Activation of hippocampal MEK1 contributes to the cumulative antinociceptive effect of electroacupuncture in neuropathic pain rats.

BACKGROUND: Electroacupuncture (EA) intervention can relieve a variety of pain; however, optimal EA protocols have not been clearly determined. In addition, although central mitogen-activated protein kinase kinase (MEK) signaling has been shown to be involved in the antinociceptive effect of acupuncture stimulation, its characteristics at different time-points of EA intervention have not been fully elucidated. Therefore, the present study investigated the relationship between the effects of different numbers of EA intervention sessions and the activation of MEK1 in the hippocampus and hypothalamus in a rat model of neuropathic pain. METHODS: After ligation of the left sciatic nerve, which induces chronic constriction injury (CCI), the acupoints Zusanli (ST36) and Yanglingquan (GB34) were applied. The thermal withdrawal latency of the hind paw was used to evaluate the effect of EA on pain thresholds. Intra-hippocampus microinjection of PD98059, a MEK inhibitor, was performed to validate the involvement of MEK in EA analgesia. The hippocampus and hypothalamus were harvested to examine the phosphorylation levels of MEK (pMEK) by western blotting. RESULTS: In CCI rats, the thermal pain threshold of the affected hind paw decreased significantly relative to the control. Following subsequent daily EA interventions, CCI-induced ipsilateral hyperalgesia was markedly improved from day 4 and the analgesic effect of EA lasted 3 days after cessation of EA. Four sessions of EA markedly suppressed CCI-induced decrease of hippocampal pMEK1 (normalized to the total MEK level). In contrast, successive sessions of EA intervention gradually down-regulated the CCI-induced up-regulation of hypothalamic pMEK1 along with the increase numbers of EA intervention. However, EA did not exert the same analgesic effect after microinjection of PD98059 into the contralateral hippocampus during the first 3 days of EA intervention. CONCLUSIONS: EA intervention can induce time-dependent cumulative analgesia in neuropathic pain rats after 4 successive sessions of daily EA intervention, which is at least in part related to the activation of hippocampal MEK1.

NK cells mediate the cumulative analgesic effect of electroacupuncture in a rat model of neuropathic pain.

BACKGROUND: Cumulating evidence has revealed the effectiveness of acupuncture therapy in relieving pain via immunoregulation. However, its underlying mechanism remains unknown. The present study was designed to determine the changes of immunogenic responses at different time-points of electroacupuncture (EA) interventions in neuropathic pain rats. METHODS: The neuropathic pain model was established by ligature of the left sciatic nerve to induce chronic constriction injury (CCI). EA was applied at Zusanli (ST36) and Yanglingquan (GB34) for the EA groups. The thermal pain threshold was detected with an algesia-detector. The subgroups of plasma and splenic lymphocytes were determined via fluorescence-activated cell sorting. Specific inflammatory cytokines were assayed using an ELISA-based bead multiplex assay. The activities of splenic natural killer (NK) cells and cytotoxic T lymphocytes were detected by methyl thiazolyl tetrazolium colorimetric method. For confirming the involvement of NK cell in EA-analgesia, anti-asialo-ganglio-N-tetraosylceramide (anti-asialo-GM1) antibody was given to CCI rats before EA. RESULTS: Following CCI, the thermal pain threshold of the affected hind footpad was significantly decreased, and increased from the 3rd day to the 12th day after EA interventions, presenting a time-dependent tendency from the 5th day on. From day 3 to 5 of EA interventions, the percentages and activity of splenic NK cells, concentrations of splenic interleukin-2 (IL-2) and beta-endorphin (ß-EP) were significantly increased. Meanwhile, the concentrations of plasma IL-2, IL-1ß and gamma-interferon (IFN-γ) were significantly decreased and returned to the normal level on day 12 following EA. Plasma transforming growth factor-ß (TGF-ß) levels were considerably upregulated on day 5 and 12 following EA. The CD4+/CD8+ T cell ratio was markedly downregulated compared with the control and CCI groups on day 5 and returned to the normal level on day 12 following EA. After depleting NK cells by anti-asialo-GM1 antibody, the increased thermal pain threshold following EA intervention was obviously reduced. CONCLUSIONS: Repeated EA interventions have a time-dependent cumulative analgesic effect in neuropathic pain rats, which is closely associated with its regulatory effects on NK cells, splenic IL-2, ß-EP, and plasma IL-2, IL-1ß, IFN-γ and TGF-ß levels.

Electroacupuncture Alleviates Pain Responses and Inflammation in Collagen-Induced Arthritis Rats via Suppressing the TLR2/4-MyD88-NF-κB Signaling Pathway.

