Gao Yh

Proteomic Analysis of Differential Proteins Related to Anti-nociceptive Effect of Electroacupuncture in the Hypothalamus Following Neuropathic Pain in Rats

Increasing evidence has been accumulated for the effectiveness of acupuncture therapy in relieving pain. However, there are limited data on regulation of protein expression after electroacupuncture (EA) intervention. Thus, the present study is designed to determine changes in protein expression following EA stimulation in rats with sciatic nerve chronic constrictive injury (CCI) induced neuropathic pain. Sixty Wistar rats were equally randomized into normal control group, CCI group, and CCI with EA stimulation (EA) group. The CCI model was established by ligature of the left sciatic nerve. EA stimulation was applied at Zusanli (ST36) and Yanglingquan (GB34) in the EA group. Differentially expressed hypothalamic proteins in the three groups were identified by 2-D gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The functional clustering and pathway of the identified proteins were analyzed by Mascot software. Results showed that, after CCI, the thermal pain threshold of the affected hind footpad was decreased and was reversed gradually by 12 sessions of EA treatment. Following EA intervention, there were 17 hypothalamic proteins identified with significant changes in the expression (>twofold). Three gene-ontologies (oxidoreductase activity, oxidation reduction, and protein binding) were enriched, while there was a significant regulation of glycolysis/gluconeogenesis/hexose metabolism pathway. These data demonstrate that EA intervention can attenuate pain via regulation of expression of multiple proteins in the hypothalamus. Further, hypothalamic glucose metabolism may be important in supporting energy and neurotransmitter homeostasis in the effects of EA intervention.

Effects of repeated electroacupuncture on β-endorphin and adrencorticotropic hormone levels in the hypothalamus and pituitary in rats with chronic pain and ovariectomy

ObjectiveTo explore the mechanism of electroacupuncture (EA)-induced cumulative analgesic effects on chronic pain in rats with or without ovariectomy (OVX).MethodsA total of 110 female Wistar rats were randomized into normal control (n=10), chronic constrictive injury (CCI, n=10), CCI+EA (n=30), OVX+CCI (n=30), and OVX+CCI+EA (n=30) groups. Each of the latter 3 groups was further divided into 2 days (2 d), 2 weeks (2 W) and 3 weeks (3 W) subgroups, respectively (n=10 in each subgroup). The CCI pain model was established by ligature of the right sciatic nerve, and the memory impairment model duplicated by OVX. The paw withdrawal latency (PWL, pain threshold) of the bilateral footplates was detected by radiant heat irradiation, and the bilateral difference in PWL (PWLD) was used to evaluate changes in the pain reaction. Morris water maze test was conducted for evaluating the rats’ learning-memory ability. EA was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) for 2 d, 2 W and 3 W, respectively. Pituitary and hypothalamic β-endorphin (EP) and adrenocorticotrophic hormone (ACTH) contents were detected by immunoradioassay.ResultsCompared with the CCI group, PWLD of the CCI+EA-3 W group decreased signifificantly (P<0.05). Compared with the OVX+CCI group, PWLD of the OVX+CCI+EA-3 W group was lowered considerably (P<0.05), but the value was markedly higher than its basal value and those of the normal control and CCI+EA groups (P<0.05). In comparison with the sham-OVX group, the escape latency, swimming distance (SD) in the target quadrant and total SD were increased remarkably in the OVX group (P<0.05), while the number of target platform crossings was decreased signifificantly (P<0.05), suggesting an impairment of the OVX rats’ learning-memory ability. In simple CCI rats, both β-EP and ACTH contents of the pituitary increased markedly (P<0.05), and those of the hypothalamus decreased obviously compared to the normal control group (P<0.05). After EA, pituitary and hypothalamic ACTH levels were signifificantly lowered at 2 d and hypothalamic ACTH and β-EP contents increased obviously at 3 W in comparison with the CCI group (P<0.05). In OVX+CCI rats, following EA, pituitary β-EP contents at 2 d, 2 W and 3 W, and hypothalamic β-EP and ACTH contents at 2 W and hypothalamic ACTH levels at 3 W increased signifificantly (P<0.05), but hypothalamic β-EP level at 3W decreased markedly (P<0.05). The effects of repeated EA in lowering pituitary ACTH and raising hypothalamic β-EP and ACTH levels disappeared after OVX+CCI.ConclusionsRepeated EA has a cumulative analgesic effect, which is closely associated with its effects in regulating pituitary and hypothalamicβ-EP and ACTH levels. OVX may weaken the analgesic effect of EA by affecting hypothalamic-pituitary axis activity.

