EGFR dependent subcellular communication was responsible for morphine mediated AC superactivation.

Compensatory adenylyl cyclase (AC) superactivation has been postulated to be responsible for the development of morphine tolerance and dependence, the underlying mechanism was demonstrated to comprise c-Src-dependent upregulation of AC5 within the lipid rafts. In the present study, we demonstrated that chronic morphine treatment sensitized EGFR signaling by augmenting EGFR phosphorylation and translocation into ER, which was essential for CRT-MOR tethering within the lipid rafts and AC5 superactivation. Intriguingly, synaptic clustering of CRT-MOR was dependent on EGFR phosphorylation and presumed to implicate in alignment and organization of synaptic compartments. Taken together, our data raised the possibility that an adaptive change in MOR and EGFR signal systems might establish CRT related subcellular communication, the signaling network within brain synaptic zone was proposed to implicate in morphine tolerance and dependence.

Age-Dependent Neuroimmune Modulation of IGF-1R in the Traumatic Mice.

BACKGROUND: Age-dependent neuroimmune modulation following traumatic stress is accompanied by discordant upregulation of Fyn signaling in the frontal cortex, but the mechanistic details of the potential cellular behavior regarding IGF-1R/Fyn have not been established. METHODS: Trans-synaptic IGF-1R signaling during the traumatic stress was comparably examined in wild type, Fyn (-/-) and MOR (-/-) mice. Techniques included primary neuron culture, in vitro kinase activity, immunoprecipitation, Western Blot, sucrose discontinuous centrifugation. Besides that, [3 H] incorporation was used to assay lymphocyte proliferation and NK cell activity. RESULTS: We demonstrate robust upregulation of synaptic Fyn activity following traumatic stress, with higher amplitude in 2-month mice than that in 1-year counterpart. We also established that the increased Fyn signaling is accompanied by its molecular connection with IGF-1R within the synaptic zone. Detained analysis using Fyn (-/-) and MOR (-/-) mice reveal that IGF-1R/Fyn signaling is governed to a large extent by mu opioid receptor (MOR), and with age-dependent manner; these signaling cascades played a central role in the modulation of lymphocyte proliferation and NK cell activity. CONCLUSIONS: Our data argued for a pivotal role of synaptic IGF-1R/Fyn signaling controlled by MOR downstream signaling cascades were crucial for the age-dependent neuroimmune modulation following traumatic stress. The result here might present a new quality of synaptic cellular communication governing the stress like events and have significant potential for the development of therapeutic approaches designed to minimize the heightened vulnerability during aging.

Neuroimmune modulation following traumatic stress in rats: evidence for an immunoregulatory cascade mediated by c-Src, miRNA222 and PAK1.

BACKGROUND: Neuroimmune modulation following traumatic stress is accompanied by cortical upregulation of c-Src expression, but the mechanistic details of the potential regulatory link between c-Src expression and immunosuppression have not been established. METHODS: We used a combination of techniques to measure temporal changes in: (i) the parallel expression of c-Src and microRNA222; (ii) levels of PAK1 (p21-activated kinase 1); and (iii) the association between PAK1 and interleukin 1ß signaling, both in cortex of rats following traumatic stress and in primary cortical neurons. Techniques included real-time PCR, immunoprecipitation, western blotting and subcellular fractionation by discontinuous centrifugation. We also measured lymphocyte proliferation and natural killer (NK) cell activity. RESULTS: We confirm robust upregulation of c-Src expression following traumatic stress. c-Src upregulation was accompanied by marked increases in levels of miRNA222; other studied miRNAs were not affected by stress. We also established that PAK1 is a primary target for miRNA222, and that increased levels of miRNA222 following traumatic stress are accompanied by downregulation of PAK1 expression. PAK1 was shown to mediate the association of IL-1RI with lipid rafts and thereby enhance IL-1 signaling. Detailed analyses in cultured neurons and glial cells revealed that PAK1-mediated enhancement of IL-1RI activation is governed to a large extent by c-Src/miRNA222 signaling; this signaling played a central role in the modulation of lymphocyte proliferation and NK cell activity. CONCLUSIONS: Our results suggest that neuroimmune modulation following traumatic stress is mediated by a cascade that involves c-Src-mediated enhancement of miRNA222 expression and downregulation of PAK1, which in turn impairs signaling via IL-1ß/IL1-RI, leading to immunosuppression. The regulatory networks involving c-Src/miRNA222 and PAK1/IL-1RI signaling have significant potential for the development of therapeutic approaches designed to promote recovery following traumatic injury.

