NMDAR2B/PKA/CREB signaling pathway contributes to esophageal neuropathic pain in gastroesophageal reflux disease

Figure 1 Immunopositive product expression of N-methyl-D-aspartate pathway-related proteins in the cingulate gyrus of rats in each group (original magnification: × 200). A: Control; B: Model; C: Model + N-methyl-D-aspartate receptor (NMDAR) agonist; D: Model + NMDAR antagonist; E: Model + protein kinase A (PKA) antagonist; F: Model + NMDAR antagonist + PKA agonist. NMDAR: N-methyl-D-aspartate receptor; PKA: Protein kinase A; CREB: cAMP-response element binding protein; BDNF: Brain-derived neurotrophic factor.

Figure 2 Immunopositive product expression of N-methyl-D-aspartate pathway-related proteins in the dorsal thalamus of rats in each group (original magnification: × 200). A: Control; B: Model; C: Model + N-methyl-D-aspartate receptor (NMDAR) agonist; D: Model + NMDAR antagonist; E: Model + protein kinase A (PKA) antagonist; F: Model + NMDAR antagonist + PKA agonist. NMDAR: N-methyl-D-aspartate receptor; PKA: Protein kinase A; CREB: cAMP-response element binding protein; BDNF: Brain-derived neurotrophic factor.

Figure 3 Immunopositive product expression of N-methyl-D-aspartate pathway-related proteins in the dorsal horn of the spinal cord of rats in each group (original magnification: × 200). A: Control; B: Model; C: Model + N-methyl-D-aspartate receptor (NMDAR) agonist; D: Model + NMDAR antagonist; E: Model + protein kinase A (PKA) antagonist; F: Model + NMDAR antagonist + PKA agonist. NMDAR: N-methyl-D-aspartate receptor; PKA: Protein kinase A; CREB: cAMP-response element binding protein; BDNF: Brain-derived neurotrophic factor.

Figure 4 Immunopositive product expression of N-methyl-D-aspartate pathway-related proteins in the distal esophageal mucosa of rats in each group (original magnification: × 200). A: Control; B: Model; C: Model + N-methyl-D-aspartate receptor (NMDAR) agonist; D: Model + NMDAR antagonist; E: Model + protein kinase A (PKA) antagonist; F: Model + NMDAR antagonist + PKA agonist. SP: Substance P; CGRP: Calcitonin gene-related peptide.

Figure 5 Results of Western blot of N-methyl-D-aspartate pathway-related proteins in the cingulate gyrus of rats in each group. A: Relative expression of N-methyl-D-aspartate (NMDA) pathway proteins in the cingulate cortex among different groups of rats (n = 6). Data are presented as mean ± SD; B: Relative expression of NMDA pathway proteins in the cingulate gyrus of rats in each group [1: Model; 2: Control; 3: Model + NMDA receptor (NMDAR) agonist; 4: Model + NMDAR antagonist; 5: Model + protein kinase A (PKA) antagonist; 6: Model + NMDAR antagonist + PKA agonist]. ^aP < 0.001, model group vs control group. ^bP < 0.05, ^cP < 0.01, ^dP < 0.001, model + NMDAR agonist group, model + NMDAR antagonist group, model + PKA antagonist group vs model group. ^eP < 0.05, ^fP < 0.001, model + NMDAR antagonist + PKA agonist group vs model + NMDAR antagonist group. NR/NMDAR: N-methyl-D-aspartate receptor; PKA: Protein kinase A; CREB: cAMP-response element binding protein; BDNF: Brain-derived neurotrophic factor.

Figure 6 Results of Western blot of N-methyl-D-aspartate pathway-related proteins in the dorsal thalamus of rats in each group. A: Relative expression of N-methyl-D-aspartate (NMDA) pathway proteins in the dorsal thalamus among different groups of rats. Data are presented as mean ± SD; B: Relative expression of NMDA pathway proteins in the dorsal thalamus of rats in each group [1: Model; 2: Control; 3: Model + NMDA receptor (NMDAR) agonist; 4: Model + NMDAR antagonist; 5: Model + protein kinase A (PKA) antagonist; 6: Model + NMDAR antagonist + PKA agonist].. NR/NMDAR: N-methyl-D-aspartate receptor; PKA: Protein kinase A; CREB: cAMP-response element binding protein; BDNF: Brain-derived neurotrophic factor. ^aP < 0.001, model group vs control group. ^bP < 0.05, ^cP < 0.01, ^dP < 0.001, model + NMDAR agonist group, model + NMDAR antagonist group, model + PKA antagonist group vs model group. ^eP < 0.05, model + NMDAR antagonist + PKA agonist group vs model + NMDAR antagonist group. NR/NMDAR: N-methyl-D-aspartate receptor; PKA: Protein kinase A; CREB: cAMP-response element binding protein; BDNF: Brain-derived neurotrophic factor.

