itpr1 inhibitor 2 apb  (MedChemExpress)

Journal: The Journal of Headache and Pain

Article Title: ER stress-induced ITPR1/ANO1 signaling drives trigeminal neuropathic pain through calcium-dependent neuronal hyperexcitability

doi: 10.1186/s10194-025-02231-9

Figure Lengend Snippet: ITPR1 is increased in the TG after pIONT and mediates Ca 2+ release. ( A ) Time course of Itpr1 mRNA expression in TG of naive, sham-operated, and pIONT-operated mice. *** P < 0.001. Student’s t-test, n = 7 or 8 mice/group. ( B ) Representative Western blot of ITPR1 protein expression in TG 10 days post-pIONT. ( C ) Quantification of ITPR1 protein levels showing significant upregulation following pIONT. * P < 0.05. Student’s t-test, n = 4 mice/group ( D ) Representative images of ITPR1 staining in the TG of naive, sham-, and pIONT-operated mice. Double staining of ITPR1 with TUJ1, IBA-1, FABP7, CGRP, NF200, and IB4. ( E ) Quantitative analysis of ITPR1 fluorescence intensity. * P < 0.05, One-way ANOVA followed by the Bonferroni test, 3 mice/group. ( F ) Percentage of ITPR1 co-localization with cellular markers (TUJ1, FABP7, and IBA-1). ( G ) Percentage of ITPR1 co-localization with different neuron type markers (CGRP, NF200, and IB4). ( H ) Example traces of neuronal Ca²⁺ dynamics during ATP bath application, comparing NC siRNA- and Itpr1 siRNA-treated pIONT groups. ( I ) Representative traces of ATP-induced Ca²⁺ signals (ΔF/F₀) in NC siRNA- and Itpr1 siRNA-treated TG neurons. ( J ) Quantification showing Itpr1 siRNA significantly attenuates ATP-induced Ca²⁺ signaling. NC: n = 277 cells; Itpr1 siRNA: n = 200 cells; *** P < 0.001. Student’s t-test. 3 mice/group

Article Snippet: The ITPR1 agonist Adenophostin A (MCE; HY-142117), ITPR1 inhibitor 2-APB (Sigma‒Aldrich; Cat. No. CAS524958), ER stress inhibitor 4-PBA (Sigma‒Aldrich; CAS1821121), and ANO1 blocker MONNA (Sigma‒Aldrich; SML0902) were dissolved in 1% DMSO.

Techniques: Expressing, Western Blot, Staining, Double Staining, Fluorescence

Journal: The Journal of Headache and Pain

Article Title: ER stress-induced ITPR1/ANO1 signaling drives trigeminal neuropathic pain through calcium-dependent neuronal hyperexcitability

doi: 10.1186/s10194-025-02231-9

Figure Lengend Snippet: ITPR1 expression is regulated by the transcription factor Runx2. ( A ) Heatmap of transcription factor expression profiles in TG following pIONT. ( B ) JASPAR database prediction of RUNX2 binding sites in the Itpr1 promoter region. ( C ) Dual-luciferase reporter assay showing RUNX2 inhibition suppresses Itpr1 promoter activity. *** P < 0.001. One-way ANOVA followed by the Bonferroni test. ( D - E ) qPCR analysis of Runx2 ( D ) and Itpr1 ( E ) mRNA levels after TM injection. * P < 0.05. Student’s t-test. ( F ) Runx2 mRNA expression in TG post-pIONT. ** P < 0.01. Student’s t-test, n = 6 mice/group. ( G ) Representative Western blot of RUNX2 protein in TG 10 days post-pIONT. ( H ) Quantification confirms pIONT-induced RUNX2 upregulation. ** P < 0.01. Student’s t-test, n = 5 mice/group. ( I - J ) Mechanical allodynia (0.02 g, 0.16 g stimuli) showing that single-dose Runx2 siRNA (3 µg, day 0) attenuates pIONT-induced hypersensitivity. * P < 0.05. Two-way ANOVA followed by the Bonferroni test, n = 7 mice/group. ( K - L ) Runx2 ( K ) and Itpr1 ( L ) mRNA levels post-pIONT with Runx2 siRNA. * P < 0.05, ** P < 0.01. Student’s t-test, n = 6 mice/group. ( M , N ) A single dose injection of Runx2 siRNA (3 µg, day 7) attenuated pIONT-induced mechanical allodynia. * P < 0.05, ** P < 0.01. Two-way ANOVA followed by the Bonferroni test, n = 7 mice/group. ( O - P ) Runx2 ( O ) and Itpr1 ( P ) mRNA after late Runx2 siRNA intervention. * P < 0.05. Student’s t-test, n = 6 mice/group

