Electroacupuncture may alleviate diabetic neuropathic pain by inhibiting the microglia P2X4R and neuroinflammation.

Diabetic neuropathic pain (DNP) is a common and destructive complication of diabetes mellitus. The discovery of effective therapeutic methods for DNP is vitally imperative because of the lack of effective treatments. Although 2 Hz electroacupuncture (EA) was a successful approach for relieving DNP, the mechanism underlying the effect of EA on DNP is still poorly understood. Here, we established a rat model of DNP that was induced by streptozotocin (STZ) injection. P2X4R was upregulated in the spinal cord after STZ-injection. The upregulation of P2X4R was mainly expressed on activated microglia. Intrathecal injection of a P2X4R antagonist or microglia inhibitor attenuated STZ-induced nociceptive thermal hyperalgesia and reduced the overexpression of brain-derived neurotrophic factor (BDNF), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the spinal cord. We also assessed the effects of EA treatment on the pain hypersensitivities of DNP rats, and further investigated the possible mechanism underlying the analgesic effect of EA. EA relieved the hyperalgesia of DNP. In terms of mechanism, EA reduced the upregulation of P2X4R on activated microglia and decreased BDNF, IL-1ß and TNF-α in the spinal cord. Mechanistic research of EA's analgesic impact would be beneficial in ensuring its prospective therapeutic effect on DNP as well as in extending EA's applicability.

Electroacupuncture alleviates the relapse of pain-related aversive memory by activating KOR and inhibiting GABAergic neurons in the insular cortex.

Pain-related aversive memory is common in chronic pain patients. Electroacupuncture has been demonstrated to block pain-related aversive memory. The insular cortex is a key region closely related to aversive behaviors. In our study, a potential mechanism underlying the effect of electroacupuncture treatment on pain-related aversive memory behaviors relative to the insular cortex was investigated. Our study used the chemogenetic method, pharmacological method, electroacupuncture intervention, and behavioral detection. Our study showed that both inhibition of gamma-aminobutyric acidergic neurons and activation of the kappa opioid receptor in the insular cortex blocked the pain-related aversive memory behaviors induced by 2 crossover injections of carrageenan in mice; conversely, both the activation of gamma-aminobutyric acidergic neurons and inhibition of kappa opioid receptor in the insular cortex play similar roles in inducing pain-related aversive memory behaviors following 2 crossover injections of carrageenan. In addition, activation of gamma-aminobutyric acidergic neurons in the insular cortex reversed the effect of kappa opioid receptor activation in the insular cortex. Moreover, electroacupuncture effectively blocked pain-related aversive memory behaviors in model mice, which was reversed by both activation of gamma-aminobutyric acidergic neurons and inhibition of kappa opioid receptor in the insular cortex. The effect of electroacupuncture on blocking pain-related aversive memory behaviors may be related to the activation of the kappa opioid receptor and inhibition of gamma-aminobutyric acidergic neurons in the insular cortex.

Electroacupuncture Inhibits Pain Memory and Related Anxiety-Like Behaviors by Blockading the GABAB Receptor Function in the Midcingulate Cortex.

Pain memory is commonly considered an underlying cause of chronic pain and is also responsible for a range of anxiety. Electroacupuncture (EA) has been shown to ameliorate pain memories and exert anti-anxiety effects. Previous research has indicated that GABAergic neurons and/or GABA receptors (GABARs) in the midcingulate cortex (MCC) have potential associations with chronic pain and anxiety. However, there is no known empirical research that has specifically studied the effects of EA on the GABAergic system in the MCC. Here, we used cross-injection of carrageenan to establish the pain memory rats model. Immunofluorescence were used to detect the excitability of GABAergic neurons within MCC. Von Frey filament, elevated zero maze, and open field tests were used to measure mechanical allodynia and anxiety-like behaviors, combined with chemogenetic and pharmacologic technologies. Finally, this study provides evidence that pain memories contribute to generalized negative emotions and that downregulating the activity of GABAergic neurons within MCC could block pain memories and reverse anxiety emotion. Specifically, GABABR is involved in pain memory and related anxiety-like behaviors. Activation of GABAergic neurons in the MCC did not reverse the effects of EA on pain memories and related anxiety-like behaviors, whereas these effects could be reversed by a GABABR agonist. These findings highlight the functional significance of GABABR in the EA-mediated attenuation of pain memories and related anxiety-like behaviors in rats.

