Acupuncture or electroacupuncture (EA) has been demonstrated to have a powerful antihypernociceptive effect on inflammatory pain. The attenuation of G protein-coupled receptor kinase 2 (GRK2) in spinal cord and peripheral nociceptor has been widely acknowledged to promote the transition from acute to chronic pain and to facilitate the nociceptive progress. This study was designed to investigate the possible role of spinal GRK2 in EA antiallodynic in a rat model with complete Freund’s adjuvant (CFA) induced inflammatory pain. EA was applied to ST36 (“
Patients diagnosed with trauma, inflammatory diseases, cancer, and diabetes often suffer from persistent pain. Chronic pain significantly reduces life quality of patients and brings a great challenge on clinical investigation. Acupuncture originated in ancient China has been proved to have a promising analgesic effect on several pain disorders, such as neuropathic pain, inflammatory pain, and cancer pain [
G protein-coupled receptor kinase 2 (GRK2) is a member of GRKs and is widely expressed in peripheral and central nervous system. GRK2 regulates cellular signaling by phosphorylating specific agonist-activated G protein-coupled receptors (GPCRs) [
The aim of present study was to investigate the possible role of spinal GRK2 in EA antiallodynic effect. For this purpose, low level of spinal GRK2 was induced by intrathecal administration with GRK2 oligonucleotides antisense, and the effect of low spinal GRK2 level on EA antiallodynic effect on inflammatory pain was evaluated in a rat model of complete Freund’s adjuvant (CFA) induced mechanical allodynia. Mismatch sense was used as control.
Experiments were performed on adult male Sprague-Dawley (SD) rats weighing 180–200 g. Animals were obtained from Shanghai Laboratory Animal Center, Chinese Academy Sciences, China. They were housed under a 12 : 12 hour light/dark cycle at a room temperature of
CFA (Sigma, suspended in an 1 : 1 oil/saline emulsion, 0.1 ml, 50
EA treatment started at 24 hours after CFA injection. The body of rats was loosely immobilized while head and four limbs were kept free to move in a special designed holder. EA was administered by using two stainless steel acupuncture needles (0.3 mm in diameter) inserted into the bilateral “
CFA injection, EA treatment, GRK2 AS(MM)-ODN i.t. injection, and behavioral test timeline for single (a) and repeated (b) treatment. The bold lines (b) were repetition of day 1.
In order to investigate whether the downregulation of spinal GRK2 would alter the antiallodynic effect of EA, GRK2 AS-ODN was intrathecally injected 30 min after EA treatment, and the mechanical threshold was measured. The GRK2 antisense oligodeoxynucleotide (AS-ODN) sequence 5′-CTTTTGGAAGATGTCG-3′, directed against nucleotides 480–495 of the rat GRK2 cDNA sequence, was synthesized by Sangong Biotech (Shanghai, CN). The mismatch (MM) ODN sequence was designed by mismatching seven bases (denoted by bold face) of the GRK2 AS sequence: 5′-GTTTACGTAGTTCTCC-3′ [
According to previous description [
Western blot was conducted to verify the effect of AS-ODN on spinal GRK2 expression in normal rats. The L4-L5 segments of the spinal cord were quickly removed and ultrasonically disrupted in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfonate, sodium orthovanadate, sodium fluoride, ethylenediaminetetraacetic acid, and leupeptin), followed by centrifugation at 12,000 ×g. The total protein level in the supernatants was measured using the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Samples were separated on 10% acrylamide gels and then transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk in tris-buffered saline with Tween (TBST) (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) for 2 h at room temperature, the membranes were incubated with the primary antibodies: rabbit anti-GRK2 (1 : 3,000, Santa Cruz) and mouse anti-GAPDH (1 : 10,000, Proteintech) at 4°C overnight. Then, the blots were washed in TBST and incubated in the appropriate secondary antibody (1 : 10,000, Abcam) for 2 h at room temperature. Western blot images were captured on an ImageQuant LAS4000 mini image analyzer (GE Healthcare, Buckinghamshire, UK), and the band levels were quantified using Quantity One version 4.62.
All data are presented as mean ± standard error of the mean (SEM). The statistical significance of differences between groups was analyzed with Student’s
CFA i.pl. injection provoked a significant reduction in paw withdrawal threshold (PWT) one day after injection (Figure
EA treatment attenuated the severity of mechanical allodynia inflammatory hypernociception induced by complete Freund’s adjuvant (CFA). Single (a) and repeated (b) EA treatment statistically raised the decreased mechanical threshold. However, sham EA treatment did not exhibit any effect on paw withdrawal threshold (PWT). The data are expressed as the mean ± SEM (
Rats after i.t. treatment with GRK2 AS-ODN for three consecutive days showed a significant decrease in GRK2 protein levels in the spinal cord as compared to MM-ODN group (Figures
The attenuation of spinal GRK2 completely eliminated the antiallodynic effect of EA treatment. (a, b) Intrathecal injection of GRK2 AS-ODN significantly reduced the expression of GRK2, while GRK2 MM-ODN injection did not alter the production of GRK2. (c, d) Single and repeated exposure of GRK2 AS-ODN completely reversed the increase in paw withdrawal threshold (PWT) by EA treatment in CFA-induced mechanical allodynia. But GRK2 MM-ODN did not change the PWT after EA treatment. (e, f) Reduction of GRK2 by GRK2 AS-ODN did not alter the PWT in normal rats during single and repeated exposure to GRK2 antisense oligodeoxynucleotide. The data are expressed as the mean ± SEM (
Acupuncture has been demonstrated to exert a neuroprotective effect on several diseases especially for painful diseases. This is the first time to investigate the possible role of spinal GRK2 in acupuncture antiallodynic effect. In a rat model of CFA-induced inflammatory pain, single EA treatment displayed a transient antiallodynic effect 2 h after treatment, while repeated EA treatments significantly reduced the severity of mechanical allodynia in the following five days. The reduction of spinal GRK2 by GRK2 AS-ODN i.t. injection inhibited the transient effect and completely eliminated the consistent antiallodynic effect of EA.
Tissue injury or inflammation caused a robust release of proalgesic mediators which targeting selected GPCRs including C-C chemokine receptor 2 [
As a component of the innate immune system, microglia samples the extracellular space of central nervous system through continuous extension, retraction, and remodeling of the cellular processes. Microglia responded quickly after injury or inflammation and took strong responsibility in neuroinflammation. Resting microglia undergo rapid morphological and functional activations [
Accumulated data demonstrated that EA attenuated pain through the inhibition of neuroinflammation by regulating spinal microglia. Previous studies have shown that nerve inflammation and nerve injury caused rapid activation of spinal microglia and elevated expression of TNF-
The attenuation of spinal GRK2 completely reversed both the transient and long-term antihypernociceptive effect by EA treatment on inflammatory pain. The results further supported that spinal GRK2 may be a key molecular target for inflammatory pain regulation and developing strategies targeting GRK2 may be a promising way for clinical pain interventions.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by The National Natural Science Foundation (nos. 81473749, 81371247, and 81171045).