Article Snippet: The ITPR1 agonist Adenophostin A (MCE; HY-142117), ITPR1 inhibitor 2-APB (Sigma‒Aldrich; Cat. No. CAS524958), ER stress inhibitor 4-PBA (Sigma‒Aldrich; CAS1821121), and ANO1 blocker MONNA (Sigma‒Aldrich; SML0902) were dissolved in 1% DMSO.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Inhibition, Activity Assay, Injection, Western Blot

Journal: The Journal of Headache and Pain

Article Title: ER stress-induced ITPR1/ANO1 signaling drives trigeminal neuropathic pain through calcium-dependent neuronal hyperexcitability

doi: 10.1186/s10194-025-02231-9

Figure Lengend Snippet: ITPR1 mediates ERK activation, inflammatory mediator expression, and neuronal hyperexcitability in the TG after pIONT . ( A , B ) Western blot analysis demonstrating pIONT-induced pERK upregulation is attenuated by Itpr1 siRNA (A) or 2-APB (B) treatment. * P < 0.05, ** P < 0.01, Student’s t-test, n = 3 mice/group. ( C ) qPCR analysis showing Itpr1 siRNA suppresses pIONT-induced upregulation of inflammatory cytokines in TG. * P < 0.05, ** P < 0.01, *** P < 0.001. Student’s t-test, n = 6 mice/group. ( D ) qPCR analysis revealing 2-APB-mediated reduction of pIONT-induced cytokine expression in TG. * P < 0.05, ** P < 0.01. Student’s t-test, n = 6 mice/group. ( E ) Representative action potential (AP) traces evoked by rheobase current in TG neurons from Sham, pIONT, pIONT-NC siRNA, and pIONT- Itpr1 siRNA at 10 days after surgery. ( F ) Itpr1 siRNA prevents pIONT-induced reduction in AP rheobase. * P < 0.05, ** P < 0.01, Student’s t-test. n = 8–9 neurons/group from 3–4 mice. ( G ) Representative traces of APs evoked by depolarizing current steps across treatment groups. ( H ) Itpr1 siRNA significantly reduces pIONT-induced increase in AP firing. *** P < 0.001. Two-way ANOVA followed by the Bonferroni test. n = 8–9 neurons/group from 3–4 mice. ( I-J ) pIONT decreases resting membrane potential and threshold potential, which are unaffected by Itpr1 siRNA. ** P < 0.01, *** P < 0.001. Student’s t-test. n = 8–9 neurons/group from 3–4 mice

Article Snippet: The ITPR1 agonist Adenophostin A (MCE; HY-142117), ITPR1 inhibitor 2-APB (Sigma‒Aldrich; Cat. No. CAS524958), ER stress inhibitor 4-PBA (Sigma‒Aldrich; CAS1821121), and ANO1 blocker MONNA (Sigma‒Aldrich; SML0902) were dissolved in 1% DMSO.