Neuron-glial cell communication in the traumatic stress-induced immunomodulation.

We have previously reported that neuron and glia could collaboratively govern the immunomodulation in traumatic rats. Herein, we characterized the sequential involvement of cortical neuron, microglia, and astrocytes in the traumatic stress-mediated neuroimmune modulation. At day 1 of trauma, transient extracellular signal related kinase 1/2 (ERK1/2) activation was initiated in neuron and microglia, which was accompanied by RSK-1 expression in the cytosol. At day 3 of trauma, persistent ERK1/2 activation occurred in astrocytes, which were destined for the nucleus leading to Elk-1 expression. Furthermore, the functional overlap of ERK1/2 and neuroligin 1 in astrocytes was strengthened at day 3 of trauma and responsible for the recovery from the immnosuppression. These effects could be disrupted by ß-neurexin blockade. Altogether, we proposed the mechanism underlying the traumatic stress-induced immunosuppression, in which local activity ensured the initial establishment of neural circuitry in the frontal cortex. ERK1/2-signaling events are required for the temporal and spatial coordination between neuron and glial cells.

Beta-adrenoceptor mediated surgery-induced production of pro-inflammatory cytokines in rat microglia cells.

Immunological changes initiated by major operative injury may result in inflammatory responses in both peripheral and central nervous system, which may lead to organ dysfunction. Recent studies indicate that beta-adrenergic receptors (beta-ARs) may mediate production of pro-inflammatory cytokines in the brain. In the present study propranolol (beta-AR antagonist), but not prazosin (alpha1-AR antagonist), antagonized surgical trauma induced pro-inflammatory cytokine production in microglia cells isolated from rats. beta-AR activation in the absence of pro-inflammatory stimuli increased IL-1beta, TNF-alpha and IL-6 mRNA and protein expressions in the primary microglia cell culture. Isoproterenol (beta-AR agonist) treatment induced a time- and concentration-dependent increase of IL-1beta in cells. Both ERK1/2 and P38 MAPK inhibitor, but not PKA and JNK1/2 inhibitor abrogated isoproterenol-induced IL-1beta and IL-6 production in microglia cells. In conclusion, the results suggest that beta-ARs possess pro-inflammatory properties by modulating the functions of microglia cell.

Electroacupuncture downregulates TLR2/4 and pro-inflammatory cytokine expression after surgical trauma stress without adrenal glands involvement.

Cumulative evidences suggest that electroacupuncture (EA) can modulate immune function, but the mechanism needs further study. In the present study, the contribution of EA on toll-like receptors 2 and 4 (TLR2/TLR4) and pro-inflammatory cytokine expression after surgical trauma stress were investigated. The mRNA level of both TLR2/4 and pro-inflammatory cytokine was measured by quantitative real-time PCR. ELISA and Western blot assay were chosen for TLR2/TLR4 protein expression and pro-inflammatory cytokine production, respectively. The results showed that surgical trauma stress increased TLR2 mRNA and TLR2/4 proteins in the spleen and augmented pro-inflammatory cytokines (e.g. IL-1beta) mRNA and protein expression in the spleen and plasma. These effects could be deteriorated by adrenalectomy (ADX). EA at "Zusanli" acupoint significantly inhibited surgical trauma-induced TLR2 mRNA and TLR2/4 protein expression in spleen and pro-inflammatory cytokine expression in the spleen and plasma. ADX, however, could not block the effect of EA. These results suggested that surgical trauma stress primes the innate immune system for enhanced TLR2 expression and pro-inflammatory cytokine production. EA inhibits TLR2/4 and pro-inflammatory cytokines to produce an anti-inflammatory effect in a surgical trauma stress model, without adrenal gland involvement.

Neurite outgrowth is dependent on the association of c-Src and lipid rafts.

