Jun-Fan Fang

Intervention of electroacupuncture on spinal p38 MAPK/ATF-2/VR-1 pathway in treating inflammatory pain induced by CFA in rats

BackgroundPrevious studies have demonstrated that p38 MAPK signal transduction pathway plays an important role in the development and maintenance of inflammatory pain. Electroacupuncture (EA) can suppress the inflammatory pain. However, the relationship between EA effect and p38 MAPK signal transduction pathway in inflammatory pain remains poorly understood. It is our hypothesis that p38 MAPK/ATF-2/VR-1 and/or p38 MAPK/ATF-2/COX-2 signal transduction pathway should be activated by inflammatory pain in CFA-injected model. Meanwhile, EA may inhibit the activation of p38 MAPK signal transduction pathway. The present study aims to investigate that anti-inflammatory and analgesic effect of EA and its intervention on the p38 MAPK signal transduction pathway in a rat model of inflammatory pain.ResultsEA had a pronounced anti-inflammatory and analgesic effect on CFA-induced chronic inflammatory pain in rats. EA could quickly raise CFA-rat’s paw withdrawal thresholds (PWTs) and maintain good and long analgesic effect, while it subdued the ankle swelling of CFA rats only at postinjection day 14. EA could down-regulate the protein expressions of p-p38 MAPK and p-ATF-2, reduced the numbers of p-p38 MAPK-IR cells and p-ATF-2-IR cells in spinal dorsal horn in CFA rats, inhibited the expressions of both protein and mRNA of VR-1, but had no effect on the COX-2 mRNA expression.ConclusionsThe present study indicates that inhibiting the activation of spinal p38 MAPK/ATF-2/VR-1 pathway may be one of the main mechanisms via central signal transduction pathway in the process of anti-inflammatory pain by EA in CFA rats.

Effect of Electroacupuncture on Activation of p38MAPK in Spinal Dorsal Horn in Rats with Complete Freund's Adjuvant-Induced Inflammatory Pain

Activation of mitogen-activated protein kinases (MAPKs), especially p38 MAPK, plays an important role in the development of central sensitization related to persistent inflammatory pain. Electroacupuncture (EA) is well known to relieve persistent inflammatory pain. However, little is known about relationship between EA and p38 MAPK. Inflammatory pain rat model was induced by intraplantar injection of complete Freunds adjuvant (CFA). Male adult SD rats were randomly divided into the saline group, CFA group, and CFA + EA group. EA (constant saquare wave, 2 Hz and 100 Hz alternating frequencies, intensities ranging from 1 to 2 mA) was applied to bilateral “Zusanli” (ST 36) and “Kunlun” acupoints (BL 60) for 30 min, once per day. The paw edema and paw withdrawal threshold (PWT) were measured at preinjection and days postinjection 1, 3, and 14. Spinal p-p38MAPK- immunoreactivty (p-p38MAPK-IR) cells were detected by immunohistochemistry at postinjection day 3 and 14. EA significantly inhibited paw edema at postinjection days 14 and increased PWT at postinjection days 3 and 14. Moreover, the increasing number of spinal p-p38MAPK-IR cells which was induced by CFA injection was suppressed by EA stimulation. These results indicate that anti-inflammatory and analgesic effect of EA might be associated with its inhibition of spinal p38 MAPK activation and thereby provide a potential mechanism for the treatment of inflammatory pain by EA.

Electroacupuncture attenuates mechanical allodynia by suppressing the spinal JNK1/2 pathway in a rat model of inflammatory pain

