Journal: Scientific Reports

Article Title: Syntabulin regulates the trafficking of PICK1-containing vesicles in neurons

doi: 10.1038/srep20924

Figure Lengend Snippet: ( A ) Neurons were infected with syntabulin shRNA lentivirus as indicated at DIV5 and examined at DIV13. Syntabulin knockdown significantly increased total ASIC2 expression but reduced surface ASIC2 levels as compared with the levels in control and scrambled-shRNA control. ( B ) Quantification of normalized ASIC2/GAPDH ratio, total ASIC2/surface ASIC2 ratio and surface ASIC2 levels. One-way ANOVA and Sidak test; ** P = 0.008, and P = 0.009 for Stb sh#1 Total ASIC2/GAPDH and Surface ASIC2/GAPDH compared to control respectively; * P = 0.044 and * P = 0.016 for Stb #1 total ASIC2/GAPDH and Surface/Total ASIC2 ratio compared to scrambled shRNA control respectively; * P = 0.020 for Stb #1 Surface/Total ASIC2 ratio compared to control; “ns” P > 0.05. n = 4 − 6 independent experiments. Error bars represent the SEM. ( C , D ) Hoechst staining of neurons after syntabulin knockdown, in pH 7.4 solution and pH6.0 solution. Knockdown of syntabulin significantly increased cell death as compared with controls in pH6.0. Scale bar = 20 μm. ( E ) Quantification of normalized total Hoechst intensity. One-way ANOVA and Sidak test; * P = 0.035 compared to control; * P = 0.043 compared to scrambled-shRNA control; “ns” P > 0.05; n = 6 independent experimental repeats. Error bars represent the SEM.

Article Snippet: The following antibodies were purchased: rabbit polyclonal ASIC2 antibody, Proteintech (17851-1-AP); mouse myc and β-tubulin antibodies, DSHB (9E10 and E7); mouse GAPDH antibody, Beyotime (GA019); mouse Map2 antibody, Sigma (M9942); and mouse β-actin antibody, Sigma (A5316).

Techniques: Infection, shRNA, Knockdown, Expressing, Control, Staining

Journal: European journal of pharmacology

Article Title: Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord.

doi: 10.1016/j.ejphar.2023.176173

Figure Lengend Snippet: Fig. 1. Changes in ASIC1, ASIC2, and ASIC3 protein expression in the spinal dorsal horn during chronic morphine exposure. A, Schematic dia gram of behavioral testing upon morphine treatment. B, During nine days of chronic morphine exposure (10 mg/kg twice daily), the MPE percentages of the hot plate test in C57 mice were recorded 30 min after treatment, and the area under the curve (AUC) of MPE % of hot plate latency over the nine days was calculated. ***P < 0.001 compared with Day 1; n = 6 per group. C, During nine days of chronic morphine exposure (10 mg/kg twice daily), the MPE percent ages of the tail flick test in C57 mice were recorded 30 min after treatment, and the area under the curve (AUC) of MPE % of tail-flick latency over the nine days was calculated. ***P < 0.001 compared with Day 1; n = 6 per group. D-I, The mRNA levels (D–F) and protein levels (G–I) of ASICs compared between the saline group and the morphine group; ***P < 0.001; n = 3 per group. Abbre viations: Mor = morphine.

Article Snippet: Antibodies against Asic2 (A04055-1) and Asic3 (BA2745-2) were purchased from Boster Biotechnology (California, USA).

Techniques: Expressing, Hot Plate Test, Tail Flick Test, Saline

Journal: European journal of pharmacology

Article Title: Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord.

doi: 10.1016/j.ejphar.2023.176173

Figure Lengend Snippet: Fig. 2. Chronic morphine administration increased ASIC3 immunoreactivity in microglia of the dorsal horn. A-B. On Day 9, the fluorescence density of ASIC3 was evaluated after chronic morphine or saline administration. Scale bar: 20 μm ***P < 0.001 compared with the saline group; n = 4 per group. C. ASIC3 merged with NeuN, Iba1, and GFAP in the dorsal horn by double immunofluorescence staining. Scale bar 20 μm. Abbreviations: Mor = morphine.

Article Snippet: Antibodies against Asic2 (A04055-1) and Asic3 (BA2745-2) were purchased from Boster Biotechnology (California, USA).