Analgesic effect of electroacupuncture on bone cancer pain in rat model: the role of peripheral P2X3 receptor.

Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 104 cells/3 µL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4-6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,ß-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca2+ imaging analysis revealed that the EA stimulation decreased the percentage of α,ß-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,ß-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA's analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic.

Electroacupuncture Alleviates Anxiety-Like Behaviors Induced by Chronic Neuropathic Pain via Regulating Different Dopamine Receptors of the Basolateral Amygdala.

Chronic pain, such as neuropathic pain, causes anxiety and other negative emotions, which aggravates the pain sensation and increases the risk of chronic pain over time. Dopamine receptor D1 (DRD1) and dopamine receptor D2 (DRD2) in the basolateral amygdala (BLA) have been implicated in mediating anxiety-related behaviors, but their potential roles in the BLA in neuropathic pain-induced anxiety have not been examined. Electroacupuncture (EA) is commonly used to treat chronic pain and emotional disorders, but it is still unclear whether EA plays a role in analgesia and anxiety relief through DRD1 and DRD2 in the BLA. Here, we used western blotting to examine the expression of DRD1 and DRD2 and pharmacological regulation combined with behavioral testing to detect anxiety-like behaviors. We observed that injection of the DRD1 antagonist SCH23390 or the DRD2 agonist quinpirole into the BLA contributed to anxiety-like behaviors in naive mice. EA also activated DRD1 or inhibited DRD2 in the BLA to alleviate anxiety-like behaviors. To further demonstrate the role of DRD1 and DRD2 in the BLA in spared nerve injury (SNI) model-induced anxiety-like behaviors, we injected the DRD1 agonist SKF38393 or the DRD2 antagonist sulpiride into the BLA. We found that both activation of DRD1 and inhibition of DRD2 could alleviate SNI-induced anxiety-like behaviors, and EA had a similar effect of alleviating anxiety. Additionally, neither DRD1 nor DRD2 in the BLA affected SNI-induced mechanical allodynia, but EA did. Overall, our work provides new insights into the mechanisms of neuropathic pain-induced anxiety and a possible explanation for the effect of EA treatment on anxiety caused by chronic pain.

Nrf2 Activation Mediates Antiallodynic Effect of Electroacupuncture on a Rat Model of Complex Regional Pain Syndrome Type-I through Reducing Local Oxidative Stress and Inflammation.

Complex regional pain syndrome type-I (CRPS-I) represents a type of neurovascular condition featured by severe pain in affected extremities. Few treatments have proven effective for CRPS-I. Electroacupuncture (EA) is an effective therapy for pain relief. We explored the mechanism through which EA ameliorates pain in a rat CRPS-I model. The chronic postischemic pain (CPIP) model was established using Sprague-Dawley rats to mimic CRPS-I. We found that oxidative stress-related biological process was among the predominant biological processes in affected hindpaw of CPIP rats. Oxidative stress occurred primarily in local hindpaw but not in the spinal cord or serum of model rats. Antioxidant N-acetyl cysteine (NAC) attenuated mechanical allodynia and spinal glia overactivation in CPIP model rats, whereas locally increasing oxidative stress is sufficient to induce chronic pain and spinal glia overactivation in naive rats. EA exerted remarkable antiallodynia on CPIP rats by reducing local oxidative stress via enhancing nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Pharmacological blocking Nrf2 abolished antioxidative and antiallodynic effects of EA. EA reduced spinal glia overactivation, attenuated the upregulation of inflammatory cytokines, reduced the enhanced TRPA1 channel activity in dorsal root ganglion neurons innervating the hindpaws, and improved blood flow dysfunction in hindpaws of CPIP rats, all of which were mimicked by NAC treatment. Thus, we identified local oxidative injury as an important contributor to pathogenesis of animal CRPS-I model. EA targets local oxidative injury by enhancing endogenous Nrf2-mediated antioxidative mechanism to relieve pain and inflammation. Our study indicates EA can be an alternative option for CRPS-I management.

Electroacupuncture Regulates Pain Transition Through Inhibiting PKCε and TRPV1 Expression in Dorsal Root Ganglion.

