Electroacupuncture suppresses spinal nerve ligation-induced neuropathic pain via regulation of synaptic plasticity through upregulation of basic fibroblast growth factor expression.

BACKGROUND: Improving synaptic plasticity is a good way to alleviate neuropathic pain. Electroacupuncture (EA) is currently used worldwide to treat this disease, but its specific mechanisms of action need further investigation. Evidence has suggested that basic fibroblast growth factor (bFGF) plays an important role in promoting nerve regeneration and can promote the expression of vascular endothelial growth factor (VEGF). OBJECTIVE: In this study, we examined the effects of EA on synaptic plasticity and its underlying mechanism. METHODS: A spinal nerve ligation (SNL) rat model was established. NSC37204 (a specific inhibitor of bFGF) was used to determine the relationship between bFGF and putative EA-mediated improvements in synaptic plasticity. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were assessed to evaluate hyperalgesia in rats with SNL. Tissue morphology was detected by hematoxylin-eosin (HE) and Nissl staining, while neural plasticity and its molecular mechanisms were examined by Western blotting, quantitative real-time polymerase chain reaction (qPCR), dual-label immunohistochemistry and transmission electron microscopy. RESULTS: We found that EA improved synaptic plasticity, consistent with higher levels of expression of bFGF and VEGF. Contrary to the beneficial effects of EA, NSC37204 promoted synaptic reconstruction. Furthermore, EA-induced improvements in the neurobehavioral state and improved synaptic plasticity were blocked by NSC37204, consistent with lower expression levels of bFGF and VEGF. CONCLUSION: These findings indicate that EA suppresses SNL-induced neuropathic pain by improving synaptic plasticity via upregulation of bFGF expression.

[Effect of electroacupuncture on angiopoietin-1 in spinal cord of rats with neuropathic pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the behavior, histomorphology and the expression of angiopoietin-1 (Angpt-1) in rats with spinal nerve injury, so as to explore its mechanism on neuropathic pain. METHODS: Forty-five male SD rats were randomly divided into sham, model and EA groups (n=15 rats in each group). Spinal nerve ligation (SNL) of the L5 lumbar vertebra was performed to establish a rat model of neuropathic pain. The rats in the EA group were given EA at "Zusanli" (ST36) and "Kunlun" (BL60) of the operation side with continuous wave at a frequency of 2 Hz and an intensity of 1.5 mA once a day, 30 minutes each time for 7 days. The sham group only exposed L5 spinal nerves without ligation. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were observed and recorded before modeling and on days 3,5,7,10,12 and 14 after modeling. L4-L6 segments of spinal cord were taken and the morphological changes of spinal dorsal horn were observed by HE staining. The changes of spinal dorsal horn nerve fiber structure were observed by silver plating staining. Angpt-1 expression was detected by Western blot and immunohistochemistry. RESULTS: Compared with the sham group, the model group had significant reductions in MWT and TWL at each time point (P<0.01); compared with the model group, the EA group had significant increases in MWT and TWL on days 10,12 and 14 after intervention (P<0.05, P<0.01). HE staining showed that in the model group, the spinal dorsal horn showed degeneration and necrosis of neurons, nuclear fixation and shrinkage, and loose surrounding tissues. The degree of tissue damage of the EA group was milder than that of the model group. The silver staining results showed the model group had obvious neuronal fibrillary tangles, while there were fewer neuronal fibrillary tangles in the EA group. Compared with the sham group, the Angpt-1 expression in the model group was significantly decreased (P<0.01), and compared with the model group, the EA group had a significant increase in the expression of Angpt-1 (P<0.01). CONCLUSION: EA can promote the recovery of nerve function in SNL rats by up-regulating Angpt-1 expression.

Electroacupuncture improves neuronal plasticity through the A2AR/cAMP/PKA signaling pathway in SNL rats.

