Electroacupuncture mediates extracellular signal-regulated kinase 1/2 pathways in the spinal cord of rats with inflammatory pain

Jian-Qiao Fang, Jun-Fan Fang, Yi Liang, Jun-Ying Du, Jian-Qiao Fang, Jun-Fan Fang, Yi Liang, Jun-Ying Du

Abstract

Background: Activation of extracellular signal-regulated kinase1/2 (ERK1/2) in dorsal horn of the spinal cord by peripheral inflammation is contributed to inflammatory pain hypersensitivity. Although electroacupuncture (EA) has been widely used to alleviate various kinds of pain, the underlying mechanism of EA analgesia requires further investigation. This study investigated the relationship between EA-induced analgesia and ERK signaling involved in pain hypersensitivity.

Methods: The rats were randomly divided into control, model, EA and sham EA groups. Inflammatory pain model was induced by injecting of 100 μl Complete Freund's adjuvant (CFA) into the plantar surface of a hind paw. Rats in the EA group were treatment with EA (constant aquare wave, 2 Hz and 100 Hz alternating frequencies, intensities ranging from 1-2 mA) at 5.5 h, 24.5 h and 48.5 h. Paw withdrawal thresholds (PWTs) were measured before modeling and at 5 h, 6 h, 25 h and 49 h after CFA injection. Rats were killed and ipsilateral side of the lumbar spinal cords were harvested for detecting the expressions of p-ERK1/2, Elk1, COX-2, NK-1 and CREB by immunohistochemistry, real-time PCR, western blot analysis and EMSA. Finally, the analgesic effect of EA plus U0126, a MEK (ERK kinase) inhibitor, on CFA rats was examined.

Results: Inflammatory pain was induced in rats by hindpaw injection of CFA and significantly increased phospho-ERK1/2 positive cells and protein levels of p-ERK1/2 in the ipsilateral spinal cord dorsal horn (SCDH). CFA up-regulated of cyclooxygenase-2 (COX-2) mRNA and protein expression at 6 h after injection and neurokinin-1 receptor (NK-1) expression at 49 h post-injection, in the SCDH. EA, applied to Zusanli (ST36) and Kunlun (BL60), remarkably increased the pain thresholds of CFA injected rats, significantly suppressed ERK1/2 activation and COX-2 protein expression after a single treatment, and decreased NK-1 mRNA and protein expression at 49 h. EA decreased the DNA binding activity of cAMP response element binding protein (CREB), a downstream transcription factor of ERK1/2, at 49 h after CFA injection. Moreover, EA and U0126 synergistically inhibited CFA-induced allodynia.

Conclusions: The present study suggests that EA produces analgesic effect by preventing the activation of ERK1/2-COX-2 pathway and ERK1/2-CREB-NK-1 pathway in CFA rats.

