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. 2019 Sep 4;12:2663-2672.
doi: 10.2147/JPR.S205987. eCollection 2019.

Inhibition of Chemokine CX3CL1 in Spinal Cord Mediates the Electroacupuncture-Induced Suppression of Inflammatory Pain

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Free PMC article

Inhibition of Chemokine CX3CL1 in Spinal Cord Mediates the Electroacupuncture-Induced Suppression of Inflammatory Pain

Yuheng Li et al. J Pain Res. .
Free PMC article

Abstract

Purpose: Chemokine CX3CL1 and its receptor CX3CR1 in the lumbar spinal cord play crucial roles in pain processing. Electroacupuncture (EA) is recognized as an alternative therapy in pain treatment due to its efficacy and safety. However, the analgesic mechanism of EA remains unclear. The aim of this study was to investigate whether EA suppressed complete Freund's adjuvant (CFA)-induced pain via modulating CX3CL1-CX3CR1 pathway.

Materials and methods: Inflammatory pain was induced by intraplantar injection of CFA to the left hind paw of Sprague-Dawley rats. EA with 2 Hz for 30 mins was given to bilateral Zusanli acupoints (ST36) on the first and third day after CFA injection. Mechanical allodynia and thermal hyperalgesia were tested with von Frey tests and Hargreaves tests, respectively. The expressions of CX3CL1, CX3CR1 and p38 mitogen-activated protein kinase (MAPK) were quantified with Western blots. The release of IL-1β, IL-6 and TNF-α were evaluated with ELISA. Recombinant CX3CL1 or control IgG were then injected through intrathecal catheters in the EA-treated CFA model rats. The behavioral tests, p38 MAPK activation and cytokine release were then evaluated.

Results: EA significantly inhibited inflammatory pain induced by CFA for 3 days. Meanwhile, EA downregulated the expression of CX3CL1 but not CX3CR1 in the lumbar spinal cord of the CFA rats. Besides, activation of p38 MAPK and the release of pain-related cytokines (IL-1β, IL-6 and TNF-α) were inhibited by EA. Intrathecal injection of CX3CL1 largely reversed the analgesic effect of EA treatment and re-activated p38 MAPK signaling, and resulted in pro-inflammatory cytokines increase in acupuncture-treated rats.

Conclusion: Our findings indicate that EA alleviates inflammatory pain via modulating CX3CL1 signaling in lumbar spinal cord, revealing a potential mechanism of anti-nociception of EA in inflammatory pain.

Keywords: CX3CL1; cytokine; electroacupuncture; inflammatory pain; p38 MAPK.

Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Diagrammatic sketch of the experiment procedures. Notes: (A) In experiment 1, rats were randomly allocated to Control, CFA, CFA+EA, CFA+sham EA groups and underwent behavioral tests on the day before modeling to obtain the baseline data. In the 3 days after modeling, behavioral tests were repeated once upon a day; EA treatment was given on day 1 and day 3 after modeling, at 1 hr before the behavioral tests. Spinal cord tissue was then collected from six animals in each group after the behavioral tests on day 1 and day 3. (B) In experiment 2, intrathecal catheter was implanted to the lumbar spinal cord 3 days before modeling. IgG and recombinant CX3CL1 were given through intrathecal catheter 30 mins before the EA treatment. The behavioral tests and EA treatments were done in the same way as that in experiment 1. Abbreviations: CFA, complete Freund’s adjuvant; EA, electroacupuncture.
Figure 2
Figure 2
Analgesic effects of EA on pain hypersensitivity in CFA model rats. Notes: CFA modeling induced inflammatory pain in CFA, CFA+EA, CFA+sham EA group rats. (A) CFA-treated rats showed the decreased paw withdrawal threshold, while sham EA did not suppress the abnormal sensation, EA treatment alleviated such allodynia. (B) The paw withdrawal latency decreased after CFA modeling, EA treatment prolonged the latency time, sham EA did not show the same effect. **P<0.01 vs Control; ##P<0.01 vs CFA; ΔP <0.05 vs CFA+EA, ΔΔP<0.01 vs CFA+EA; n=10 in each group. Abbreviations: CFA, complete Freund’s adjuvant; EA, electroacupuncture.
Figure 3
Figure 3
Expression of CX3CL1 and CX3CR1 in the lumbar spinal cord. Notes: (A) The expression of CX3CL1 in CFA group increased 1 day after modeling. (B) The CX3CL1 level in CFA group was upregulated significantly, but decreased to be as low as the Control in CFA+EA group at day 3 after modeling, sham EA did not prevent upregulation of CX3CL1 at day 3. (C and D) The expression of CX3CL1 receptor CX3CR1 was not affected by CFA or EA treatment. *P<0.05 vs Control, **P<0.01 vs Control; ##P<0.01 vs CFA; ΔΔP<0.01 vs CFA+EA; n=6 in each group. Abbreviations: CFA, complete Freund’s adjuvant; EA, electroacupuncture; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 4
Figure 4
EA blocks the activation of p38 MAPK and the release of cytokines in spinal cord. Notes: (A) The total amount of spinal cord p38 MAPK was not changed by either CFA modeling or real/sham EA treatments. (B) CFA activated the phosphorylation of p38 MAPK on the first and third day after modeling, EA inhibited the activation of p38 MAPK at day 3 after modeling, sham EA failed to suppress p38 MAPK activation at day 3. (C–E) EA suppressed the release of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) on the day 3 after CFA injection, sham EA exerts no effect on suppressing cytokine release. *P<0.05 vs Control, **P<0.01 vs Control; ##P<0.01 vs CFA; ΔΔP<0.01 vs CFA+EA; n=6 in each group. Abbreviations: p38 MAPK, p38 mitogen-activated protein kinase; phosph-p38 MAPK, phosphorylated p38 MAPK; CFA, complete Freund’s adjuvant; EA, electroacupuncture.
Figure 5
Figure 5
Intrathecal injection of CX3CL1 blocked the analgesic effect of EA treatment. Notes: (A) Intrathecal injection of CX3CL1 partially decreased the PWT improved by EA treatment, but did not aggravate the pain in CX3CL1 group. (B) The PWL of the CX3CL1+EA group was lower than it of the EA treatment group (IgG+EA group), but higher than IgG group, the PWL of CX3CL1 group was not affected by drug administration. (C) The amount of p38 MAPK kept a similar level among four groups. (D) The p38 MAPK signaling suppressed by EA treatment was reactivated by CX3CL1 administration in CX3CL1+EA group; but CX3CL1 did not further activate p38 MAPK signaling in CX3CL1 group. (E–G) Intrathecal injection of CX3CL1 led to a higher level of pro-inflammatory cytokine release in the spinal cord of the EA-treated rats. *P<0.05 vs IgG, **P<0.01 vs IgG; #P<0.05 vs IgG+EA, ##P<0.01 vs IgG+EA; n=8 in each group in behavioral tests, n=6 in each group in Western blots and ELISA assay. Abbreviations: PWT, paw withdrawal threshold; PWL, paw withdrawal latency; p38 MAPK, p38 mitogen-activated protein kinase; phosph-p38 MAPK, phosphorylated p38 MAPK; CFA, complete Freund’s adjuvant; EA, electroacupuncture; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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