A Study Of Substance P On Expression Of Endogenous Growth Factors In Fibroblasts And Its Signal Transduction Mechanisms

With the development of neuroimmunology,it has been well known that sensory neuropeptides released from the ends of the peripheral sensory nerves in response to traumatic stimuli have participated in modulation of wound healing,in which substance P (SP) plays an important role. Several lines of evidence have indicated that SP,as an important mediator of neurogenic inflammation,elicits various biologic effects,including capillary dilatation and increased vascular permeability,activation and regulation of various immune cells and inducing the synthesis of cytokines and growth factors,modulation of transference and proliferation of the repair cells,by which SP participates in the wound healing in many ways. However,little knowledge has been acquired upon certain precise interaction mechanisms between SP and endogenous growth factors during wound healing and signal transduction mechanisms of SP at present. Therefore,it is necessary to carry out further studies so as to provide some theoretical bases for a novel clinical therapy to promot wound healing by means of improving the nerve function and regulating the secretion of sensory neuropeptides.Our preliminary studies in vivo have showed that SP can promote wound healing in rats,and the expressions of SP and growth factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) in fibroblasts and endothelial cells of cut wound are in phase. These results indicate that SP may exert its effects through regulating the expressions of endogenous growth factors on repair cells during the course of wound healing. In order to confirm this hypothesis and further clarify the important effects of SP on wound healing,the network regulative characteristics of cytokines in wound healing,the regulative effects and signal transduction mechanisms of SP on expression of growth factors,we have chosen a primary fibroblast culture of rat granulation tissue,investigated the effects of SP on proliferation of fibroblasts with MTT assay and detected the gene expression and protein syntheses changes of fibroblasts intrinsic EGF and bFGF with reverse transcriptional polymerase chain reaction (RT-PCR) and western blot analysis. Upon the regulation of gene transcription,electriphoretic mobility shift assay (EMSA) hasbeen used to detect the activation of NF-kappa B in fibroblasts stimulated with SP and probe its signal transduction mechanisms. finally,with transcription factor decoy strategy,we have verified the effects of NF-kappa B on expressions of EGF and bFGF induced by SP.The main results and conclusions are as follows:1. SP can markedly promote the proliferation of rat granulation tissue fibroblasts in vitro with a dose-dependent fashion,which is probably correlated with syntheses of EGF and bFGF induced by SP.2. SP can induce the gene expressions and protein syntheses of EGF and bFGF of rat granulation tissue fibroblasts,and presents some dose and time fashions.3. SP can activate NF-kappa B in a dose-dependent fashion,which is mediated by neurokinin-1 receptor (NK-1R) and correlated with mobilization of intercellular calcium and formation of reactive oxygen free radicals intermediates as second messengers,but not with PKC .4. Transfecting NF-kappa B decoy oligodeoxynucleotide into the fibroblasts before the stimuli of SP can down-regulate the expression of bFGF,but can't completely block its expression,indicating that some other transcription factors are also involved in the transcription regulation of bFGF. NF-kappa B decoy oligodeoxynucleotide has no significant effect on the expression of EGF,showing that the activation of NF-kappa B is not involved in SP-induced expression of EGF.