Many cases of acute pain can be resolved with few side effects. However, some cases of acute pain may persist beyond the time required for tissue injury recovery and transit to chronic pain, which is hard to treat. The mechanisms underlying pain transition are not entirely understood, and treatment strategies are lacking. In this study, the hyperalgesic priming model was established on rats to study pain transition by injection of carrageenan (Car) and prostaglandin E2 (PGE2). The expression levels of protein kinase C epsilon (PKCε) and transient receptor potential vanilloid 1 (TRPV1) in the L4-L6 dorsal root ganglion (DRG) were investigated. Electroacupuncture (EA) is a form of acupuncture in which a small electric current is passed between a pair of acupuncture needles. EA was administrated, and its effect on hyperalgesia and PKCε and TRPV1 expression was investigated. The PKCε-TRPV1 signaling pathway in DRG was implicated in the pain transition. EA increased the pain threshold of model animals and regulated the high expression of PKCε and TRPV1. Moreover, EA also regulated hyperalgesia and high TRPV1 expression induced by selective PKCε activation. We also found that EA partly increased chronic pain threshold, even though it was only administered between the Car and PGE2 injections. These findings suggested that EA could prevent the transition from acute to chronic pain by inhibiting the PKCε and TRPV1 expression in the peripheral nervous system.

Electroacupuncture Ameliorates Chronic Inflammatory Pain-Related Anxiety by Activating PV Interneurons in the Anterior Cingulate Cortex.

Chronic inflammatory pain is a common clinical disease that tends to be associated with negative emotions such as anxiety and depression. The anterior cingulate cortex (ACC) is involved in pain and pain-related anxiety, and γ-aminobutyric acid (GABA)-ergic interneurons play an important role in chronic pain and anxiety. Electroacupuncture (EA) has good analgesic and antianxiety effect, but the underlying mechanisms have not yet been fully elucidated. In this study, we established a chronic inflammatory pain model and observed that this model induced anxiety-like behaviors and decreased the numbers of parvalbumin (PV) and somatostatin (SOM) positive cells. Activation of PV but not SOM interneurons by chemogenetic techniques alleviated anxiety-like behaviors and pain sensation. EA treatment improved pain sensation, anxiety-like behaviors and increased the number of PV- positive cells in the ACC, but did not affect on the number of SOM-positive cells in the ACC. Moreover, specific inhibition of PV interneurons by chemogenetic methods reversed the analgesic and antianxiety effects of EA. These results suggest that EA ameliorates chronic inflammatory pain and pain-related anxiety by upregulating PV but not SOM interneurons in the ACC.

Role of GABAAR in the Transition From Acute to Chronic Pain and the Analgesic Effect of Electroacupuncture on Hyperalgesic Priming Model Rats.

Chronic pain is a costly health problem that impairs health-related quality of life when not effectively treated. Regulating the transition from acute to chronic pain is a new therapeutic strategy for chronic pain that presents a major clinical challenge. The underlying mechanisms of pain transition are not entirely understood, and strategies for preventing this transition are lacking. Here, a hyperalgesic priming model was used to study the potential mechanism by which γ-aminobutyric acid receptor type A (GABAAR) in the dorsal root ganglion (DRG) contributes to pain transition. Furthermore, electroacupuncture (EA), a modern method of acupuncture, was administered to regulate pain transition, and the mechanism underlying EA's regulatory effect was investigated. Hyperalgesic priming was induced by intraplanar injection of carrageenan (Car)/prostaglandin E2 (PGE2). The decrease in mechanical withdrawal threshold (MWT) induced by PGE2 returned to baseline 4 h after injection in NS + PGE2 group, and still persisted 24 h after injection in Car + PGE2 group. Lower expression of GABAAR in the lumbar DRG was observed in the model rats. Furthermore, activating or blocking GABAAR could reversed the long-lasting hyperalgesia induced by Car/PGE2 injection or produced a persistent hyperalgesia. In addition, GABAAR may be involved in Protein Kinase C epsilon (PKCε) activation in the DRG, a mark molecular of pain transition. EA considerably increased the mechanical pain thresholds of hyperalgesic priming model mammals in both the acute and chronic phases. Furthermore, EA upregulated the expression of GABAAR and inhibited the activation of PKCε in the DRG. In addition, peripheral administration of picrotoxin blocked the analgesic effect of EA on the model rats and abolished the regulatory effect of EA on PKCε activation. These findings suggested that GABAAR plays a key role in both the transition from acute to chronic pain and the analgesic effect of EA on hyperalgesic priming.