Improvements in neuronal plasticity are considered to be conducive to recovery from neuropathic pain. Electroacupuncture (EA) is regarded as an effective rehabilitation method for neuropathic pain. However, the effects and potential mechanism associated with EA-induced repair of hyperesthesia are not fully understood. Evidence has suggested that the adenosine A2A receptor (A2AR) and the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway play an important role in improving neuropathic pain. Here, we examined the function of EA in promoting neuronal plasticity in spinal nerve ligation (SNL) rats. The A2AR antagonist SCH58261, A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-50-N-ethylcarboxamido adenosine HCl (CGS21680) and A2AR siRNA were used to confirm the relationship between A2AR and the cAMP/PKA pathway as well as the effects of A2AR on EA-induced improvements in neurobehavioral state and neuronal plasticity. Mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), HE staining, Western blotting, RT-PCR, immunofluorescence, enzyme-linked immunosorbent assay, Nissl staining, silver staining, Golgi-Cox staining and transmission electron microscopy were used to evaluate the changes in neurobehavioral performance, protein expression, neuronal structure and dendrites/synapses. The results showed that EA and CGS21680 improved the behavioral performance, neuronal structure and dendritic/synaptic morphology of SNL rats, consistent with higher expression levels of A2AR, cAMP and PKA. In contrast to the positive effects of EA, SCH58261 inhibited dendritic growth and promoted dendritic spine/synaptic remodeling. In addition, the EA-induced improvement in neuronal plasticity was inhibited by SCH58261 and A2AR siRNA, consistent with lower expression levels of A2AR, cAMP and PKA, and worse behavioral performance. These results indicate that EA suppresses SNL-induced neuropathic pain by improving neuronal plasticity via upregulating the A2AR/cAMP/PKA signaling pathway.

Electroacupuncture may alleviate neuropathic pain via suppressing P2X7R expression.

Neuropathic pain is a severe problem that is difficult to treat clinically. Reducing abnormal remodeling of dendritic spines/synapses and increasing the anti-inflammatory effects in the spinal cord dorsal horn are potential methods to treat this disease. Previous studies have reported that electroacupuncture (EA) could increase the pain threshold after peripheral nerve injury. However, the underlying mechanism is unclear. P2X7 receptors (P2X7R) mediate the activation of microglia and participate in the occurrence and development of neuropathic pain. We hypothesized that the effects of EA on relieving pain may be related to the downregulation of the P2X7R. Spinal nerve ligation (SNL) rats were used as a model in this experiment, and 2'(3')-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a P2X7R agonist. We found that EA treatment decreased dendritic spine density, inhibited synaptic reconstruction and reduced inflammatory response, which is consistent with the decrease in P2X7R expression as well as the improved neurobehavioral performance. In contrast to the beneficial effects of EA, BzATP enhanced abnormal remodeling of dendritic spines/synapses and inflammation. Furthermore, the EA-mediated positive effects were reversed by BzATP, which is consistent with the increased P2X7R expression. These findings indicated that EA improves neuropathic pain by reducing abnormal dendritic spine/synaptic reconstruction and inflammation via suppressing P2X7R expression.

Electroacupuncture effects on the P2X4R pathway in microglia regulating the excitability of neurons in the substantia gelatinosa region of rats with spinal nerve ligation.

Electroacupuncture (EA) has been used to treat neuropathic pain induced by peripheral nerve injury (PNI) by applying an electrical current to acupoints with acupuncture needles. However, the mechanisms by which EA treats pain remain indistinct. High P2X4 receptor (P2X4R) expression levels demonstrate a notable increase in hyperactive microglia in the ipsilateral spinal dorsal horn following PNI. In order to demonstrate the possibility that EA analgesia is mediated in part by P2X4R in hyperactive microglia, the present study performed mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests in male Sprague­Dawley rats that had undergone spinal nerve ligation (SNL). The expression levels of spinal P2X4R were determined using reverse transcription­quantitative PCR, western blotting analysis and immunofluorescence staining. Furthermore, spontaneous excitatory postsynaptic currents (sEPSCs) were recorded using whole­cell patch clamp to demonstrate the effect of EA on synaptic transmission in rat spinal substantia gelatinosa (SG) neurons. The results of the present study demonstrated that EA increased the MWT and TWL and decreased overexpression of P2X4R in hyperactive microglia in SNL rats. Moreover, EA attenuated the frequency of sEPSCs in SG neurons in SNL rats. The results of the present study indicate that EA may mediate P2X4R in hyperactive spinal microglia to inhibit nociceptive transmission of SG neurons, thus relieving pain in SNL rats.

[Effect of electroacupuncture on the morphological changes of the spinal dorsal horn and the expression of p38 mitogen-activated protein kinase in the injured spinal cord of rats with neuropathic pain].

