| Backgroud:Diabetes is one of the common diseases threatening human health,and diabetic neuropathy pain caused by diabetes mellitus is one of the most common chronic complications of diabetes.It is characterized by spontaneous pain,induced pain,hypersensitivity,hypersensitivity and abnormal pain.Which seriously affect the quality of patients’ life and the comfort of patients’ life.However,it is is still know little about the pathogenesis of diabetic neuropathy,and there is no effective treatment for this chronic neuropathic disease.Therefore,to research the pathogenesis of diabetic neuropathy pain,and find a more effective treatment measures,reducing diabetic complications and mortality rate,improve patients quality of life,reducing the medical cost,has a great significance.NLRP3 is the downstream of P2X4,as an intracellular receptors called dangerous associated molecular patterns(DAMPs).P2X4 receptor ligand gating of endogenous ATP nonselective cation channels receptor,which is widely distributed in brain,spinal cord and ganglion,P2X4 express not only activation of microglia,but also mature and secretion of cytokines which can damage body.However,in diabetic,it is not clear that the signal transduction mechanism of the activated microglia cell.P2X4,as a neuropathy pain of participants,whether can be mediated by activation of ATP and other endogenous DAMPs activate the NLRP3,regulating the IL-1β?The diabetic neuropathy pain model is built in this study,to discuss the P2X4/NLRP3 signaling pathways in diabetic neuropathy pain and its molecular mechanism,and observe the role of Ghrelin in diabetic neuropathy pain.Providing theoretical basis and new strategies for the prevention and treatment of diabetic neurologic pain in patients.Part One:Ghrelin and P2X4 rational preliminary probe into mechanism of pain in diabetic neuropathyObjective:To explore the rat spinal cord P2X4 in diabetic neuropathy pain course of and the expression of Ghrelin.Methods:Twenty-four male Sprague-Dawley rats were randomly allocated into 4 groups(n=6 each):normal group(group N),diabetes 2 week group(group DM 2W),diabetes 4 week group(group DM 4W),diabetes 6 week group(group DM 6W).Diabetes mellitus was induced by intraperitoneal 1%streptozotocin 60 mg/kg.All rats using normal diet feeding.Mechanical withdrawal threshold(MWT),motor nerve conduction velocity(MNCV)were detected at the time of 2,4,6 week,then the rats were sacrificed,the spinals were removed for Ghrelin and P2X4.(by Western blot).Results:Compared with group N,the MWT was significantly down-regulated in DM 2W group(P<0.01);in DM 4W group and DM 6W group,the MWT was further down-regulated.Compared with group N,the MNCV was significantly down-regulated in DM 6W group(P<0.05);Compared with group N,group DM 2w Ghrelin and P2X4 expression of rat spinal cord were up-regulated,in group DM 6w,the expression to the peak.Conclusions:In the the process of diabetic neuropathic pain there are compensatory increase of Ghrelin and the excessive expression of P2X4.Part Two:Role of P2X4/NLRP3 on diabetic neuropathic pain and the protective effect of GhrelinObjective:To evaluate the role of P2X4/NLRP3 on diabetic neuropathic pain and the protective effect of Ghrelin.Methods:Twenty-four male Sprague-Dawley rats were randomly allocated into 3 groups(n=8 each):diabetes group(group DM),diabetes+Ghrelin group(group DG),diabetes+Ghrelin+[D-Lys3]-GHRP-6 group(group DGG).Diabetes mellitus was induced by intraperitoneal 1%streptozotocin 60 mg/kg.After the diabetes model was succeed,group DG was giving Ghrelin 200μg/kg from 1d to 42d,group DGG was giving Ghrelin 200μg/kg and[D-Lys3]-GHRP-6 50mg/kg from 1d to 42d.Mechanical withdrawal threshold(MWT),motor nerve conduction velocity(MNCV)were detected at the time of 2,4,6 week,then the rats were sacrificed,the spinals were removed for determination of pathology change(by Nissl staining),the pathology change of sural nerve(by Electron microscopy,EM).and Ghrelin,P2X4,NLRP3,IL-1β(by Western blot).Results:Compared with group DM,the structure of spine of Nissl and EM of sural nerve were normal,the MWT and MNCV was significantly up-regulated,the expression of Ghrelin was up-regulated,the expression of P2X4,NLRP3 and IL-1β was down-regulated in DG group(P<0.05).Compared with group DG,the structure of spine of Nissl and EM of sural nerve were damaged seriously,the MWT and MNCV was significantly down-regulated,the expression of Ghrelin was down-regulated,the expression of P2X4,NLRP3 and IL-1β was up-regulated in DGG group(P<0.05).Conclusions:Ghrelin involved in the diabetic neuropathic pain may be by the signaling pathway of P2X4/NLRP3. |
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