| Objective:Our team’s previous study found that the level of CXCL1 in the blood of AIA rats induced by CFA was increased after acupuncture in bilateral ST36,and the CXCL1 in blood was involved in acupuncture analgesia.The Level of CXCL1 were also increased in the spinal cord.This study is based on the platform that acupuncture on bilateral ST36 is effective in relieving pain in AIA rats,aiming to explore the effect target of CXCL1 in blood and the source of CXCL1 increased in spinal cord after acupuncture on AIA rats ST36.To clarify whether the increased CXCL1 in the spinal cord mediates acupuncture analgesia,and to elucidate the mechanism of CXCL1 and its receptor CXCR2 mediating acupuncture analgesia in the spinal cord.In order to provide experimental basis for acupuncture anti-inflammatory pain.Method:1.Effect of acupuncture on CXCL1 gene and protein in spinal cord :The AIA model of rats was established using the 0.1ml CFA subcutaneous injection of right paw.The paw withdrawal thermal latency(PWL)was observed.The spinal cord was taken 7 days after acupuncture.ELISA method was used to detect the changes in CXCL1,and RT-q PCR was used to detect CXCL1 m RNA in the spinal cord.Immunofluorescence staining was used to measure the mean fluorescence intensity of CXCL1 in the spinal cord.2.Screen the active organs of CXCL1 in blood : Using small animal in vivo imaging technology,CXCL1 recombinant protein was labeled with Alexa Fluor 750 and then injected into rats through the tail vein.The Perkin Elmer3 D live animal imaging device was used to dynamically observe the enrichment of CXCL1 in the whole body after the increase of CXCL1 of blood.And the rats organs were dissected and observed in vitro after tail vein injection immediately and living observation.3.Regulate CXCL1 in spinal cord to mediate the analgesic effect of acupuncture: Intrathecal injection of CXCL1 neutralizing antibody and CXCL1 recombinant protein reduced the content of CXCL1 in the spinal cord of the acupuncture group and increased the concentration of CXCL1 in the spinal cord of the model group in order to observe the effect of heat radiation on pain and the content of COX2 in the spinal cord.4.Spinal cord cxcl1-cxcr2 desensitization is involved in acupuncture analgesia mechanism : ELISA was used to detect the contents of CXCR2 in the whole protein and membrane protein,and the contents of COX2 and PGE2 of the spinal cord.RT-q PCR was used to detect the level of CXCR2 m RNA in the spinal cord.The change of CXCR2 content in spinal dorsal horn was detected by immunofluorescence method.CXCL1 and CXCR2 were respectively co-stained with the astrocyte marker GFAP and the neuron marker Neu N by immunofluorescence method,to determine the expression cells of CXCL1 and CXCR2 in the spinal cord.Western-Blot was detected the contents of GRK2 and GRK6 and β-Arrestin1/2 in the spinal cord.To explore the analgesic mechanism of acupuncture in regulating the cxcl1-cxcr2 signaling pathway.Results:Experiment one: Acupuncture on ST36 can improve the inflammatory pain of the AIA rat,and found that the spinal cord CXCL1,which was raised by the acupuncture,was mainly derived from the blood.1.The analgesic effect of acupuncture: After modeling,the PWL were reduced in model group,compared to the saline group(P<0.01),and acupuncture increased the PWL of inflamed paw,from 1day after acupuncture(P<0.01),and lasted until 7 days(P<0.01).It suggested that acupuncture could improve the inflammatory pain of AIA rats.2.The content of CXCL1 in spinal cord: Compared with the saline group,CXCL1(P<0.05)was increased in model group,CXCL1(P<0.01)was increased in acupuncture group;Compared with the model group,CXCL1(P<0.01)was increased in acupuncture group.It suggested that acupuncture could up-regulate CXCL1 protein in spinal cord.3.The CXCL1 mean fluorescence intensity in the dorsal horn of spinal cord: Compared with the saline group,the CXCR2 in the model and acupuncture group increased(P<0.01).Compared with the model group,CXCL1 in the acupuncture group increased(P<0.01).It suggested that acupuncture could up-regulate CXCL1 protein in spinal cord dorsal horn.4.The content of CXCL1 m RNA in spinal cord: Compared with the saline group,CXCL1 m RNA in the model group with no statistically significant difference(P>0.05),and in the acupuncture group increased(P>0.05).Compared with the model group,CXCL1 m RNA in the acupuncture group showed an increasing trend,with no statistical difference(P>0.05).It suggested that acupuncture have little effect on CXCL1 production in the spinal cord.5.Live imaging of small animals: CXCL1 in the blood can enter the spinal cord and brain through the blood-spinal cord barrier and blood-brain barrier with the blood circulation.so the increased CXCL1 in the spinal cord mainly comes from the blood.It suggested that CXCL1 in the blood can enter the central nervous system.Experiment two: The CXCL1 of spinal cord mediated acupuncture treatment of inflammatory pain6.The regulation results of neutralizing CXCL1 in the spinal cord:(1)PWL: Compared with the acupuncture group,the PWL was decreased in acupuncture + low-dose CXCL1 neutralizing antibody group in 1h,6h,20 h after the first day of acupuncture and 1h after the second day of acupuncture(P<0.01);The PWL was no significant difference in 1h,6h and 20 h after the first day of acupuncture in the acupuncture + high-dose CXCL1 neutralizer group(P>0.05),and 1h after acupuncture on the second day decreased(P<0.05).