| Background:Acupuncture-moxibustion is an important part of traditional Chinese medicine, and it is effective to relieve various kinds of pain. The present therapy can be used to treat various kinds of acute, chronic, and cancer pain in clinic. Thyroidectomy is one of the best indications for acupuncture anesthesia. Postoperative pain is a kind of acute pian, clinically,acupuncture can relieve many kinds of postoperative pain, promote recovery of the traumatic tissues, reduce nausea and vomitting and improve prognosis.It has been demonstrated by the animal experiments that EA can alleviate the reaction of pain of foot incision in Rats. However, the underlying mechanism of acupuncture in relieving cervical incision pain is still on researching and no reports have been seen up to now. The spinal cord is the first station where the nociceptive information enters the central nervous system, the dorsal horn, especially its superficial layer contains various kinds of neurotransmitters, mediators which play an important role in transmitting and regulating pain information in the spinal cord level. The present study is aimed to explore the spinal mechanism of electroacupuncture of Futu (LI18), etc. underlying relieving pain in neck-incision pain rats by observing the expression of N-methyl D-aspartate receptor subtype2B (NMDA2B), metabotropic glutamate receptor subtype5(mGluR5), γ-aminobutyric acid B1receptor(GABAB1R), intracellar cyclic adenosine monophosphate(cAMP), mitogen-activated protein kinases(MAPK), cAMP-response element binding protein(CREB) genes and proteins in the cervical segments (C1-C4) of the spinal cord. In addition, this study is also designed to observe the effects of EA of different acupoints on neck-incision pain reactions, providing experimental evidence for clinical application of acupuncture analgesia in the operation of thyroid gland.Materials and Methods:One hundred and twenty two male Wistar rats were randomized into normal control (n=22), model (n=34), EA-Futu (LI18)(n=22), EA-Hegu-Neiguan (L14-PC6)(n=22) and EA-Zusanli-Yanglingquan (ST36-GB34)(n=22) groups. Rats were given with a longitudinal incision (1.5cm) in the cervical thyroid gland regions under anesthesia (isoflurane) to establish neck-incision pain model. Each group was treated by EA (2/15HZ,1-2mA,30min) of the acupoints mentioned above4h,24h and48h. The threshold of the pain reaction was detected by heat radiation before and after the surgery, and the post-acupuncture treatment (n=8/group). The back part of the cervical segment of the spinal cord (C1-C4) was removed for assaying the expression of NMDAR2B, mGluR5, GABAB1R, MAPK,cAMP and CREB of the spinal dorsal horn by RT-PCR and western blot techniques respectively(n=8/group).Results:1Effects of EA on pain behaviorCompared with the pain threshold of pre-operation(model groupl7.80±1.52sec, Futu(LI18) group17.88+1.89sec,Hegu(LI4)-Neiguan(PC6) group18.77±3.22sec, Zusanli(ST36)-Yanglignquan(GB34)group17.63±2.18sec,) the pain threshold (PT) of the rats decreased significantly after cervical incision (model group11.29±0.99sec, Futu group11.45±2.02sec, Hegu-Neiguan groupl2.01±1.38sec, Zusanli-Yanglignquan group11.20± 1.72sec)(P<0.05).. Compared with that of model group (post-operation4hll.12±1.00sec, post-operation1d11.64±0.97sec, post-operation2d11.68±1.40sec), the PTs of EA-Futu(LIl8)group(16.7±1.97sec,17.76±1.94sec,16.98±1.84sec)and EA-Neiguan-Hegu(L14-PC6) group (17.3±2. lsec,18.15±1.28sec,17.91±2.31sec) increased markedly after EA treatment (P<0.05),. While the PT of EA Zusanli-Yanglingquan(ST36-GB34) group(12.15±2.53sec,11.98±1.72sec,12.18±1.79sec)had no apparent changes in comparison with that of model group(P>0.05).2Effects of EA on expression of GABA/Glu receptors in the cervical spinal cordResult of RT-PCR showed that in the superficial dorsal horn the expressions of mGluR mRNA in model group (1.01±0.01) were signi f icantly higher than those in normal group (0.56±0.04)(P<0.05). Compared with model group, the expressions mGluR mRNA in Futu (LI18) group (0.83±0.17) were lower than those in model group(P>0.05), while the effects were obviously better than Neiguan-Hegu (L14-PC6) group (1.04±0.13) and Zusanli-Yanglingquan(ST36-GB34) group (1.03±0.05)(P<0.05); The expressions of mGluR mRNA in spinal cord dorsal horn in EA-Neiguan-Hegu(L14-PC6)group and EA-Zusanli-Yanglingquan(ST36-GB34) group changed little.