OBJECTIVE: To investigate the effect of electroacupuncture stimulation at "Zusanli"(ST36) and "Kunlun"(BL60) on the morphological changes of the spinal dorsal horn and the expression of p38 mitogen-activated protein kinase (p38MAPK) in the injured spinal cord of rats with neuropathic pain. METHODS: Male Sprague-Dawley rats were randomly divided into sham model group, model group, electroacupuncture group, and medication group, with 10 rats in each group. Spinal nerve ligation of the L5 lumbar vertebra was performed to establish a rat model of neuropathic pain. The rats in the electroacupuncture group were given electroacupuncture at ST36 and BL60 of the operation side with dilatational wave at a frequency of 2 Hz/100 Hz and an intensity of 1.5 mA once a day, 30 minutes each time, and those in the medication group were given intraperitoneal injection of 100 mg/mL Gabapentin solution (100 mg/kg) once a day; the one-week intervention was started at one week after surgery. Mechanical withdraw threshold (MWT) and thermal withdrawal latency (TWL) were observed and recorded before modeling and on days 1,3,5,7,10,12 and 14 after modeling, and the motor function of the affected hindlimb was scored. Methenamine silver stain was used to observe the morphological changes of the spinal dorsal horn, and Western blot was used to measure the relative protein expression of p38MAPK and phospho-p38MAPK(p-p38MAPK) in L4-L6 spinal segments. RESULTS: Compared with the sham model group, the model group had significant reductions in MWT and TWL at each time point (P<0.001) and a significant increase in motor function score (P<0.001); compared with the model group, the electroacupuncture group and the medication group had significant increases in MWT and TWL and a significant reduction in motor function score after treatment (P<0.05). The model group had obvious neuronal fibrillary tangles, particle vacuolar degeneration, and vacuoles containing argyrophilic grains in the cytoplasm of neural cells under a light microscope, while there were fewer neuronal fibrillary tangles in the electroacupuncture group and reduced vacuolar degeneration in the medication group. Compared with the sham model group, the model group had significant increases in the protein expression of p-p38MAPK in the spinal dorsal horn (P<0.001), and compared with the model group, the electroacupuncture group and the medication group had significant reductions in the protein expression of p-p38MAPK in the spinal dorsal horn(P<0.05). CONCLUSION: Electroacupuncture stimulation at ST36 and BL60 can increase pain threshold, improve the motor function of the affected hindlimb, and improve the necrosis of neurofibrils in the spinal dorsal horn in rats with neuropathic pain, possibly by regulating the expression of p-p38MAPK in the spinal dorsal horn.

Effect of electroacupuncture on the expression of P2 × 4, GABAA γ 2 and long-term potentiation in spinal cord of rats with neuropathic pain.

OBJECTIVE: To observe the impacts of electroacupuncture (EA) stimulation at "Zusanli and Kunlun Points" on spinal dorsal horn microglia activation in L5 spinal nerve ligation (SNL) rats and BNDF, P2 × 4 and GABAAγ2, and the changes in spinal dorsal horn synaptic plasticity in model rats. METHODS: Adult male SD rats (180-220 g) were selected and randomly divided into 6 groups, including the sham group, the SNL group, the SNL + EA group, the SNL+5-BDBD group, the SNL + EA + 5-BDBD group and the SNL + FEA group. The changes in the Iba-1, BDNF, P2 × 4 and GABAAγ2 in the spinal cord of rats were observed by Western blotting, immunofluorescence, RT-PCR and other techniques; the long-term changes in the potential after the excitatory synapse of the spinal dorsal horn in rats were observed by in vivo electrophysiological technique. RESULTS: After 7 days of intervention, the fluorescence intensity (FI) of P2 × 4 and Iba-1 in the SNL + EA group was lower than that in the SNL group and higher than that in the sham group(P < 0.01), but the FI of GABAAγ2 was higher than that in the SNL group(P < 0.01); the expression of Iba-1, BDNF and P2 × 4 proteins in the SNL + EA group, the SNL+5-BDBD group and the SNL + EA + 5-BDBD group was significantly lower than that in the SNL + FEA group(P < 0.05), but the expression of GABAAγ2 protein was higher (P < 0.05); after treatment with EA, the expression levels of Iba-1 mRNA and P2 × 4 mRNA in the SNL + EA group were lower than those in the SNL group(P < 0.01), but the expression levels of GABAAγ2 mRNA were higher (P < 0.01). Meanwhile, long-term potentiation changes could not be induced in the SNL + EA group. CONCLUSION: The EA stimulation at "Zusanli" and "Kunlun" points can improve the pain threshold of rats with neuropathic pain (NP), inhibit the excitatory postsynaptic potential (EPSP), and weaken the excitatory transmission efficiency between synapses during NP.