Regulation of neurite outgrowth is an important aspect not only for proper development of the nervous system but also for tissue regeneration after nerve injury and the treatment of neuropathological conditions. Here, we report that neurite outgrowth in cortical neuron and neuro 2A (N2A) cell was dependent on intact lipid rafts, as well as the enhanced localization of c-Src in the lipid rafts. Src inhibition or lipid rafts disruption could specifically block c-Src phosphorylation profile, pY416 Src increase and pY529 Src decrease, they also resulted in pY529 Src and c-terminal Src kinase (Csk) partition out of lipid rafts. Thus, we concluded that c-Src signal cascades within the lipid rafts is crucial for efficient neurite outgrowth.

Can electroacupuncture affect the sympathetic activity, estimated by skin temperature measurement? A functional MRI study on the effect of needling at GB 34 and GB 39 on patients with pain in the lower extremity.

The Purpose of this study was to investigate the possible needling effect on sympathetic activity by using functional MRI (fMRI). Twelve patients with left lower extremity pain were enrolled in our study. Each was given deep needling at left GB34 (yanglingquan) and GB39 (xuanzhong) points simultaneously. All patients got the strong DeQi sensation by manipulating the needles, and then electroacupucture (EA) was given and lasted for thirty minutes before fMRI scan. Then the fMRI scan was performed by scanning the whole brain with five blocks lasting 2 minutes each. The patients' palm skin temperatures were tested every five minutes as indication of the sympathetic activity from the beginning of EA to the end of our fMRI scan. Functional images were processed by using FEAT software at different levels including the simple single subject analysis, multi-subjects group mean analysis, and multi-subjects unpaired two difference analysis. Finally, 9 of 12 patients' palm temperatures increased gradually but the other three decreased. In comparison of two different palm skin temperature change groups, more significant activation over bilateral caudate head, right lentiform and periaqueductal gray (PAG) were found in the temperature-increased group, but palm temperature-decreased patients revealed more significant activation over bilateral anterior cingulated cortex (ACC), insula, primary somatosensory gyrus (SI), orbitofrontal cortex, occipital cortex, hippocampus and amygdala formation. Our study suggested that needling at analgesic points may modulate the sympathetic activity and such evident difference on brain responses may correlate with the clinical analgesia effects.

Characterization of Fyn signaling on the age-dependent immuno-modulation on traumatic rats.

Traumatic stress is well characterized to develop immuno-depression in our previous report. Here, we provide evidence that adult and aged rats showed similar decrease in lymphocyte proliferation and natural killer (NK) cell activity. However, compared with beginning recovering from traumatic stress after 3 day and fully recovered by 7 day in adult rats, aged rats begin the recovery phage later than 3 day and do not fully recovered by 7 day. In parallel, Fyn expression in cerebral cortex was augmented with the highest level at 3 day of trauma in both age groups of rats, although aged rats exhibited lower level than the younger cohorts. Immune consequences were consequently modified by intracerebroventricular (i.c.v.) injection of Fyn antibody or recombinant adenovirus expressing active Fyn. Finally, the increase in Fyn expression was converged on ERK1/2 (extracellular signal regulated kinase 1/2) activation. Taken together, the data indicated that immunological processes in response to traumatic stress was age dependent, Fyn-ERK1/2 signal pathway was required to convey the recovery signals.

Electroacupuncture combined with clomipramine enhances antidepressant effect in rodents.

The present study was designed to evaluate the antidepressant effect of electroacupuncture (EA) and the potential additive or synergistic effects of EA and clomipramine (CLO, a tricyclic antidepressant) in the mouse forced swimming test (FST) and chronic mild stress (CMS) induced depression-model rats. The FST is an antidepressant screening procedure performed initially to observe the immediate effects of EA and/or CLO on the immobility time. CLO (2.5, 5, 10, 20 and 60mg/kg intraperitoneally) were administered at 23, 6 and 1h respectively prior to each test. EA was given at the 'Bai-Hui' (Du 20) and unilateral 'An-Mian' (EX 17) acupoints 1h before each test. Immobility time was significantly reduced by EA and CLO at 2.5, 5, 10, 20 or 60mg/kg, respectively. EA combined with 2.5mg/kg CLO exhibited additive effects on the immobility time. In addition, rats were exposed chronically (1st-11th week) to a variety of mild unpredictable stressors. Depressed mood and anhedonia were recognized as a decrease in sucrose intake in the CMS rats. CLO at 2.5, 5mg/kg and EA at the same acupoints and parameters were administrated on the CMS rats once every other day for 6 weeks (5th-11th week). The intake of 1% sucrose solution was reduced by CMS, which was restored to normal level after 6 weeks treatment with 5mg/kg CLO or EA combined with 2.5mg/kg CLO. However, neither the sucrose intake nor the sucrose preference in the depressive rats was significantly changed by the treatment with EA or 2.5mg/kg CLO alone. These results demonstrated that EA combined with CLO at low doses has an additive or synergistic antidepressant action, and this combination may provide an effective strategy for depression management.