Results: EA intervention and OxPAPC injection could relieve mechanical allodynia and thermal hyperalgesia caused by CIA. Paw edema and pathological damage of synovium were significantly ameliorated after EA intervention and OxPAPC injection. Furthermore, EA intervention and OxPAPC injection markedly reduced the contents of serum TNF-α, IL-1ß, and IL-6, as well as the protein expression levels of synovial TLR2, TLR4, MyD88, and NF-κB p-p65. In particular, the expression of TLR2 and TLR4 on synovial fibroblasts and macrophages in synovium was significantly reduced by EA intervention. Conclusions: Repeated EA stimulation at ST36 and SP6 can effectively relieve joint pain and synovial inflammation caused by RA in CIA rats. The analgesic and anti-inflammatory effect of EA may be closely related to the inhibition of innate immune responses driven by the TLR2/4-MyD88-NF-κB signaling pathway in the synovium.

[Effects of transcutaneous auricular vagus nerve stimulation on bone and cartilage destruction in rats with rheumatoid arthritis].

OBJECTIVE: To observe the alleviating effect of transcutaneous auricular vagus nerve stimulation (taVNS) on articular cartilage and bone destruction in rats with collagen-induced arthritis (CIA), and explore the cellular and molecular mechanisms of taVNS against rheumatoid arthritis (RA). METHODS: The male SD rats were randomly divided into normal control group (n=12), model group (n=12), and taVNS group (n=12). The CIA rat model was established by multi-point injection of emulsion prepared from type Ⅱ bovine collagen and Freund's incomplete adjuvant into the root of rat tail. The rats in the taVNS group were treated with taVNS at bilateral auricular conchae, 30 min per time, once a day, for consecutive 28 d. The cartilage destruction of the ankle joint was observed by safranin O-fast green staining, the production of osteoclasts in the joint tissue by tartrate-resistant acid phosphatase (TRAP) staining, and the bone erosion by X-ray and Micro-CT imaging. The protein expression levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG) in the synovial tissues were detected by Western blot. RESULTS: Compared with the normal control group, the CIA rats presented with typical RA symptoms and elevated arthritis index (AI,P<0.05). After intervention with taVNS, the AI remarkably declined in comparison with that in the model group (P<0.05). Compared with the control group, the model group displayed loss of cartilage matrix in the ankle joint, thinned cartilage layer, obvious cartilage damage, and increased number of osteo-clasts in the joint (P<0.01); the imaging results showed bone loss and three-dimensional structural destruction of ankle joint and aggravated bone erosion (P<0.01); the expression levels of MMP-1, MMP-3 and MMP-13, and RANKL/OPG ratio were significantly elevated in the synovial tissue of ankle joint (P<0.01, P<0.05), while the expression level of OPG was decreased (P<0.05). Compared with the model group, taVNS resulted in relatively intact cartilage layer of ankle joint, alleviated cartilage destruction, decreased number of osteoclasts (P<0.01), improved bone erosion, loss, and three-dimensional structural destruction (P<0.01), and diminished MMP-1, MMP-3, and MMP-13 expression and RANKL/OPG ratio in the synovial tissue of ankle joint (P<0.05, P<0.01), while the expression level of OPG was increased (P<0.05). CONCLUSION: taVNS effectively relieves bone and cartilage destruction in CIA rats, which might be related to its efficacy in reducing the production of osteoclasts in joint tissues and down-regulating the expression levels of MMP-1, MMP-3 and MMP-13, and RANKL/OPG ratio.

[Effect of electroacupuncture on articular morphological changes and serum inflammatory factors and synovial MMPs in collagen-induced arthritis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Zusanli"(ST36) and "Sanyinjiao"(SP6) on serum TNF-α, IL-1ß, and IL-6 and expression of synovial matrimetalloproteinases (MMPs) and articular morphology in collagen-induced arthritis (CIA) rats, so as to explore its mechanisms underlying relief of arthritis. METHODS: Thirty male SD rats were randomly divided into normal control, CIA model and EA groups (n=10 rats per group). The arthritis model was induced by multi-point intradermal injection of bovine type Ⅱ collagen emulsion. EA (2 Hz/100 Hz, 1 mA) was applied to bilateral ST36 and SP6 for 30 min, once a day for 28 days. The hind-limb paw volume was measured and the arthritis index (AI) score given according to the swelling degree, rigidity and deformity of the ankle joint (0-4 points). After EA intervention, the morphological damage of the affected ankle joints was revealed by H.E. staining, safranin O-fast green staining, and tartrate-resistant acid phosphatase (TRAP) staining, separately. The levels of serum TNF-α, IL-1ß, and IL-6 were measured by ELISA, and the expression levels of MMP-1, MMP-3, MMP-13, and receptor activator of nuclear factor Kappa B ligand (RANKL) in the synovial tissue were detected by Western blot. RESULTS: Compared with the normal control group, the paw volume, AI score, TRAP-revealed number of osteoclasts, contents of serum TNF-α, IL-1ß and IL-6, and expression levels of MMP-1, MMP-3, MMP-13 and RANKL proteins were significantly increased in the model group (P<0.01, P<0.05). Following the intervention, the paw volume, AI score, number of osteoclasts, contents of serum TNF- α, IL-1ß and IL-6, and expression levels of MMP-1, MMP-3, MMP-13 and RANKL proteins were significantly decreased in the EA group (P<0.05, P<0.01) in contrast to the model group. H.E. and safranin O-fast green staining showed rough articular cartilage surface with thinned cartilage layer, obvious hyperplasia of the synovial tissue with many inflammatory cells, and serious damage and degradation of the cartilage matrix in the model group, these situations were relatively milder in the EA group. CONCLUSION: EA of ST36 and SP6 can reduce the articular damage in collagen-induced arthritis rats, which is associated with its function in reducing inflammatory response and down-regulating the expression of synovial MMP-1, MMP-3, MMP-13 and RANKL proteins.