Atractylenolide III ameliorates cerebral ischemic injury and neuroinflammation associated with inhibiting JAK2/STAT3/Drp1-dependent mitochondrial fission in microglia.

BACKGROUND: Inflammation is a major contributor to stroke pathology, making it a promising strategy for intervention. Microglia, the resident macrophages in the brain, play essential roles in both the generation and resolution of neuroinflammation. In particular, mitochondrial homeostasis is critical for microglial function and its dysregulation is involved in the pathogenesis of ischemic stroke. Atractylenolide III (A III), a sesquiterpene lactone found in Atractylodes macrocephala Koidz, has been shown to have an inhibitory effect on inflammation. However, its effect specifically on neuroinflammation and microglial mitochondrial homeostasis following stroke remains elusive. HYPOTHESIS: We hypothesized that A III protects against brain ischemia through inhibition of neuroinflammation mediated by JAK2/STAT3/Drp1-dependent mitochondrial fission. METHODS: The neuroprotective and anti-neuroinflammatory effects of A III were investigated in vivo in mice with transient occlusion to the middle cerebral artery (MCAO) and in vitro in oxygen glucose deprivation-reoxygenation (OGDR)-stimulated primary microglia from mice. RESULTS: A III and AG490, an inhibitor of JAK2, treatment reduced brain infarct size, restored cerebral blood flow (CBF), ameliorated brain edema and improved neurological deficits in MCAO mice. Furthermore, A III and AG490 inhibited mRNA and protein expressions of proinflammatory (IL-1ß, TNF-α, and IL-6) and anti-inflammatory cytokines in both MCAO mice and OGDR-stimulated primary microglia. The JAK2/STAT3 pathway was effectively suppressed by A III, similar to the effect of AG490 treatment. In addition, A III and AG490 treatments significantly decreased Drp1 phosphorylation, translocation and mitochondrial fission in primary microglia stimulated with OGDR for 24 h. CONCLUSION: Our study demonstrated that A III was able to reduce complications associated with ischemia through inhibiting neuroinflammation, which was mediated in part by JAK2/STAT3-dependent mitochondrial fission in microglia.

Shengmai injection attenuates the cerebral ischemia/reperfusion induced autophagy via modulation of the AMPK, mTOR and JNK pathways.

Context Shengmai injection (SMI) is a patented Chinese medicine originated from the ancient Chinese herbal compound Shengmai san, which is used extensively for the treatment of cardiovascular and cerebrovascular disease in the clinic. Objective To determine the neuroprotective effect of SMI, we investigated the effect of SMI on cerebral ischemia/reperfusion (I/R) injury in mice as well as the mechanisms underlying this effect. Materials and methods Right middle cerebral artery was occluded by inserting a thread through internal carotid artery for 1 h, and then reperfused for 24 h in mice. The neuroprotective effects were determined using transmission electron microscopic examination, the evaluation of infarct volume, neurological deficits and water brain content. Related mechanisms were evaluated by immunofluorescence staining and western blotting. SMI was injected intraperitoneally after 1 h of ischemia at doses of 1.42, 2.84 and 5.68 g/kg. The control group received saline as the SMI vehicle. Results Results showed that SMI (1.42, 2.84 and 5.68 g/kg) could significantly reduce the infarct volume, SMI (5.68 g/kg) could also significantly improve the neurological deficits, decreased brain water content, as well as the neuronal morphological changes. SMI (5.68g/kg) could significantly inhibit the expression of autophagy-related proteins: Beclin1 and LC3. It also reduced the increase in LC3-positive cells. SMI (5.68 g/kg) remarkably inhibited the phosphorylation of adenosine monophosphate activated protein kinase (AMPK), and down-regulated the phosphorylation of mammalian target of rapamycin (mTOR) and Jun N-terminal kinase (JNK) after 24 h of reperfusion. Discussion and conclusion The results indicate that SMI provides remarkable protection against cerebral ischemia/reperfusion injury, which may be partly due to the inhibition of autophagy and related signalling pathways.

