Hindawi Neural Plasticity Volume 2021, Article ID 5597163, 10 pages https://doi.org/10.1155/2021/5597163 Research Article Differential Proteomic Analysis of the Hippocampus in Rats with Neuropathic Pain to Investigate the Use of Electroacupuncture in Relieving Mechanical Allodynia and Cognitive Decline Degui Gong,1 Xiangmei Yu,2 Menghong Jiang,2 Changzheng Li,2 and Zhifu Wang 2,3 1Affiliated Rehabilitation Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou, China 2Fujian University of Traditional Chinese Medicine, Fuzhou, China 3Key Laboratory of Orthopedics & Traumatology of Traditional Chinese Medicine and Rehabilitation, Ministry of Education, China Correspondence should be addressed to Zhifu Wang; [email protected] Received 27 February 2021; Revised 22 June 2021; Accepted 25 July 2021; Published 5 August 2021 Academic Editor: Jia-Bao Guo Copyright © 2021 Degui Gong et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abnormal changes in hippocampal function and neuroplasticity are involved in neuropathic pain, which induces hyperalgesia and learning and memory deficits. Previous studies from our group have shown that electroacupuncture at Huantiao (GB30) and Yanglingquan (GB34) has an obvious analgesic effect on neuropathic pain. However, the central regulatory mechanism occurring in the hippocampus remains to be investigated. In this study, behavioral and proteomic analyses were performed to identify differentially expressed hippocampal proteins involved in electroacupuncture-induced analgesia. Our results showed both upregulated (TMEM126A, RDH13, and Luc7L) and downregulated proteins (Mettl7A, GGA1 RTKN, RSBN1, and CDKN1B). Further protein verification revealed for the first time that hippocampal TMEM126A plays an important anti- inflammatory role in the treatment of neuralgia by electroacupuncture. 1. Introduction tization and cognitive decline associated with neuropathic pain in humans and rodents [11–13]. Spinal opioids, seroto- Cognitive impairment is commonly associated with the expe- nin, norepinephrine, amino acids, and glial cells/cytokines rience of pain, with approximately 73%–81% of patients with are the primary mediators of EA-induced analgesia of neuro- chronic pain suffering from a memory deficit [1, 2]. Recent pathic pain [14]. Recent studies have focused on the role of clinical studies have reported objective cognitive impair- hippocampal functional changes in the beneficial effect of ments in patients with peripheral neuropathic pain [3, 4]. EA on the pain condition. Regulation of the hippocampal Several studies have provided evidence suggesting that struc- proteins related to amino acid metabolism and activation of tural and functional abnormalities in the hippocampus the MAPK signaling pathway is involved in the analgesic underlie the cognitive deficits associated with neuropathic effect of EA in neuropathic pain [15, 16]. However, few stud- pain [5–8]. The hippocampus receives complex integrated ies to date have focused on the role of the molecular function sensory and cognitive information, including pain signals. of the hippocampus in EA-induced analgesia and cognitive The changes of some gene and proteins such as the upregula- improvement. tion of TNF-α, IL-1, and MCP-1 in the hippocampus play Proteomics analysis provides a valuable strategy for key roles in the neuropathic pain accompanied with cognitive exploring the pathogenesis of pain mellitus, as well as impairment in rodents [9, 10]. However, the hippocampal therapeutic targets in this condition. We hypothesize that proteins and their related signaling pathways involved in some key genes or proteins in the hippocampus play an neuropathic pain still remain largely elusive. essential role in the neuropathic pain with EA treatment. Electroacupuncture (EA) at acupoints Huantiao (GB30) Based on the prior work outlined above, this study was and Yanglingquan (GB34) is effective in relieving pain sensi- aimed at exploring the molecular mechanisms underlying 2 Neural Plasticity EA-induced analgesia in the hippocampus using proteomic tion group and model group underwent the experimental and behavioral analyses. protocol at the same time each day. 2.3. Behavioral Testing. The mechanical paw withdrawal 2. Materials and Methods threshold (PWT) was assessed on day 0, 7, 14, 21, and 28 fi 2.1. Animals and Materials. Male Sprague-Dawley (SD) rats postsurgery after EA stimulation using von Frey laments. fi (8–10 weeks, 200–220 g) were obtained from the Shanghai According to the up-down method, von Frey laments of dif- Slack Laboratory Animal Co. (Shanghai, China). The rats ferent intensities (0.6 g, 1.0 g, 1.4 g, 2.0 g, 4.0 g, 6.0 g, 8.0 g, were housed under stable temperature conditions (22°C) 15.0 g, and 26.0 g) were used to determine the 50% PWT in and a 12/12 h light/dark cycle with free access to food and rats. A quick paw withdrawal or licking of the paw in water at the Experimental Animal Center of Fujian Univer- response to the stimulus was considered to be a positive