Experimental study on acupuncture activating the gonadotropin-releasing hormone neurons in hypothalamus.

OBJECTIVE: To probe into the most effective site, extra-ordinary point, acupoint and channel for regulating reproductive endocrine function by means of the study on acupuncture activating the gonadotropin-releasing hormone neurons (GnRH) in hypothalamus. METHODS: Female SD rats of reproductive age were used, and the in vivo study on GnRH neurons in hypothalamus was made with mimic sexual stimulation and feedback regulation. The neuron-activating effects of the acupoints on the channels pertaining to the zang- and fu-organs related with reproductive endocrine and the extra-ordinary points in different regions were studied using the discharge of the neuron as index, and then the neurons were labeled with horseradish peroxidase (HRP). RESULTS: Acupuncture was given at two acupoints each on the three yin channels of foot, the three yang channels of foot, the Conception Vessel and the Governor Vessel. The order of the mean increasing percentage in the hypothalamic GnRH neuron electric activity was: the Gallbladder Channel > the Spleen Channel > the Stomach Channel/the Bladder Channel/the Conception Vessel > the Liver Channel > the Kidney Channel > the Governor Vessel; for different acupoints, it was: Guanyuan (CV 4) > Sanyinjiao (SP 6) > Zusanli (ST 36) > Daimai (GB 26)/Yanglingquan (GB 34) > Shenshu (BL 23) > Weizhong (BL 40) > Yaoyangguan (GV 3)/Liangmen (ST 21)/Fujie (SP 14) > Qimen (LR 14)/Yingu (KI 10) > Tangzhong (CV 17)/Zhiyang (GV 9); for different positions, it was: the lower abdominal part/the lower limb part > the thoracodorsal part; for the extra-ordinary points, it was: Zigong (EX-CA1) > Dannang (EX-LE6)/Yaoyan (EX-B7) > Baichongwo (EX-LE3)/Qianzheng > Jingbi/Bizhong/Taiyang (EX-HN5) > Erbai (EX-UE2)/ Dingchun; and for the distribution sites of the extra-ordinary points: lower abdominal region > the lower limb region > the craniofacial region > the upper limb region/the thoracodorsal region. CONCLUSION: For regulating the reproductive endocrine function, the acupoints located at the same neural segment with the reproductive organ should be selected as the main points, and it is necessary to combine with syndrome differentiation of the viscera and channels.

Contribution of GABAergic modulation in DRGs to electroacupuncture analgesia in incisional neck pain rats.

PURPOSE: Acupuncture therapy is effective for relieving postoperative pain. Our previous study showed that electroacupuncture (EA) at Futu (LI18) and Hegu (LI4)-Neiguan (PC6) could alleviate incisional neck pain, which was related with its effect in upregulating γ-aminobutyric acid (GABA) expression in cervical (C3-6) dorsal root ganglions (DRGs); but whether its receptor subsets GABAAα2R and GABABR1 in C3-6 DRGs are involved in EA analgesia or not, it remains unknown. MATERIALS AND METHODS: Seventy-five male Sprague Dawley rats were randomized to normal control, model, LI18, LI4-PC6, and Zusanli (ST36)-Yanglingquan (GB34) groups. The incisional neck pain model was established by making a longitudinal incision along the midline of the rats' neck, followed by repeated mechanical stimulation. EA was applied to bilateral LI18, LI4-PC6, or ST36-GB34 for 30 minutes at 4, 24, and 48 hours after operation. The thermal pain threshold of the neck was detected by a tail-flick unit, and the C3-6 DRGs were removed for assaying the immunoactivity of substance P (SP), GABAAα2R, glial fibrillary acidic protein (GFAP; a marker of satellite glial cells [SGCs]), and GABABR1 and the expression of GABAAα2R and GABABR1 mRNA and proteins using immunofluorescence, real-time PCR, and Western blotting, respectively. RESULTS: The cervical thermal pain threshold was significantly lower in the model group than the normal group (P<0.001), indicating hyperalgesia after neck incision, and was considerably increased in both EA-LI18 and LI4-PC6 groups (P<0.001), but not in ST36-GB34 group compared with model group (P>0.05). Immunofluorescence staining showed that GABAAα2 R expressed on SP+ neurons, and GABABR1 on SGCs. EA of LI18 and LI4-PC6 markedly suppressed the modeling-induced upregulation of the immunoactivity of SP (P<0.001 and P<0.01, respectively) and GFAP (P<0.01 and P<0.001, respectively) and significantly reversed neck incision-induced downregulation of the expression of GABAAα2R and GABABR1 mRNAs and proteins (P<0.05). CONCLUSION: EA of LI18 and LI4-PC6 has an analgesic effect in incisional neck pain rats, which is related to its effects in upregulating GABAergic inhibitory modulation on nociceptive peptidergic neurons and SGCs in cervical DRGs.