Analgesic Effects of Danggui-Shaoyao-San on Various "Phenotypes" of Nociception and Inflammation in a Formalin Pain Model.

Danggui-Shaoyao-San (DSS) is a traditional Chinese medicine, which has long been used for pain treatment and has been demonstrated to possess anti-oxidative, cognitive enhancement, and anti-depressant effects. In the present study, the effects of aqueous extracts of DSS on spontaneous pain behaviors and long-term hyperalgesia were examined to investigate the anti-nociceptive effects and underlying mechanisms. Single pretreatment of DSS dose-dependently reduced spontaneous flinches/licking time in the second, rather than the first, phase after subcutaneous injection of 5 % formalin into one hindpaw, in doses of 2.4 and 9.6 g/kg. DSS also dose-dependently inhibited FOS and cyclooxygenase-2 (COX-2) expression in both superficial and deep layers within the spinal dorsal horn. Further, DSS reduced hypoalgesia in the injected paw from 1 to 3 days and produced anti-hyperalgesic actions in both the injected paw after 3 days and non-injected paw. These data suggest involvement of enhancement of descending pain inhibition by suppression of 5-HTT levels in the spinal dorsal horn and reduction of peripheral long-term inflammation, including paw edema and ulcers. These findings suggest that DSS may be a useful therapeutic agent for short- and long-term inflammation induced pain, through both anti-inflammatory and suppression of central sensitization mechanisms.

Beneficial effect of Danggui-Shaoyao-San, a traditional Chinese medicine, on drowsiness induced by chronic restraint stress.

Danggui-Shaoyao-San (DSS), a famous Chinese complex prescription, has a long history of treating dysmenorrhea in China. Recent studies showed that DSS had its therapeutic efficacy on depression. In the present study, the effects of aqueous extract of DSS were examined to investigate the pharmacological basis for its therapeutic efficacy on chronic restraint stress (CRS)-induced sleep disorder. Mice were treated with DSS by gavage administration at doses of 0.625g/kg, 1.25g/kg and 2.5g/kg for 10 days. Sleep time and sleep latency were measured 24h after the CRS procedures using the sodium pentobarbital-induced sleep test. Open field test was used to evaluate the mice locomotion and anxiety/depression levels. Body weight and food intake were determined as well. The results showed that DSS dose-dependently prolonged latency and decreased sleep time of CRS mice, reversed CRS-induced decrease of mean velocity, the percentage of center distance and time in central area, as well as decreased body weight and food intake. In conclusion, our results suggest that DSS has exerted measurable therapeutic effects in hypersomnia induced by chronic restraint stress.

Danggui-shaoyao-san, a traditional Chinese medicine prescription, alleviates the orthodontic pain and inhibits neuronal and microglia activation.

BACKGROUND: The pain caused by orthodontic treatment has been considered as tough problems in orthodontic practice. Danggui-shaoyao-san (DSS) is a traditional Chinese medicine (TCM) prescription which has long been used for pain treatment and possesses antioxidative, cognitive enhancing and antidepressant effects. We raise the hypothesis that DSS exerts analgesic effect for orthodontic pain via inhibiting the activations of neuron and microglia. METHODS: DSS was given twice a day from day 5 prior to experimental tooth movement (ETM). Directed face grooming and vacuous chewing movements (VCM) were evaluated. Immunofluorescent histochemistry and Western blot analysis were used to quantify the Iba-1 (microglia activation) and Fos (neuronal activation) expression levels in the trigeminal spinal nucleus caudalis (Vc). RESULTS: ETM significantly increased directed face grooming and VCM which reached the peak at post-operative day (POD) 1 and gradually decreased to the baseline at POD 7. However, a drastic peak increase of Fos expression in Vc was observed at 4 hours and gradually decreased to baseline at POD 7; while the increased Iba-1 level reached the peak at POD 1 and gradually decreased to baseline at POD 7. Furthermore, pre-treatment with DSS significantly attenuated the ETM induced directed face grooming and VCM as well as the Fos and Iba-1 levels at POD 1. CONCLUSION: Treatment with DSS had significant analgesic effects on ETM-induced pain, which was accompanied with inhibition of both neuronal and microglial activation.