Electroacupuncture inhibits the interaction between peripheral TRPV1 and P2X3 in rats with different pathological pain.

Chronic pain is regarded to be one of the common and refractory diseases to cure in the clinic. One hundred Hz electroacupuncture (EA) is commonly used for inflammatory pain and 2 Hz for neuropathic pain possibly by modulating the transient receptor potential vanilloid subtype 1 (TRPV1) or the purinergic P2X3 related pathways. To clarify the mechanism of EA under various conditions of pathological pain, rats received a subcutaneous administration of complete Freund's adjuvant (CFA) for inflammatory pain and spared nerve injury (SNI) for neuropathic pain. The EA was performed at the bilateral ST36 and BL60 1 d after CFA or SNI being successfully established for 3 consecutive days. The mechanical hyperalgesia test was measured at baseline, 1 d after model establishment, 1 d and 3 d after EA. The co-expression changes, co-immunoprecipitation of TRPV1 and P2X3, and spontaneous pain behaviors (SPB) test were performed 3 d after EA stimulation. One hundred Hz EA or 2Hz EA stimulation could effectively down-regulate the hyperalgesia of CFA or SNI rats. The increased co-expression ratio between TRPV1 and P2X3 at the dorsal root ganglion (DRG) in two types of pain could be reduced by 100Hz or 2Hz EA intervention. While 100Hz or 2Hz EA was not able to eliminate the direct physical interaction between TRPV1 and P2X3. Moreover, EA could significantly inhibit the SPB induced by the co-activation of peripheral TRPV1 and P2X3. All results indicated that EA could significantly reduce the hyperalgesia and the SPB, which was partly related to inhibiting the co-expression and indirect interaction between peripheral TRPV1 and P2X3.

Phosphoproteomic Profiling of Rat's Dorsal Root Ganglia Reveals mTOR as a Potential Target in Bone Cancer Pain and Electro-Acupuncture's Analgesia.

Bone cancer pain (BCP) is a clinical refractory mixed pain involving neuropathic and inflammatory pain, with the underlying mechanisms remaining largely unknown. Electro-acupuncture (EA) can partly alleviate BCP according to previous research. We aim to explore the proteins and major pathways involved in BCP and EA treatment through phosphoproteomic profiling. BCP rat model was built by tibial inoculation of MRMT-1 mammary gland carcinoma cells. Mechanical hyperalgesia determined by paw withdrawal thresholds (PWTs) and bone destruction manifested on the radiographs confirmed the success of modeling, which were attenuated by EA treatment. The differentially expressed phosphorylated proteins (DEPs) co-regulated by BCP modeling and EA treatment in rat dorsal root ganglions (DRGs) were analyzed through PEX100 Protein microarray. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEPs were significantly enriched in mammalian target of rapamycin (mTOR) signaling pathway. The phosphorylations of mTOR at Ser2448 and Thr2446 were increased in BCP and downregulated by EA. In addition, the phosphorylation of S6K and Akt, markers of the mTOR complex, were also increased in BCP and downregulated by EA. Inhibition of mTOR signaling alleviated the PWTs of BCP rats, while the mTOR agonist impaired the analgesic effect of EA. Thus, our study provided a landscape of protein phosphorylation changes in DRGs of EA-treated BCP rats and revealed that mTOR signaling can be potentially targeted to alleviate BCP by EA treatment.

Electroacupuncture Attenuates Morphine Tolerance in Rats with Bone Cancer Pain by Inhibiting PI3K/Akt/JNK1/2 Signaling Pathway in the Spinal Dorsal Horn.

