| Objective:Previous studies have shown that BDNF(Brain derived neurotrophic factor)and its high affinity receptor TrkB(Tropomyosin related kinase B)play an important role in the formation of pain.Regulation of BDNF and TrkB can alleviate the development of pain.In this study,we applied an inflammatory pain mouse model induced by CFA(Complete Freund’s Adjuvant),and based on the stable analgesia experimental platform of acupuncture at bilateral Zusanli(ST36),we explored the potential mechanisms of acupuncture analgesia through BDNF-TrkB pathway,so as to provide evidence for the study of the effective mechanism of acupuncture analgesia.Method:1.In the first part of this study,50μL CFA was subcutaneously injected into the right plantar of mice to induce inflammatory pain and acupuncture at bilateral ST36.The analgesic effect of acupuncture was evaluated by measuring the Paw Withdrawal Thermal Latency and Paw Withdrawal Mechanical Threshold of the plantar.The effects of acupuncture on spontaneous movement and pain emotion of CFA mice were evaluated by open field test.The expression of COX1(Cyclo-oxygen-ase 1)in spinal cord was detected by Western blot,the expression of PGE2(Prostaglandin E2)in spinal cord was detected by ELISA.2.In the second part of this study,COX1 gene-knockout mice were used and 50μL CFA was subcutaneously injected into the right plantar to induce inflammatory pain.The changes of the Paw Withdrawal Thermal Latency and Paw Withdrawal Mechanical Threshold were observed.The expression of PGE2 in spinal cord was detected by ELISA,and the expression of BDNF and TrkB in spinal cord was detected by Western blot.3.In the third part of this study,on the premise of confirming the effectiveness of acupuncture analgesia,the expression of EP2(Prostaglandin receptor 2),EP4(Prostaglandin receptor 4),BDNF and TrkB in spinal cord were detected by Western blot.The colocalization of EP2 and neuron,BDNF and neuron,TrkB and microglia was observed by immunofluorescence.4.In the fourth part of this study,the downstream signaling pathways(PI3K-Akt signaling pathway,NF-κB signaling pathway)of BDNF-TrkB were explored,on the premise of confirming the effectiveness of acupuncture analgesia,the expression of p-PI3K p85 and PI3K p85,p-Akt and Akt,p-IκBα and IκBα,p-NF-κB p65 and NF-κB p65 in spinal cord were detected by Western blot.Results:1.The results of the first part revealed that compared with the saline group,the Paw Withdrawal Thermal Latency and Paw Withdrawal Mechanical Threshold of plantar in the model group and acupuncture group decreased significantly(P < 0.01)after 24 hours of modeling,and lasted until the 7th day(P < 0.01).Compared with the model group,the Paw Withdrawal Thermal Latency and Paw Withdrawal Mechanical Threshold in the acupuncture group increased from 1st day after acupuncture(P < 0.01),and lasted to the 7th day(P <0.01).The results of open field test showed that after 24 hours of modeling,compared with the saline group,total sport distance,average speed,sport distance and sport time in the central area of the model group and the acupuncture group were lower than those in the saline group(all P<0.01 in the model group and P<0.05 in the acupuncture group).After acupuncture on 7th day,compared with the saline group,total sport distance,average speed,sport distance and sport time in the central area of the model group were still lower than those of the saline group(P<0.05 or P<0.01),and the four indexes of the acupuncture group were lower than those of the saline group,but there was no statistical difference(P > 0.05).Compared with the model group,total sport distance,average speed,sport distance and sport time in the central area of the acupuncture group were significantly higher than those in the model group(P<0.05).On the 7th day,the expression of COX1(P<0.01)and PGE2(P<0.05)in spinal cord in model group were higher than those in saline group,but there was no significant difference in COX1 and PGE2 in acupuncture group(P > 0.05).Compared with the model group,there was significant difference in the decrease of COX1 in the acupuncture group(P<0.05),but there was no significant difference in the decrease of PGE2 in spinal cord(P>0.05).2.The results of the second part showed that after injection of CFA,compared with the wild type saline group,the Paw Withdrawal Thermal Latency and Paw Withdrawal Mechanical Threshold of plantar in the wild type model group and COX1-knockout model group decreased(P<0.01),and this trend continued in D3,D5,D7(P<0.01).Compared with the wild type model group,the Paw Withdrawal Thermal Latency and Paw Withdrawal Mechanical Threshold of plantar in COX1-knockout model mice increased at D5 and D7(P <0.01).Compared with the wild type saline group,the expression of PGE2 increased with no significant difference(P>0.05),and the expression of BDNF(P<0.05)and TrkB(P<0.01)in wild type model group on the 7th day after modeling increased siginificantly;while PGE2 decreased and BDNF and TrkB showed no evident changes in COX1-knockout model group(P>0.05).Compared with the wild type model group,PGE2(P<0.05),BDNF(P<0.05),TrkB(P<0.01)decreased in COX1-knockout model group.3.According to the results of the third part,after seven days of acupuncture intervention,the expression of EP4 in spinal cord of saline group,model group and acupuncture group showed no significant change(P>0.05).Compared with the saline group,the expression of EP2(P<0.01),BDNF(P<0.01)and TrkB(P<0.01)of spinal cord in model group increased,but there was no significant difference in acupuncture group(P > 0.05).Compared with the model group,the expression of EP2(P<0.01),BDNF(P<0.01)and TrkB(P<0.05)of spinal cord in acupuncture group decreased.The colocalization of EP2 with neurons,BDNF with neurons were observed by immunofluorescence,but the colocalization of TrkB with microglia was not observed.4.The results of the fourth part suggested that after seven days of acupuncture intervention,the p-PI3K p85/PI3K p85 in spinal cord of saline group,model group and acupuncture group showed no significant change(P>0.05).Compared with the saline group,the p-Akt/Akt(P<0.01),p-IκBα/IκBα(P<0.05)and p-NF-κB p65/NF-κB p65(P<0.01)of spinal cord in the model group were significantly higher,and in the acupuncture group,the p-NF-κB p65/NF-κB p65 was significantly higher than those in the saline group(P < 0.01),but there was no significant difference in p-Akt/Akt and p-IκBα/IκBα(P > 0.05).Compared with the model group,the p-Akt/Akt(P<0.05),p-IκBα/IκBα(P<0.05)and p-NF-κB p65/NF-κB p65(P<0.05)decreased in the acupuncture group.Conclusion:1.Acupuncture on ST36 showed analgesia effect on inflammatory mice,as well as improvement of motor function and alleviation of negative emotions.2.Acupuncture may exert analgesic effect by reducing COX1 in spinal cord tissue,EP2 and BDNF of neurons,inhibiting TrkB and the activation of PI3K-Akt and NF-κB signal pathway,but it has not been proved that the action cells of BDNF-TrkB was microglias. |
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