Acupuncture effects on the hippocampal cholinergic system in a rat model of neuropathic pain

The present study observed the effects of repeated electroacupuncture of Zusanli (ST36) and Yanglingquan (GB34) on expression of hippocampal acetylcholinesterase, vesicular acetylcholine transporter, and muscarinic M1 receptor mRNA in chronic constrictive injury (neuropathic pain) and/or ovariotomy rats. Results demonstrated increased expression of hippocampal acetylcholinesterase, vesicular acetylcholine transporter, and muscarinic M1 receptor mRNA, as well as decreased pain threshold, in a rat model of chronic neuropathic pain after electroacupuncture. The effects of electroacupuncture increased with prolonged time, but the above-mentioned effects decreased in memory-deficient animals. Results indicated that repeated electroacupuncture has a cumulative analgesic effect, which is closely associated with upregulation of acetylcholinesterase and vesicular acetylcholine transporter activity, as well as M1 receptor mRNA expression and memory.

Correlation between the cumulative analgesic effect of electroacupuncture intervention and synaptic plasticity of hypothalamic paraventricular nucleus neurons in rats with sciatica.

In the present study, a rat model of chronic neuropathic pain was established by ligation of the sciatic nerve and a model of learning and memory impairment was established by ovariectomy to investigate the analgesic effect of repeated electroacupuncture stimulation at bilateral Zusanli (ST36) and Yanglingquan (GB34). In addition, associated synaptic changes in neurons in the paraventricular nucleus of the hypothalamus were examined. Results indicate that the thermal pain threshold (paw withdrawal latency) was significantly increased in rats subjected to 2-week electroacupuncture intervention compared with 2-day electroacupuncture, but the analgesic effect was weakened remarkably in ovariectomized rats with chronic constrictive injury. 2-week electroacupuncture intervention substantially reversed the chronic constrictive injury-induced increase in the synaptic cleft width and thinning of the postsynaptic density. These findings indicate that repeated electroacupuncture at bilateral Zusanli and Yanglingquan has a cumulative analgesic effect and can effectively relieve chronic neuropathic pain by remodeling the synaptic structure of the hypothalamic paraventricular nucleus.

The cumulative analgesic effect of repeated electroacupuncture involves synaptic remodeling in the hippocampal CA3 region.

In the present study, we examined the analgesic effect of repeated electroacupuncture at bilateral Zusanli (ST36) and Yanglingquan (GB34) once a day for 14 consecutive days in a rat model of chronic sciatic nerve constriction injury-induced neuropathic pain. In addition, concomitant changes in calcium/calmodulin-dependent protein kinase II expression and synaptic ultrastructure of neurons in the hippocampal CA3 region were examined. The thermal pain threshold (paw withdrawal latency) was increased significantly in both groups at 2 weeks after electroacupuncture intervention compared with 2 days of electroacupuncture. In ovariectomized rats with chronic constriction injury, the analgesic effect was significantly reduced. Electroacupuncture for 2 weeks significantly diminished the injury-induced increase in synaptic cleft width and thinning of the postsynaptic density, and it significantly suppressed the down-regulation of intracellular calcium/calmodulin-dependent protein kinase II expression in the hippocampal CA3 region. Repeated electroacupuncture intervention had a cumulative analgesic effect on injury-induced neuropathic pain reactions, and it led to synaptic remodeling of hippocampal neurons and upregulated calcium/calmodulin-dependent protein kinase II expression in the hippocampal CA3 region.

Analysis on interrelation between electroacupuncture-induced cumulative analgesic effect and hypothalamic cholinergic activities in chronic neuropathic pain rats