Sympathetic nervous system mediates surgical trauma stress-induced splenocyte apoptosis in rats.

Surgical trauma stress has been reported to induce immunosuppression. The mechanisms involved are still unclear. The present study was designed to assess the role of the sympathetic nervous system in regulating splenocyte apoptosis induced by surgical trauma stress. Our results showed that the rats that underwent surgical trauma stress exhibited a significant reduction in splenic cellularity, the loss of splenocytes was likely mediated by apoptosis, for a substantial increase in apoptosis was observed by using DNA gel electrophoresis and TUNEL assay. At the same time, an increase in Fas(CD95/Apo-1) protein expression in splenocytes was also observed. These effects were significantly abolished by either chemical sympathectomy or beta-adrenergic receptor antagonist propranolol. The data clearly revealed that the sympathetic nervous system especially beta-adrenergic receptors was involved in surgical trauma-induced immune alterations via a mechanism of apoptotic cell death.

Neuronal specificity of needling acupoints at same meridian: a control functional magnetic resonance imaging study with electroacupuncture.

The purpose of this study was to investigate the neuronal specificity of needling acupoints at same meridian by functional Magnetic Resonance Imaging (fMRI). The selected acupoints GB34 (Yanglinquan) and GB39 (Xuanzhong) were at the same gallbladder meridian based on traditional Chinese medicine. In our study we devise three distinct EA (electroacupuncture) manipulations: real EA (deep needling at acupoints), sham EA (deep needling at no-meridian points) and shallow EA (subcutaneous needling at acupoints). Twelve healthy volunteers with right-handiness were enrolled and received three different EA manipulations in counter-balanced orders. DeQi scores were used to evaluate the degree of needling sensation. We found real EA can induce significant stronger needling sensation than sham EA and shallow EA. Multisubjects group mean analysis showed that pain-related cortex including primary and secondary somatosensory cortex (SI and S II), anterior cingulated cortex (ACC), insula were involved in three EA stimulation. Bilateral activation of prefrontal gyrus and occipital cortex were exclusively found in real EA. Deactivation over the rostral segment of ACC was also shown in real and shallow EA. Further paired two difference analysis indicated that real EA induced higher activation than sham EA over bilateral prefrontal gyrus, right-side occipital gyrus and deactivation over the rostral segment of ACC. In the comparing with real EA versus shallow EA, there was right-side activation over the SI, S II, motor cortex, ACC, insula, thalamus, hippocampus, occipital cortex, and cerebellum; also activation over bilateral prefrontal gyrus, caudate and pons. Although no significant activation was found over periaqueductal gray (PAG), further analysis showed the mean and maximal signal changes were different under three EA manipulations. We concluded that EA at analgesic acupoints of same meridian maybe involved the pain-related neuromatrix especially the hypothalamus-limbic system; deep EA at meridian points could elicit stronger needling sensation and modulate the pain-related neuromatrix more effectively than EA at nonmeridian points or shallow EA at meridian points.

Sucrose preference is restored by electro-acupuncture combined with chlorimipramine in the depression-model rats.

The present study was to investigate the effect of electroacupuncture (EA) combined with chlorimipramine on the sucrose preference of depressive rats induced by chronic mild stress (CMS). Rats were exposed chronically (1st - 4th week) to a variety of mild unpredictable stresses. The tricyclic antidepressant chlorimipramine and EA were administrated on these depressive rats for 6 weeks (5th - 11th week). EA was applied at points of "Bai-Hui" (Du 20) and "An-Mian" (EX 17) (right side), by EA apparatus (Model 6805-2, Shanghai). Dense (60Hz/5sec)-sparse (4Hz/2.5sec) frequency of the wave was selected and the current intensity (< or = 1mA) was adjusted to provoke slight twitches of the rat's ear. The preference for 1% sucrose solution and the immobility time in the forced swimming test were measured as the symptoms of anhedonia and depressed mood, which were central features of major depression. The preference for 1% sucrose solution was reduced by CMS, but could be restored to normal level after 6 weeks treatment with chlorimipramine at 5mg/kg or EA combined with chlorimipramine at 2.5mg/kg. In the forced swimming test, the immobility time of depressive rats was decreased in both groups. However, the preference for sucrose and the immobility time in the depressive rats were not significantly changed by the treatment with only EA or chlorimipramine at 2.5 mg/kg. The results suggested that EA could potentiate the antidepressant effect of chlorimipramine in low dose, and EA combined with antidepressant might be a better method in treating depression.

