| Background:Since acupuncture was proposed by NIH consensus as a therapeutic intervention of complementary medicine, the acupuncture-induced analgesic effect has been used widely to alleviate diverse pains, particularly chronic pain, and is termed "acupuncture analgesia" Acupuncture signals ascend mainly through the spinal ventrolateral funiculus to the brain. Many brain nuclei composing a complicated network are involved in processing acupuncture analgesia, including the periaqueductal grey (PAG),nucleus raphe magnus (NRM), arcuate nucleus (Arc), locus coeruleus, nucleus submedius, preoptic area, habenular nucleus, accumbens nucleus, septal area, amygdala, caudate nucleus, etc, however, the action of hippocampus in acupuncture analgesia has been seldom reported. Our research results have revealed that repeated EA at Zusanli (ST36)-Yanglingquan(GB34) could provide an accumulative analgesia effect in CCI induced chronic neuropathic pain in rats, which were closely related to the up-regulation of SYN, CaMKII and PKA expression in the CA3and CA1regions of hippocampus and improvement of increases synaptic gap.The chronic neuropathic and inflammatory pain induced by neuronal injury could produce plastic changes in central nervous system. It has been proved that the morphology, neuronal synaptic plasticity and related gene and protein changes in hippocampus has occurred in pain statement. In the past decades, it has reported that NO involves in the pain mediation in the level of peripheral and central nervous systems. In the spinal level, NO participates in the formation and the development of hyperalgesia, and it might be a comprehensive effect which consists of not only the mediation of cGMP but also the action of Ca2+even some unknown mechanisms. In the cellular, the inner Ca2+coupling with CaM target on NOS together to produce NO with the material of L-Arg. The NO which acts on the adjacent cells or the cells of themselves by the action of diffusing conducts the nociceptive signals to the brain center by activating sGC, converting GTP to cGMP, further activating PKG, finally exciting the nociceptive neurons in spinal cord dorsal horn, and is termed NO of spinal level participates in the conduction of nociceptive signals by NO-cGMP-PKG signaling pathway. However, whether the NO-cGMP-PKG signaling pathway in the hippocampus of the limbic system of upper spinal cord participates in the chronic neuropathic pain has not been reported yet. The present study was designed to further explore the mechanism underlying EA-induced accumulative analgesic effect from the changes of nNOS, cGMP, PKG mRNA expression of NO-cGMP-PKG signaling pathway in the hippocampus of limbic system in rats with chronic neuropathy pain, providing experimental evidence for clinical application of acupuncture therapy in the chronic neuropathic pain.Materials and MethodsForty male Wistar rats were randomized into normal control (n=22), model (n=8), EA-2Hz (n=8), EA-2/15Hz (n=8) and EA-100Hz (n=8) groups. CCI pain model was established by ligature of the left sciatic nerve with surgical suture. EA (2Hz,2/15Hz,100Hz,1mA,30min) was daily applied to bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for2weeks respectively. Hyperalgesic scores (HS) were detected with radiation-heat irradiation and VON FREY test. After deeply anesthetized (25%urethane and1.5%cholorase,0.5ml/100g), the hippocampal tissue of rats was dissected out. nNOS, iNOS and PKG mRNA expression was detected by reversed transcription-polymerase chain reaction (RT-PCR) after homogenate.The other fifty six male Wistar rats were