showing phosphorylated and total levels of ER stress sensors and apoptosis-related proteins. (C) Representative western blots showing levels of puromycin. (D–K) Quantitative analysis of the normalized p-PERK/PERK (D, n = 3), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 5), CHOP (J, n = 5), and cleaved-caspase-3 (K, n = 4). GAPDH and α-tubulin were used as loading controls. ** P < 0.01, *** P < 0.001, OGD/R vs . control; # P < 0.05, ## P < 0.01, OGD/R + NSP vs . OGD/R, unpaired t -test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; i.c.v.: intracerebroventricular; NSP: neuroserpin; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase.

    Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

    Techniques: Transduction, Western Blot, Control, Binding Assay, In Vitro

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

    Journal: Neural Regeneration Research

    Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

    doi: 10.4103/NRR.NRR-D-24-00044

    Figure Lengend Snippet: OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

    Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

    Techniques: Activation Assay, Cell Culture, Control, Western Blot, MTT Assay, Comparison, Binding Assay, In Vitro

    Neuroserpin inhibits ER stress–mediated signaling transduction induced by OGD/R. (A) Schematic representation of the timing of the experimental procedures. Neuroserpin (NSP; 20 ng/mL) was added to cortical neurons (7 DIV) 4 hours before OGD and during OGD, followed by reperfusion. Cell lysates were collected at the indicated time points of reperfusion for the examination of ER stress signaling molecules. (B) Representative western blots showing phosphorylated and total levels of ER stress sensors and apoptosis-related proteins. (C) Representative western blots showing levels of puromycin. (D–K) Quantitative analysis of the normalized p-PERK/PERK (D, n = 3), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 5), CHOP (J, n = 5), and cleaved-caspase-3 (K, n = 4). GAPDH and α-tubulin were used as loading controls. ** P < 0.01, *** P < 0.001, OGD/R vs . control; # P < 0.05, ## P < 0.01, OGD/R + NSP vs . OGD/R, unpaired t -test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; i.c.v.: intracerebroventricular; NSP: neuroserpin; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase.

    Journal: Neural Regeneration Research

    Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

    doi: 10.4103/NRR.NRR-D-24-00044

    Figure Lengend Snippet: Neuroserpin inhibits ER stress–mediated signaling transduction induced by OGD/R. (A) Schematic representation of the timing of the experimental procedures. Neuroserpin (NSP; 20 ng/mL) was added to cortical neurons (7 DIV) 4 hours before OGD and during OGD, followed by reperfusion. Cell lysates were collected at the indicated time points of reperfusion for the examination of ER stress signaling molecules. (B) Representative western blots showing phosphorylated and total levels of ER stress sensors and apoptosis-related proteins. (C) Representative western blots showing levels of puromycin. (D–K) Quantitative analysis of the normalized p-PERK/PERK (D, n = 3), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 5), CHOP (J, n = 5), and cleaved-caspase-3 (K, n = 4). GAPDH and α-tubulin were used as loading controls. ** P < 0.01, *** P < 0.001, OGD/R vs . control; # P < 0.05, ## P < 0.01, OGD/R + NSP vs . OGD/R, unpaired t -test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; i.c.v.: intracerebroventricular; NSP: neuroserpin; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase.

    Article Snippet: The following primary antibodies were used (with an overnight incubation at 4°C): neuroserpin (mouse, 1:1000; Santa Cruz Biotechnology, Cat# sc-48360, RRID: AB_628245), phosphorylated-PERK (p-PERK, rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179S, RRID: AB_2095853), PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phosphorylated-eIF2α (p-eIF2α, rabbit, 1:1000, Cell Signaling Technology, Cat# 3398S, RRID: AB_2096481), eIF2α (rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phosphorylated-IRE1 (p-IRE1, rabbit, 1:1000, Abcam, Cat# ab48187, RRID: AB_873899), IRE1 (rabbit, 1:1000, Abcam, Cat# ab37037, RRID: AB_775780), ATF6 (rabbit, 1:1000, Abcam, Cat# ab203119, RRID: AB_2650448), ATF4 (rabbit, 1:1000; Cell Signaling Technology, Cat# 11815S, RRID: AB_2616025), CHOP (rabbit, 1:1000, Cell Signaling Technology, Cat# 2895S, RRID: AB_2089254), Cleaved caspase-3 (rabbit, 1:1000, Cell Signaling Technology, Cat# 9664S, RRID: AB_2070042), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, rabbit, 1:2000, Proteintech, Rosemont, IL, USA, Cat# 10494-1-AP, RRID: AB_2263076), and α-tubulin (mouse, 1:2000, Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, RRID: AB_184122).

    Techniques: Transduction, Western Blot, Control, Binding Assay, In Vitro