Figures

Figure 1
Figure 1
The paw withdrawal thresholds (PWTs) of rats in each group at different time-points. PWTs were measured at pre-injection, 5, 6, 25 and 49 h after NS/CFA injections. Values represent mean ± SEM; n = 10 per group for each time point. **P < 0.01 versus control group at the corresponding time point. ▲▲P < 0.01 versus model group at the corresponding time point. ##P < 0.01 versus sham EA group at the corresponding time point.
Figure 2
Figure 2
The expressions of p-ERK1/2-IR cells, COX-2 mRNA and NK-1mRNA in spinal cord dorsal horn of rats in control group and at 6, 25 and 49 h after subcutaneous injection of CFA into the plantar hindpaw region. A-D. Immunohistochemistry shows p-ERK1/2-IR cells in the L4-6 ipsilateral spinal cord dorsal horn in control rats (A), model rats at 6 h (B), 25 h (C), and 49 h (D). E-G. Quantification of p-ERK1/2-IR cells, COX-2 mRNA and NK-1 mRNA expression in L4-6 ipsilateral spinal cord dorsal horn, respectively. Results are mean ± SEM; n = 6. *P < 0.05, **P < 0.01 versus control group at the corresponding time point. The scale bar is 100 μm.
Figure 3
Figure 3
Effect of EA on p-ERK1/2 expression in the spinal cord dorsal horn in inflammatory pain. Immunohistochemiscal analysis shows p-ERK1/2-IR cells in the L4-6 ipsilateral spinal cord dorsal horn in control rats (A), model rats (B), EA-treated rats (C) and sham EA-treated rats (D) at 6 h after CFA injection. (E). Quantification of p-ERK1/2-IR cells in the L4-6 ipsilateral spinal cord dorsal horn of the control group and at 6 and 49 h after CFA injection. (F). The expression of p-ERK1/2 protein in the L4-6 ipsilateral spinal cord dorsal horn by western blot. (G). The quantification of p-ERK1/2 protein normalized against β-actin. Results are mean ± SEM; n = 6. **P < 0.01 versus control group at the corresponding time point. ▲P < 0.05, ▲▲P < 0.01 versus model group at the corresponding time point. ##P < 0.01 versus sham EA group at the corresponding time point. The scale bar is 100 μm.
Figure 4
Figure 4
Effect of EA on COX-2 expression in the ipsilateral spinal dorsal horn at 6 h after CFA injection. (A). The expression of COX-2 mRNA (relative to GAPDH) in L4-6 ipsilateral spinal dorsal horn was measured by real-time PCR. **P < 0.01 versus control group. ▲P < 0.05 versus model group. #P < 0.05 versus sham EA group. Results are mean ± SEM; n = 6. (B). The expression of COX-2 protein measured by western blotting analysis. **P < 0.01 versus control group. ▲P < 0.05 versus model group. #P < 0.05 versus sham EA group. Results are mean ± SEM; n = 6. (C). COX-2 protein in normalized against β-actin.
Figure 5
Figure 5
Effect of EA on NK-1 expression in the ipsilateral spinal dorsal horn at 49 h after CFA injection. (A). The expression of NK-1 mRNA (relative to GAPDH) in L4-6 ipsilateral spinal dorsal horn measured by real-time PCR. **P < 0.01 versus control group. ▲▲P < 0.01 versus model group. ##P < 0.05 versus sham EA group. Results are mean ± SEM; n = 6. (B). The expressions of NK-1 protein in the L4-6 ipsilateral spinal cord dorsal horn by western blotting analysis. **P < 0.01 versus control group. ▲P < 0.05 versus model group. Results are mea n ± SEM; n = 6. C. NK-1 protein in normalized against β-actin.
Figure 6
Figure 6
The expression of p-Elk1-IR cells in spinal cord dorsal horn of rats in the control group and at 6, 25 and 49 h after subcutaneous injection of CFA into the plantar hind paw region. A-D. Immunohistochemical analysis shows p-Elk1-IR cells in the L4-6 ipsilateral spinal cord dorsal horn of control rats (A), model rats at 6 h (B), 25 h (C) and 49 h (D). E. Quantification of p-Elk1-IR cells in L4-6 ipsilateral spinal cord dorsal horn, respectively. Results are mean ± SEM; n = 6. *P < 0.05, **P < 0.01 versus control group at the corresponding time point. The scale bar is 100 μm.
Figure 7
Figure 7
Effect of EA on p-CREB DNA binding activity in L4-6 ipsilateral spinal cord dorsal horn. (A). EMSA experiment showing binding to CREB probe in nuclear extracts prepared from control and model, EA, and sham EA groups at 6 and 49 h after CFA injection. The binding of CREB in the control group was considered for comparison (lane 2). EA regulated CREB protein binding activity at 49 after CFA-treated (lane 7). (B). EMSA experiment showing the binding of proteins present in the nuclear extracts prepared from the model group at 49 h after CFA injection. Specificity of the DNA-protein interaction was assessed by competition EMSA using a 100× molar excess of unlabeled doubled-stranded oligonucleotide CREB probe (lane 3), unlabeled mutant doubled-stranded oligonucleotide CREB probe (lane 4) and biotin-labeled mutant doubled-stranded oligonucleotide CREB probe (lane 5).
Figure 8
Figure 8
Effect of MEK inhibitor (U0126) on PWTs to mechanical stimuli in CFA-injected rats with EA stimulation. PWTs were measured at pre-injection, 5, 6, 25 and 49 h after CFA injection. Values represent mean ± SEM; n = 10 per group for each time point. ○○P < 0.01 versus DMSO group at the corresponding time point. △△P < 0.01 versus EA plus DMSO group at the corresponding time point.

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