BACKGROUND Electroacupuncture (EA) has a substantial analgesic effect on inflammatory pain induced by complete Freunds adjuvant (CFA). The activation of the c-Jun N-terminal kinase 1/2 (JNK1/2) signal transduction pathway in the spinal cord is associated with inflammatory pain. However, the relationship between EAs analgesic effect and the JNK1/2 signal transduction pathway in the inflammatory pain remain unclear. In the present study, we used the established rat model of CFA-induced inflammatory pain to investigate the role of the spinal JNK1/2 pathway in EA-mediated analgesia. RESULTS We observed a decrease in paw withdrawal thresholds and an increase in paw edema at 1 and 3 days after injecting CFA into the right hindpaw. CFA, 3 days after injection, upregulated expression of phospho-c-Jun N-terminal kinase1/2 (p-JNK1/2) protein and its downstream targets, the transcriptional regulators p-c-Jun and activator protein-1 (AP-1), as well as cyclooxygenase-2 (COX-2) and the transient receptor potential vanilloid 1 (TRPV1). EA significantly alleviated CFA-induced inflammatory pain. In addition, EA reduced p-JNK1/2 protein levels and COX-2 mRNA expressions, a degree of down-regulated p-c-Jun protein level and AP-1 DNA binding activity in the spinal dorsal horn of CFA-administered animals, but it had no effect on TRPV1 mRNA expression. Furthermore, EA and the JNK inhibitor SP600125 synergistically inhibited CFA-induced hyperalgesia and suppressed the COX-2 mRNA expression in the spinal dorsal horn. CONCLUSIONS Our findings indicate that EA alleviates inflammatory pain behavior, at least in part, by reducing COX-2 expression in the spinal cord via the JNK1/2 signaling pathway. Inactivation of the spinal JNK1/2 signal transduction pathway maybe the potential mechanism of EAs antinociception in the inflammatory pain model.

Inhibition of spinal microglia and astrocytes contributes to the anti-allodynic effect of electroacupuncture in neuropathic pain induced by spinal nerve ligation

Objective Besides neurons, activated microglia and astrocytes in the spinal cord dorsal horn (SCDH) contribute to the pathogenesis of chronic pain. Electroacupuncture (EA) has been used widely to treat various chronic pain diseases, however, the underlying mechanisms of EA are still not fully understood. Methods Male Sprague-Dawley rats were randomly divided into four groups, including an untreated healthy Control group (n=14), a True-spinal nerve ligation (SNL) group that underwent SNL and remained untreated (n=25), a True-SNL+EA group that underwent SNL followed by EA treatment (n=25), and a Sham-SNL group that underwent sham surgery and remained untreated (n=15). SNL was performed unilaterally at L5 and EA was applied to ST36 and BL60 bilaterally once per day. Paw withdrawal thresholds (PWTs) were measured ipsilaterally at baseline and 1, 3, 7, and 14 days after ligation. Activation of microglia and astrocytes in the SCDH were examined bilaterally by immunofluorescence staining, and concentrations of interleukin-1β (IL-1β) and interleukin (IL-6) were measured in the ipsilateral SCDH by ELISA. Results SNL significantly decreased PWTs and activated glial cells in the superficial laminae of the ipsilateral SCDH. In rats with SNL, glial fibrillary acidic protein (GFAP) immunoreactivity peaked at 7 days and was maintained until 14 days post-ligation, while anti-integrin alphaM (OX-42) immunoreactivity peaked at 3 days and declined gradually. EA significantly alleviated SNL-induced mechanical allodynia. Furthermore, EA reduced microglial activation (OX-42 positive ratios) in the lumbar SCDH at 3 days post-ligation and suppressed astrocyte activation (GFAP positive ratios) at all time points observed. Conclusions EA stimulation alleviates SNL-induced neuropathic pain, at least in part through inhibition of spinal glial activation. Moreover, inhibition of spinal microglia and astrocyte activation may contribute to the immediate effects and maintenance of EA analgesia, respectively.

Electroacupuncture attenuates spinal nerve ligation-induced microglial activation mediated by p38 mitogen-activated protein kinase