PURPOSE: Morphine is often used for the treatment of moderate and severe cancer pain, but long-term use can lead to morphine tolerance. Methods for effectively inhibiting morphine tolerance and the related mechanism of action are of great significance for the treatment of cancer pain. Previous studies have shown that electroacupuncture (EA) can inhibit the occurrence of morphine tolerance, but the mechanism is not yet clear. The aim of the present study was to explore the signaling pathway by which EA attenuates the development of bone cancer pain (BCP)-morphine tolerance (MT). MATERIALS AND METHODS: Changes in the paw withdrawal threshold (PWT) of rats with bone cancer pain-morphine tolerance were observed in a study of EA combined with intrathecal injection of a PI3K inhibitor (LY294002) or agonist (insulin-like growth factor-1 [IGF-1]). We also tested the protein expression of phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-Akt), phosphorylated c-Jun NH2-terminal kinase 1/2 (p-JNK1/2), and ß-arrestin2 in the L4-6 spinal dorsal horn of rats. RESULTS: The protein expression of p-PI3K, p-Akt, p-JNK1/2, and ß-arrestin2 was upregulated in the L4-6 spinal dorsal horn of rats with bone cancer pain and bone cancer pain-morphine tolerance. EA delayed the occurrence of morphine tolerance in rats with bone cancer pain and downregulated the protein expression of p-PI3K, p-Akt, p-JNK1/2, and ß-arrestin2 in the L4-6 spinal dorsal horn of rats with bone cancer pain-morphine tolerance. Intrathecal injection of LY294002 attenuated the development of morphine tolerance and downregulated the protein expression of p-Akt, p-JNK1/2, and ß-arrestin2 in the spinal dorsal horn of rats with bone cancer pain-morphine tolerance. In addition, the inhibitory effect of EA on morphine tolerance was reversed by IGF-1. CONCLUSION: The mechanism underlying the ability of EA to attenuate morphine tolerance may be associated with inhibition of the PI3K/Akt/JNK1/2 signaling pathway.

Rostral Anterior Cingulate Cortex-Ventrolateral Periaqueductal Gray Circuit Underlies Electroacupuncture to Alleviate Hyperalgesia but Not Anxiety-Like Behaviors in Mice With Spared Nerve Injury.

Neuropathic pain is a common cause of chronic pain and is often accompanied by negative emotions, making it complex and difficult to treat. However, the neural circuit mechanisms underlying these symptoms remain unclear. Herein, we present a novel pathway associated with comorbid chronic pain and anxiety. Using chemogenetic methods, we found that activation of glutamatergic projections from the rostral anterior cingulate cortex (rACC Glu ) to the ventrolateral periaqueductal gray (vlPAG) induced both hyperalgesia and anxiety-like behaviors in sham mice. Inhibition of the rACC Glu -vlPAG pathway reduced anxiety-like behaviors and hyperalgesia in the spared nerve injury (SNI) mice model; moreover, electroacupuncture (EA) effectively alleviated these symptoms. Investigation of the related mechanisms revealed that the chemogenetic activation of the rACC Glu -vlPAG circuit effectively blocked the analgesic effect of EA in the SNI mice model but did not affect the chronic pain-induced negative emotions. This study revealed a novel pathway, the rACC Glu -vlPAG pathway, that mediates neuropathic pain and pain-induced anxiety.

Effects of low- and high-frequency electroacupuncture on protein expression and distribution of TRPV1 and P2X3 in rats with peripheral nerve injury.

BACKGROUND: Whether electroacupuncture (EA) stimulation at different frequencies has a similar effect on spared nerve injury (SNI) as other neuropathic pain models, and how EA at different frequencies causes distinct analgesic effects on neuropathic pain is still not clear. METHODS: Adult male Sprague-Dawley rats were randomly divided into sham SNI, SNI, 2 Hz, 100 Hz and sham EA groups. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured. EA was performed once a day on days 1 to 14 after SNI. The expressions of transient receptor potential cation subfamily V member 1 (TRPV1) and peripheral purinergic P2X receptor 3 (P2X3) were determined by western blotting and immunofluorescence. TRPV1 siRNA and P2X3 siRNA were administered by intrathecal injection. TRPV1 or P2X3 agonists were combined with EA. RESULTS: There were significant decreases in PWT, but no changes in PWL in the 14 days after SNI. EA using 2- or 100-Hz stimulation similarly increased PWT at every time point. The cytosol protein expression of P2X3 in the L4-L6 dorsal root ganglia (DRG) increased, but the expression of TRPV1 decreased in the SNI model. Both these effects were ameliorated by EA, with 2-Hz stimulation having a stronger effect than 100-Hz stimulation. Blocking either TRPV1 or P2X3 specific siRNAs attenuated the decreased PWT induced by SNI. Administration of either a TRPV1 or P2X3 agonist inhibited EA analgesia. CONCLUSION: 2- and 100-Hz EA similarly induced analgesic effects in SNI. This effect was related to up-regulation and down-regulation, respectively, of cytosol protein expression of P2X3 and TRPV1 in L4-L6 DRG, with 2 Hz having a better effect than 100 Hz.

