Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A Representative immunofluorescence image for ASIC1a (red) in the PVN from a naïve mouse. Scale bar, 100 μm. B Schematic of bilateral cannula injection sites into the paraventricular nucleus (PVN) (coordinates: anterior-posterior = –0.79, medial-lateral = ±0.26, dorsal-ventral = –4.35). C Schematic of pharmacological experimental procedure. D The bar graph illustrates the behavioral analysis, showing that the PcTx1 group exhibited a significant increase in the time spent in the center of the open-field test (OFT) compared to the ACSF group (n = 7 per group). * p < 0.05 (unpaired t -test). E The bar graph illustrates the behavioral analysis, showing that the PcTx1 group exhibited a significant increase in the time spent in the open arms of the elevated plus maze (EPM) compared to the ACSF group (n = 7 per group). * p < 0.05 (unpaired t -test). F The bar graph illustrates the results of the tail suspension test (TST), showing that the PcTx1 group exhibited a significant reduction in immobility time compared to the ACSF group (n = 7 per group). * p < 0.05 (unpaired t-test). G The bar graph illustrates the results of the forced swimming test (FST), showing that the PcTx1 group exhibited a significant reduction in immobility time compared to the ACSF group (n = 7 per group). * p < 0.05 (unpaired t -test). H Experimental paradigm for blood collection after forced swimming. I The bar graph illustrates plasma ACTH levels in the PcTx1 group and ACSF group following stress stimulation (n = 6 per group). * p < 0.05 (unpaired t-test). J The bar graph illustrates plasma corticosterone levels in the PcTx1 group and ACSF group following stress stimulation (n = 6 per group). * p < 0.05 (unpaired t-test). See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Immunofluorescence, Injection, Suspension, Clinical Proteomics

Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A Experimental paradigm for viral injection of AAV-DIO-mCherry in Crh-Cre mice. B, C Representative images ( B ) and statistical data ( C ) showing ASIC1a (green) neurons in the PVN co-localized with CRH (red) neurons. Scale bars, 50 μm. D Experimental paradigm for viral injection of AAV-DIO-shRNA in Crh-Cre mice. E Schematic illustration of the construction strategy for ASIC1a knockdown. F, G Representative Western blot images ( F ) and quantification ( G ) demonstrating that AAV-DIO-shASIC1a injection reduced ASIC1a expression in the PVN (n = 6 per group). * p < 0.05 (unpaired t-test). H Immunofluorescence images showing mCherry-positive neurons co-labeled with ASIC1a in the AAV-mCherry and AAV-shASIC1a groups. Scale bars, 50 μm. See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Injection, shRNA, Knockdown, Western Blot, Expressing, Immunofluorescence, Labeling

Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A-D Behavioral effects of the genetic knockdown of ASIC1a in CRH PVN neurons. Figure 3A shows the open-field test (OFT), Fig. 3B shows the elevated plus maze (EPM), Fig. 3C shows the tail suspension test (TST), and Fig. 3D shows the forced swimming test (FST), demonstrating significant behavioral differences between the AAV-mCherry group and the AAV-shASIC1a group (n = 10 per group). * p < 0.05, ** p < 0.01 (unpaired t-test). E Experimental paradigm for blood collection following forced swimming. F, G Plasma ACTH levels ( F ) and corticosterone levels ( G ) were significantly reduced in the AAV-shASIC1a group compared to the AAV-mCherry group after forced swimming (n = 6 per group). * p < 0.05, ** p < 0.01 (unpaired t-test). H Schematic of the real-time optical fiber photometry assay. I Experimental paradigm for viral injection of AAV-shRNA and AAV-DIO-GCaMP6s in Crh-Cre mice. J, K Representative images ( J ) and statistical data ( K ) showing AAV-shRNA and AAV-DIO-GCaMP6s expression in the PVN. Scale bars, 50 μm. L-O Calcium activity analysis of CRH PVN neurons in the AAV-shASIC1a group compared to the AAV-mCherry group. Figure 3L shows average calcium activity, Fig. 3M shows the area under the curve, Fig. 3N shows the maximum peak value, and Fig. 3O shows calcium signals at specific time points (n = 5 per group). * p < 0.05, ** p < 0.01 (unpaired t-test and two-way ANOVA). See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Knockdown, Suspension, Clinical Proteomics, Injection, shRNA, Expressing, Activity Assay

Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A The heatmap shows the release levels of CRH in BE (2)-C cell culture supernatants detected by ELISA. The BE (2)-C cells were treated with HBSS with a pH of 6.5 for 5 min or pretreated with 100 nM PcTx1 or 200 nM amiloride for 5 min and maintained in HBSS pH 6.5. The supernatants were collected at 1, 15, 30, 60, 90, 120, 180 min, 12 h, and 24 h for detection. The experiment was conducted with three independent biological replicates, ensuring the presence of the drug throughout both the culture and acid treatment stages (n = 3 per group). * p < 0.05, *** p < 0.001, **** p < 0.0001 (two-way ANOVA). B The bar graph shows CRH release levels in neuronal culture supernatants detected by ELISA. The experimental treatments were conducted as in Fig. 4A, and supernatants were collected at 3-h time point for detection (n = 3 per group). ** p < 0.01 (one-way ANOVA). C The bar graph shows CRH levels in the supernatant of ASIC1a knockdown neuronal culture medium detected by ELISA after HBSS pH 6.5 treatment and the supernatants were collected at 3-h time point for detection (n = 3 per group). * p < 0.05 (unpaired t- test). D Representative confocal images (scale bar = 5 μm) of CRH in BE (2)-C cells overexpressing GFP/ASIC1a-GFP for 48 h, before or 30 min after treatment with HBSS at pH 6.5. E The bar graph illustrates the relative fluorescence intensity of CRH shown in Fig. 4D (n = 3 per group). * p < 0.05, **** p < 0.0001 (one-way ANOVA). F Representative western blot assay showing the protein expression levels of CRH in primary hypothalamic neurons. Experimental treatments were conducted as in Fig. 4A, and the cells were detected at the 3-h time point. G The bar graph illustrates the grayscale scanning analysis of Fig. 4F, showing the relative expression levels of CRH (n = 3 per group). ** p < 0.01 (one-way ANOVA). H The bar graph shows the mRNA expression levels of CRH in primary hypothalamic neurons detected by qPCR. Experimental treatments were conducted as in Fig. 4F (n = 3 per group). **** p < 0.0001 (one-way ANOVA). See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Knockdown, Fluorescence, Western Blot, Expressing

Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A Acid (pH 6.5)-induced changes in cytosolic Ca 2+ signal, indicated by GCaMP6 fluorescence, in cultured mouse hypothalamic neurons (n = 77 in Vector group, n = 63 in OE ASIC1a group, n = 48 in shASIC1a group, n = 73 in OE ASIC1a + PcTx1 group, n = 60 in OE ASIC1a + Amiloride group). B Representative western blot assay showing the expression of calcium signaling pathway-related proteins in primary hypothalamic neurons. Experimental treatments were conducted as in Fig. 4F. C The bar graph illustrates the grayscale scanning analysis of Fig. 5B, showing the relative expression levels of P-CaMKII (n = 3 per group). ** p < 0.01 (one-way ANOVA). D The bar graph illustrates the grayscale scanning analysis of Fig. 5B, showing the relative expression levels of c-Fos (n = 3 per group). * p < 0.05 (one-way ANOVA). E The bar graphs show the mRNA expression levels of c-Fos in primary hypothalamic neurons detected by qPCR. Experimental treatments were conducted as in Fig. 4F (n = 3 per group). **** p < 0.0001 (one-way ANOVA). F Representative confocal images (scale bar = 10 μm) of c-Fos and CRH in primary hypothalamic neurons overexpressing GFP/ASIC1-GFP or knockdown of ASIC1a for 48 h, before or 15 min after treatment with HBSS at pH 6.5 with or without 100 nM PcTx1 or 200 nM amiloride. G The bar graph illustrated the relative fluorescence intensity of c-Fos shown in Fig. 5F (n = 3 per group). **** p < 0.0001 (one-way ANOVA). H The bar graph shows the secretion levels of CRH in primary hypothalamic neuron culture supernatants detected by ELISA, after treatment with pH 6.5 HBSS or simultaneous treatment with 80 μM T-5224 for 30 min following 48-h overexpression of ASIC1a (n = 3 per group). * p < 0.05 (one-way ANOVA). I Representative western blot assay showing the protein expression levels of CRH, ASIC1a, and c-Fos in primary hypothalamic neurons. Experimental treatments were conducted as in Fig. 5H. J The bar graph illustrates the grayscale scanning analysis of Fig. 5I, showing the relative expression levels of c-Fos (n = 3 per group). * p < 0.05 (one-way ANOVA). K The bar graph illustrates the grayscale scanning analysis of Fig. 5I, showing the relative expression levels of CRH (n = 3 per group). * p < 0.05 (one-way ANOVA). See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Fluorescence, Cell Culture, Plasmid Preparation, Western Blot, Expressing, Knockdown, Enzyme-linked Immunosorbent Assay, Over Expression

