Impact of electro-acupuncture on EAAT2 and NMDAR-2B expression in goats with visceral hypersensitivity

Abstract

Objective: This study evaluates the effect of electro-acupuncture (EA) on visceral hypersensitivity (VH) and the expression of N-methyl-D-aspartate receptor-2B (NMDAR-2B) and glutamate transporter EAAT2 in goats.

Methods: Twenty-four goats were divided into four groups: saline, 2, 4, 6-Trinitrobenzenesulfonic acid (TNBS), TNBS + EA, and sham EA. EA was administered at Zusanli (ST36) with 60 Hz and 1-3 mA on specified days. Electromyography (EMG) recorded visceromotor response to colorectal distention (CRD). Spinal cords were collected for immunohistochemistry, western blotting, and RT-PCR. The ileum was examined histologically.

Results: The repeated EA administration significantly attenuated VH (P < 0.05) in TNBS-treated goats without similar effects in the sham group. NMDAR-2B expression increased (P < 0.01), and EAAT2 expression decreased (P < 0.01) in the TNBS group compared to saline. EA increased the EAAT2 and decreased the NMDAR-2B expression (P < 0.01) compared to TNBS, with no change in the sham-EA group.

Conclusion: EA may alleviate VH by upregulating EAAT2 and downregulating NMDAR-2B in the spinal cord of TNBS-treated goats, indicating its potential for treating chronic visceral pain in gastrointestinal disorders.

Keywords: Electro-acupuncture; Glutamate transporter EAAT2; Goat; Ileitis; Inflammatory bowel disease; NMDAR-2B; TNBS; Visceral hypersensitivity.

Fig. 1

Illustration of the experimental design EMG = Electromyography, EA = Electro-acupuncture.

Fig. 2

Microscopic lesions and microscopic lesion scores in the ileum (stained with hematoxylin and Eosin) of goats on day 22 after treatments. The representative images of the different groups were taken at 10×. There was no significant inflammation in the Saline group, moderate infiltration of inflammatory cells and ulceration, and wall thickness in the TNBS, EA, and Sham groups. The values are means ± SD, n = 6. ∗∗P < 0.01 TNBS, EA, and sham group vs. Saline group.

Fig. 3

Visceromotor responses (VMR) to goats' colorectal distension (CRD) at days 7, 10, 13, 16, 19, and 22 after surgery. The values are means ± SD, n = 6. ∗P < 0.05, ∗∗P < 0.01 TNBS, EA, and sham group vs. Saline group, ⁺P < 0.05, ⁺⁺P < 0.01; EA, sham group vs. TNBS ^#P < 0.05, ^##P < 0.01; TNBS, sham vs. EA.

Fig. 4

Effects of Electro-acupuncture (EA) on excitatory amino acid transporter 2 (EAAT2) expression in the spinal cord dorsal horn of the goats. Samples were collected from the spinal cord of the T11 segment on day 22 after surgery. (A) Western blotting EAAT2, in response to EA in the spinal cord of the goats. Electrophoresis was performed in the same conditions for all the samples. (B) The expression of EAAT2 was represented by Quantity One software (Bio-Rad) on scanned membrane images. (C) Relative gene expression of EAAT2 using q PCR. The values mean ± SD, n = 6. ∗∗P < 0.01 TNBS, EA, and sham group vs. Saline group, ⁺⁺P < 0.01; EA vs. TNBS ^#P < 0.05, ^##P < 0.01; EA vs sham. For uncropped pictures of gels, please see Supplementary Fig. 4.

Fig. 5

Excitatory amino acid transporter 2 (EAAT2) positive immunoreactivity in the dorsal horn of the spinal cord at day 22 (20 × ) in Saline, TNBS, EA, and Sham groups. Brown stain shows the nuclei of positive cells or cytoplasm. Scale bars = 50 μm.

Fig. 6

Effect of EA on N-methyl-D-aspartate receptors 2B (NR2B) expression in the spinal cord dorsal horn. Samples were collected from the spinal cord of the T11 segment on day 22. (A) Western blotting of NR2B and p-NR2B in response to EA in the spinal cord of the goats. (B) The representative expression of NR2B was measured using Quantity One software (Bio-Rad) on scanned membrane images. (C) The representative expression of p-NR2B was measured using Quantity One software (Bio-Rad) on scanned membrane images. (D) Relative gene expression of NR2B using q PCR. The values are means ± SD, n = 6. ∗∗P < 0.01 TNBS, EA, and sham group vs. Saline group, ⁺P < 0.05, ⁺⁺P < 0.01; EA vs. TNBS ^#P < 0.05, ^#P < 0.05, ^##P < 0.01; EA vs Sham. For uncropped pictures of gels, please see Supplementary Fig. 6.

Fig. 7

N-methyl-D-aspartate receptors 2B (NR2B) immunoreactivity in the spinal cord dorsal horn at day 22 (20×) in Saline, TNBS, EA, and Sham groups. Brown stain shows the nuclei of positive cells or cytoplasm. Scale bars = 50 μm.

Fig. 8

Phosphorylated N-methyl-D-aspartate receptors 2B (p-NR2B) positive immunoreactivity in the spinal cord dorsal horn at day 22 (20×) in Saline, TNBS, EA, and Sham groups. Brown stain shows the nuclei of positive cells or cytoplasm. Scale bars = 50 μm.