Analgesic Effect Of Acupuncture On Adenosine A1 Receptor Signal Pathway In Caudate Putamen Of Striatum

Objective:Acupuncture analgesia has been widely used in clinic,its analgesic effect is definite but its mechanism has not been fully elucidated.In order to further clarify its analgesic mechanism,this study took the rat model of arthritis inflammatory pain induced by Complete Freund’s adjuvant(CFA)as the research object,and to observe the effect of acupuncture at bilateral Zusanli on Adenosine receptor(ADOR),cyclic adenosine Monophosphate(cAMP)protein kinase A(PKA)-cAMP-response element binding protein(CREB)signaling pathway in the caudate putamen brain region and excitability of adenosine A1 receptor(ADORA1)neurons in the caudate putamen brain region.To explore the role of acupuncture regulation center ADORA1 signaling pathway and some molecular mechanisms,providing scientific basis for acupuncture analgesia.Method:Experiment 1:Effects of acupuncture on adenosine receptor expression in painrelated brain region of CFA rats.In this part of the experiment,screened male Wistar rats were randomly divided into three groups:Saline group,model group(CFA group)and acupuncture group(CFA+MA group).Saline group was given right plantar intradermal injection of normal saline(0.1ml);CFA group and CFA+MA group were given complete freund’s adjuvant(0.1ml,lmg/ml)intradermal injection into the right plantar to make CFA model rats.On the first day after model building,the CFA+MA group began to manual acupuncture the rats at bilateral Zusanli,using twisting technique,with a frequency of 180 times/min,and each acupoint was twisted for 2 minute with a 5 min-interval,a total of 4 times and 30 min during an manual acupuncture session,manual acupuncture intervention once a day,a total of 7 days.The main effect index was the right plantar thermal pain threshold of rats in each group.After the thermal radiation pain threshold was measured on day 7,the nucleus groups of rat brain tissue were collected,including caudate putamen(CPu),arcuate nucleus(Arc),ventral tegmental area(VTA),periaqueductal gray(PAG),rostral ventromedial medulla(RVM),and Quantitative real-time PCR(q-PCR)was used to detect the mRNA expression levels of Adenosine A1 receptor(ADORA1),Adenosine A2a receptor(ADORA2a),Adenosine A2b receptor(ADORA2b)and Adenosine A3 receptor(ADORA3)in each nuclear group.Western blot(WB)was used to detect the protein content of ADORA1 and the protein content of ADORA2a,ADORA2b and ADORA3 in CPu brain region.And multiple immunofluorescence staining was further used to detect the effect of acupuncture on the expression of ADORA1 in CPu brain region.Experiment 2:Effect of acupuncture on cAMP-PKA-CREB signaling pathway in caudate putamen brain region of CFA rats.In this part of the experiment,Enzyme-Linked immunosorbent assay(ELISA)method was used to detect the changes of cAMP protein content and activity in CPu brain region by in brain tissue samples of rats in Saline group,CFA group and CFA+MA group.Western blot was used to detect the changes of PKA and CREB protein content and phosphorylation of PKA and CREB protein in CPu brain region.And multiple immunofluorescence staining was further used to co-stained ADORA1 and p-CREB,and observe the changes of ADORA1 and p-CREB co-expression in CPu brain region of each group.Experiment 3:Effect of acupuncture on excitability of ADORA1 neurons in caudate putamen brain region of CFA rats.In this part of the experiment,in order to investigate whether ADORA1 in CPu brain region is expressed on neurons,and by using multiple immunofluorescence staining technique,NeuN was used as a marker of neuron cells,and NeuN and ADORA1 were co-stained,and the co-expression of NeuN and ADORA1 was observed in each group.Further,by using multiple immunofluorescence staining technique,c-FOS was used as a marker of neuronal excitability,and c-FOS and ADORA1 were co-stained,and the excitability of ADORA1 neurons was observed in each group.Results:Experiment 1:Acupuncture at bilateral Zusanli can up-regulate ADORA1 gene and protein content in CPu brain region of CFA rats.This experiment replicated the analgesic effect platform of acupuncture:there was no significant change in the basal heat pain threshold of the right plantar of rats in each group before modeling,but the heat pain threshold of rats after modeling was significantly reduced,which showed a significant statistical difference compared with the Saline group,indicating that modeling was successful.After acupuncture,the threshold of heat pain in the acupuncture group was significantly higher than that in the model group,and the difference was statistically significant from day 1 to day 7,indicating that acupuncture at bilateral Zusanli had analgesic effect.The results of q-PCR showed that:In CPu brain region,the ADORA1 mRNA in the CFA group was higher than that in the Saline group,and there was statistical difference;After acupuncture,the ADORA1 mRNA further increased,with an increasing trend compared with the CFA group,but there was no statistical difference,and significantly increased compared with the Saline groups and there was significant statistical difference.In PAG brain region,the ADORA1 mRNA in CFA group was higher than that in Saline group,and there was statistical difference;Compared with CFA group,the ADORA1 mRNA decreased significantly after acupuncture,and there was significant statistical difference.In RVM brain region,compared with the Saline group,the ADORA1 mRNA in the CFA group showed an increasing trend,but there was no statistical difference;After acupuncture,the ADORA1 mRNA in the CFA group was further increased,and there was significant statistical difference;Compared with model group,the ADORA1 mRNA in CFA+MA group showed a slight increase trend,with no statistical difference.There was no significant difference in the ADRA2a mRNA and ADRA2b mRNA in five pain related brain regions of rats in Saline group,CFA group and CFA+MA group.In CPu brain region,compared with the Saline group,the ADORA3 mRNA in the CFA group increased and there was no statistical difference;After acupuncture,the ADORA3 mRNA in the CFA group increased further and there was significant statistical difference;Compared with the CFA group,the ADORA3 mRNA in the CFA+MA group increased,but there was