| Objective:Although acupuncture is widely used in clinic as an analgesic strategy,the biological mechanism of analgesia has not been fully clarified.Therefore,to clarify its mechanism is helpful to improve clinical efficacy and international promotion.So,this study explored the effect of acupuncture on the analgesic effect of Complete Freund’s Adjuvant(CFA)in mice,and used the phosphorylated proteomics technique to screen the possible key proteins and important signaling pathways involved in acupuncture analgesia in mouse spinal cord tissue.Meanwhile,based on the previous study of our team,we found that CXC chemokine ligand 1(CXCL1)and its receptor CXC chemokine receptor 2(CXCR2)mediate acupuncture analgesia,but the specific mechanism remains unclear.Therefore,combined with the results of phosphorylated proteomics analysis to further explore the possible molecular mechanism of spinal cord CXCL1-CXCR2 mediates acupuncture analgesia.Method:First,three different modeling doses and two different manual acupuncture methods were used,the paw thermal withdrawal threshold(PTWT)and paw mechanical withdrawal threshold(PMWT)as the main effect indexes to optimize the appropriate dose of mouse inflammatory pain model and manual acupuncture method for the study of acupuncture analgesia.At the same time,to clarify the analgesic effect of acupuncture,the content of cyclooxygenase 2(COX2)and prostaglandin E2(PGE2)in spinal cord were detected by Western Blotting(WB)and Enzyme-linked immunosorbent assay(Elisa).Second,on the basis of the effectiveness of acupuncture analgesia,using the phosphoproteomics technology to screen the key proteins and important pathways involved in acupuncture analgesia in the spinal cord.Finally,combined with previous studies on the mechanism of acupuncture increased CXCL1,and CXCR2 decreased the analgesic,the selected endocytosis pathway was verified,the content of CXCL1 in spinal cord was detected by Elisa,the content of CXCR2,Rab5,Rab11,Rab7,Lamp1 in spinal cord was detected by WB,and double immunofluorescence staining was applied to detect the expression and co-localization of CXCR2 with Neu N,Rab7,Lamp1.Results:1.Experimental study on analgesic effect of acupuncture on CFA mice(1)Results of different modeling doses: Different modeling doses(20μL,50μL,100μL)could reduce the threshold of thermal radiation pain and mechanical pain in mice(P<0.01),and induce the inflammatory pain model in mice.However,the combined results of thermal radiation pain and mechanical pain showed that the 50μL model group was stable as a whole,so the modeling dose of 50 ul was selected.(2)Results of different acupuncture methods:The PTWT compared with the saline group,the model group,acupuncture(1)group and acupuncture(2)group were all decreased from24 h to 7 days after modeling(P<0.05 or P<0.01);compared with the model group,the PTWT in the acupuncture group(1)and acupuncture group(2)increased(P<0.01 or P<0.05)after 1-7 days of acupuncture treatment;compared with acupuncture group(2),the PTWT in the acupuncture group(1)was increased on the 1day and 2day(P<0.05 or P<0.01).The PMWT compared with the saline group,model group decreased from 24 h to 7 days(P<0.05 or P<0.01),acupuncture group(1)was decreased on the 1day and 7day(P<0.01),acupuncture group(2)was decreased on the1 day,3day,6day and 7day after modeling(P<0.05 or P<0.01);compared with the model group,after acupuncture intervention,the PMWT of acupuncture group(1)was increased on the 2day,4day and 7day(P<0.05 or P<0.01),and the acupuncture group(2)was increased on the 2day and 7day(P<0.05 or P<0.01);there was no statistical significance between acupuncture groups(P>0.05).In conclusion,compared with the model group,the pain threshold of the acupuncture group(1)was increased more obviously than that of the acupuncture group(2),and the number of days was more.Therefore,the acupuncture group(1)was used for the later study.(3)Spinal COX2 and PGE2 results: There was no significant difference in COX2 protein content among all groups(P>0.05).Compared with the saline group,the content of PGE2 protein in the model group was increased(P<0.05),while that in the acupuncture group was decreased(P<0.05).2.Screening results of key proteins and pathways for acupuncture analgesia based on spinal cord phosphoproteomics(1)Acupuncture has analgesic effect: Compared with the saline group,1-5 days after modeling,the PTWT and the PMWT in the model group and the acupuncture group were decreased(P<0.01);compared with the model group,the PTWT and the PMWT on the modeling side were both increased 1-5 days after acupuncture intervention(P<0.05 or P<0.01).(2)Results of protein differential modification analysis: Comparison among different groups showed that the differentially modified proteins and sites were as follows: model group VS saline group up-regulated 403 phosphorylation sites corresponding to 301 proteins,downregulated 439 phosphorylation sites corresponding to 339 proteins.The acupuncture group VS model group up-regulated 432 phosphorylation sites,corresponding to 318 proteins,and downregulated 363 phosphorylation sites,corresponding to 273 proteins.The acupuncture group VS the saline group up-regulated 424 phosphorylation sites,corresponding to 298 proteins,and down-regulated 434 phosphorylation sites,corresponding to 322 proteins.(3)The functional annotation of the differentially modified protein GO showed that the protein of model and acupuncture in the biological processes involved were mainly related to cellular process,biological regulation,stimulus response,developmental metabolic and signal transduction.Cell component is mainly located in cell,organelle,membrane,macromolecular complex,membrane-enclosed lumen.Molecular function is mainly related to binding function and catalytic activity,but also related to molecular function regulator,structural molecular activity and transporter activity.(4)Results of KEGG enrichment and clustering analysis: Under the model state,there were four pathways related to pain: c AMP signaling pathway,Gap junction,Axon guidance and Calcium signaling pathway.Under the acupuncture state,there were two pathways related to pain: Endocytosis signaling pathway and GABAergic synapse signaling pathway.(5)Results of the interaction between differentially modified proteins: In the model and in the acupuncture condition,the key target proteins functions were mainly related to biological transport and endocytosis.3.The results of Acupuncture modulates the CXCL1-CXCR2 endocytosis pathway in the spinal cord(1)Changes of CXCL1-CXCR2 endocytic pathway protein in spinal cord: Compared with the saline group,CXCL1 protein content in model group was up-regulated,and there was no statistical difference(P>0.05),but compared with the model group,acupuncture group increased(P<0.05).The results of CXCR2,Rab5,Rab11,Rab7 and Lamp1 protein contents showed no statistical difference among groups(saline,model and acupuncture)(P>0.05).(2)Results of double immunofluorescence staining: Semi-quantitative analysis showed that there was no statistical difference in the co-expression rate of CXCR2 and Neu N among all groups(saline,model and acupuncture)(P>0.05).Compared with the model group,the coexpression rate of CXCR2 and Rab7 and the co-expression rate of CXCR2 and Lamp1 showed an up-regulated trend after acupuncture,but there was no statistical difference(P>0.05).Conclusion:1.Acupuncture has analgesic effect on mice with inflammatory pain induced by CFA.2.The functions of the key phosphorylated proteins involved in acupuncture analgesia in the spinal cord are mainly related to binding function and catalytic activity.It also participates in biological processes such as biological regulation,transport and endocytosis.The pathways related to acupuncture analgesia mainly include Endocytosis signaling pathway and GABAergic synapse signaling pathway.3.It has not been proved that CXCL1-CXCR2 endocytosis signaling pathway is the downstream signaling pathway of CXCL1 involvement in acupuncture analgesia in the spinal cord,which needs to be further confirmed by multiple methods. |
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