Techniques: Activation Assay, Expressing, Western Blot, Membrane

Journal: The Journal of Headache and Pain

Article Title: ER stress-induced ITPR1/ANO1 signaling drives trigeminal neuropathic pain through calcium-dependent neuronal hyperexcitability

doi: 10.1186/s10194-025-02231-9

Figure Lengend Snippet: ITPR1 contributes to pIONT–induced mechanical allodynia and chewing disorder . ( A , B ) Mechanical allodynia assessment (0.02 g, 0.16 g stimuli) showing single-dose Itpr1 siRNA (3 µg, day 0) attenuates pIONT-induced hypersensitivity. * P < 0.05, ** P < 0.01. Two-way ANOVA followed by the Bonferroni test, n = 6 mice/group. ( C , D ) Delayed intervention with Itpr1 siRNA (3 µg, day 7 post-pIONT) similarly reverses mechanical allodynia. * P < 0.05, ** P < 0.01. Two-way ANOVA followed by the Bonferroni test, n = 6 mice/group. ( E , F ) Early intra-TG 2-APB administration (2 µg, day 0) ameliorates pIONT-induced mechanical hypersensitivity. * P < 0.05. Two-way ANOVA followed by the Bonferroni test, n = 8 mice/group. ( G - H )Therapeutic 2-APB treatment (2 µg, day 7) maintains analgesic efficacy against established allodynia. * P < 0.05, ** P < 0.01. Two-way ANOVA followed by the Bonferroni test, n = 8 mice/group. ( I ) Representative pine wood blocks showing gnawing patterns in sham vs. pIONT groups. ( J ) Quantification of the weight reduction in sham-operated and pIONT-operated mice. *** P < 0.001. Two-way ANOVA followed by the Bonferroni test, n = 6 mice/group. ( K ) Wood block specimens from NC siRNA vs. Itpr1 siRNA-treated pIONT mice. ( L ) Quantification of the weight reduction in Itpr1 siRNA- or NC siRNA-treated mice. ** P < 0.01. Two-way ANOVA followed by the Bonferroni test, n = 6 mice/group

Article Snippet: The ITPR1 agonist Adenophostin A (MCE; HY-142117), ITPR1 inhibitor 2-APB (Sigma‒Aldrich; Cat. No. CAS524958), ER stress inhibitor 4-PBA (Sigma‒Aldrich; CAS1821121), and ANO1 blocker MONNA (Sigma‒Aldrich; SML0902) were dissolved in 1% DMSO.

Techniques: Blocking Assay

Journal: The Journal of Headache and Pain

Article Title: ER stress-induced ITPR1/ANO1 signaling drives trigeminal neuropathic pain through calcium-dependent neuronal hyperexcitability

doi: 10.1186/s10194-025-02231-9

Figure Lengend Snippet: ITPR1 couples with the ANO1 channel . ( A ) Co-immunoprecipitation analysis of ITPR1-ANO1 complexes in TG lysates. Top: ITPR1 pulldown using ANO1 antibody; Bottom: ANO1 pulldown using ITPR1 antibody. Controls: Input (positive), IgG (negative). ( B ) Representative Western blot of ANO1 protein expression in TG 10 days post-pIONT. ( C ) Quantification reveals significant ANO1 upregulation in the pIONT group. * P < 0.05. Student’s t-test. n = 4 mice/group. ( D ) Double staining of ITPR1 with ANO1 in TG 10 days post-pIONT. ( E ) Bar charts showed the percentage of double-stained TG neurons among total numbers of ITPR1 + labeled neurons. n = 4 mice. ( F ) Representative AdA-induced inward currents in TG neurons from sham, pIONT, pIONT + veh, and pIONT + MONNA group mice. ( G ) Current amplitude quantification. ** P < 0.01, *** P < 0.001, Student’s t-test. n = 13 neurons/group from 3–4 mice. ( H - I ) A single dose of AdA in TG of naïve mice induced mechanical allodynia. ( J-K ) Mechanical allodynia after repeated injections of AdA or AdA + MONNA in TG of naive mice. * P < 0.05, ** P < 0.01, *** P < 0.001. Two-way ANOVA followed by the Bonferroni test. n = 7 mice/group

Article Snippet: The ITPR1 agonist Adenophostin A (MCE; HY-142117), ITPR1 inhibitor 2-APB (Sigma‒Aldrich; Cat. No. CAS524958), ER stress inhibitor 4-PBA (Sigma‒Aldrich; CAS1821121), and ANO1 blocker MONNA (Sigma‒Aldrich; SML0902) were dissolved in 1% DMSO.