[Electroacupuncture of Multiple Acupoints Is Significantly Superior to That of Single Acupoint in Improving Sleep by Regulating Serum Sleep-promoting and Awakening-promoting Factors in Insomnia Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of single and multiple acupoints on sleep and concentrations of interlukin-1 ß(IL-1 ß), brain-derived neurotrophic factor (BDNF), prostaglandin D2(PGD2) and melatonin (MLT, sleep-promoting factors) and corticosterone (CORT, awakening-promoting factor) in the serum in insomnia rats, so as to explore its efficacy difference and the mechanism underlying improving sleep. METHODS: Fifty-four male SD rats were randomly divided into control, model, EA-Baihui (GV 20), EA-Shenmen (HT 7), EA-Sanyinjiao (SP 6) and EA-GV 20＋HT 7＋SP 6 groups (n＝9 rats in each group). The insomnia model was established by intraperitoneal injection of para-chlorophenylalanine (PCPA, 300 mg/kg) once daily for 2 days. In the EA-GV 20, EA-HT 7, EA-SP 6 and EA-GV 20＋HT 7＋SP 6 groups, EA stimulation was administrated for 30 min, once a day for 4 days. The sleep onset latency and sleep duration were measured after intraperitoneal injection of pentobarbital sodium (35 mg/kg). The concentrations of IL-1 ß， BDNF, MLT, PGD2and CORT in the serum were detected by ELISA. RESULTS: After EA stimulation of GV 20, HT 7, SP 6 and GV 20＋HT 7＋SP 6, the sleep latency was significantly shortened (P<0.05, P<0.01, except SP 6), and the sleep duration was remarkably prolonged in comparison with the model group (P<0.05, P<0.01), and the therapeutic effects of EA-GV 20＋HT 7＋SP 6 were significantly superior to those of EA-GV 20, EA-HT 7 and EA-SP 6 in shortening the sleep latency and lengthening the sleep duration (P<0.05). Following modeling, the concentrations of IL-1 ß， BDNF, PGD2 and MLT were significantly down-regulated, and the CORT level was markedly up-regulated in the model group relevant to the control group (P<0.05). Following EA，modeling induced dramatic decrease of serum IL-1 ß， BDNF, PGD2 and MLT was considerably up-regulated, and the increased CORT level markedly down-regulated in the EA-GV 20, EA-HT 7, EA-SP 6 and EA-GV 20＋HT 7＋SP 6 groups (P<0.05). The effects of EA-GV 20＋HT 7＋SP 6 were evidently superior to those of EA-GV 20 and EA-SP 6 in up-regulating serum IL-1 ß， BDNF and PGD2levels, and to those of HT 7, GV 20 and SP 6 in up-regulating serum MLT level, and significantly superior to those of EA-ST 7 and EA-SP 6 in down-regulating serum CORT (P<0.05). CONCLUSION: EA stimulation of HT 7, GV 20, SP 6 and GV 20＋HT 7＋ SP 6 can significantly improve the sleep in insomnia rats, which is closely associated with its effects in regulating serum sleep-promoting factors and awakening-promoting factor. Joint administration of EA of GV 20＋HT 7＋ SP 6 has a better effect than the single acupoint mentioned above.

