The Experimental Study Of Electroacupuncture Alleviating Thalamic Pain In Rats By Suppressing Adenylyl Cyclase Subtype 1 Upregulation

Objective:Thalamic pain（TP）,also known as central post-stroke pain（CPSP）,is the most common kind of central neuropathic pain corresponding to the lesion site after cerebrovascular accident（ischemia or hemorrhage）or brain injury,and the thalamus is the most frequently affected area.It is characterized by hyperalgesia,allodynia spontaneous pain,resulting in a serious impact on the prognosis and quality of life of patients with TP.The pathogenesis of TP is complex and the treatment is difficult.Pharmacotherapy as the first choice is ineffective,and the side effects of long-term application are often unbearable for patients.Non-pharmacological therapies,which focus on neuromodulation,are gradually becoming widely recognized for their small damage,less side effects,and good comfort.As a therapy combining Chinese traditional medicine and modern technology,electro-acupuncture（EA）has the characteristics of simple operation,safety and reliability,low cost,etc.,and has a good therapeutic effect on a variety of chronic pains,including peripheral neuropathic pain,but the analgesic effect and molecular mechanism of TP still need to be further studied.In this study,a new rat model of TP induced by cobra venom was established.After EA treatment,the mechanical pain thresholds in rats were detected and the changes of their behaviors were evaluated.In this study,the original rat model of TP in recent years was used by our research group to detect the change of pain threshold in rats after EA intervention and observe spontaneous pain.Then,transcriptomics and bioinformatics methods were applied to screen the differentially expressed genes and signal transduction pathways between groups,analyze and determine key genes and pathways,and use molecular biology methods to verify and explore the possible mechanism of EA in the treatment of TP in rats,so as to provide new targets and schemes for the treatment of TP.Methods:1.According to the original method of the research group in recent years,a rat model of TP was established and the effect of EA analgesia was observed.Sixty Sprague Dawley rats were randomly divided into four groups:sham operation group（SH）,thalamic pain group（TP）,thalamic pain+electroacupuncture group（EA）and thalamic pain+sham electroacupuncture group（SE）,with 15 rats in each group.Trace amount of cobra venom solution was injected into the ventroposterior lateral（VPL）nucleus of the right thalamus to construct TP model in TP group,EA group and SE group,while the SH group were injected the same amount of sterile normal saline at the same position with the same method.In each group,the mechanical withdrawal thresholds（MWT）of the rats’four paws and bilateral faces were measured before creating the model and the 3^rd,7^th,14^th,21^st,and 28^thday after surgery.The behavioral manifestations of rats,including the changes of exploratory behavior and grooming,were observed and recorded by video recording.The rats of EA group were treated with EA on the 7^thday after modeling.The acupoints of"Quchi"and"Shousanli"on the right forelimb of rats were selected once every other day for 30 minutes each time until the end of the 27^thday after modeling.The location and duration of acupuncture in SE group were the same as those in EA group,but only acupuncture without current.2.Transcriptomics and bioinformatics methods were used to screen key differentially expressed genes.The subjects were determined according to the results of MWT and behavioral video analysis of rats in each group.The right thalamus tissue of rats was taken out on the 28^thday after operation.After the total RNA in the sample was extracted and identified as qualified,the whole transcriptome was sequenced by RNA-seq and the expression profile of the obtained genes was analyzed to screen the differentially expressed genes among the groups.Then we used the analytical methods of GO and KEGG pathways to understand the biological process,molecular function or cellular component of differential gene enrichment,and describe the signal transduction pathways of gene enrichment.Important differentially expressed genes and signal pathways were found by comparative analysis.The key genes were obtained by protein-protein interaction analysis using string database and Cytoscape software.3.Key genes and signal pathways were verified by molecular biological methods.Another 60 rats were randomly divided into sham operation group（SH group）,thalamic pain group（TP group）,thalamic pain+electroacupuncture group（EA group）and thalamic pain+inhibitor group（ST group）,with 15 rats in each