randomized into normal control (n=22), model (n=8), EA-2/15Hz (n=8), EA-7-NI (n=8),EA-DMSO (n=8), EA-LY83583(n=8), and EA-Ethanol (n=8) groups. CCI pain model was established by ligature of the left sciatic nerve with surgical suture. EA (2/15Hz,1mA,30min) was daily applied to bilateral "Zusanli"(ST36)-’Yanglingquan"(GB34) for2weeks respectively. Hyperalgesic scores (HS) were detected with radiation-heat irradiation and VON FREY test. After deeply anesthetized (25%urethane and1.5%cholorase,0.5ml/100g), the hippocampus of the brain tissue of rats was dissected out. nNOS and sGC protein expression was detected by Western Blot after homogenate.Results:1) Effect of EA on pain threshold in CCI ratsCompared with the pain threshold of pre-operation (model group0.35±0.05sec, EA-2Hz group0.37±0.08sec, EA-2/15Hz group0.37±0.08sec, EA-100Hz group0.30±0.05sec)the pain threshold (PT) of radiation-heat of the rats decreased significantly after CCI (model group6.82+0.35sec, EA-2Hz group7.07±0.53sec, EA-2/15Hz group6.28±1. llsec, EA-100Hz group6.53±0.10sec)(P<0.05). Compared with that of model group (EA3d:6.78±0.47sec, EA7d:6.02±0.75sec, EA10d:3.92±0.29sec, EA14d2.98±0.39sec), EA-2Hz group (3.82±0.89sec,3.55±0.92sec,2.32±0.65sec,2.02±0.35sec), EA-2/15Hz group (3.65±0.75sec,3.05±0.98sec,2.03±0.46sec,1.88±0.31sec) increased markedly after EA treatment(P<0.05). While EA-100Hz group (4.72±1.20sec,4.40±1.24sec,3.38±0.73sec,2.13±0.38sec) had no apparent changes in comparison with that of model group(P>0.05)except EA3d and EA14d.Compared with the pain threshold of pre-operation(model group0.43 ±0.07sec, EA-2Hz group0.40+0.11sec, EA-2/15Hz group0.45+0.06sec, EA-100Hz group0.47+0.15sec)the pain threshold (PT) of VON FREY of the rats decreased significantly after CCI (model group4.20+0.81sec, EA-2Hz group3.58+0.29sec, EA-2/15Hz group5.00+0.67sec, EA-100Hz group4.57+0.88sec)(P<0.05). Compared with that of model group (EA3d:4.53+0.59sec, EA7d:3.00+0.61sec, EA10d:2.92+0.39sec, EA14d2.70+0.27sec), EA-2Hz group (2.27+0.46sec,2.12+0.37sec,2.47+0.36sec,1.50+0.18sec), EA-2/15Hz group (2.22+0.27sec,2.00+0.23sec,1.47+0.08sec,1.15+0.14sec) increased markedly after EA treatment (P<0.05). While EA-100Hz group (2.50+0.34sec,2.38+0.28sec,2.52+0.22sec,1.13+0.26sec) had no apparent changes in comparison with that of model group(P>0.05)except EA3d and EA14d.2) Involvement of changes of relative mRNA expression of Hippocampal NO/cGMP/PKG Signaling Pathway in the Accumulative Analgesic Effect of ElectroacupunctureResult of RT-PCR showed that in the hippocampus the expressions of nNOS mRNA in model group (1.80+0.36) were significantly higher than those in normal group (0.86+0.07)(P<0.05). Compared with model group, the expressions nNOS mRNA in EA-2Hz group (1.16+0.09), EA-2/15Hz group (1.20+0.07), EA-100Hz group (1.00+0.17) were significantly lower (P <0.05).After CCI, compared with normal group (0.92+0.05), the expressions of PKG mRNA in the hippocampus were a little higher in model group (1.13+0.11)(P>0.05). Compared with the model group, the expressions of PKG mRNA in EA-2Hz group (0.81+0.09), EA-2/15Hz group (0.77+0.07), EA-100Hz group (0.65+0.11) were significantly lower(P<0.05).After CCI, compared with normal group (0.62+0.11), the expressions of iNOS mRNA in the hippocampus had almost no changes in the model group (1.13+0.11)(P>0.05). Compared with the model group, the expressions of iNOS mRNA in EA-2Hz group(0.67±0.03) were a little higher(P>0.05),while in EA-2/15Hz group(0.50±0.02),EA-100Hz group(0.52±0.12)were a little lower(P>0.05).3)Validation of the role of NO in the accumulative analgesia