[High Mobility Group Box 1/ CD 24 Receptor/ß-EP Signaling in "Zusanli" (ST 36) Region Contributes to Electroacupuncture Analgesia in Rats with Neuropathic Pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of high mobility group box 1 (HMGB 1) and its receptor CD 24 proteins and ß-endorphin (ß-EP) content in "Zusanli" (ST 36) region in rats with chronic constriction injury (CCI), so as to explore its mechanisms underlying pain relief. METHODS: Thirty male Wistar rats were rando-mized into control, CCI model and EA groups (n＝ 10 rats in each). The neuropathic pain model was established by ligature of the left sciatic nerve to induce CCI in the model and EA groups, and sham operation was performed in rats of the control group. Paw with drawal latency (PWL, thermal pain threshold) of the bilateral hind-limbs was detected by using an algesia-detector. Eight days after CCI operation, EA was applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 30 min, once daily for 5 days. The acetylated-HMGB 1 expression was determined by immunoprecipitation, and the expression of HMGB 1 and toll like receptor 4 (TLR 4) proteins and CD 24 mRNA were detected using Western blot and fluorescent quantitative real time-PCR, respectively, and the content of ß-EP in the acupoint region was assayed by enzyme linked immunosorbent assay (ELISA). Anti-CD 24 neutralizing antibody (200 µL, 100 µg/mL) was injected into ST 36 region once daily for 3 days for verifying the involvement of HMGB 1/CD 24 signaling in EA analgesia. RESULTS: Compared with the control group, the bilateral PWL difference values in the other two groups were significantly increased (P<0.05), meaning an occurrence of hyperalgesia after CCI. In comparison with the CCI model group, the hyperalgesia in the EA group was obviously decreased (P<0.05). After CCI, the expression levels of HMGB 1 and TLR 4 proteins were considerably increased compared with those in the control group (P<0.05). After 5-times' EA, the acetylated-HMGB 1, the expression of CD 24 mRNA, and the content of ß-EP were notably up-regulated (P<0.05), and there were no obvious changes in the expression levels of HMGB 1 and TLR 4 proteins (P>0.05). After local injection of anti-CD 24 antibody, EA-induced increases of ß-EP content and reduction of thermal pain threshold were significantly suppressed (P<0.05). CONCLUSION: EA of ST 36 and GB 34 can alleviate neuropathic pain in CCI rats, which is associated with its effects in up-regulating ß-EP content, and HMGB 1 protein and CD 24 mRNA expression levels in ST 36 region. The activated HMGB 1/CD 24/ß-EP signaling contributes to EA-ST 36 induced analgesia.

Effect of electroacupuncture on thermal pain threshold and expression of calcitonin-gene related peptide, substance P and γ-aminobutyric acid in the cervical dorsal root ganglion of rats with incisional neck pain.

OBJECTIVE: Acupuncture therapy effectively reduces post-surgical pain, but its mechanism of action remains unclear. The aim of this study was to investigate whether expression of γ-aminobutyric acid (GABA) and the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) in the primary sensory neurons of cervical dorsal root ganglia (DRG) are involved in electroacupuncture (EA)-induced analgesia in a rat model of incisional neck pain. METHODS: The pain model was established by making a longitudinal midline neck incision in 60 rats. Another 15 rats underwent sham surgery (normal group). Post-incision, 15 rats remained untreated (model group) and 45 rats underwent EA (frequency 2/100 Hz, intensity 1 mA) at bilateral LI18, LI4-PC6 or ST36-GB34 (n=15 each) for 30 min at 4 hours, 24 hours, and 48 hours post-surgery, followed by thermal pain threshold (PT) measurement. 30 min later, the rats were euthanased and cervical (C3-6) DRGs removed for measurement of immunoreactivity and mRNA expression of SP/CGRP and the GABAergic neuronal marker glutamic acid decarboxylase 67 (GAD67). RESULTS: Thermal PT was significantly lower in the model group versus the normal group and increased in the LI18 and LI4-PC6 groups but not the ST36-GB34 group compared with the model group. Additionally, EA at LI18 and LI4-PC6 markedly suppressed neck incision-induced upregulation of mRNA/protein expression of SP/CGRP, and upregulated mRNA/protein expression of GAD67 in the DRGs of C3-6 segments. CONCLUSIONS: EA at LI18/LI4-PC6 increases PT in rats with incisional neck pain, which is likely related to downregulation of pronociceptive mediators SP/CGRP and upregulation of the inhibitory transmitter GABA in the primary sensory neurons of cervical DRGs.

[Transcutaneous Electrostimulation of Auricular Otopoints Reduces Epileptic Attack Possibly by Suppressing Hippocampal Gliocyte Proliferation and Regulating IL-6 and IL-10 Expression in Chronic Temporal Lobe Epilepsy Rats].

