Electroacupuncture Alleviates Spared Nerve Injury-Induced Neuropathic Pain And Modulates HMGB1/NF-κB Signaling Pathway In The Spinal Cord

Abstract

Background: Neuropathic pain with complications greatly affects patients worldwide. High mobility group box 1 (HMGB1) has been shown to contribute to the pathogenesis of neuropathic pain; thus, suppression of HMGB1 may provide a novel therapeutic option for neuropathic pain. Electroacupuncture (EA) has been indicated to be effective in attenuating neuropathic pain, but the underlying mechanism remains to be fully clarified. We aim to explore whether 2Hz EA stimulation regulates the spinal HMGB1/NF-κB signaling in neuropathic pain induced by spared nerve injury (SNI).

Materials and methods: Paw withdrawal threshold and CatWalk gait analysis were used to assess the effect of 2Hz EA on pain-related behaviors in SNI rats. Administration of 2Hz EA to SNI rats once every other day lasting for 21 days. Expression of spinal protein molecules were detected using Western blot and immunofluorescence staining.

Results: It was found that SNI significantly induced mechanical hypersensitivity and decrease of gait parameters, and subsequently increased the levels of HMGB1, TLR4, MyD88, and NF-κB p65 protein expression. 2Hz EA stimulation led to remarkable attenuation of mechanical hypersensitivity, upregulation of spinal HMGB1, TLR4, MyD88, and NF-κB p65 protein expressions induced by SNI, and significant improvement in gait parameters. Furthermore, immunofluorescence staining also confirmed that 2Hz EA obviously suppressed the co-expression of microglia activation marker CD11b and TLR4 or MyD88, as well as the activation of NF-κB p65 in SNI rats.

Conclusion: This study suggested that blockade of HMGB1/NF-κB signaling in the spinal cord may be a promising therapeutic approach for 2Hz EA management of SNI-induced neuropathic pain.

Keywords: HMGB1/NF-κB signaling; TLR4; electroacupuncture; neuropathic pain; spared nerve injury.

Figure 1

The schematic diagram of the experimental procedures. Notes: (A) The PWT was assessed after 2Hz EA stimulation on day 21 post-SNI surgery. Rats received 2Hz EA treatment once every other day lasting for 21 days. The CatWalk gait analysis was performed on the same day as the examination of PWT. (B) Following 2Hz EA treatment for 21 days, rats were sacrificed after the measurement of PWT, and the samples were collected from the L4–L6 segment of spinal cord for immunofluorescence staining and Western blot experiments. Abbreviations: PWT, paw withdrawal threshold; EA, electroacupuncture; SNI, spared nerve injury.

Figure 2

Effect of 2Hz EA on paw withdrawal threshold (PWT) induced by spared nerve injury. Notes: Rats were treated with 2Hz EA once every other day for 21 days post-surgery. Sham represents the sham group; SNI represents rats treated with the spared nerve injury; SNI+EA represents rats receiving spared nerve injury and 2Hz EA treatment. ***P<0.001, compared to the sham group, ^###P<0.001, compared to the SNI group. All data were expressed as the mean±SEM, n =13 per group. Abbreviations: PWT, paw withdrawal threshold; EA, electroacupuncture; SNI, spared nerve injury; SEM, standard error of the mean.

Figure 3

Effect of 2Hz EA on Catwalk gait parameters in SNI rats. Notes: Rats were treated with 2Hz EA once every other day for 21 days post-surgery. Sham represents the sham group; SNI represents rats treated with the spared nerve injury; SNI+EA represents rats receiving spared nerve injury and 2Hz EA treatment. The measurement of Catwalk gait parameters of stand(s), print length (cm), print area (cm²), stride length (cm), and swing speed (cm/s) is shown in (A–E). ***P<0.001, compared to the sham group, ^#P<0.05, ^##P<0.01, ^###P< 0.001, compared to the SNI group. All data were expressed as the mean±SEM, n=10–14 per group. Abbreviations: EA, electroacupuncture; SNI, spared nerve injury; SEM, standard error of the mean.