group.The method of making model is the same as before.The intervention methods of EA group were the same as before.The rats of ST group were intraperitoneally injected with the specific inhibitor of the selected key proteins from the 7^thday after operation.The MWT of the rats’right faces were measured 1 hour after each injection.The rats of ST group were continuously administered for 5 days.Western blot（WB）and immunofluorescence（IF）were used to detect the expression of key proteins in the right thalamus of SH group,TP group and St group on the 11^thday after operation.On the 28th day after operation,the differentially expressed m RNA was detected qualitatively and quantitatively by quantitative real-time reverse transcription polymerase chain reaction（q RT-PCR）;WB test and IF staining were used to detect the expression of key proteins and signal pathways in the right thalamus of rats in each group.Results:1.There was no significant difference in the MWT of all tested sites and behavioral video results between groups before operation（P>0.05）.During the whole experiment,there was no significant change in the MWT of paws and left face of rats among the 4 groups（P>0.05）.On the 3rd day after operation,the MWT of the right face in TP group,EA group and SE group decreased and the duration of the exploration behavior decreased and the duration of the grooming increased,which was significantly different from that in SH group（P<0.01）.This phenomenon continued until the end of the experiment in TP group and SE group.In EA group,EA treatment was applied from the 7th day after operation,then the MWT of the right face gradually increased,the duration of exploration behavior gradually increased,and the duration of grooming gradually reduced.There was no significant difference between EA and SH group on the 28^thday after operation（P>0.05）.2.The right thalamus tissues of rats in SH group,TP group and EA group were taken and analyzed by RNA-seq technology.It was found that there were 1113 differentially expressed genes in TP group compared with SH group and 377 differentially expressed genes in EA group compared with TP group,of which 112 genes were common.Taking these112 genes as further research objects,the results of GO analysis showed that the differentially expressed genes are mainly concentrated in the biological processes such as axogenesis,regulation of synaptic structure and activity,neuronal projection,neuronal signal transduction,etc.The results of KEGG analysis showed that the differentially expressed genes are mainly concentrated in c AMP signal pathway,oxytocin signal pathway,chemokine signal pathway,GABAergic synapse,cholinergic synapse and neuroactive ligand receptor interaction,etc.According to bioinformatics and protein-protein interaction network analysis,ADCY1 is the key protein for EA to exert analgesic effect on thalamic pain rats,and c AMP signal pathway is the key pathway closely related to the analgesia of thalamic pain with EA treatment.3.The MWT of the right face in ST group increased gradually from the7^thday after treatment with ST034307,which is a specific inhibitor of ADCY1,and recovered to no difference compared with SH group on the 11^thday（P>0.05）,but reduced gradually from the 12^thday,and there was significant difference compared with SH group and no significant difference with TP group on the 14^thand 21^stdays.On the 11^thday after operation,the expression of key proteins in the right thalamus of rats in SH group,TP group and ST group was detected.The results showed that the expression of ADCY1 and PKA in TP group increased,which was significantly different from that in SH group（P<0.01）;Compared with TP group,the expression of ADCY1 and PKA in ST group decreased obviously（P<0.01）.On the 28^thday after operation,the content of key genes and protein expression in the right thalamus of rats in SH group,TP group and EA group were detected.The results showed that the content of ADCY1 m RNA in TP group was significantly higher than that in SH group（P<0.01）,while that in EA group was significantly lower than that in TP group（P<0.05）,and there was no significant difference between EA group and the SH group,（P>0.05）.The expression of ADCY and PKA in TP group increased significantly compared with SH group（P<0.01）;Compared with TP group,the expression of ADCY1 and PKA in EA group decreased significantly（P<0.01）.Conclusion:1.Intermittent and repeated EA stimulation can alleviate the mechanical hyperalgesia and spontaneous pain of the right face caused by the injection of cobra venom into the right VPL nucleus in rats.2.EA can alleviate thalamic pain in rats by suppressing adenylyl cyclase subtype 1 and c AMP signal pathway upregulation.