| Objective:In the previous research platform of acupuncture at bilateral Zusanli points to treat AIA(Adjuvant Induced Arthritis)inflammatory pain model induced by CFA(Complete Freund’s Adjuvant),we found that acupuncture can increase the content of CXCL1 in blood and participate in acupuncture analgesia.At the same time,CXCL1 in blood can reach the brain through blood circulation.However,the mechanism of CXCL1 involved in acupuncture analgesia in the brain is not clear.This study is based on a stable and repeatable acupuncture platform to explore whether acupuncture up-regulating CXCL1 in the blood plays an analgesic effect on the brain,and to clarify the mechanism of CXCL1 and its receptor CXCR2 in the brain involved in acupuncture analgesia.Methods:1.Experimental study on the action site of serum CXCL1 in AIA mice: Using the in vivo imaging technique of small animals,AIA mice were taken as the research object,CXCL1 recombinant protein was labeled with dye AF750,and simulated acupuncture was injected into the tail vein to increase blood CXCL1,through Perkin Elmer3 D small animal living imager to dynamically observe whether the increased blood CXCL1 was enriched in the brain.2.Effect of Acupuncture on CXCL1 protein and Gene content in brain: Acupuncture at bilateral Zusanli points of AIA rats was taken as the platform of acupuncture effect,and the pain threshold of plantar thermal radiation was taken as the main effect index.After 7 days of acupuncture,S1(the primary somatosensory cortex),mPFC(medial prefrontal cortex,A cerebral cortical structure,including ACC(anterior cingulate cortex),PrLC(prelimbic cortex),ILC(infralimbic cortex)),PrLC(prelimbic cortex),ILC(infralimbic cortex),NAC(nucleus accumbens),AMY(Amygdala),PAG(periaqueductal gray)and RVM(rostral ventromedial medulla),of brain tissue were taken to detect the changes of CXCL1 protein content in each nucleus by ELISA method,and the percentage of CXCL1 positive cells in each nucleus was detected by immunofluorescence staining.The changes of CXCL1 mRNA content in S1 brain area were detected by RT-q PCR method.3.Study on the mechanism of CXCL1-CXCR2 in S1 brain area involved in acupuncture analgesia: Detection of CXCR2 protein content in S1 by ELISA method and the changes of mRNA contents of CXCR2 and mRNA content of MOR,KOR,DOR in S1 region were detected by RT-q PCR.CXCR2 was co-stained with astrocyte marker GFAP and neuron marker Neu N by immunofluorescence double labeling method to determine the expression cells of CXCR2 in S1 region.Results:1.It was verified that the serum CXCL1 of acupuncture AIA mice could reach the brain through the blood-brain barrier.In vivo imaging results of small animals: After AIA mice were injected with AF750-labeled CXCL1 recombinant protein through tail vein to simulate acupuncture to increase the content of CXCL1 in blood,it was found that the elevated CXCL1 in blood could enter the brain through the blood-brain barrier with the blood circulation.2.Acupuncture at Zusanli point of AIA rats can increase the protein content of CXCL1 in S1 region.2.1 Acupuncture can improve inflammatory pain in AIA rats:After the establishment of the model,compared with the saline group,the plantar thermal radiation pain threshold of the AIA group was lower than that of the saline group(P<0.01),it shows that the AIA model is successful.Acupuncture can increase the thermal radiation pain threshold of the model side,which takes effect from the 2nd day of acupuncture and lasts until the 7th day.Acupuncture showed significant difference on the 2nd day(P=0.01)and 3rd-7th day(P < 0.01),suggesting that acupuncture can improve inflammatory pain in AIA rats.2.2 The results of ELISA protein detection in multiple encephalic region : the content of CXCL1 protein increased after acupuncture in the S1region(P < 0.01).And there was no significant difference in the content of CXCL1 protein in mPFC,NAc,AMY,PAG,RVM regions(P>0.05).It is suggested that acupuncture can up-regulate the content of CXCL1 protein in S1 region.2.3 Percentage of CXCL1 positive cells in S1 region of cerebral cortex: The results showed that CXCL1 in S1 region was expressed on cytoplasmic membrane,suggesting that nerve cells in S1 region have the ability to produce CXCL1.and there was no significant difference after acupuncture in the percentage of CXCL1 positive cells(P>0.05).Combined with ELISA results the fact that acupuncture can up-regulate the CXCL1 in S1 region,it is speculated that the increase of CXCL1 in S1 region by acupuncture may come from blood circulation.2.4 The changes of CXCL1 mRNA content in S1 region: Compared with the AIA model group,there was no change in CXCL1 mRNA in the acupuncture group,which further confirmed that the CXCL1 in the S1 region increased by acupuncture came from the blood circulation.3 Acupuncture increasing CXCL1 in S1 region may exert analgesic effect by upregulating neuronal CXCR2 receptor and neuronal opioid peptide receptor.3.1 The effect of Acupuncture on CXCR2 protein in S1 brain region: Compared with saline group,the content of CXCR2 protein in AIA model group increased(P =0.085),and the content of CXCR2 protein in acupuncture group increased(P < 0.05).Compared with the AIA model group,the content of CXCR2 protein in the acupuncture group was significantly higher than that in the AIA model group(P < 0.01).It is suggested that acupuncture can up-regulate the content of CXCR2 protein in S1 region3.2 The effect of Acupuncture on CXCR2 Gene in S1 region: Compared with the saline group,the content of CXCR2 gene in the AIA model group and acupuncture group was significantly higher than that in the saline group(P < 0.01).Compared with the AIA model group,the content of CXCR2 gene in the acupuncture group was significantly higher than that in the AIA model group(P < 0.01).It is suggested that acupuncture can up-regulate the gene content of CXCR2 in S1 region.3.3 CXCR2 expression cells in S1 region: CXCR2 co-stained with neuron(Neu N),but not with astrocyte(GFAP),suggesting that neurons in S1 region could express CXCR2.3.4 Effect of Acupuncture on MOR,KOR and DOR genes in S1 brain region: Compared with the AIA model group,the KORmRNA in the acupuncture group was significantly higher than that in the AIA model group,and the MORmRNA in the acupuncture group was significantly higher than that in the AIA model group.There was no significant difference in DORmRNA in the acupuncture group.It is suggested that acupuncture can up-regulate the gene contents of KOR and MOR in S1 region of AIA rats.Conclusion:1.In the AIA rat model,the CXCL1 in the S1 region increased by acupuncture mainly came from the blood circulation.2.Acupuncture improving inflammatory pain in AIA rats may be related to the upregulation of CXCR2 receptor and the expression of opioid receptor in S1 region neurons.The mechanism needs to be further explored. |
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