ObjectiveTo investigate whether analgesic effect of electroacupuncture (EA) is affected by p38 mitogen-activated protein kinase (p38 MAPK) on microglia.MethodsThere were two experiments. The experiment 1: 40 male Sprague-Dawley (SD) rats were randomly divided into the normal, surgery, EA and sham EA groups, and the L5 spinal nerve ligation (SNL) on the right side was used to establish neuropathic pain model. EA was applied to bilateral Zusanli (ST36) and Kunlun (BL60) at 24, 48 and 72 h after SNL for 30 min, once per day. The paw withdrawal thresholds (PWTs) were measured before surgery (as base) and at 24, 25, 49 and 73 h after surgery. Phospho-p38 MAPK (p-p38 MAPK), oxycocin-42 (OX-42, marker of microglia), and glial fibrillary acidic protein (GFAP, marker of astrocyte) in bilateral spinal cord dorsal horn (SCDH) were detected by immunofluorescence, respectively. The experiment 2: 40 male SD rats were cannulated for SNL-induced neuropathic pain, and then were randomly divided into the dimethyl sulfoxide (DMSO), EA plus DMSO, 4-(4-fluorophenyl)-2-(4-methylsulfonylpheny)-5-(4-pyridyl)-1H-imidazole (SB203580) and EA plus SB203580 groups. SB203580 (30 nmol/L) was administered 5 min prior to EA treatment. The PWTs and OX-42 in bilateral SCDH were measured as mentioned above.ResultsSNL-induced neuropathic pain reduced PWTs and increased the expression of p-p38 MAPK and OX-42 in bilateral lumbar SCDH of rats (P<0.01). Spinal p-p38 MAPK was only co-localized with OX-42 in our study. EA treatment significantly alleviated SNL-mediated mechanical hyperalgesia, and suppressed the expression of p-p38 MAPK and OX-42 in lumbar SCDH (P<0.05 or P<0.01). Intrathecal injection of low dose SB203580 had no influence on PWTs (P>0.05), but significantly inhibited the expression of OX-42 positive cells in bilateral SCDH (P<0.01 or P<0.05). EA plus SB203580 synergistically increased PWTs, and reduced the expression of bilateral spinal OX-42 (P<0.01 or P<0.05).ConclusionsThe central mechanism of EA-induced anti-hyperalgesia may be partially associated with the reduced expression of p-p38 MAPK, and subsequently reducing the activation of OX-42 in neuropathic pain. Therefore, EA may be a new complementary and alternative therapy for neuropathic pain.

Electroacupuncture treatment partly promotes the recovery time of postoperative ileus by activating the vagus nerve but not regulating local inflammation.

Postoperative ileus (POI) after abdominal surgery significantly lowers the life quality of patients and increase hospital costs. However, few treatment strategies have successfully shortened the duration of POI. Electroacupuncture (EA) is a modern way of administering acupuncture and widely used in various gastrointestinal (GI) diseases in the world. Here, we studied the effect of EA on POI and its underlying mechanisms. Intestinal manipulation resulted in significant delays of GI transit, colonic transit and gastric emptying. Surgery also up-regulated c-fos in nucleus of the solitary tract (NTS) and induced inflammation response in the small intestine. Further, operation and inhale anesthesia inhibited NTS neuron excitation duration for the whole observation time. EA administered at ST36 indeed shortened the recovery time of GI and colonic transit, and significantly increased the gastric emptying. EA also significantly activated the NTS neurons after operation. However, there was no anti-inflammation effect of EA during the whole experiment. Finally, atropine blocked the regulatory effect of EA on GI function, when it was injected after surgery, but not before surgery. Thus, the regulatory effect of EA on POI was mainly mediated by exciting NTS neurons to improve the GI tract transit function but not by activating cholinergic anti-inflammatory pathway.

Strong Manual Acupuncture Stimulation of “Huantiao” (GB 30) Reduces Pain-Induced Anxiety and p-ERK in the Anterior Cingulate Cortex in a Rat Model of Neuropathic Pain

Persistent neuropathic pain is associated with anxiety. The phosphorylation of extracellular signal-regulated kinase (p-ERK) in the anterior cingulate cortex (ACC) plays an important role in pain-induced anxiety. Acupuncture is widely used for pain and anxiety. However, little is known about which acupuncture technique is optimal on pain-induced anxiety and the relationship between acupuncture effect and p-ERK. The rat model was induced by L5 spinal nerve ligation (SNL). Male adult SD rats were randomly divided into control, SNL, strong manual acupuncture (sMA), mild manual acupuncture (mMA), and electroacupuncture (EA) group. Bilateral “Huantiao” (GB 30) were stimulated by sMA, mMA, and EA, respectively. The pain withdrawal thresholds (PWTs) and anxiety behavior were measured, and p-ERK protein expression and immunoreactivity cells in ACC were detected. PWTs increased significantly in both sMA and EA groups. Meanwhile, anxiety-like behavior was improved significantly in the sMA and mMA groups. Furthermore, the overexpression of p-ERK induced by SNL was downregulated by strong and mild manual acupuncture. Therefore, strong manual acupuncture on bilateral “Huantiao” (GB 30) could be a proper therapy relieving both pain and pain-induced anxiety. The effect of different acupuncture techniques on pain-induced anxiety may arise from the regulation of p-ERK in ACC.