(2)Spinal cord pain factor COX2: compared with the acupuncture group,the COX2 content in the acupuncture + low-dose CXCL1 neutralizing antibody group increased(P<0.01),the COX2 content in the acupuncture + high-dose CXCL1 neutralizing antibody group showed no significant change(P>0.05).Low dose CXCL1 neutralizing antibody can significantly antagonize the analgesic effect of acupuncture.It suggested that low-dose CXCL1 neutralizing antibody could significantly antagonize the analgesic effect of acupuncture.7.The regulation results of recombining CXCL1 in the spinal cord: PWL:(1)Compared with the model group,the model + low-dose CXCL1 recombinant protein group showed no significant change at 1h,6h and 20 h after acupuncture on the first day(P>0.05),and decreased at 1h after acupuncture on the second day(P<0.05).In the model + high-dose CXCL1 recombinant protein group,the PWL was increased in 1h after acupuncture(P<0.01),and increased in 6h(P<0.05),but have no difference in 20 h after acupuncture on the first day(P>0.05),it was increase in 1h after acupuncture on the second day(P<0.05).(2)Pain factor COX2 in Spinal cord: Compared with the model group,the content of COX2 in the model + high-dose CXCL1 recombinant protein group showed a decreasing trend,but there was no statistical difference(P>0.05).Compared with the acupuncture group,the content of COX2 increased in the high-dose group of model + recombinant protein(P<0.05).High dose CXCL1 recombinant protein can simulate the analgesic effect of acupuncture.It suggested that high dose CXCL1 recombinant protein could simulate the analgesic effect of acupuncture.Experiment three: The increased CXCL1 of spinal cord by acupuncture may prompt the spinal cord CXCR2 receptor desensization.8.The analgesic effect of acupuncture: After modeling,the PWL were reduced in model group,compared to the saline group(P<0.01),and acupuncture increased the PWL of inflamed paw,from 1 day after acupuncture(P<0.05),and lasted until 7 days.It suggested that acupuncture could improve the inflammatory pain of AIA rats9.The content of CXCR2 in spinal cord whole protein: Compared with the saline group,CXCR2(P<0.01)was increased in model group and acupuncture group;Compared with the model group,CXCR2(P<0.05)was decreased in acupuncture group.It suggested that acupuncture could down-regulate the content of CXCR2 in the whole spinal cord protein.10.The content of CXCR2 in spinal cord membrane protein: Compared with the saline group,CXCR2(P<0.01)was increased in model group and CXCR2(P<0.05)was increased in acupuncture group;Compared with the model group,CXCR2(P<0.01)was decreased in acupuncture group.It suggested that acupuncture could down-regulate the content of CXCR2 in spinal membrane protein.11.The CXCR2 mean fluorescence intensity in the dorsal horn of spinal cord: Compared with the saline group,the CXCR2 in the model group increased(P<0.01)and CXCR2 in acupuncture group did not change significantly(P>0.05).Compared with the model group,CXCR2 in the acupuncture group decreased(P<0.05).It suggested that acupuncture could down-regulate CXCR2 content in spinal dorsal horn.12.The CXCR2 mRNA in Spinal cord: Compared with the saline group,CXCR2(P<0.01)was increased in model group and acupuncture group;Compared with the model group,CXCR2 m RNA with no statistically significant difference(P>0.05).It suggested that acupuncture have little effect on CXCR2 production in the spinal cord.13.The expression cells of CXCL1 and CXCR2 in the spinal cord: CXCL1 co-stained with astrocytes(GFAP)and neurons(Neu N),CXCR2 co-stained with neurons(Neu N),but not astrocytes(GFAP).These results suggested that CXCL1 was expressed in spinal astrocytes and neurons,and CXCR2 was expressed in neurons.14.Spinal cord desensitization protein: Compared with the saline group,GRK2,GRK6,β-Arrestin1/2 were increased,but there was no statistical differences in acupuncture group(P>0.05),Compared with the model group,GRK2,β-Arrestin1/2 were increased,but there was no statistical differences in acupuncture group(P>0.05)in acupuncture group,GRK6 with no statistically significant difference(P>0.05)in acupuncture group.It is suggested that acupuncture does not affect the content of desensitization protein in spinal cord.15.Pain factor COX2 and PGE2 in Spinal cord:(1)COX2: Compared with the saline group,COX2 was increased in model group(P<0.01)and increased in acupuncture group,but with no statistically significant difference(P>0.05);Compared with the model group,COX2(P<0.01)was decreased in acupuncture group.(2)PGE2: Compared with the saline group,PGE2 was increased in model group and decreased in acupuncture group,both with no statistically significant difference(P>0.05);Compared with the model group,PGE2(P<0.01)was decreased in acupuncture group.It suggested that acupuncture could down-regulate the contents of spinal pain factors COX2 and PGE2.Conclusion:1.Based on acupuncture to improve the inflammatory pain effect of AIA rats,it was found that the up-regulated CXCL1 of spinal cord mainly came from blood.2.CXCL1 in the spinal cord is involved in acupuncture analgesia.3.Acupuncture elevation of CXCL1 in the spinal cord can desensitize the CXCR2 receptor in the spinal cord,and lower the downstream pain-causing factors COX2 and PGE2,thus playing an analgesic role.4.The mechanism of CXCL1-CXCR2 receptor desensitization in the spinal cord caused by acupuncture has nothing to do with the GRK6 pathway,and its mechanism still needs to be further explored. |
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