Korean J Pain.  2022 Oct;35(4):423-432. 10.3344/kjp.2022.35.4.423.

Perioperative stress prolong post-surgical pain via miR-339-5p targeting oprm1 in the amygdala

• Zhu Y
• Sun M
• Liu P
• Shao W
• Xiong M
• Xu B

• KMID: 2533982
• DOI: http://doi.org/10.3344/kjp.2022.35.4.423

Abstract

Background
The decreased expression of mu-opioid receptors (MOR) in the amygdala may be a key molecular in chronic post-surgical pain (CPSP). It is known that miR-339-5p expression in the amygdala of a stressed rat model was increased. Analyzed by RNAhybrid, miR-339-5p could target opioid receptor mu 1 (oprm1) which codes MOR directly. So, the authors hypothesized that miR-339-5p could regulate the expression of MOR via targeting oprm1 and cause the effects to CPSP.
Methods
To simulate perioperative short-term stress, a perioperative stress prolongs incision-induced pain hypersensitivity without changing basal pain perception rat model was built. A pmiR-RB-REPORT™ dual luciferase assay was taken to verify whether miR-339-5p could act on oprm1 as a target. The serum glucocorticoid level of rats was test. Differential expressions of MOR, GFAP, and pERK1/2 in each group of the rats’ amygdala were tested, and the expressions of miR-339-5p in each group of rats’ amygdalas were also measured.
Results
Perioperative stress prolonged the recovery time of incision pain. The expression of MOR was down-regulated in the amygdala of rats in stress + incision (S + IN) group significantly compared with other groups (P < 0.050). miR-339-5p was up-regulated in the amygdala of rats in group S + IN significantly compared with other groups (P < 0.050). miR-339-5p acts on oprm1 3’UTR and take MOR mRNA as a target.
Conclusions
Perioperative stress could increase the expression of miR-339-5p, and miR-339-5p could cause the expression of MOR to decrease via targeting oprm1. This regulatory pathway maybe an important molecular mechanism of CPSP.

Keyword

Amygdala; Glucocorticoids; MicroRNAs; Models; Animal; Pain Perception; Pain; Postoperative; Physiopathology; Receptors; Opioid; mu; Stress; Physiological

Figure

• 

  Fig. 1 (A–C) The mechanical pain threshold, thermal withdrawal latency, and cold withdrawal latency of the rats did not change within 3 days of stress (sleep deprived and movements restriction) compared with the control group (*P > 0.050). The mechanical pain and cold withdrawal latency significantly declined from the 4th day (*P < 0.050), and the thermal withdrawal latency is significantly declined from the 5th day (**P < 0.050). (D–F) The recovery time of postoperative incision pain is significantly delayed in group stress + incision (S + IN). The mechanical pain threshold, and cold withdrawal latency are significantly lower than that in group IN from the 5th day after operation (***P < 0.050). The thermal withdrawal latency of the rats in group S + IN is significantly lower than that in group IN from the 5th day after operation (***P < 0.050). At the end of the experiment (9th day after operation), the mechanical pain threshold, cold withdrawal latency, and thermal withdrawal latency are significantly lower than that in other groups. The error bars indicate mean ± standard deviation.

• 

  Fig. 2 (A, C) Western blotting assays of GFAP, p-ERK1/2, and MOR were performed in the amygdala tissue of rats. The results show that the expression of GFAP and p-ERK1/2 are up-regulated in group S + IN. The expression of MOR is down-regulated in group S + IN. (B) Both stress and operation could increase the level of serum cortisol in rats. The level of serum cortisol in the rats of group S + IN is significantly higher than that in other groups (*P < 0.050), but there was no significant difference between group IN and group S (**P < 0.050). The error bars indicate mean ± standard deviation. GFAP: glial fibrillary acidic protein, p-ERK1/2: phosphorylated extracellular regulatory protein kinase, MOR: mu-opioid receptors, S: stress, IN: incision.

• 

  Fig. 3 miR-339-5p directly targets the oprm1 3’UTR. (A) pmiR-RB-REPORTTM Double luciferase reporter vector. (B) The predicted sequences for target site of r-oprm1-WT and r-oprm1-MUT (indicated by red letters). (C) Relative luciferase activity. After the transfection of rno-mir-339-5p, the reported fluorescence expression of r-oprm1-MUT increased significantly compared with that of r-oprm1-WT (*P < 0.050). This significant difference does not exist in the NC (**P > 0.050). The error bars indicate mean ± standard deviation. 3’UTR: 3’untranslated region, NC: negative control.

• 

  Fig. 4 (A) The expression of GFAP, p-ERK1/2, and MOR by immunohistochemistry staining in amygdala tissue of rats (magnification: 200×). The positive expressing proteins appeared brown in color. (B) The results showed that GFAP and p-ERK1/2 are significantly up-regulated in group S + IN (*P < 0.050), the expression of MOR is significantly down-regulated in group S + IN (*P < 0.050). There were no significant differences between group IN and group S (**P > 0.050). The expression of GFAP and p-ERK1/2 are significantly up-regulated in group S and group IN (*P < 0.050) compared with the sham group. The expression of MOR is significantly down-regulated in group S and group IN (*P < 0.050) compared with the sham group. The error bars indicate mean ± standard deviation. GFAP: glial fibrillary acidic protein, p-ERK1/2: phosphorylated extracellular regulatory protein kinase, MOR: mu-opioid receptors, S: stress, IN: incision, IOD: integrated optical density.

• 

  Fig. 5 (A, B) Immunofluoresence assays indicate that the expression of MOR is significantly down-regulated in group S + IN (*P < 0.050). Under the laser-scanning confocal microscope the MOR appeared with a green fluorescence (magnification: 400×). (C) The results of qRT-PCR show that the expression of miR-339-5p is significantly up-regulated in group S + IN (*P < 0.050). There were no significant differences between group IN and group S (**P > 0.050). The error bars indicate mean ± standard deviation. MOR: mu-opioid receptors, S: stress, IN: incision, qRT-PCR: quantitative real-time polymerase chain reaction.

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