Effect of Electroacupuncture on Pain Perception and Pain-Related Affection: Dissociation or Interaction Based on the Anterior Cingulate Cortex and S1.

Electroacupuncture (EA) can effectively modulate pain perception and pain-related negative affect; however, we do not know whether the effect of EA on sensation and affect is parallel, or dissociated, interactional. In this study, we observed the effects of the anterior cingulate cortex (ACC) lesion and the primary somatosensory cortex (S1) activation on pain perception, pain-related affection, and neural oscillation in S1. ACC lesions did not affect pain perception but relieved pain-paired aversion. S1 activation increased pain perception and anxious behavior. EA can mitigate pain perception regardless of whether there is an ACC lesion. Chronic pain may increase the delta and theta band oscillatory activity in the S1 brain region and decrease the oscillatory activity in the alpha, beta, and gamma bands. EA intervention may inhibit the oscillatory activity of the alpha and beta bands. These results suggest that EA may mitigate chronic pain by relieving pain perception and reducing pain-related affection through different mechanisms. This evidence builds upon findings from previous studies of chronic pain and EA treatment.

Electroacupuncture Regulates Pain Transition by Inhibiting the mGluR5-PKCε Signaling Pathway in the Dorsal Root Ganglia.

BACKGROUND: Acute pain can transition to chronic pain, presenting a major clinical challenge. Electroacupuncture (EA) can partly prevent the transition from acute to chronic pain. However, little is known about the mechanisms underlying the effect of EA. This study investigated the effect of EA on pain transition and the activation of metabotropic glutamate receptor 5 (mGluR5)-protein kinase C epsilon (PKCε) signaling pathway in the dorsal root ganglia (DRG). METHODS: The hyperalgesic priming model was established by the sequential intraplantar injection of carrageenan (1%, 100 µL) and prostaglandin E2 (PGE2) into the left hind paw of rats. EA treatment (2/100 Hz, 30 min, once/day) was applied at bilateral Zusanli (ST36) and Kunlun (BL60) acupoints in rats. Von Frey filaments were used to investigate the mechanical withdrawal threshold (MWT) at different time points. The protein expression levels of mGluR5 and PKCε in the ipsilateral L4-L6 DRGs of rats were detected by Western blot. Some pharmacological experiments were performed to evaluate the relationship between mGluR5, PKCε and the MWT. It was also used to test the effects of EA on the expression levels of mGluR5 and PKCε and changes in the MWT. RESULTS: Sequential injection of carrageenan and PGE2 significantly decreased the MWT of rats and up-regulated the expression level of mGluR5 and PKCε in the ipsilateral L4-L6 DRGs. EA can reverse the hyperalgesic priming induced by sequential injection of carrageenan/PGE and down-regulate the protein expression of mGluR5 and PKCε. Glutamate injection instead of PGE2 can mimic the hyperalgesic priming model. Pharmacological blocking of mGluR5 with specific antagonist MTEP can prevent the hyperalgesic priming and inhibit the activation of PKCε in DRGs. Furthermore, EA also produced analgesic effect on the hyperalgesic priming rats induced by carrageenan/mGluR5 injection and inhibited the high expression of PKCε. Sham EA produced none analgesic and regulatory effect. CONCLUSION: EA can regulate pain transition and it may relate with its inhibitory effect on the activation of mGluR5-PKCε signaling pathway in the DRGs.

[Early intervention of bone-nearby acupuncture combined with electroacupuncture on morphine tolerance in bone cancer pain rats and its effect on the expression of HDAC and MOR in dorsal root ganglia].

