Electroacupuncture treatment upregulates α7nAChR and inhibits JAK2/STAT3 in dorsal root ganglion of rat with spared nerve injury

Abstract

Background: Neuropathic pain with complicated mechanism severely disrupts patient quality of life. The novel approaches and more effective management should be further investigated. It was reported that alpha-7 nicotinic acetylcholine receptor (α7nAChR) and janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling in dorsal root ganglion (DRG) contributed to the pathogenesis of neuropathic pain. Our previous study has shown that electroacupuncture (EA) alleviated neuropathic pain via activating α7nAChR in the spinal cord. However, whether the effect of 2 Hz EA on spared nerve injury (SNI)-induced neuropathic pain is mediated through modulation of α7nAChR and JAK2/STAT3 pathway in the DRG remains unclear.

Materials and methods: The SNI-induced neuropathic pain rat model was used in this study. After application of 2 Hz EA treatment to SNI rats on day 3, 7, 14 and 21 post-surgery, the expression levels of α7nAChR, JAK2/STAT3 and some cytokines in DRG were determined by qRT-PCR and Western blot analysis.

Results: We found that SNI induced significant down-regulation of α7nAChR mRNA and protein expression. SNI also obviously elicited the decrease in anti-inflammatory cytokine IL-10 protein expression. The enhancement of p-JAK2, p-STAT3, pro-inflammatory cytokines IL-1β and IL-6 protein levels induced by SNI were also observed. However, 2 Hz EA treatment to SNI rats distinctly improved α7nAChR and IL-10 levels and reduced p-JAK2, p-STAT3, IL-1β and IL-6 expression in the DRG.

Conclusion: Our present study suggested that 2 Hz EA treatment indeed activated α7nAChR, suppressed JAK2/STAT3 signaling and re-balanced the relationship between pro-inflammatory and anti-inflammatory cytokines in DRG of SNI rat, which provided insight into our understanding of the mechanism for 2 Hz EA to attenuate neuropathic pain.

Keywords: JAK2/STAT3; dorsal root ganglion; electroacupuncture; neuropathic pain; α7nAChR.

Figure 1

Effect of 2 Hz EA on α7nAChR expression in DRG of SNI rat. Notes: (A) Relative level of α7nAChR mRNA expression. (B) Western blotted band of α7nAChR and relative level of α7nAChR protein expression. Samples were collected from the L4–L6 segment of DRG on day 3, 7, 14 and 21 after surgery. *P<0.05, **P<0.01, ***P<0.001, compared with Sham group, ^##P<0.01, ^###P<0.001, compared with SNI group. All data were expressed as the mean ± SEM, n=4–6 per group. One-way ANOVA followed by Newman–Keuls post-hoc test. Abbreviations: EA, electroacupuncture; SNI, spared nerve injury; α7nAChR, alpha-7 nicotinic acetylcholine receptor; DRG, dorsal root ganglion; d, day; SEM, standard error of the mean.

Figure 2

Effect of 2 Hz EA on JAK2 and STAT3 protein expression in DRG of SNI rat. Notes: (A) Western blotted band of p-JAK2 and JAK2. (B) Relative level of p-JAK2/JAK2 protein expression. (C) Western blotted band of p-STAT3 and STAT3. (D) Relative level of p-STAT3/STAT3 protein expression. Samples were collected from the L4–L6 segment of DRG on day 3, 7, 14 and 21 after surgery. *P<0.05, ***P<0.001, compared with Sham group, ^#P<0.05, ^##P<0.01, ^###P<0.001, compared with SNI group. All data were expressed as the mean ± SEM, n=4–5 per group. One-way ANOVA followed by Newman–Keuls post-hoc test. Abbreviations: EA, electroacupuncture; SNI, spared nerve injury; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; DRG, dorsal root ganglion; d, day; SEM, standard error of the mean.

Figure 3

Effect of 2 Hz EA on IL-1β and IL-6 protein expression in DRG of SNI rat. Notes: (A) Western blotted band of IL-1β. (B) Relative level of IL-1β protein expression. (C) Western blotted band of IL-6. (D) Relative level of IL-6 protein expression. Samples were collected from the L4–L6 segment of DRG on day 3, 7, 14 and 21 after surgery. *P<0.05, **P<0.01, ***P<0.001, compared with Sham group, ^#P<0.05, ^##P<0.01, ^###P<0.001, compared with SNI group. All data were expressed as the mean ± SEM, n=4–6 per group. One-way ANOVA followed by Newman–Keuls post-hoc test. Abbreviations: EA, electroacupuncture; SNI, spared nerve injury; IL-1β, Interfenon-1beta; IL-6, Interfenon-6; DRG, dorsal root ganglion; d, day; SEM, standard error of the mean.

Figure 4

Effect of 2 Hz EA on IL-10 protein expression in DRG of SNI rat. Notes: (A) Western blotted band of IL-10. (B) Relative level of IL-10 protein expression. Samples were collected from the L4–L6 segment of DRG on day 3, 7, 14 and 21 after surgery. *P<0.05, **P<0.01, compared with Sham group, ^##P<0.01, ^###P<0.001, compared with SNI group. All data were expressed as the mean ± SEM, n=6 per group. One-way ANOVA followed by Newman–Keuls post-hoc test. Abbreviations: EA, electroacupuncture; SNI, spared nerve injury; IL-10, Interfenon-10; DRG, dorsal root ganglion; d, day; SEM, standard error of the mean.