no significant statistical difference.In VTA brain region,compared with Saline group,the ADORA3 mRNA in CFA group did not change significantly,and the ADORA3 mRNA increased after acupuncture,and there was a significant statistical difference;Compared with CFA group,the ADORA3 mRNA in the CFA+MA group increased,and there was statistical difference.The results of WB showed:in CPu brain region,compared with Saline group,there was no significant change in the ADORA1/Tubulin in the model group,but the in ADORA1/Tubulin the CFA+MA group increased and there was statistical difference;Compared with the CFA group,the ADORA1/Tubulin increased after acupuncture,and there was significant difference;There was no significant change in ADORA1/Tubulin in the other four pain brain regions after acupuncture.In CPu brain region,the protein content of ADORA2a,ADORA2b and ADORA3 in each group was further detected by WB,and the results showed that:there were no significant differences in ADORA2a/Tubulin,ADORA2b/Tubulin,and ADORA3/Tubulin in CPu brain regions after acupuncture.Afterwards,immunofluorescence staining was used to further detect the expression of ADORA1 in CPu brain area after acupuncture,and the results showed that:in CPu brain region,Compared with Saline group,the ADORA1 expression of CFA group and CFA+MA group increased,and there was significant statistical difference in CFA group and CFA+MA group;Compared with CFA group,the ADORA1 expression increased significantly after acupuncture,and there was significant statistical difference.Experiment 2:Acupuncture at bilateral Zusanli can down-regulate CREB phosphorylation level and co-expression of ADORA1 and p-CREB in CPu brain region.The results of ELISA showed that:in CPu brain region,cAMP protein content in the CFA group showed a downward trend compared with that in the Saline group,but there was no statistical difference;Compared with CFA group,acupuncture group had a downward trend,but no statistical difference.In CPu brain region,compared with Saline group,cAMP protein activity of CFA group and CFA+MA group decreased,and there was statistical different between CFA group and Saline group,and there was significant statistical different between CFA+MA group and saline group;Compared with CFA group,CFA+MA group had a downward trend,but there was no statistical difference.The results of WB showed that p-PKA/Tubulin,PKA/Tubulin and p-PKA/PKA in CPu brain region of rats in Saline group,CFA group and CFA+MA group had no significant differences in general.In CPu brain region,compared with Saline group,p-CREB/Tubulin in CFA group and CFA+MA group were decreased,and there was statistical difference;Compared with the CFA group,there was no significant difference in p-CREB/Tubulin after acupuncture.In CPu brain region,compared with Saline group,CREB/Tubulin in CFA group and CFA+MA group were decreased,and there was statistical difference;Compared with the CFA group,there was no significant difference in CREB/Tubulin after acupuncture.In CPu brain region,the p-CREB/CREB of the CFA group and the CFA+MA group showed a downward trend compared with the Saline group,but there was no significant difference between the saline group,the CFA group and the CFA+MA group.The co-staining of ADORA1(labeled red fluorescence)and pCREB(labeled green fluorescence)was performed by multiple immunofluorescence staining technique,and it was found that ADORA1 was co-located with p-CREB.Further semiquantitative analysis was conducted on p-CREB expression and co-expression of ADORA1 and p-CREB in CPu brain area of rats in each group:In the CPu brain region,compared with the Saline group,the p-CREB expression in the CFA group was significantly decreased and there was significant statistical difference,while that in the CFA+MA group was further decreased and there was significant statistical difference;Compared with the CFA group,there was a significant decrease after acupuncture,and there was significant statistical difference.In the CPu brain region,compared with the Saline group,the co-expression of ADORA1 and p-CREB in the CFA group was significantly decreased and there was significant statistical difference,while that in the CFA+MA group was further decreased and there was significant statistical difference;Compared with the CFA group,there was a significant decrease after acupuncture,and there was significant statistical difference.Experiment 3:Acupuncture at bilateral Zusanli could up-regulate the expression of ADORA1 in neurons and inhibit the excitability of ADORA1 neurons in CPu brain region.The multiple immunofluorescence staining was used to detect the co-expression of ADORA1 and NeuN in the CPu brain region of rats,and the data were analyzed semi-quantitatively:in CPu brain region,compared with the Saline group,the co-expression of ADORA1 and NeuN in the CFA group showed an upward trend,but there was no statistical difference.After acupuncture,the co-expression of ADORA1 and NeuN was further increased and there was statistical difference;Compared with CFA group,CFA+MA group increased,and there was significant statistical difference.The co-staining of ADORA1 and c-fos in CPu brain region of rats in each group was further detected by multiple immunofluorescence staining,and the data were analyzed semi-quantitatively:in CPu brain region,compared with Saline group,the coexpression of ADORA1 and c-fos in the CFA group and the CFA+MA group decreased significantly,and the co-expression of ADORA1 and c-fos in the CFA group and the CFA+MA group decreased significantly and there was significant statistical difference,while the coexpression of ADORA1 and c-fos in the CFA+MA group decreased further and there was significant statistical difference;Compared with the CFA group,the CFA+MA group was significantly decreased and there was significant statistical difference.Conclusion:1.Acupuncture at bilateral Zusanli points had analgesic effect on CFA rats;2.The improvement of inflammatory pain in CFA rats by acupuncture may be related to the up-regulation of ADORA1 expression in CPu brain region by acupuncture,and the inhibition of cAMP-PKA-CREB signaling pathway;3.Acupuncture may exert analgesic effect by inhibiting excitability of ADORA1 neurons in CPu brain region,but its analgesic mechanism still needs to be further verified.