In the present study, we demonstrated that in CCI-induced neuropathic pain rats, 12 sessions of EA could effectively suppress CCI-induced pain reactions. This result is identical to those reported in our previous studies
[26, 32]. We also compared global changes of acupuncture-associated proteins in the hippocampus under CCI condition, and found 19 differential expressed proteins that returned to the normal control levels following repeated EA interventions. These differential proteins are involved in physiological, metabolic, and cellular processes, with major pathways including “cysteine metabolism”, “valine, leucine and isoleucine degradation”, and “MAPK signaling”.
There is strong evidence that treatment of sciatica patients using thermal acupuncture can result in a progressive increase in the pain threshold with increasing number of treatment sessions, and an accompanying gradual improvement in symptoms
. In this study, we found that stress-induced-phosphoprotein 1 (STIP1), which participates in short-term memory formation and long-term memory consolidation in the hippocampus
[34, 35], was involved in the analgesic effect of EA interventions. In rats with neuropathic pain, Xing et al.
 demonstrated that EA treatment had a regulatory effect on long-term synaptic plasticity in the spinal dorsal horn. Experimental studies have demonstrated that under pain condition, changes in hippocampal synaptic plasticity are accompanied by changes in learning, memory, and adaptive processes. Interestingly, recent studies have independently shown hippocampal abnormalities in animal models of chronic pain including short-term recognition memory
, long-term potentiation deficits, abnormal cytokine expression, and impaired enriched-environment neurogenesis
. Therefore, Changes of STIP1 following EA may suggest a direct link between information storage and the analgesic effect of EA.
The functional changes in the hippocampus were accompanied by changes of protein expression, particularly those proteins involving amino acid metabolism. For example, we found that expression of 3-mercaptopyruvate sulfurtransferase (MPST), aspartate aminotransferase 1 (GOT1), and lactate dehydrogenase B (LDHB), which are involved in cysteine metabolism, was decreased after CCI. Among them, GOT1 is especially important as it also plays a key role in glutamate metabolism. It has been well established that under pain condition, glutamate level in the central never system was increased rapidly, so the decrease of GOT1 protein level following CCI suggests a shift in the equilibrium of GOT1 towards glutamate. Our results demonstrated that EA can apparently reverse the decrease of GOT1. This may be a critical mechanism of analgesic effect of EA. Chronic pain is also a type of chronic stress. Under chronic stress condition, significant decreases in hippocampal synaptic density and surface density have been reported
. Remodeling of the synaptic compartments involves a variety of physiological processes, including enhanced translational activity and related protein synthesis. In the nervous system, neuronal activity is strongly correlated with the levels of amino acid metabolism, and a substantial amount of amino acids are required for changes in protein expression and production of cellular defense mechanisms. Many forms of long-lasting behavioral and synaptic plasticity also require the synthesis of new proteins
. Thus, it is feasible that the regulatory activities of many cellular enzymes, especially those in amino acid metabolism found in the present study, can change after CCI.
We also observed a decrease in the expression of valine, leucine, and isoleucine degradation related proteins, isovaleryl-CoA dehydrogenase (IVD), 3-hydroxyisobutyrate dehydrogenase (HIBADH), and aldehyde dehydrogenase 2 family (ALDH2). Karpova et al.
 reported that hippocampal synaptic activity related to induction of long-term potentiation (LTP) led to a rapid increase in the rate of protein synthesis as well as accelerated protein degradation. Therefore, our study may lend new support to the concept that complementary processes of enhanced protein synthesis and activity-dependent protein degradation is a mechanism underlying control of synaptic protein components
Glutathione (GSH), the major endogenous antioxidant produced by cells, has been shown to modulate the activity of N-methyl-D-aspartate receptors (NMDARs) via its reducing effects
. In the hippocampus, NMDAR-dependent LTP, a form of synaptic plasticity, is thought to represent a cellular model of memory. The NMDAR is the predominant glutamate receptor involved in hippocampal synaptic plasticity, and is critical for LTP, memory, and learning. Cysteine, a precursor for the formation of glutathione, reverses the L-type calcium channel-dependent LTP seen in the aged animals to NMDAR-dependent LTP
. Thus as such, modulating cysteine levels in the hippocampus by acupuncture stimulation may be a therapeutic strategy for treatment of impairments in plasticity and synaptic transmission function in CCI rats.
In response to stress or nerve injury, intracellular pathways that are normally inactive under steady-state condition are activated, while some of the normal pathways may be inhibited or bypassed. In a mouse model of pain induced by subcutaneous injection of formalin, Seo et al.
 reported that intrathecal and intraperitoneal injection of glutamate or acetic acid resulted in up-regulation of hippocampal phosphorylated Ca2+/calmodulin-dependent protein kinase II alpha or phosphorylated extracellular signal-regulated protein expression. In repeated stress stimulation-induced depression rats, EA also increased the number of p-CREB-positive neurons in the hippocampus and restored hippocampal BDNF mRNA expression induced by immobilization stress
. These data suggest a role of EA in regulating cellular signal transduction in the hippocampus. We also found evidence of activation of MAPK signal transduction pathway proteins, mitogen-activated protein kinase kinase 1 (MAPKK1), stathmin 1 (STMN1), and myelin basic protein (MBP) following EA
[47, 48]. The MAPKs are highly conserved across all eukaryotes, and their roles extend beyond the cytoplasm to the nucleus, where they can directly modulate gene transcription. The MAPK signal transduction pathway is also an important signaling event for the induction of hippocampal LTP. Activation of this pathway is necessary for both the transcriptional and translational events underlying long-term memory formation in the hippocampus.
In the nervous system, nerve injury and stress condition can cause oxidative damage to the cytoskeleton and membrane structures. In addition, significant energy production is required for protein expression and cellular defense mechanisms. This is supported by the increased expression of proteins involving antioxidative processes, neuronal integrity, and glycolysis/gluconeogenesis observed in the present study.
The goal of our study was to examine for proteins involved in the analgesic effect of EA. Although there are few comparable studies, using 2-DE-based proteomics, Sung et al.
 reported differential changes in 36 hypothalamic proteins in a model of rat-tail neuropathic pain, which were all restored to control levels following EA of ST36. Similar to our results, twenty-one of these proteins are involved in biological processes including enzyme metabolism and signal transduction, suggesting common mechanisms of EA-induced analgesia.
The expression of three differential proteins (PITPNA, GOT1, and MBP) was further examined using quantitative real-time PCR and Western blot. Although changes in PITPNA and MBP mRNA were similar to that for 2-DE, there was no change in GOT1 mRNA. It is likely that this relates to mRNA-independent post-translational modification, trafficking, and degradation, as a large number of modification sites and domains exist in GOT1.