Figure 7 Results of Western blot of N-methyl-D-aspartate pathway-related proteins in the dorsal horn of the spinal cord of rats in each group. A: Relative expression of N-methyl-D-aspartate (NMDA) pathway proteins in the dorsal horn of the spinal cord among different groups of rats. Data are presented as mean ± SD; B: Relative expression of NMDA pathway proteins in the dorsal horn of the spinal cord of rats in each group [1: Model; 2: Control; 3: Model + NMDA receptor (NMDAR) agonist; 4: Model + NMDAR antagonist; 5: Model + protein kinase A (PKA) antagonist; 6: Model + NMDAR antagonist + PKA agonist]. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, model group vs control group. ^dP < 0.01, ^eP < 0.001, model + NR agonist group, model + NR antagonist group, model + PKA antagonist group vs model group. ^fP < 0.01, model + NR antagonist + PKA agonist group vs model + NR antagonist group. NR/NMDAR: N-methyl-D-aspartate receptor; PKA: Protein kinase A; CREB: cAMP-response element binding protein; BDNF: Brain-derived neurotrophic factor.

Figure 8 Results of Western blot of substance P and calcitonin gene-related peptide in the distal esophageal mucosa of rats in each group. A: Relative expression of substance P and calcitonin gene-related peptide in the distal esophageal mucosa among different groups of rats. Data are presented as mean ± SD; B: Relative expression of N-methyl-D-aspartate (NMDA) pathway proteins in the distal esophageal mucosa of rats in each group [1: Model; 2: Control; 3: Model + NMDA receptor (NMDAR) agonist; 4: Model + NMDAR antagonist; 5: Model + protein kinase A (PKA) antagonist; 6: Model + NMDAR antagonist + PKA agonist]. ^aP < 0.001, model group vs control group. ^bP < 0.01, ^cP < 0.001, model + NR agonist group, model + NR antagonist group, model + PKA antagonist group vs model group. ^dP < 0.01, model + NR antagonist + PKA agonist group vs model + NR antagonist group. NR: N-methyl-D-aspartate receptor; PKA: Protein kinase A; SP: Substance P; CGRP: Calcitonin gene-related peptide.

Figure 9 Expression of mRNA for N-methyl-D-aspartate pathway-related proteins in the cingulate cortex among different groups of rats. Data are presented as mean ± SD. ^aP < 0.001, model group vs control group. ^bP < 0.001, model + N-methyl-D-aspartate receptor (NMDAR) agonist group, model + NMDAR antagonist group, model + protein kinase A (PKA) antagonist group vs model group. ^cP < 0.01, ^dP < 0.001, model + NMDAR antagonist + PKA agonist group vs model + NR antagonist group. NR/NMDAR: N-methyl-D-aspartate receptor; PKA: Protein kinase A.

Figure 10  Expression of mRNA for N-methyl-D-aspartate pathway-related proteins in the dorsal thalamus among different groups of rats. Data are presented as mean ± SD.

Figure 11  Expression of mRNA for N-methyl-D-aspartate pathway-related proteins in the dorsal horn of the spinal cord among different groups of rats. Data are presented as mean ± SD. ^aP < 0.01, ^bP < 0.001, model group vs control group. ^cP < 0.01, ^dP < 0.001, model + N-methyl-D-aspartate receptor (NMDAR) agonist group, model + NMDAR antagonist group, model + protein kinase A (PKA) antagonist group vs model group. ^eP < 0.001, model + NMDAR antagonist + PKA agonist group vs model + NMDAR antagonist group. NR/NMDAR: N-methyl-D-aspartate receptor; PKA: Protein kinase A.

Figure 12  Expression of mRNA for substance P and calcitonin gene-related peptide in the distal esophageal mucosa among different groups of rats. Data are presented as mean ± SD. ^aP < 0.001, model group vs control group. ^bP < 0.01, ^cP < 0.001, model + N-methyl-D-aspartate receptor (NMDAR) agonist group, model + NMDAR antagonist group, model + protein kinase A (PKA) antagonist group vs model group. ^dP < 0.05, ^eP < 0.01, model + NMDAR antagonist + PKA agonist group vs model + NMDAR antagonist group. NR: N-methyl-D-aspartate receptor; PKA: Protein kinase A; SP: Substance P; CGRP: Calcitonin gene-related peptide.

Figure 13  Serum brain-derived neurotrophic factor concentration among different groups of rats. Data are presented as mean ± SD. ^aP < 0.001, model group vs control group. ^bP < 0.01, ^cP < 0.001, model + N-methyl-D-aspartate receptor (NMDAR) agonist group, model + NMDAR antagonist group, model + protein kinase A antagonist group vs model group. NR: N-methyl-D-aspartate receptor; PKA: Protein kinase A; BDNF: Brain-derived neurotrophic factor.