Figure 4

Effect of 2Hz EA on SNI-induced HMGB1 protein expression in the spinal cord. Notes: (A) Western blotted band of HMGB1. (B) Relative level of HMGB1/β-tubulin protein expression. Samples were collected from the L4–L6 segment of rat spinal cord on day 21 post-surgery. ***P<0.001, compared to the sham group, ^##P<0.01, compared to the SNI group. All data were expressed as the mean±SEM, n=6 per group. Abbreviations: HMGB1, high mobility group box 1; EA, electroacupuncture; SNI, spared nerve injury; SEM, standard error of the mean.

Figure 5

Effect of 2Hz EA on TLR4 protein expression and activation of microglia in the spinal cord following SNI. Notes: (A) Western blotted band of TLR4. (B) Relative level of TLR4/β-tubulin protein expression. (C) TLR4 and CD11b were double labeled in the dorsal horn of spinal cord (scale bar=20μm). 2Hz EA treatment significantly downregulated the co-expression of TLR4 (red) and microglial activation indicator CD11b (green). (D) Intensity mean value for TLR4 or CD11b. Samples were collected from the L4–L6 segment of rat spinal cord on day 21 post-surgery. ***P<0.001, compared to the sham group, ^##P<0.01, ^###P<0.001, in comparison with the SNI group. All data were expressed as the mean ±SEM, n=4 per group. Arrows indicate expressions of TLR4 and CD11b as well as co-localization of TLR4 with CD11b in the spinal dorsal horns. Abbreviations: TLR4, toll-like receptor 4; EA, electroacupuncture; SNI, spared nerve injury; SEM, standard error of the mean.

Figure 6

Effect of 2Hz EA on MyD88 protein expression and activation of microglia in the spinal cord following SNI. Notes: (A) Western blotted band of MyD88. (B) Relative level of MyD88/β-tubulin protein expression. (C) MyD88 and CD11b were double labeled in the dorsal horn of spinal cord (scale bar=20μm). 2Hz EA treatment significantly downregulated the co-expression of MyD88 (red) and microglial activation indicator CD11b (green). (D) Intensity mean value for MyD88 or CD11b. Samples were collected from the L4–L6 segment of rat spinal cord on day 21 post-surgery. *P< 0.05, ***P<0.001, compared to the sham group, ^#P<0.05, ^###P<0.001, compared to the SNI group. All data were expressed as the mean±SEM, n=4 per group. Arrows indicate expressions of MyD88 and CD11b as well as co-localization of MyD88 with CD11b in the spinal dorsal horns. Abbreviations: MyD88, myeloid differentiation factor-88; EA, electroacupuncture; SNI, spared nerve injury; SEM, standard error of the mean.

Figure 7

Effect of 2Hz EA on SNI-induced NF-κB activation in the spinal cord of rats. Notes: (A) Western blotted bands of p-NF-κB and NF-κB. (B) Relative levels of p-NF-κB/β-tubulin and NF-κB/β-tubulin protein expression. (C) p-NF-κB and DAPI were double labeled in the dorsal horn of spinal cord (scale bar=20μm). 2Hz EA treatment significantly downregulated the distribution of p-NF-κB (red) but not DAPI (blue). (D) Intensity mean value for p-NF-κB and DAPI. Samples were collected from the L4–L6 segment of rat spinal cord on day 21 post-surgery. *P<0.05, **P<0.01, compared to the sham group, ^#P<0.05, compared to the SNI group. All data were expressed as the mean±SEM, n=4 per group. Arrows indicate expressions of p-NF-κB and DAPI as well as co-localization of p-NF-κB with DAPI in the spinal dorsal horns. Abbreviations: NF-κB, nuclear factor kappa beta; EA, electroacupuncture; SNI, spared nerve injury; SEM, standard error of the mean.