OBJECTIVE: To observe the effect of transcutaneous otopoint electrostimulaiton (TCOES) on seizure frequency, immunoreactivity of hippocampal gliocytes and expression of proinflammatory cytokine interleukin-6(IL-6) and anti-inflammatory cytokine IL-10 in chronic temporal lobe epilepsy (CTLE) rats, so as to investigate its antiepileptic mechanism. METHODS: Thirty-six SD rats were randomly divided into control, model and TCOES groups (n=12 in each group). The CTLE model was established by intraperitoneal injection (i.p.i.) of lithium chloride (127.2 mg/kg), scopolamine (1 mg/kg, 20 h after the 1st injection) and pilocarpine (10 mg/kg, 30 min after scopolamine injection). Rats of the control group were treated by i.p.i. of normal saline. TCOES (1 mA, 20 Hz) was applied to bilateral otopoint "Heart"-"Lung"-"Subcortex" region for 20 min, once daily for 6 weeks. The epileptic attack was observed by a video monitoring system. The numbers of ionized calcium-binding adapter molecule-1 (Iba 1)-labeled microgliacytes and glial fibrillary acidic protein (GFAP)-labeled astrocytes in the CA 1 and CA 3 regions of hippocampus were counted under light microscope after immunostaining, and the expression levels of hippocampal IL-6 and IL-10 proteins and genes were determined by immunofluorescence and quantitative real-time PCR, respectively. RESULTS: After TCOES intervention, the seizure frequency was significantly decreased in comparison with pre-treatment(P<0.05), modeling-induced dramatic increase of the numbers of microgliacytes and astrocytes,IL-6 immunoactivity in the hippocampal CA 1 and CA 3 regions, and IL-6 mRNA expression in the hippocampus were significantly suppressed (P<0.05), and hippo-campal IL-10 immunoactivity and mRNA expression were considerably up-regulated in comparison with the model group (P<0.05). CONCLUSIONS: TCOES intervention has an antiepileptic effect in CTLE rats, which may be associated with its effects in suppressing gliocyte proliferation, suppressing the expression of proinflammatory cytokine IL-6, and up-regulaiting the expression of anti-inflammatory cytokine IL-10 in the hippocampus.

[Effects of Electroacupuncture on Activities of Satellite Glial Cells of Dorsal Root Ganglia in Rats with Neck Incision Pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Futu"(LI 18), etc. on activities of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) in rats with neck-incision pain so as to explore its mechanism underlying reduction of post-surgical pain of thyroidectomy. METHODS: Male SD rats were randomly divided into control, model, EA-Futu (LI 18), EA-Hegu (LI 4)-Neiguan (PC 6), and EA-Zusanli (ST 36)-Yanglingquan (GB 34) groups, with 20 rats in each group. The neck-incision pain model was established by making a longitudinal incision and repeated mechanical stimulation. In the EA-LI 18, EA-LI 4-PC 6 and EA-ST 36-GB 34 groups, EA stimulation was administrated for 30 min, once a day,continuously for 3 days. The thermal pain threshold (PT) of the neck-incision region was detected. The immunoactivity of glial fibrillary acidic protein (GFAP,a specific marker for SGCs) and connexin 43 (Cx 43) of DRGs (C 2-C 6) was determined by fluorescent immunohistochemistry. The expression levels of GFAP, IL-1 ß, IL-6, and TNF-α mRNAs were determined by quantitative Real-time PCR, and the contents of IL-1 ß,IL-6,TNF-α assayed by enzyme linked immunosorbent assay (ELISA) and the expression of Cx 43 protein was detected by Western blot. RESULTS: After EA intervention at LI 18 and LI 4-PC 6 (but not ST 36-GB 34), neck incision-induced reduction of the thermal PT was obviously prolonged in comparison with the model group (P<0.05),suggesting a pain relief. The expression levels of GFAP, IL-1 ß, IL-6 and TNF-α mRNAs and Cx 43 protein, and the contents of IL-1 ß, IL-6 and TNF-α in C 2-C 6 DRGs were all significantly up-regulated in the model group relevant to those of the control group (P<0.05). Following EA, modeling induced dramatic increase of expression of GFAP, IL-1 ß, IL-6 and TNF-α mRNAs and Cx 43 protein in both EA-LI 18 and EA-LI 4-PC 6 groups, and the contents of IL-1 ß and TNF-α in the EA-LI 18 group, IL-6 in the EA-LI 4-PC 6 group was considerably down-regulated (P<0.05). In comparison with the model group, no significant changes were found in all the abovementioned indexes of EA-ST 36 -GB 34 group except the down-regulated IL-1 ß and TNF-α mRNAs, in the contents of IL-1 ß and TNF-α of the EA-LI 4-PC 6 group, and in the IL-6 content of the EA-LI 18 group (P>0.05). CONCLUSIONS: EA stimulation of LI 18 and LI 4-PC 6 can significantly suppress pain reaction of neck incision in the rat, which is closely associated with its effects in down-regulating the activity of SGCs, decreasing the release of pro-inflammatory cytokines and in weakening the expression of Cx 43 in the cervical DRGs.

[Effects of Transcutaneous Electrostimulation of Auricular Points on Behavior and Hippocampal IL-1 ß and TNF-α Expression in Temporal Lobe Epilepsy Rats].