sity of Chinese Medicine. SD rats were randomly divided into response. The protocol for assessing the PWT was based on a Sham operation group (Sham), a spared nerve injury model the one described in our previous study [20]. group (SNI), and an electroacupuncture group (EA). Ten The novel-object recognition test was performed in a animals per group underwent behavioral testing and analysis, Plexiglas box (60 mm × 60 mm × 50 mm) on day 28 postsur- and four animals per group were used for Enzyme-Linked gery. After habituation, on day 1, the rats were placed in the Immunosorbent Assay (ELISA) and western blot testing after box for 20 min without objects being presented. Four hours all behavioral testing. All animal experimental protocols were later, the rats were placed in the box again and allowed to approved by the Animal Ethics Committee of Fujian Univer- explore objects A and B for 5 min. After a retention interval sity of Traditional Chinese Medicine (No. FJTCM2019-006). of 24 h, a cognitive test was performed in the same box by All rats were humanely sacrificed according to the care guide- replacing object B with a novel object C. The rats were then lines. The rats were anesthetized and euthanized with more allowed to explore the objects freely for 5 min. The ratio of ff isoflurane to minimize the pain experienced by the animals. the di erence between the time spent exploring the familiar The following reagents and instruments were used: von object and that spent exploring the novel object over the total Frey filament (North Medical, CA, USA); acupuncture nee- time spent exploring both objects was used to evaluate cogni- dle, 0:30 mm × 25 mm (Suzhou Medical Supplies Factory, tive function. China); Tmem126a ELISA kit (Wuhan Boster Biological 2.4. Proteomic Detection and Analysis Technology, China); TMEM126A rabbit pAb (A12823; ABclonal Technology, China); and GAPDH goat pAb 2.4.1. Protein Extraction and Trypsin Digestion. All methods (ab9483; Abcam, USA). of proteomic detection and analysis were according to our team’s previous research as follows [21]. 2.2. Spared Nerve Injury-Induced Neuropathic Pain Model Proteins were extracted from the hippocampal tissue and EA Stimulation. The spared nerve injury of sciatic nerve samples in lysis buffer (8 M urea, 1% protease inhibitor) was performed under anesthesia according to the procedures using a high-intensity ultrasonic processor. Remaining debris described previously [17]. In the SNI and EA groups, the was removed by centrifugation at 12,000 × g for 10 min at right sciatic nerve branches of rats were exposed and per- 4°C. The protein concentration was detected with a Bicinch- fl formed selective cutting, or ligation. Brie y, the common oninic Acid (BAC) kit. peroneal and the tibial nerves were separated, tightly ligated For trypsin digestion, protein solution was reduced with with 5-0 silk, and transected distal to the ligation. A 2 mm dithiothreitol for 30 min at 56°C, then incubated with iodoa- length of each nerve was removed, while preserving the integ- cetamide for 15 min at 37°C in darkness. Secondly, protein rity of the sural nerve. In the Sham operation group, only the samples were diluted with millimolar Triethylammonium sciatic nerve branch was exposed, without performing any bicarbonate (TEAB), until the urea concentration was < nerve severance or ligation. Rats were monitored for any sign 2 M. Finally, trypsin was added at a mass ratio (trypsin : pro- of infection or distress after surgery. tein) of 1 : 50 for the first digestion at 37°C overnight, then at Seven days after spared nerve injury surgery, EA group a ratio of 1 : 100 for the second digestion. rats were loosely fixed on a wooden stand that permitted free movement of their head and limbs. The acupoints Huantiao 2.4.2. Tandem Mass Tag (TMT) Labeling and LC-MS/MS (GB30) and Yanglingquan (GB34) on the right side were Analysis. After trypsin digestion, peptides were desalted and selected for acupuncture and electrical stimulation. In the vacuum-dried with the Strata X C18 SPE column. Peptides rat, GB30 is located at the junction of the lateral third and were reconstituted in 0.5 M TEAB and marked according to medial two-thirds of the line connecting the prominence of the TMT kit protocols. greater trochanter of the femur with the sacral hiatus, while Separated peptides were subjected to sodium/iodide sym- GB34 is located in the depression anterior and distal to the porter sources. Tandem mass spectrometry (MS/MS) was head of the fibula [18, 19]. EA stimulation was performed performed using a Q Exactive Plus (Thermo) instrument, for 30 min, once a day for three consecutive weeks. Needles coupled online to an ultraperformance liquid chromatogra- inserted at GB30 and GB34 were connected to a G6805-1A phy system. multifunctional EA apparatus (Shanghai Medical Electronic Using an electrospray voltage of 2.0 kV, intact peptides Apparatus Company, Shanghai, China), using a stimulation were then detected in the Orbitrap at 70,000 mass resolution.