ObjectiveTo observe the effects of repeated electroacupuncture (EA) of Zusanli (ST36)- Yanglingquan (GB34) on hypothalamic acetylcholinesterase (AchE) and vesicular acetylcholine (ACh) transporter (VAChT) activities and choline acetyltransferase (ChAT) mRNA and muscarinic M1 receptor (M1R) mRNA expression in chronic constrictive injury (CCI) and/or ovariectomy (OVX) rats so as to reveal its underlying mechanism in cumulative analgesia.MethodsA total of 103 female Wistar rats were randomly divided into normal control (n =15), CCI (n =15), CCI+EA2d (n =15), CCI+EA2W (n =15), OVX+CCI =13), OVX+CCI+EA2d (n =15), and OVX+CCI+EA2W groups (n =15). CCI model was established by ligature of the unilateral sciatic nerve with surgical suture. Memory impairment model was established by removal of the bilateral ovaries. Morris water test was conducted to evaluate the OVX rats’ memorylearning ability, and the thermal pain threshold (PT) of the bilateral paws was detected the next morning after EA. EA (2/15 Hz, 1 mA) was applied to bilateral ST36-GB34 for 30 min, once daily for 2 days or 2 weeks, respectively. Hypothalamic AChE activity was detected by histochemistry, VAChT immunoactivity was determined by immunohistochemistry, and ChAT mRNA and M1R mRNA expressions were assayed by reverse transcription-polymerase chain reaction (RT-PCR).ResultsIn comparison with the normal control group, the AChE activity in hypothalamic arcuate nucleus (ARC) and supraoptic nucleus (SON) regions of CCI group, AChE activity in paraventricular nucleus (PVN), ARC, and SON regions of OVX+CCI group, and hypothalamic muscarinic M1R mRNA expression levels in both CCI and OVX+CCI groups were down-regulated significantly (P <0.05). Compared with the CCI group, the AChE activities in hypothalamic ARC and SON regions of CCI+EA2d and CCI+EA2W groups and PVN region of CCI+EA2W group and hypothalamic ChAT mRNA and M1R mRNA expression levels in CCI+EA2W group were up-regulated considerably (P <0.05). In comparison with the OVX+CCI group, the AChE activities in PVN, ARC, and SON regions and the expressions of hypothalamic ChAT mRNA and VAChT in ARC region of OVX+CCI+EA2W group were up-regulated remarkably (P <0.05). The effects in rats of CCI+EA2W group were evidently superior to those of OVX+CCI+EA2d group in up-regulating AChE activities in PVN, ARC, and SON regions, VAChT immunoactivity in ARC region, and expression levels of hypothalamic ChAT mRNA and M1R mRNA (P <0.05). Similar situations were found in OVX+CCI rats after EA2W. It suggested a cumulative effect after repeated EA of ST36-GB34. Comparison between CCI+EA2W and OVX+CCI+EA2W groups showed that the effects in rats of the former group were evidently better than those of the latter group in up-regulating AChE activity in ARC and SON regions and the expressions of hypothalamic ChAT mRNA and M1 mRNA (P <0.05), suggesting a reduction of EA2W effects after OVX.ConclusionRepeated EA can significantly up-regulate AChE and VAChT activities and ChAT mRNA and M1R mRNA expressions in the hypothalamus of CCI and OVX+CCI rats, which may contribute to the cumulative analgesic effects of repeated EA and be closely related to the animals’ neuromemory ability.

Effect of Electroacupuncture Intervention on Expression of CGRP, SP, COX-1, and PGE2 of Dorsal Portion of the Cervical Spinal Cord in Rats with Neck-Incision Pain

The present study was aimed to determine if cervicospinal substance P (SP) and its neurokinin-1 receptor (NK-1R), calcitonin gene-related peptide (CGRP), cyclooxygenase-1 (COX-1), and prostaglandin E2 (PGE2) were involved in electroacupuncture (EA) analgesia in neck-incision pain rats. EA intervention was applied to bilateral Futu (LI18), Hegu (LI4)-Neiguan (PC6), and Zusanli (ST36)-Yanglingquan (GB34) for 30 min. Cervicospinal SP and CGRP immunoactivity was detected by immunofluorescence technique, NK-1R and COX-1 protein and mRNA expression levels were determined using Western blot and real-time PCR, respectively, and PGE2 content was measured using ELISA. Outcomes indicated that EA of EA-LI18 and LI4-PC6 (not ST36-GB34) significantly suppressed neck-incision induced decrease of thermal pain threshold (P < 0.05). EA stimulation of LI18 and LI4-PC6 markedly inhibited neck-incision induced upregulation of SP and CGRP immunoactivity, NK-1 R and COX-1 mRNA and protein expression levels, as well as the increase of PGE2 content in the dorsal cervicospinal cord (P < 0.05). These findings showed that LI18 and LI4-PC6 EA stimulation-induced downregulation of SP, CGRP, NK-1R, COX-1, and PGE2 levels in the dorsal cervicospinal cord may contribute to their effects in relieving neck-incision pain. This study highlights the targets of EA intervention for reducing post-thyroid-surgery pain for the first time.