Electroacupuncture suppresses surgical trauma stress-induced lymphocyte apoptosis in rats.

Cumulative evidences suggested that electroacupuncture (EA) could modulate immune function, but the mechanism needs further study. In the present study, the effect of EA on surgical trauma stress-induced lymphocyte apoptosis was investigated by using DNA gel electrophoresis, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and Western blot for Fas protein expression. The results showed that rats with surgical trauma stress exhibited a significant reduction in splenic cellularity. Increase in apoptotic cell death and Fas (CD95/Apo-1) expression in splenic lymphocytes was also observed. EA could suppress the increase of apoptosis and Fas protein expression in splenic lymphocytes induced by the surgical trauma stress. These results implied that EA could decrease splenic lymphocytes apoptosis via inhibiting Fas protein expression; consequently prevent deleterious immunological changes in the post-operative state.

Modulation of the gene expression in the protective effects of electroacupuncture against cerebral ischemia: a cDNA microarray study.

In the present study, a cDNA microarray technology was used to analyze the gene expression profile in ischemia and in electroacupuncture (EA)-treated ischemia. A focal cerebral ischemia/reperfusion model on Macaca mulatta was performed with a modified middle cerebral artery occlusion method. Among the nearly 8000 genes, approximately 8% of the total number of genes examined were affected after ischemia/reperfusion injury. Major altered genes were downregulated. In EA-treated monkeys, approximately 10% of the total number of genes examined were affected. Major altered genes were upregulated, including signal transduction-, cell-cycle-, metabolism-, stress response-, DNA repair-related genes. One of the representative upregulated genes encodes insulin-like growth factor-1 (IGF-1) was confirmed using in situ hybridization. Results showed that after ischemia/reperfusion injury, IGF-1 mRNA expression decreased in ipsilateral striatum, whereas increased in ipsilateral hippocampus. No expression changes were observed in cortex. EA treatment could obviously upregulate the IGF-1 mRNA expression in striatum, and further enhance its expression in hippocampus. Therefore, the data presented suggest a possible mediator underlying the mechanisms of anti-ischemic effect of acupuncture. In conclusion, the protective mechanisms of EA against stroke include several related pathways and gene expressions. Microarray analysis may provide a framework for understanding these complicated mechanisms and yield valuable, clinically relevant insights and potentially therapeutic targets of stroke.

Melatonin enhances lymphocyte proliferation and decreases the release of pituitary pro-opiomelanocortin-derived peptides in surgically traumatized rats.

The present study was undertaken to evaluate the effects of melatonin (MT) on spleen lymphocyte proliferation and release of pituitary pro-opiomelanocortin (POMC)-derived peptides in surgically traumatized rats. MT prevented the depression of lymphocyte proliferation induced by trauma in vivo and in vitro, and prevented the decrease of beta-endorphin and adrenocorticotropin in the pituitary in vivo, but dose-dependently inhibited the release of POMC-derived peptides from the pituitary in vitro. The culture media of the pituitaries derived from the traumatized rats inhibited lymphocyte proliferation of normal rats. These results suggest that MT can improve the trauma-induced depression of lymphocyte proliferation and inhibit the release of POMC-derived peptides. The neuroimmunomodulatory role of MT and POMC-derived peptides deserves further study.

[History and causes of acupuncture advancing towards the world].

Acupuncture therapy is one of the original innovations in traditional Chinese medicine. The study of acupuncture has gradually advanced towards the world since 1970s. In these years the most important events might be the influence of acupuncture anesthesia, the attention of the World Health Organization, the establishment and development of the World Federation of Acupuncture and Moxibustion Societies, and the consensus development conference on acupuncture held by the National Institutes of Health in America, and so on. The reasons why acupuncture is accepted by the world are both the convinced curative effects and the scientific foundations, due to the great efforts of medical stuff in China guided by the principle of integration of traditional Chinese and western medicine. This article poses several proposals about further extending the influence of acupuncture. In brief, acupuncture research is one of the few fields that authentically impact the western scientific technology. It is of very important responsibility in the new century to impulse the acupuncture study to further progress and expand the international influence of acupuncture.