Techniques: Fluorescence, Saline, Double Immunofluorescence Staining

Journal: European journal of pharmacology

Article Title: Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord.

doi: 10.1016/j.ejphar.2023.176173

Figure Lengend Snippet: Fig. 3. Amiloride effects on morphine-induced antinociception tolerance and ASIC3 expression. A Graphical depiction of the treatment protocol. B–C, Amiloride alleviates chronic morphine tolerance. The hot plate test (B) and tail-flick test (C) were conducted on Days 1, 3, 5, 7, and 9. ***P < 0.001 compared with the morphine group; n = 6 per group. D-E, The area under the curve (AUC) for the %MPE of hot plate latency (D) and tail flick latency (E) was calculated over nine days. ***P < 0.001 compared with the morphine group; n = 6 per group. F-G, Western blot results (F) and immunofluorescence images (G) showing that amiloride prevented the upregulation of ASIC3 caused by morphine in the spinal cord. ***P < 0.001 compared with the saline group, #P < 0.05, ##P < 0.01 and ###P < 0.001 compared with the morphine group; n = 3 per group. Scale bar 20 μm. Abbreviations: Mor = morphine and Ami = amiloride.

Article Snippet: Antibodies against Asic2 (A04055-1) and Asic3 (BA2745-2) were purchased from Boster Biotechnology (California, USA).

Techniques: Expressing, Hot Plate Test, Tail Flick Test, Western Blot, Immunofluorescence, Saline

Journal: European journal of pharmacology

Article Title: Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord.

doi: 10.1016/j.ejphar.2023.176173

Figure Lengend Snippet: Fig. 4. APETx2 effects on morphine-induced antinociception tolerance and ASIC3 expression. A. Graphical depiction of the treatment protocol. B–C, APETx2 alleviates chronic morphine tolerance. The hot plate test (B) and tail-flick test (C) were conducted on Days 1, 3, 5, 7, and 9. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the morphine group; n = 8 per group. D-E, The area under the curve (AUC) for the %MPE of hot plate latency (D) and tail flick latency (E) was calculated over nine days. *P < 0.05 and **P < 0.01 compared with the morphine group; n = 8 per group. F-G, Western blot results (F) and immunofluorescence images (G) showing that APETx2 prevented the upregulation of ASIC3 caused by morphine in the spinal cord. ***P < 0.001 compared with the saline group, #P < 0.05 and ###P < 0.001 compared with the morphine group; n = 3 per group. Scale bar 20 μm. Abbreviations: Mor = morphine and APE = APETx2.

Article Snippet: Antibodies against Asic2 (A04055-1) and Asic3 (BA2745-2) were purchased from Boster Biotechnology (California, USA).

Techniques: Expressing, Hot Plate Test, Tail Flick Test, Western Blot, Immunofluorescence, Saline

Journal: European journal of pharmacology

Article Title: Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord.

doi: 10.1016/j.ejphar.2023.176173

Figure Lengend Snippet: Fig. 6. In BV-2 cells, amiloride reduces morphine-induced inflammatory responses in an ASIC3-dependent manner. A-B. Real-time PCR showed that ami loride decreased the mRNA levels of IL-1β (A) and TNF-α (B) in BV-2 cells stimulated with morphine. C-D. Amiloride’s anti-inflammatory effects on IL-1β (C) and TNF-α (D) mRNA in BV-2 cells were sufficiently reduced by overexpression of ASIC3. E. Amiloride inhibited ASIC3 expression in morphine-stimulated BV-2 cells. F. Amiloride’s effect on p38 MAPK phosphorylation in morphine-stimulated BV-2 cells. G-H. BV-2 cells were transfected with 2.5 μg oeASIC3 or vector for 48 h, followed by coculture with morphine (200 μM) with or without amiloride (10 μM) for 12 h. The immunoblot assay was used to determine the efficacy of ASIC3 overexpression. (A–H The data came from three separate experiments). I. Amiloride (10 μM) decreased NF–B translocation from the cytoplasm to the nucleus in BV-2 cells following a 4-h morphine (200 μM) treatment (n = 4). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. the vehicle group; #P < 0.05 and ###P < 0.001 vs. the morphine-treated group or amiloride-treated group; &&&P < 0.001 vs. the morphine and amiloride-cotreatment group. Scale bar 10 μm. Abbreviations: Mor = morphine and Ami = amiloride.

Article Snippet: Antibodies against Asic2 (A04055-1) and Asic3 (BA2745-2) were purchased from Boster Biotechnology (California, USA).

Techniques: Real-time Polymerase Chain Reaction, Over Expression, Expressing, Phospho-proteomics, Transfection, Plasmid Preparation, Western Blot, Translocation Assay

Journal: European journal of pharmacology

Article Title: Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord.

doi: 10.1016/j.ejphar.2023.176173

Figure Lengend Snippet: Fig. 7. The schematic model indicates that ASIC3-dependent neuroinflammation suppression by amiloride potentiates morphine tolerance. Morphine activates microglia and increases the production of proinflammatory cytokines such as IL-1β and TNF-α through the p38/NF-κB pathway. Amiloride suppresses microglial activation and improves morphine tolerance by downregulating ASIC3 in the spinal cord.

Article Snippet: Antibodies against Asic2 (A04055-1) and Asic3 (BA2745-2) were purchased from Boster Biotechnology (California, USA).

Techniques: Activation Assay