The Effect of Electroacupuncture on PKMzeta in the ACC in Regulating Anxiety-Like Behaviors in Rats Experiencing Chronic Inflammatory Pain

Chronic inflammatory pain can induce emotional diseases. Electroacupuncture (EA) has effects on chronic pain and pain-related anxiety. Protein kinase Mzeta (PKMzeta) has been proposed to be essential for the maintenance of pain and may interact with GluR1 to maintain CNS plasticity in the anterior cingulate cortex (ACC). We hypothesized that the PKMzeta-GluR1 pathway in the ACC may be involved in anxiety-like behaviors of chronic inflammatory pain and that the mechanism of EA regulation of pain emotion may involve the PKMzeta pathway in the ACC. Our results showed that chronic inflammatory pain model decreased the paw withdrawal threshold (PWT) and increased anxiety-like behaviors. The protein expression of PKCzeta, p-PKCzeta (T560), PKMzeta, p-PKMzeta (T560), and GluR1 in the ACC of the model group were remarkably enhanced. EA increased PWT and alleviated anxiety-like behaviors. EA significantly inhibited the protein expression of p-PKMzeta (T560) in the ACC, and only a downward trend effect for other substances. Further, the microinjection of ZIP remarkably reversed PWT and anxiety-like behaviors. The present study provides direct evidence that the PKCzeta/PKMzeta-GluR1 pathway is related to pain and pain-induced anxiety-like behaviors. EA treatment both increases pain-related somatosensory behavior and decreases pain-induced anxiety-like behaviors by suppressing PKMzeta activity in the ACC.

Alleviating Mechanical Allodynia and Modulating Cellular Immunity Contribute to Electroacupuncture’s Dual Effect on Bone Cancer Pain

Hypothesis: Electroacupuncture (EA) has been used as an alternative analgesic therapy for hundreds of years, yet its analgesic potency and therapeutic advantage against bone cancer pain (BCP) in comparison with morphine remains unclear. This study aimed to investigate the effects of EA on mechanical allodynia and cellular immunity of BCP rats, and to further explore the potential mechanism. Methods: The BCP model was established by implanting Walker 256 mammary gland carcinoma cells into the left tibia of adult female Sprague-Dawley rats. EA (dilatational wave, 2/100 Hz, 0.5 mA–1mA–1.5 mA for 10 minutes each intensity) was applied bilaterally to Zusanli (ST 36) and Kunlun (BL 60) for 30 minutes. Both EA stimulation and morphine (10 mg/kg, intraperitoneally) was given once every other day. Naloxone (0.3 mg/kg, intraperitoneally) was injected at 30 minutes prior to EA. Mechanical allodynia were demonstrated by paw withdrawal thresholds (PWTs) which measured by dynamic plantar aesthesiometer. T cell proliferation, percentage of CD3+, CD4+ and CD8+ T lymphocytes in spleen as well as expression of interleukin-2 (IL-2) in plasma were detected by WST-8, flow cytometry, and enzyme-linked immunosorbent assay technique, respectively. Results: An intratibial inoculation of Walker 256 mammary gland carcinoma cells significantly decreased PWTs to mechanical stimuli. EA stimulation alleviated mechanical allodynia in BCP rats, and the analgesic potency of EA was weaker than that of morphine. In contrast to morphine, EA stimulation of BCP rats increased splenic concanavalin A (Con A)-induced T cell proliferation and plasma IL-2 content, as well as increased the percentages of splenic CD3+CD4+ and CD3+CD8+ T cell subsets. Moreover, both the analgesic effect and the partial immunomodulation of EA were suppressed by an intraperitoneal injection of naloxone. Conclusion: EA could significantly alleviate BCP-induced mechanical allodynia. Although the analgesic effect of EA was weaker than that of morphine, EA had an immunomodulation effect on cellular immunity. Both analgesic and immunomodulatory effect of EA might share the same mechanism via the opioid-mediated pathway, which needs further investigation.