effect of electroacupuncture by microinjection, of inhibitors of nNOS and sGC into hippocampusCompared with the pain threshold of pre-operation(model group0.54±0.13sec,EA-2/15Hz group0.23±0.09sec,EA-7-NI group0.21±0.05sec, EA-LY83583group0.37±0.10sec,EA-DMSO group0.51±0.13sec,EA-ethanol group0.42±0.11sec)the pain threshold(PT)of radiation-heat of the rats decreased significantly after CCI(model group6.55±1.80sec, EA-2/15Hz group7.05±0.87sec,EA-7-NI group6.16±1.76sec,EA-LY83583group6.16±0.59sec,EA-DMS0group6.58±0.80sec,EA-ethanol group5.57±0.82sec)(P<0.05).Compared with that of model group(EA7d:5.18±1.24sec, EA10d:4.58±1.14sec,EA14d4.58±0.68sec),mode1+2/15Hz group(4.00±1.18sec,2.92±0.51sec,2.00±0.48sec),model+7-NI group G.04±0.28sec,1.77±0.53sec,1.46±0.31sec),EA-LY83583group(2.57±0.69sec,1.56±0.2lsec,0.94±0.18sec),EA-DMSO group(2.82±0.54sec,1.80±0.65sec,1.17±0.37sec),EA-ethanol group β.52±0.91seG2.19±0.61sec,1.83±0.63sec)increased markedly after EA treatment(P<0.05).Compared with the pain threshold of pre-operation(model group0.43±0.07sec,EA-2/15Hz group0.45±0.11sec,EA-7-NI group0.40±0.06sec, model+LY83583group0.50±0.13sec,EA-DMSO group0.47±O.15sec,model+ethanol groupO.70±0.19sec)the pai n threshold(PT)of VON FREY of the rats decreased significantly after CCI(model group5.45±0.90sec, model+2/15Hz group5.72±0.35sec,EA-7-NI group4.56±1.28sec,EA-LY83583group4.93±0.61sec,EA-DMSO group4.57±0.20sec,EA-ethanol group4.77±0.66sec)(P<0.05).Compared with that of model group(EA7d:4.10±1.42sec,EA10d:4.25±1.60sec,EA14d4.33±1.15sec), EA-2/15Hz group6.03±0.61sec2.58±0.85sec2.53±0.89sec), EA-7-NI group2.00±0.95sec,1.66±0.99sec,1.48±0.71sec), EA-LY83583group (2.32±0.59sec,1.28±0.15sec,1.48±0.20sec), EA-DMSO group (2.33±0.26sec,2.51±0.58sec,1.93±0.34sec), EA-ethanol group (2.76±1.10sec,2.09±0.59sec,1.98±0.65sec) increased markedly after EA treatment (P<0.05).Result of Western Blot showed that in the hippocampus the expressions of nNOS protein in model group (0.97±0.22) were a little higher than those in normal group (1.12±0.20)(P>0.05). Compared with model group, the expressions of nNOS protein in EA-2/15Hz group (1.01±0.22), EA-7-NI (the inhibitor of nNOS) group (1.02±0.23) were a little lower (P>0.05).After CCI, compared with normal group (0.27±0.03), the expressions of sGC protein in the hippocampus were a little higher in model group0.37±0.04)(P>0.05). Compared with model group, the expressions of sGC protein in EA-2/15Hz group (0.25±0.10), EA-LY-83583(the inhibitor of sGC) group (0.35±0.05) were a little lower (P>0.05).Conclusion1) Chronic constrictive injury (CCI) can induce a remarkable pain reaction (decrease of pain threshold) in rats which is characterized by the up regulation of the expression of nNOS、PKG mRNA. Therefore it shows that CCI induced injury can produce pain reaction and increase the action of nNOS, PKG in hippocampus in rats.2)EA of "Zusanli"(ST36)-"Yanglingquan"(GB34) is able to significantly alleviate pain sensitivity caused by CCI, and the analgesia effect induced by EA-2/15Hz group is obviously better than EA-2Hz and EA-100Hz group.3)EA of "Zusanli"(ST36)-"Yanglingquan"(GB34) is able to significantly down regulate the increased expression of nNOS and PKG mRNA and nNOS, sGC protein in the hippocampus induced by CCI which indicate that nNOS, PKG, sGC participate in the effect of analgesia of EA.4)EA-2Hz,EA-2/15Hz, EA-100Hz of "Zusanli"(ST36)-"Yanglingquan"(GB34) fail to affect the expression of iNOS mRNA in the hippocampus, suggesting that iNOS may not play a role in analgesic effect of EA. |
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