(P>0.05).Result of Western blot showed that in the superficial dorsal horn the expressions of mGluR mRNA in model group (1.43±0.04) were significantly higher than those in normal group (0.98±0.04)(P<0.05). The protein expression of mGluR in EA groups (1.22±0.04,1.06±0.04,1.14±0.04) became clearly lower (P<0.05). Compared with normal group (0.44±0.04) the protein expression of NMDAR2B (0.46±0.04) became higher, but variated a little (P>0.05). And in all the EA groups (0.41±0.03,0.45±0.04,0.43±0.05) the expression of mGluR variate a little(P>0.05) After the cervical incision operation, compared with normal group (0.64±0.15), the protein expression of GABAB1R became lower in model group(0.56±0.01) became lower, in EA Putu(LI18) group (0.61±0.03) were higher,and in EA-Neiguan-Hegu(L14-PC6)group(0.54±0.04),and EA-Zusanli-Yanglingquan(ST36-GB34) group (0.56±0.05) variated a little (P>0.05)3Effects of EA on activities of intracellular cAMP/MAPK/CREB signal pathway in the cervical spinal cord dorsal hornAfter the cervical incision operation, compared with the normal control group (0.21±0.06,0.54±0.11), the expression of cAMP mRNA and CREBmRNA in spinal cord dorsal horn in model group (0.53±0.11,3.32±0.68) was upregulated significantly (P<0.05), while compared with the model group, the expression levels of cAMP mRNA and CREB mRNA in the EA Futu(LI18) group (0.13±0.02,0.39±0.01) were down-regulated remarkablely (P<0.05). Both cAMP mRNA and CREB mRNA expression in the EA-L14-PC6(0.47±0.06,2.97±0.57) and EA-ST36-GB34(0.51±0.09,3.71±0.64) groups had no marked changes (P>0.05). The effects of Futu (LI18) on down-regulating cAMP and CREB mRNA were obviously better than those of EA-L14-PC6and EA-ST36-GB34(P<0.05). Compared with the normal group (1.38±0.1) the expression level of MAPK mRNA was slightly higher in the model group (1.72±0.36)(P>0.05).Such is the case in all the3EA groups (1.68±0.17,1.77±0.4,1.91±0.33) in the expression of MAPK mRNA (P>0.05).After the cervical incision operation, compared with the normal group (0.64±0.03) the expression of CREB protein was higher in the model group (0.65±0.16). In comparison with the model group, the expression of CREB protein was decreased mildly in the EA Futu(LI18) group (0.54±0.13)(P>0.05), but obviously decreased in the EA-L14-PC6group (0.43±0.04) and EA-ST36-GB34group (0.33±0.03)(P<0.05). Compared with the model group, the expression of p-CREB protein in EA Futu(LI18) group (0.31±0.05) and EA-L14-PC6group (0.29±0.02) were obviously lower(P<0.05), and no remarkable changes were found in the EA-ST36-GB34group (0.34±0.05)(P>0.05)Conclusions:1Neck-incision injury can induce remarkable pain reaction (decrease of pain threshold) in rats, which lasts for5days. EA at"Futu"(LI18)and Hegu-Neiguan(L14-PC6) acupoint can obviously inhibit the pain reaction in cervical incision pain rats.2EA at"Futu"(LH8) acupoint can obviously inhibit the upregulated expression of excitatory amino acid receptor mGluR5mRNA and mGluR5protein in the cervical spinal cord dorsal horns (C1-C4)48h after modeling in neck-incision pain rats.3EA at"Futu"(LI18) acupoint can obviously down-regulate the expression of intracellular cAMP mRNA, CREB mRNA, p-CREB protein, inhibiting the activities of cAMP/MAPK/CREB signal pathway in the cervical spinal cord dorsal horn (C1-C4). |
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