The Mechanism Of NGF/TrKA Signaling Pathway In Knee Osteoarthritis Pain And The Intervention Effect Of "Warming Channel And Activating Blood Circulation" External Therapy

ObjectiveTo clarify the relationship between NGF/TrKA signaling pathway and peripheral hyperalgesia in KOA;to explore the therapeutic mechanism of "warming channel and activating blood circulation"external therapy(represented by "YiCeng")in improving peripheral hyperalgesia of KOA based on NGF/TrKA signaling pathway.Methods(1)Use HE staining,immunofluorescence,Western Blot,PCR,ELISA and other methods to observe whether the NGF/TrKA signaling pathway is activated in clinical KOA synovial samples;(2)To observe the intervention effect of "YiCeng" based on serum pain mediators and peripheral hyperalgesia by ELISA,peripheral hyperalgesia threshold tests and other methods;(3)The fibroblast-like synoviocytes(FLS)inflammation model was constructed by LPS in vitro,and Western Blot,PCR and other methods were used to explore the expression of pain mediators and inflammatory factors in FLS based on the NGF/TrKA signaling pathway;(4)The rat KOA model was constructed by ACLT method,and HE staining,immunohistochemistry,immunofluorescence,Western Blot,PCR,peripheral hyperalgesia threshold tests were used to clarify the mechanism of NGF/TrKA signaling pathway regulating peripheral hyperalgesia;(5)The rat KOA model was constructed by ACLT method,and HE staining,Western Blot,PCR,and peripheral pain threshold tests were used to explore the intervention effect mechanism of"YiCeng" in improving peripheral hyperalgesia of KOA.Results(1)There were significant increased protein and gene expressions of NGF,TrKA,TRPV1,IL-1β,CGRP,PGP 9.5 in the synovial tissue of the TKA group compared with the arthroscopy group;(2)There was a significant decreased concentration of IL-6 and TNF-α of KOA patients after applying "YiCeng" compared with those before application;NGF and SP concentration of KOA patients after applying "YiCeng" were decreased compared with those before application,although the difference was not statistically significant;and peripheral hyperalgesia thresholds were improved after applying "YiCeng" compared with those before use,and the difference was statistically significant;(3)There were significant increased expressions of NGF,TrKA,TRPV1 and IL-1β in the FLS inflammation model of the LPS group compared with the normal group;and there were significant decreased expressions of NGF,TrKA,TRPV1 and IL-1β after inhibition of TrKA using siRNA TrKA compared with the LPS group;(4)There were significant increased protein and gene expressions of NGF,TRPV1,IL-1β and PGP 9.5 in the synovial tissue of KOA model rats compared with the normal group;and the peripheral hyperalgesia threshold in the KOA group was lower than that of the normal group;these proteins and genes expressions decreased and peripheral hyperalgesia improved after the KOA model rats were intra-articular injected with siRNA TrKA,and the difference was statistically significant.(5)"YiCeng" can down-regulate the protein and gene expressions of NGF,TrKA,TRPV1,IL-1β,PGP 9.5 in the synovial tissue of KOA rats,and the difference is statistically significant;it can also down-regulate TRPV1 and neural markers S100 proteins and genes expressions in DRG tissue,and the difference was statistically significant;and the nerve fiber density in the synovial tissue was reduced and the peripheral hyperalgesia state of the rats was improved.Conclusion(1)The activation of NGF/TrKA signaling pathway in KOA synovium can promote the release of pain mediators and inflammatory factors,and aggravate local pain and inflammation;(2)Activation of the NGF/TrKA signaling pathway can up-regulate the expression of TRPV1 in the synovium and DRG tissues,may increase the density of synovial nerve fibers,and aggravate the peripheral hyperalgesia state;inhibiting this pathway can down-regulate the expression of TRPV1 in synovium and DRG tissues,reduce the density of synovial nerve fibers and improve peripheral hyperalgesia;(3)"YiCeng" may reduce the expression of pain mediators,inflammatory factors and neural markers in synovial and DRG tissues by inhibiting NGF/TrKA signaling pathway in synovial tissue of KOA model rats,and may reduce the nerve fiber density in synovial tissue,improving peripheral hyperalgesia.