Electroacupuncture participates in pain transition through the KCC2/GABAAR pathway in the spinal dorsal horn of male rats

Abstract

Objective: Preventing the transition from acute to chronic pain (pain transition) is a new strategy for treating chronic pain. The present study aimed to investigate the role of K⁺-Cl⁻ Cotransporter Isoform 2 (KCC2) and γ-aminobutyric acid receptor type A (GABAAR) in the spinal cord dorsal horn (SCDH) in pain transition and the intervention effect of electroacupuncture (EA), and to understand the mechanism of EA in preventing acute and chronic pain transition in the spinal center.

Methods: A rat model of hyperalgesic priming (HP) was established by injecting carrageenan (Car) into the plantar area of rats, followed by the injection of prostaglandin E₂ (PGE₂) into the dorsal foot 7 days later. The GABAAR agonist (muscimol) and KCC2 activator (CLP257) were intrathecally injected for three consecutive days after PGE₂ injection. EA was applied at a frequency of 2/100 Hz to the bilateral foot Zusanli (ST36) and Kunlun (BL60). A von Frey filament was used to detect the pain threshold in each group of rats. Western blotting (WB) and immunofluorescence (IF) were used to detect GABAAR and KCC2 expression in each rats group. By combining EA intervention with a KCC2 inhibitor (VU0240551), we explored the mechanism of pain transition of EA regulation of GABAAR and KCC2 expression in SCDH.

Results: The HP model was established by injecting mice with Car/PGE₂. Compared to the normal saline (NS) + NS and NS + PGE₂ groups, the pain threshold of the Car + PGE₂ group decreased significantly 48 hours after PGE₂ injection (P < 0.01). The WB results indicated that intrathecal injection of a GABAAR agonist upregulated GABAAR expression in the SCDH of HP model rats (P < 0.05). WB and IF results revealed that intrathecal injection of the KCC2 activator significantly increased GABAAR and KCC2 expression in the SCDH of HP model rats (P < 0.01) and that GABAAR and KCC2 were co-expressed in the same SCDH cells. Compared to the Car + PGE₂ group, EA intervention significantly increased MWTs from 48 to 72 hours after the first injection and 4, 24, and 48 hours after the second injection (P < 0.01). EA upregulated GABAAR and KCC2 expression in the SCDH of rats with HP (P < 0.05). Intrathecal injection of the KCC2 inhibitor blocked the analgesic effect of EA in HP model rats (P < 0.01).

Conclusions: In SCDH, KCC2 expression was downregulated, causing downregulation of GABAAR expression and resulting in pain transition. EA upregulates KCC2 and GABAAR expression and prevents pain transition.

Keywords

γ-Aminobutyric acid receptor type A / Electroacupuncture / Hyperalgesic priming / K⁺-Cl^-cotransporter isoform 2 / Spinal dorsal horn

Conflict of interest statement

The authors declare no conflict of interest.

Funding

This work was supported by the Natural Science Foundation of Zhejiang Province (No. LY23H270007).

Author contributions

Mengting Shi and Yangkun Liu wrote the manuscript. Yin Jin revised the manuscript. Junfan Fang and Yi Liang completed the data analysis and image processing. Ruijie Ma and Jie Zhou designed this project. All authors shared the raw data of this experimental study. Furthermore, all authors contributed to and approved the final version of the manuscript accepted for publication.

Ethical approval of studies and informed consent

All experimental procedures were approved by the Animal Care and Welfare Committee of Zhejiang University of Chinese Medicine, Zhejiang Province, China (approval number IACUC-20180319-12).

Acknowledgments

None.

Data availability

The data that support the findings of this study are available on request from the corresponding author Jie Zhou upon reasonable request.

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