Effect of melatonin and electroacupuncture (EA) on NK cell activity, interleukin-2 production and POMC-derived peptides in traumatic rats.

The present study was to evaluate the effect of melatonin (MT) and EA on the cytotoxic activity of natural killer (NK) cells, the dynamic changes of the induction of interleukin-2(IL-2) and the content of POMC-derived peptides, beta-endorphins (betaE) and ACTH in spleen lymphocytes and in plasma of traumatic rats. The results showed that intraperitioneal (i.p.) injection of MT was able to recover the lower levels of NK cell activity and the induction of IL-2 production; MT could also decrease the higher betaE and ACTH levels induced by trauma in spleen lymphocytes and plasma. EA needling of Zusanli (St.36) and Lanwei (Extra.37) points obviously improved the immunosuppression produced by trauma and antagonized the elevation of betaE and ACTH contents induced by trauma stress in spleen lymphocytes and plasma. MT + EA could further modulate the depressed immune function, and there was a significant difference compared with MT (i.p.) or EA alone. MT + EA group further decreased the betaE and ACTH contents in immune cells and plasma. Yet, the mechanisms of the attenuation of MT and EA on immunosuppression induced by trauma need further study.

Inhibitory effects of intrathecally administered interleukin-1beta on carrageenan-induced hyperalgesia and spinal c-Fos expression in rats.

The present study investigated the effects of interleukin-1beta (IL-1beta) injected intrathecally (i.t.) on carrageenan-induced thermal hyperalgesia and spinal c-Fos expression. The paw withdrawal latency (PWL) by the thermal stimulus was taken as an index of the thermal hyperalgesia of rats. I.t. injection of 10 ng IL-1beta significantly increased the PWL of the carrageenan-injected paw. Expression of c-Fos induced by intraplantar (i.pl.) injection of carrageenan was examined in the spinal cord with immunohistochemical methods. Three hours after i.pl. injection of carrageenan, the number of c-Fos-like immunoreactive (c-Fos-LI) neurons was significantly increased in laminae I-II, III-IV and V-VI of the ipsilateral spinal cord at L4-5 with the higher density in laminae I-II and V-VI. I.t. pre-injected IL-1beta significantly decreased the number of carrageenan-induced c-Fos-LI neurons in laminae I-II in the ipsilateral spinal cord and also inhibited the hyperalgesia induced by i.pl. carrageenan. These results suggested that i.t. injection of IL-1beta suppressed the central nociceptive input into laminae I-II and produced an antinociceptive effect.

Scientific bases of acupuncture analgesia.

The present paper was to review the physiological bases of acupuncture analgesia (AA) on normal subjects, patients, and animals. Effect of acupuncture on pain perception in normal subjects was studied and compared with sham acupuncture. It was shown that the analgesic effect of acupuncture has its physiological basis. Using neurophysiological, neuropharmacological, neurobiochemical and neuromorphological methods, the neurohumoral mechanism of AA was studied from the peripheral neural pathway of acupuncture sensation (De-Qi sensation in Chinese traditional medicine) to the central neuromodulatory effect of AA. It was indicated that needling of acupuncture point could activate the afferent fibers of peripheral nerves to elicit De-Qi sensation, then ascended mainly through the ventro-lateral funiculi, which conducted pain and temperature sensation upward to the brain, activated the antinociceptive system including certain brain nuclei. modulators (opioid peptides), neurotransmitters, through the descending inhibitory pathway resulting in analgesia. Especially the clinical and laboratory results indicated that the endogenous opiate peptides (EOP) participated in AA from the presynaptic level to the receptor sites, which provided a scientific basis for understanding the mechanism of AA. Substantial evidences have been accumulated that acupuncture has prominent analgesic effect; but it fails to give sufficient analgesia during operation. Some effective measures to improve the therapeutic effect of acupuncture, such as the combination of acupuncture with drugs, the selection of suitable EA parameters and optimal time spacing should be adopted.