OBJECTIVE: To observe the effect of transcutaneous otopoint electrostimulation (TCOES) on behavior and expression of hippocampal interleukin-1 ß (IL-1 ß) and tumor necrosis factor-α (TNF-α) expression in lithium-pilocarpine induced chronic spontaneous temporal lobe epilepsy (TLE) rats, so as to investigate its antiepileptic mechanism. METHODS: Thirty-six SD rats were randomly divided into control, model and TCOES groups (n=12 in each group). The epilepsy model was established by intraperitoneal injection of lithium chloride (127.2 mg/kg), scopolamine(1 mg/kg, 20 h after the 1st injection) and pilocarpine (10 mg/kg, 30 min after scopolamine injection). Rats of the control group were treated by injection of normal saline(i.p.i.). Transcutaneous electrostimulation (1 mA, 20 Hz) was applied to bilateral otopoints "Heart" "Lung" and "Subcortex" for 20 min, once daily for 6 weeks except the weekends. The behavior reactions were observed by a video monitoring system. The expression of IL-1 ß and TNF-α proteins and genes in the hippocampus were determined by immunofluorescence and quantitative real-time PCR, separately. RESULTS: Behavioral observation showed that after TCOES intervention, the frequency of epilepsy attack was significantly decreased in comparison with pre-treatment (P<0.05). Immunofluorescence and real-time PCR showed that compared with the control group, the immunoactivity of IL-1 ß and TNF-α in both hippocampal CA 1 and CA 3 regions and hippocampal IL-1 ß and TNF-α gene expression were obviously increased in the model group (P<0.05, P<0.01). Following TCOES, the increased hippocampal IL-1 ß and TNF-α and IL-1 ß mRNA and TNF-α mRNA expression levels were all suppressed (P<0.05, P<0.01). CONCLUSIONS: TCOES intervention has an antiepileptogenic effect in temporal lobe epilepsy rats, which may be related to its effects in down-regulating expression of proinflammatory cytokine IL-1 ß and TNF-α in the hippocampus.

[Effect of electroacupuncture at different acupoints on expression of cervico-spinal GDNF and BDNF and their receptor genes in neck-incision pain rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Futu" (LI 18), etc. on the expression of genes of glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and their receptors in the cervico-spinal cord in neck-incision pain rats, so as to study its mechanism underlying incision-pain relief. METHODS: A total of 50 Sprague Dawley male rats were randomly divided into normal, model (incision pain), EA-Futu (LI 18), EA- Hegu (LI 4) - Neiguan (PC 6, LI 4-PC 6) and EA-Zusanli (ST 36)-Yanglingquan (GB 34, ST 36-GB 34) groups (n = 10/group). A 1.5 cm long longitudinal incision was made along the midline of the neck under anesthesia to duplicate neck-incision pain model. Thermal pain threshold (PT) was measured before and after modeling and after the treatment, respectively. EA (1-2 mA, 2 Hz/100 Hz) was applied to bilateral LI 18, PC 6-LI 4 and ST 36-GB 34 for 30 min. The expression of genes of GDNF and its receptor GFRalpha-1, and BDNF and its receptors TrkA, TrkB in the spinal cord (C1 - C4) tissue was detected by quantitative real-time-PCR. RESULTS: In comparison with pre-modeling in the same one group, the thermal PT levels were decreased obviously in the model, LI 18, LI 4-PC 6 and ST 36-GB 34 groups after neck incision (P < 0.05). Compared with the model group, the PT levels were increased markedly in LI 18, LI 4-PC 6 and ST 36-GB 34 groups after the EA treatment (P < 0.05). Correspondingly, the expression levels of spinal GDNF mRNA and GFRalpha-1 mRNA were obviously lower, as well as BDNF mRNA was markedly higher in the model group than in the control group (P < 0.05), and those of TrkA mRNA and TrkB mRNA were increased slightly in the model group than in the control group (P > 0.05). In comparison with the model group, the expression levels of spinal GDNF mRNA and GFRalpha-1 mRNA were up-regulated considerably (P < 0.001), whereas those of BDNF mRNA, TrkA mRNA and TrkB mRNA were decreased slightly (P > 0.05) in the LI 18, LI 4-PC 6 and ST 36-GB 34 groups after EA treatment. CONCLUSION: EA stimulation can significantly suppress pain reaction of neck incision, which is closely associated with its effects in up-regulating the expression of GDNF and its receptor GFRalpha-1 genes in the cervical spinal cord (C1 - C4).

