Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: Knockdown of KIBRA in contralateral ACC prevents the upregulation of KIBRA, p -PKMζ/GluR1 signaling pathways, and neuroinflammation in CPTP rats. (A-F) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, TNF-α, and IL-1β levels in ACC analyzed 21 days after the sham operation, thoracotomy, or KIBRA − + thoracotomy. The data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4∼8 rats in each group. ACC, anterior cingulate gyrus; CPTP, chronic post-thoracotomy pain; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Knockdown, Protein-Protein interactions

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: Overexpression of KIBRA in ACC causes allodynia and activates p -PKMζ/GluR1 signaling pathways and neuroinflammation. (A) Experiment designs are shown. (B) Mechanical hyperalgesia ratio on 7, 14, and 21 days after thoracotomy or KIBRA overexpression. **** P < 0.0001 vs Thoracotomy pain group; the data were analyzed by Fisher exact test. (C and D) The threshold% and mechanical pain threshold at different time points for pain rats after injection of rAAV-CMV-wwc1-3xFLAG-WPREs or thoracotomy, * P < 0.05, *** P < 0.001, and **** P < 0.0001 vs Sham group, &&&& P < 0.00001 vs KIBRA + pain group, the data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons test, n = 8 in Sham, n = 13 in Thoracotomy pain, n = 12 in KIBRA + pain, n = 8 in KIBRA + no pain group. (E-J) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, GluR1, TNF-α, and IL-1β levels in ACC analyzed 21 days in Sham, KIBRA + pain, and KIBRA + no pain rats. The data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4 in each group. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Over Expression, Protein-Protein interactions, Injection

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: ACC overexpression KIBRA combined with thoracotomy induced CPTP in all rats. (A) Experiment designs are shown. (B) Incidence of pain on 7, 14, and 21 days after thoracotomy in KIBRA overexpression rats. ** P < 0.01 vs Thoracotomy pain group; the data were analyzed by Fisher exact test. (C and D) The threshold% and mechanical pain threshold at different time points in KIBRA + no pain + Thoracotomy pain, Thoracotomy pain, or Sham group, **** P < 0.0001 vs Sham group, & P < 0.05 vs Thoracotomy pain group. The data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons test, n = 8 in Sham, n = 13 in Thoracotomy pain, n = 14 in KIBRA + no pain + Thoracotomy pain group. (E-J) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, GluR1, TNF-α, and IL-1β levels in ACC analyzed on POD21 in Sham, Thoracotomy pain, or KIBRA + no pain + Thoracotomy pain rats, n = 4 rats in each group. The data were analyzed by 1-way ANOVA followed by Tukey post hoc test. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; CPTP, chronic post-thoracotomy pain; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Over Expression

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: The KIBRA and p -PKMζ are activated in contralateral ACC neurons after CPTP. (A-F) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, and PKMζ levels in contralateral ACC at different time points in Thoracotomy pain (A-C) or Thoracotomy no pain (D-F), * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs Sham group; the data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4∼7 rats in each group. (G-H) The photographs of the double immunofluorescence staining show that KIBRA and p -PKMζ are only colocalized with neuron markers (NeuN) but not with astrocyte marker (GFAP) and microglia marker (Iba1) in the Sham and CPTP groups. The white arrowheads indicate the co-immunostaining. Scale bar, 100 μm. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; CPTP, chronic post-thoracotomy pain; GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adapter molecule 1; KIBRA, kidney and brain-expressed protein; NeuN, neuron-specific nuclear protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Double Immunofluorescence Staining, Marker, Immunostaining, Binding Assay

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: Knockdown of KIBRA in contralateral ACC prevents the upregulation of KIBRA, p -PKMζ/GluR1 signaling pathways, and neuroinflammation in CPTP rats. (A-F) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, TNF-α, and IL-1β levels in ACC analyzed 21 days after the sham operation, thoracotomy, or KIBRA − + thoracotomy. The data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4∼8 rats in each group. ACC, anterior cingulate gyrus; CPTP, chronic post-thoracotomy pain; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Knockdown, Protein-Protein interactions

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: Overexpression of KIBRA in ACC causes allodynia and activates p -PKMζ/GluR1 signaling pathways and neuroinflammation. (A) Experiment designs are shown. (B) Mechanical hyperalgesia ratio on 7, 14, and 21 days after thoracotomy or KIBRA overexpression. **** P < 0.0001 vs Thoracotomy pain group; the data were analyzed by Fisher exact test. (C and D) The threshold% and mechanical pain threshold at different time points for pain rats after injection of rAAV-CMV-wwc1-3xFLAG-WPREs or thoracotomy, * P < 0.05, *** P < 0.001, and **** P < 0.0001 vs Sham group, &&&& P < 0.00001 vs KIBRA + pain group, the data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons test, n = 8 in Sham, n = 13 in Thoracotomy pain, n = 12 in KIBRA + pain, n = 8 in KIBRA + no pain group. (E-J) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, GluR1, TNF-α, and IL-1β levels in ACC analyzed 21 days in Sham, KIBRA + pain, and KIBRA + no pain rats. The data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4 in each group. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Over Expression, Protein-Protein interactions, Injection

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: ACC overexpression KIBRA combined with thoracotomy induced CPTP in all rats. (A) Experiment designs are shown. (B) Incidence of pain on 7, 14, and 21 days after thoracotomy in KIBRA overexpression rats. ** P < 0.01 vs Thoracotomy pain group; the data were analyzed by Fisher exact test. (C and D) The threshold% and mechanical pain threshold at different time points in KIBRA + no pain + Thoracotomy pain, Thoracotomy pain, or Sham group, **** P < 0.0001 vs Sham group, & P < 0.05 vs Thoracotomy pain group. The data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons test, n = 8 in Sham, n = 13 in Thoracotomy pain, n = 14 in KIBRA + no pain + Thoracotomy pain group. (E-J) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, GluR1, TNF-α, and IL-1β levels in ACC analyzed on POD21 in Sham, Thoracotomy pain, or KIBRA + no pain + Thoracotomy pain rats, n = 4 rats in each group. The data were analyzed by 1-way ANOVA followed by Tukey post hoc test. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; CPTP, chronic post-thoracotomy pain; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Over Expression