The Effect of Repeated Electroacupuncture Analgesia on Neurotrophic and Cytokine Factors in Neuropathic Pain Rats

Chronic pain is a common disability influencing quality of life. Results of previous studies showed that acupuncture has a cumulative analgesic effect, but the relationship with spinal cytokines neurotrophic factors released by astrocytes remains unknown. The present study was designed to observe the effect of electroacupuncture (EA) treatment on spinal cytokines neurotrophic factors in chronic neuropathic pain rats. The chronic neuropathic pain was established by chronic constrictive injury (CCI). EA treatment was applied at Zusanli (ST36) and Yanglingquan (GB34) (both bilateral) once a day, for 30 min. IL-1β mRNA, TNF-α mRNA, and IL-1 mRNA were detected by quantitative real-time PCR, and the proteins of BDNF, NGF, and NT3/4 were detected by Western blot. The expression levels of cytokines such as IL-1β mRNA, TNF-α mRNA, IL-6 mRNA, and neurotrophic factors such as BDNF, NGF, and NT3/4 in the spinal cord were increased significantly after CCI. The astrocytes released more IL-1β and BDNF after CCI. Repeated EA treatment could suppress the elevated expression of IL-1β mRNA, TNFα mRNA, and BDNF, NGF, and NT3/4 but had no effect on IL-6 mRNA. It is suggested that cytokines and neurotrophic factors which may be closely associated with astrocytes participated in the process of EA relieving chronic pain.

Effects of Electroacupuncture at Different Points on Colorectal Distention-induced Changes in Blood Pressure, Electrogastrogram, Gastric Tension and Gastric Blood Flow

OBJECTIVE To investigate the specificity of the effects of electroacupuncture (EA) at different acupoints on gastric functional activity and gastric blood flow after colorectal distension (CRD) in the rat. METHODS Fifty Wistar rats were randomly divided into 5 groups: a control group, a Zusanli group, a non-point group, a Taichong group and a Neiguan group. Rats were anesthetized after 18 h of fasting, and a rat model of nociceptive blood pressure elevation and abnormal electrogastrogram (EGG) and gastric tension (GT) was prepared by gasbag-induced CRD. EA was given bilaterally, and its effects on gastric blood flow (GBF) of the arcus vasculosi of the greater omentum, blood pressure, EGG fast wave properties, and gastric smooth muscle tension (GT) were quantified. RESULTS CRD induced an increase in blood pressure that was significantly inhibited by EA at all points (all P < 0.05), and the inhibitory effects were greater in the Zusanli and Neiguan groups compared to the non-point group (both P < 0.05). CRD also caused reductions in GT and GBF, and in the amplitude, frequency and duration of EGG waves (all P < 0.05). These effects were also reversed by EA. The effects of EA on EGG wave amplitude, GT, and GBF were superior in the Zusanli group compared to the Taichong group, Neiguan group, and the non-point group. CONCLUSION EA significantly counteracted CRD-induced changes in blood pressure, GBF, EGG, and GT. The effects of stimulation at Zusanli (ST 36) were significantly greater compared to other points, indicating relative specificity of this acupoint.

Electroacupuncture Reduces the Effects of Acute Noxious Stimulation on the Electrical Activity of Pain-Related Neurons in the Hippocampus of Control and Neuropathic Pain Rats

To study the effects of acupuncture analgesia on the hippocampus, we observed the effects of electroacupuncture (EA) and mitogen-activated protein kinase (MEK) inhibitor on pain-excited neurons (PENs) and pain-inhibited neurons (PINs) in the hippocampal area CA1 of sham or chronic constrictive injury (CCI) rats. The animals were randomly divided into a control, a CCI, and a U0126 (MEK1/2 inhibitor) group. In all experiments, we briefly (10-second duration) stimulated the sciatic nerve electrically and recorded the firing rates of PENs and PINs. The results showed that in both sham and CCI rats brief sciatic nerve stimulation significantly increased the electrical activity of PENs and markedly decreased the electrical activity of PINs. These effects were significantly greater in CCI rats compared to sham rats. EA treatment reduced the effects of the noxious stimulus on PENs and PINs in both sham and CCI rats. The effects of EA treatment could be inhibited by U0126 in sham-operated rats. The results suggest that EA reduces effects of acute sciatic nerve stimulation on PENs and PINs in the CA1 region of the hippocampus of both sham and CCI rats and that the ERK (extracellular regulated kinase) signaling pathway is involved in the modulation of EA analgesia.