[Influence of electroacupuncture on efficacy of estrogen in regulating hypothalamic reproductive endocrine activity in rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the efficacy of estrogen-induced regulation of reproductive endocrine in ovariectomized (OVX) rats. METHODS: Thirty SD rats were randomly divided into control, model (OVX), EA-Guanyuan(CV 4), Estrogen (E) and EA+ E groups (n = 6 in each group). OVX model was duplicated by removing the bilateral ovaries. EA (5 Hz/20 Hz, 1- 2 mA) was applied to "Guanyuan" (CV 4) for 30 min, once every other day and for 3 sessions. Estrogen at doses of 0.5 mg/kg and 0. 25 mg/kg was intraperitonealy given to rats in the E group and EA+ E group, respectively. Serum estrogen and cortisol contents were assayed by enzyme linked immunosorbent assay and the expression of hypothalamic gonadotrophin releasing hormone(GnRH)mRNA, G protein-coupled receptor 54(GPR 54)mRNA, kisspeptin-1 (Kiss-l) mRNA and corticotropin releasing hormone (CRH) mRNA were detected by using real time polymerase chain reaction (RT-PCR). RESULTS: Compared with the control group, the expression levels of GnRH mRNA, GPR 54 mRNA and Kiss-1 mRNA in hypothalamus in the model group were increased significantly (P<0. 05), while the expression of hypothalamic CRH mRNA was down-regulated obviously (P<0. 05), and serum cortisol and estrogen levels were reduced significantly (P<0. 05). In comparison with the model group, the expression levels of hypothalamic GnRH mRNA in the EA, E and EA+ E groups, and those of hypothalamic GPR 54 mRNA and Kiss-1 mRNA in the E and EA+ E groups were down-regulated considerably (P<0. 05); whereas hypothalamic CRH mRNA expression levels in the E and EA+ E groups were up-regulated obviously (P<0. 05),and serum cortisol and estrogen contents in the E and EA+ E groups were increased remarkably (P<0.05). Comparison among the EA and EA+ E groups showed that the effects of EA+ E group were significantly superior to those of EA group in down-regulating hypothalalmic GnRH mRNA and GPR 54 mRNA expression, and in up-regulating hypothalamic CRH mRNA expression and serum cortisol and estrogen contents (P<0.05). No significant differences were found between E and EA + E groups in down-regulating hypothalamic GnRH mRNA, GPR 54 mRNA and Kiss-1 mRNA expression, and in up-regulating hypothalamic CRH mRNA expression and serum cortisol and estrogen levels (P>0. 05). CONCLUSION: EA-CV 4 is able to potentiate the effects of low-dose of estrogen in down-regulating hypothalamic GnRH mRNA,GPR 54 mRNA and Kiss-1 mRNA expression, and up-regulating hypothalamic CRH mRNA expression and raising serum cortisol and estrogen contents in OVX rats, showing a favorable modulation effect on reproductive endocrine activity.

[Application of molecular biological techniques to the study on acupuncture and moxibustion].

In the present paper, the authors introduce recent progress of application of molecular biological techniques in the studies on the underlying mechanism of acupuncture therapy for 1) resisting inflammation and relieving pain, 2) resisting tumors, 3) improving ischemic cerebral and myocardial injury, and 4) delaying aging from gene transcription and expression (synthesis of mRNA); and for relieving sciatic nerve injury, spinal traumatic injury, and cerebral ischemia from protein expression or phosphorylation. In addition, the authors also make a preliminary analysis on the extant problems of the application of techniques of both genomics and proteomics in acupuncture research at the present and predict their applicable prospect.

[Changes of cutaneous temperature and the activity of Ca2+- and Mg2+ -ATPase in the tissues along the running course of meridians in rabbit].

OBJECTIVE: To study the interrelation between the change of temperature and Ca2+-, Mg2+ -ATPase activity in the tissues along the running course of meridians in the rabbit. METHODS: Seven healthy mongrel rabbits were used in this study. Before and after moxibustion of "Zusanli" (ST36) and "Yinlingquan" (SP9), the cutaneous temperature along the Stomach Meridian and Spleen Meridian was detected with an infra-red thermography. The tissues showing higher temperature (including skin, subcutaneous tissue and muscle, 100 mg) and non-higher temperature (skin, subcutaneous tissue and muscle about 0.5 cm beside the higher temperature region, 100 mg) after moxibustion were sampled respectively for detecting the activity of Ca2+- and Mg2+ -ATPase with enzymologic method. RESULTS: After moxibustion of "Zusanli" (ST36) and 'Yinlingquan" (SP9), the temperature of the skin along the Stomach Meridian and Spleen Meridian on the same side increased significantly (P<0.05, 0.01); correspondingly, the activity of Mg2+ -ATPase in the higher temperature regions (lateral side of the left thigh, and the medial side of the right hindlimb) increased moderately and significantly separately in comparison with the control regions (P<0.05). No marked changes were found in the activity of Ca2+ -ATPase in the same tissues of the higher temperature regions (P>0.05). CONCLUSION: In the tissues of higher temperature region after moxibustion of SP9, Mg2+ -ATPase activity increased evidently, that may contribute to the increase of temperature along the skin of the Spleen Meridian after moxibustion, while Ca2+ -ATPase may play a minor role therein.