OBJECTIVE: To observe the effect of early intervention of bone-nearby acupuncture (BNA) combined with electroacupuncture (EA) on the expression of histone deacetylase1(HDAC1), histone deacetylase 2 (HDAC2) andµ-opioid recepter (MOR) in dorsal root ganglia (DRG) of bone cancer pain-morphine tolerance (BCP-MT) rats, and to explore its possible mechanism. METHODS: A total of 35 SD rats were randomized into a sham BCP group (n=6), a BCP group (n=7), a MT group (n=7), a BNA+EA group (n=8) and a shame BNA group (n=7). Except of the sham BCP group, cancer cell inoculation operation at left tibia was given in the other 4 groups to establish the bone cancer pain model. In the MT group, the BNA+EA group and the shame BNA group, intraperitoneal injection of morphine hydrochloride was given to establish the morphine tolerance model. After the operation, bone-nearby acupuncture combined with electroacupuncture was applied at "Zusanli" (ST 36) and "Kunlun" (BL 60) in the BNA+EA group, with dilatational wave, 2 Hz/100 Hz in frequency, 0.5 to 1.5 mA in intensity. Intervention in the shame BNA group was applied at the same time and acupoints as those in the BNA+EA group, the needles were pierced the skin without any electrical stimulation. The needles were retained for 30 min, once a day for continuous 7 days in both BNA+EA and shame BNA groups. Before and 10, 11, 15, 22 days after the operation, the left paw withdrawal threshold (PWT) was measured in the 5 groups. The levels of HDAC1, HDAC2 and MOR in DRG were detected by Western blot. RESULTS: Ten days after the cancer cell inoculation operation, the PWT of the BCP, MT, BNA+EA and sham BNA groups was decreased compared with the sham BCP group (P<0.01). Eleven days after the operation, the PWT of the MT, BNA+EA and sham BNA groups was increased compared with the BCP group (P<0.01). Twenty-two days after the operation, the difference was no significant between the BCP group and MT group (P>0.05); the PWT of the BNA+EA group was increased compared with the MT and sham BNA group (P<0.01). In the BCP group, the DRG levels of HDAC1 and HDCA2 were increased, while the level of MOR was decreased compared with the sham BCP group (P<0.05, P<0.01). In the MT group, the DRG level of HDAC1 was increased compared with the BCP group (P<0.05). In the BNA+EA group, the DRG level of HDAC1 was decreased compared with the MT group and the sham BNA group (P<0.01, P<0.05), while the level of MOR was increased (P<0.01). CONCLUSION: Early intervention of bone-nearby acupuncture combined with electroacupuncture can relieve the morphine tolerance in bone cancer pain rats, it may relate to down-regulating the expression of HDAC1 and up-regulating the expression of MOR in the dorsal root ganglia.

Electroacupuncture Alleviates Pain-Related Emotion by Upregulating the Expression of NPS and Its Receptor NPSR in the Anterior Cingulate Cortex and Hypothalamus.

OBJECTIVE: Electroacupuncture (EA) is reported effective in alleviating pain-related emotion; however, the underlying mechanism of its effects still needs to be elucidated. The NPS-NPSR system has been validated for the involvement in the modulation of analgesia and emotional behavior. Here, we aimed to investigate the role of the NPS-NPSR system in the anterior cingulate cortex (ACC), hypothalamus, and central amygdala (CeA) in the use of EA to relieve affective pain modeled by complete Freund's adjuvant- (CFA-) evoked conditioned place aversion (C-CPA). Materials and Methods. CFA injection combined with a CPA paradigm was introduced to establish the C-CPA model, and the elevated O-maze (EOM) was used to test the behavioral changes after model establishment. We further explored the expression of NPS and NPSR at the protein and gene levels in the brain regions of interest by immunofluorescence staining and quantitative real-time PCR. RESULTS: We observed that EA stimulation delivered to the bilateral Zusanli (ST36) and Kunlun (BL60) acupoints remarkably inhibited sensory pain, pain-evoked place aversion, and anxiety-like behavior. The current study showed that EA significantly enhanced the protein expression of this peptide system in the ACC and hypothalamus, while the elevated expression of NPSR protein alone was just confined to the affected side in the CeA. Moreover, EA remarkably upregulated the mRNA expression of NPS in CeA, ACC, and hypothalamus and NPSR mRNA in the hypothalamus and CeA. CONCLUSIONS: These data suggest the effectiveness of EA in alleviating affective pain, and these benefits may at least partially be attributable to the upregulation of the NPS-NPSR system in the ACC and hypothalamus.

