Study On The Mechanism Of Acupuncture Analgesia Mediated By Local NF-?B Signaling Pathway At Acupoints

ObjectiveTaking the analgesic effect of Zusanli acupuncture on adjuvant arthritis rats as the research platform,to verify the NF-?B signal pathway activated by acupuncture,to clarify the expression cell types of NF-?B signal pathway,and to further explore the neurobiological mechanism of NF-?B signal pathway mediated acupuncture analgesia,so as to provide experimental basis for elucidating the initial regulation mechanism of acupuncture analgesia effect.MethodsTo observe the analgesic effect of acupuncture "Zusanli" on normal rats and adjuvant arthritis rats with the threshold of foot heat radiation pain as an effective index,immunohistochemistry and Western blot were used to detect the expression of phosphorylated P50(P-P50)and P65(P-P65)in the nucleus to verify whether acupuncture can activate the NF-?B signal pathway in the acupoints;the expression cell types of NF-?B signal pathway in the acupoints were observed by immunofluorescence CO staining;on this basis,the upstream and downstream mechanisms of the local NF-?B signal pathway mediated acupuncture analgesia were explored: the changes of HMGB1,TLR2,TLR4,My D88 and P-P65 were detected by WB technique to observe the effect of HMGB1 neutralizing antibody on the local HMGB1-NF-?B signal pathway,to explore the upstream signal molecules of NF-?B signal pathway,to inject NF-?B inhibitor into the local acupoint,to observe its effect on analgesia,and the changes of enkephalin,dynorphin and ?-endorphin in spinal cord were detected by radioimmunoassay,the changes of COX2 content detected by ELISA,to explore the downstream mechanism of NF-?B signaling pathway mediated acupuncture analgesia.Results1.Effect of acupuncture on thermal radiation pain threshold of ratsOn the first day of acupuncture,the pain threshold of CFA rats was increased(P < 0.05),and the effect lasted until the seventh day of acupuncture(P < 0.01).The heat radiation threshold of the soles of the normal rats was only increased on the third day(P < 0.05),and no significant effect was observed at the other observation time points.2.Effect of acupuncture on local NF-?B signaling pathway at acupointsAfter acupuncture,the expression of P-P50 and P-P65 in the nucleus of the local tissues of acupoints increased(P < 0.01),indicating that the NF-?B signal pathway in the local acupoints was activated after acupuncture.3.Cell types of NF-?B signaling pathwayThere were a large number of P-P50 and P-P65 expression in the nucleus of saline +acupuncture group and model + acupuncture group,and a large number of CO staining with S100A4 labeled fibroblasts,indicating that acupuncture can activate Cell types of NF-?B signaling pathway signaling pathway on fibroblasts.4.Effect of HMGB1 neutralization antibody injection on the upstream factors of NF-?B signaling pathwayTLR2,TLR4 and My D88 decreased after local injection of HMGB1 neutralizing antibody,which indicated that the signal pathway of HMGB1-TLR-NF-?B was partially inhibited and HMGB1 might be the upstream of NF-?B signal pathway.5.Effect of injection of NF-?B inhibitor at acupoint on thermal radiation pain threshold of plantar in ratsCompared with the model group,the acupuncture group showed analgesic effect from the second day(P < 0.05)and lasted to the seventh day(P < 0.01);compared with the acupuncture group,the acupuncture effect of acupuncture + NF-?B inhibitor group decreased from the second day(P < 0.01)and lasted to the seventh day(P < 0.01),and the acupuncture analgesic effect was significantly inhibited.6.Effect of injection of NF-?B inhibitor at acupoint on analgesic substances and pain factors of spinal cordCompared with the model group,the content of COX2 decreased after acupuncture(P <0.05),and there was no significant change in opioid peptides.There was a rising trend of COX2 in the acupuncture group after local injection of NF-?B inhibitor,indicating that the mechanism of analgesic effect mediated by NF-?B signal pathway may be related to the release of COX2 in the spinal cord.Conclusion1.Acupuncture at Zusanli can activate NF-?B signal pathway,which is one of the key signal pathways.2.Acupuncture at Zusanli can activate NF-?B signal pathway on the local fibroblasts of acupoints.3.Acupuncture at Zusanli can activate HMGB1-TLR-NF-?B signal pathway,HMGB1,TLR2,TLR4,My D88 may be the upstream of NF-?B signal pathway.4.NF-?B signaling pathway in acupoint area may mediate acupuncture analgesia by inhibiting the expression of COX2 in spinal cord.