Techniques: Immunoprecipitation, Western Blot, Expressing, Double Staining, Staining, Labeling

Journal: The Journal of Headache and Pain

Article Title: ER stress-induced ITPR1/ANO1 signaling drives trigeminal neuropathic pain through calcium-dependent neuronal hyperexcitability

doi: 10.1186/s10194-025-02231-9

Figure Lengend Snippet: ITPR1 couples with ANO1 to regulate neuronal excitability and TNP pathogenesis. ( A ) Representative traces of APs evoked by rheobase current in TG neurons from NC siRNA- or Ano1 siRNA-treated mice (10 days post-surgery). ( B ) Ano1 siRNA significantly increased AP rheobase vs. NC siRNA. * P < 0.05. Student’s t-test. n = 9–10 neurons/group from 3–4 mice. ( C ) Resting membrane potential depolarization following Ano1 siRNA. ** P < 0.01. Student’s t-test. n = 9–10 neurons/group from 3–4 mice. ( D ) Representative AP trains evoked by depolarizing current steps. ( E ) Ano1 siRNA reduced the number of APs. *** P < 0.001. Two-way ANOVA followed by the post hoc Bonferroni test. n = 9–10 neurons/group from 3–4 mice. ( F ) The thresholds were not affected by Ano1 siRNA treatment. ( G , H ) A single dose of Ano1 siRNA (3 µg) injection on day 0 post-pIONT significantly attenuated mechanical allodynia. ( I , J ) A single dose of Ano1 siRNA (3 µg) injection on day 7 post-pIONT significantly attenuated mechanical allodynia. ( K, L ) Intra-TG injection of MONNA (2 µg) on pIONT day 0 attenuated mechanical allodynia. ( M, N ) Intra-TG injection of MONNA (2 µg) on pIONT day 7 attenuated mechanical allodynia. ** P < 0.01, *** P < 0.001. Two-way ANOVA followed by the post hoc Bonferroni test. n = 7 mice/group

Article Snippet: The ITPR1 agonist Adenophostin A (MCE; HY-142117), ITPR1 inhibitor 2-APB (Sigma‒Aldrich; Cat. No. CAS524958), ER stress inhibitor 4-PBA (Sigma‒Aldrich; CAS1821121), and ANO1 blocker MONNA (Sigma‒Aldrich; SML0902) were dissolved in 1% DMSO.

Techniques: Membrane, Injection

Journal: The Journal of Headache and Pain

Article Title: ER stress-induced ITPR1/ANO1 signaling drives trigeminal neuropathic pain through calcium-dependent neuronal hyperexcitability

doi: 10.1186/s10194-025-02231-9

Figure Lengend Snippet: Schematic summarizing the mechanism by which ITPR1 promotes trigeminal neuropathic pain . Following trigeminal nerve injury, ER stress is induced in TG neurons, which upregulates ITPR1 expression via the transcription factor RUNX2, leading to enhanced Ca²⁺ release from ER stores. These localized Ca²⁺ signals activate the ERK signaling pathway, resulting in hyperexcitability of sensory neurons and increased expression of pro-inflammatory mediators, including TNF-α, IL-1β, IL-6, and CCL2. Additionally, ITPR1 functionally couples with ANO1, where ITPR1-mediated Ca²⁺ release potentiates ANO1 activation. This interaction promotes Cl⁻ efflux and subsequent membrane depolarization, further lowering the activation threshold of sensory neurons and perpetuating trigeminal neuropathic pain. The illustration was generated using BioRender

Article Snippet: The ITPR1 agonist Adenophostin A (MCE; HY-142117), ITPR1 inhibitor 2-APB (Sigma‒Aldrich; Cat. No. CAS524958), ER stress inhibitor 4-PBA (Sigma‒Aldrich; CAS1821121), and ANO1 blocker MONNA (Sigma‒Aldrich; SML0902) were dissolved in 1% DMSO.

Techniques: Expressing, Activation Assay, Membrane, Generated