[Effect of electroacupuncture on expression of GABAA receptor mRNA in different brain regions in rats with chronic inflammatory pain].

OBJECTIVE: To observe the expression of GABAA receptor mRNA in different brain regions of the central nervous system in chronic inflammatory pain rats and the intervention effect of electroacupuncture (EA). METHODS: A total of 48 SPF male SD rats were randomly divided into a blank control group, a model control group, an EA group and a sham EA group, 12 rats in each group. The model of chronic inflammatory pain was established by injecting Freund's complete adjuvant into the foot. The EA group was treated with EA 28 days after the model establishment. The "Housanli" (ST 36) and "Kunlun" (BL 60) were selected and treated with dilatational wave, 2 Hz/100 Hz in frequency, 0.5-1.5 mA for 30 min; EA was given only once. In the sham EA group, the same acupoints were selected but the needles were only inserted into subcutaneous area; EA was connected for 30 min without electrical stimulation. The behavior changes of mechanical pain threshold and thermal pain threshold before model establishment, 1 day, 3 days, 7 days, 14 days, 21 days and 28 days after the model establishment as well as emotional behavior 29 days after the model establishment were observed; the relative expressions of GABAA receptor mRNA in anterior cingulate cortex, amygdala and hypothalamus were observed. RESULTS: Compared with the blank control group, the change rates of mechanical pain threshold and thermal pain threshold in the model control group were decreased significantly 1 day, 3 days, 7 days, 14 days, 21 days, 28 days after model establishment (P<0.01); 29 days after model establishment, the movement distance and staying time in the central area of open field test in the model control group were decreased significantly (P<0.05). After EA intervention, compared with the model control group and the sham EA group, the change rates of mechanical pain threshold and thermal pain threshold, as well as the movement distance and the staying time of central area were significantly increased in the EA group (P<0.01, P<0.05). Twenty-nine days after model establishment, the expression of GABAA receptor mRNA in anterior cingulate cortex and hypothalamus was not significantly different among all groups (P>0.05). Compared with the blank control group, the expression of GABAA receptor mRNA in the amygdala was decreased significantly in the model control group (P<0.01); compared with the model control group and the sham EA group, the expression of GABAA receptor mRNA in amygdala was increased after intervention in the EA group (P<0.01). CONCLUSION: Single treatment of EA could significantly increase the mechanical pain threshold and thermal pain threshold, improve abnormal emotional behavior in rats with chronic inflammatory pain, which may be related to the increasing of expression of GABAA receptor mRNA in the amygdala.

Electroacupuncture suppresses the pain and pain-related anxiety of chronic inflammation in rats by increasing the expression of the NPS/NPSR system in the ACC.

BACKGROUND: The neuropeptide S/Neuropeptide S receptor (NPS/NPSR) system is involved in the regulation of anxiety in rodents. Chronic inflammation can induce anxiety. Our lab has observed that electroacupuncture (EA) has a beneficial effect on chronic inflammatory pain and pain-related anxiety; however, the mechanism should be further clarified. In the present study, we used an inflammatory pain model to investigate the role of the NPS/NPSR system in the anterior cingulate cortex (ACC) in the analgesic and antianxiety effects of EA. RESULTS: In an inflammatory pain model, the paw withdrawal thresholds (PWTs) were decreased, pain-related anxiety-like behaviors were induced, and the ipsilateral protein expression of NPS and NPSR was decreased in the ACC. EA stimulation increased the PWTs, reduced pain-related anxiety-like behavior, and enhanced the ipsilateral protein expression of NPS and NPSR in the ACC. NPS microinjection increased the PWTs and decreased pain-related anxiety-like behaviors. Furthermore, an NPSR inhibitor combined with EA reversed the effect of EA on the PWTs and pain-related anxiety-like behaviors. CONCLUSIONS: Our results suggest that EA suppresses pain and pain-related anxiety-like behavior of chronic inflammation in rats by increasing the expression of the NPS/NPSR system in the ACC.