Journal: The American Journal of Pathology

Article Title: Endothelial Hypoxia-Inducible Factor-1α Is Required for Vascular Repair and Resolution of Inflammatory Lung Injury through Forkhead Box Protein M1

doi: 10.1016/j.ajpath.2019.04.014

Figure Lengend Snippet: Endothelial regeneration and vascular repair after endotoxemia-induced injury is also HIF-1α dependent. A: Pulmonary transvascular Evans Blue Dye–conjugated albumin (EBA) flux assay demonstrating defective vascular repair in Hif1af/f/Tie2Cre+ (CKO) mouse lungs after lipopolysaccharide (LPS) challenge (2.5 mg/kg, intraperitoneally). B: Lung wet/dry weight ratio analysis revealing lung edema in Hif1af/f/Tie2Cre+ mice at 72 hours after LPS.C: Time course of lung myeloperoxidase (MPO) activity after LPS challenge. D: Representative micrographs of hematoxylin and eosin staining of lung sections showing perivascular leukocyte sequestration in Hif1af/f/Tie2Cre+ mouse lungs at 72 hours after LPS. The boxed areas are shown at higher magnification in the insets in the left lower corners. E: Real-time quantitative RT-PCR analysis showing increased expression of proinflammatory mediators in Hif1af/f/Tie2Cre+ mouse lungs at 72 hours after LPS. F: Representative micrographs showing endothelial cell (EC) proliferation. Cryosections of lungs (5 μm thick) collected at 72 hours after LPS were immunostained with anti–5-bromo-2-deoxyuridine (BrdU) antibody to identify proliferating cells (green) and with anti-CD31/von Willebrand factor (vWF) antibodies to identify ECs (red). Arrows indicate proliferating Ecs. The boxed areas in the top panels are shown at higher magnification in the three bottom panels. G: Quantification of cell proliferation in mouse lungs. Three consecutive cryosections from each mouse lung were examined, and the average number of BrdU+ nuclei was used for each mouse. Data are expressed as means (A and C) or means ± SD (B, E, and G). n = 5 mice per group (B); n = 4 (E and G). ∗∗P < 0.01 (t-test). Scale bars = 50 μm (D and F). B, basal; Br, bronchiole; Icam-1, intercellular adhesion molecule 1; Nos-2, inducible nitric oxide synthase; Tnf-α, tumor necrosis factor–α; V, vessel; WT, wild type.

Article Snippet: To induce endotoxemia, mice received a single dose of i.p. lipopolysaccharide (LPS; 2.5 mg/kg body weight; Escherichia coli 055:B5; Santa Cruz Biotechnology, Dallas, TX).

Techniques: Flux Assay, Activity Assay, Staining, Quantitative RT-PCR, Expressing