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. 2023 Feb 2:17:1104862.
doi: 10.3389/fnins.2023.1104862. eCollection 2023.

Motilin and its receptor are expressed in the dorsal horn in a rat model of acute incisional pain: Intrathecal motilin injection alleviates pain behaviors

Yu Zhang  1 , Jun Zhao  2 , Nan Hu  1 , Jing Wang  1 , Xi Chen  1 , Kaiyuan Wang  1 , Yiqing Yin  1
Affiliations

Motilin and its receptor are expressed in the dorsal horn in a rat model of acute incisional pain: Intrathecal motilin injection alleviates pain behaviors

Yu Zhang et al. Front Neurosci. .

Abstract

Aims: To observe the effects of intrathecal administration of motilin on pain behavior and expression of motilin (MTL)/motilin receptor (MTLR) in the spinal cord of a rat model of acute incisional pain.

Methods: An incisional pain model was established in rats using a unilateral plantar incision. The rats were also injected intrathecally with 1, 5, or 25 μg of motilin. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were determined. MTL/MTLR expression in the spinal cord was detected by western blotting and immunofluorescence. The expression of MTL in the spinal cord, stomach, duodenum, and plasma was determined by enzyme-linked immunosorbent assay (ELISA).

Results: Motilin/motilin receptor were detected in the spinal cord. Spinal cord MTL/MTLR expression peaks at 2 h after modeling (P < 0.05) and start to decrease at 24 h (P < 0.05) to almost reach baseline levels at 72 h. The changes in gastric, duodenal, plasma, and spinal cord motilin levels correlated with MWT and TWL (all R 2 > 0.82). The intrathecal injection of 1, 5, or 25 μg of motilin could increase the pain threshold of rats with incisional pain within 72 h in a dose-dependent manner.

Conclusion: This study showed for the first time that MTL/MTLR are expressed in rats' spinal dorsal horn. Acute pain increased MTL/MTLR expression in the spinal dorsal horn. Also, for the first time, this study showed that motilin intrathecal injection alleviates pain in rat models of acute incisional pain. These results suggest that MTL/MTLR could be a novel target for the management of acute pain.

Keywords: acute pain; animal model; motilin; motilin receptors; spinal cord.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Changes in mechanical withdrawal threshold (MWT) measured as paw withdrawal latency (PWL) and thermal withdrawal latency (TWL) of rats at different time points after incisional pain modeling (group P). n = 6 rats/group/time point. (A) MWT. (B) TWL. *P < 0.05, **P < 0.01, and ***P < 0.001, P group vs. C group.
FIGURE 2
FIGURE 2
(A) Motilin (MTL) and MTL receptor (MTLR) fluorescence co-labeling in the spinal dorsal horn 2 h after acute incisional pain. (B) Co-labeling of MTLR and β-tubulin III fluorescence in the spinal dorsal horn. MTL (red), MTLR (green), β-tubulin III (violet), and DAPI (blue). Scale bar = 100 μm/20 μm.
FIGURE 3
FIGURE 3
Motilin receptor (MTLR) fluorescence in the spinal dorsal horn before surgery (T0), 2 h after acute incisional pain (T1), and 24 h after surgery (T2). (A) Immunofluorescence images. Scale bar = 200 μm/50 μm. (B) MTLR relative signal intensity. (C) MTLR positive percentage. n = 6 rats/group/time point. ns, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001.
FIGURE 4
FIGURE 4
Dynamic changes in motilin (MTL) levels before surgery (T0) and at 2 h (T1), 24 h (T2), 48 h (T3), and 72 h (T4) after surgery in the acute incisional pain model (P group) and controls (C group). (A) Stomach. (B) Duodenum. (C) Plasma. (D) Spine cord. n = 6 rats/group/time point. ns, not significant; *P < 0.05, ***P < 0.001, and ****P < 0.0001.
FIGURE 5
FIGURE 5
Regression curves of the correlation between mechanical withdrawal threshold (MWT) measured as paw withdrawal latency (PWL) and thermal withdrawal latency (TWL) and motilin (MTL) expression in (A) gastric body, (B) duodenum, (C) plasma, and (D) spinal cord in rats with incisional pain.
FIGURE 6
FIGURE 6
Dynamic changes of motilin (MTL) and MTL receptor (MTLR) in the dorsal horn in rats with acute incisional pain. (A) Western blot. (B) MTLR. (C) MTL. n = 6 rats/group/time point. ns, not significant; *P < 0.05. P0, 24 h before surgery; P1, 2 h after surgery; P2, 24 h after surgery; and P4, 72 h after surgery.
FIGURE 7
FIGURE 7
Effect of intrathecal injection of motilin (MTL) on the mechanical withdrawal threshold (MWT) measured as paw withdrawal latency (PWL) and thermal foot withdrawal response latency (TWL) in the control (C) and pain model groups (P) at 24 h before operation [T0; (A)], 2 h after the operation [T1; (B)], 24 h after the operation [T2; (C)], 48 h after the operation [T3; (D)], and 72 h after the operation [T4; (E)]. 1MP, pain with 1 μg of MTL; 5MP, pain with 5 μg of MTL; and 25MP, pain with 25 μg of MTL. ns, not significant; *P < 0.05, **P < 0.01.
FIGURE 8
FIGURE 8
(A) Trajectory chart of rats with intrathecal injection of 25 μl of normal saline (group Vehicle) and 25 μg of MTL (group MTL) during 30-min period. (B) Distance (mm) and mean speed (mm/s) of rats during 30-min duration in the control (Vehicle) and MTL group (MTL) in the open field testing 2 h after injection. n = 6 rats/group. Group Vehicle vs. group MTL, P < 0.05, significant.
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Grants and funding

This research was supported by the Science Foundation of Tianjin Health Bureau (grant #2015KR17) to JZ, the Tianjin “Project + Team” Key Cultivation Program (grant #XC202034) to KW, and the National Natural Science Foundation of China (grant #81701081) to NH.

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