| Background:Diabetic peripheral neuropathic pain(DNP)is the most common microvascular complication of diabetes,a lot of diabetic patients are suffering from this pain,it may lead to significant morbidity and poor life quality.Because of its complex pathogenesis,there is no specific treatment,even the effect of using strong opioid is poor,and there are significant side effects of long-term using opioid.Data shows that more than 39%of patients without any treatment.So the focus of current research is to find an effective method for clinical treatment,and the future research direction is mainly for mechanism combination and development of new effective drugs.Recent researches show that Neuregulin-1(NRG1),an essential axoglial signal in neural development,is required for peripheral nerve development and effective in nerve repair.Some studies have confirmed that NRG1 could improve the glucose tolerance,could alleviate neuropathic pain caused by spinal cord injury and peripheral nerve injury,but the neuropathic pain model used in these studies were all related to the spinal nerve ligation model,diabetic neuropathic pain model,the most similar to human diabetic pain,has not been reported.So at the present stage,to investigate whether there is association between NRG 1 and diabetic neuropathic pain,meanwhile reveal the possible mechanism,is a new direction for the treatment of DNP.Part One:Establish the neuropathic pain model of streptozotocin-induced diabetic ratsObjective:To establish the neuropathic pain model of STZ-induced diabetic rats and to observe the changes of PWT and pathological changes in dorsal root ganglion and sural nerve.Methods:Male SD rats were randomly divided into two groups,the STZ-induced group(n=40)and the control group(n=10).The STZ-induced rat model was established by injecting STZ(Sigma,60 mg/kg body weight)intraperitoneally.72 hours later,the plasma glucose levels were determined by the glucose oxidase method using the one touch ultra blood glucose meter,and rats with the blood glucose more than 16.7 mmol/L were considered to be successfully induced for diabetes.Then we used the Von Frey filaments to measure the paw withdrawal threshold(PWT)every week after the successful modeling,meanwhile monitored the blood glucose.After 2 weeks,4 weeks,6 weeks,some rats were sacrificed,and the dorsal root ganglion and sural nerve were taken away to observe the pathological changes.Results:All of the rats were survived,and three days after administration of STZ,82.5%rats had blood glucose more than 16.7 mmol/L,and the hyperglycemia remained during the whole experimental process.The PWT in the diabetic group was lower than in the normal group at the second week after the successful modeling,and it gradually decreased with the prolongation of time,there was statistical significance between the two groups(P<0.05).Pathological results showed that the lesion of dorsal root ganglion cells gradually increased with the course of disease,the nuclear membrane was shrunk,the normal structure of the endoplasmic reticulum was disappeared at 6 weeks;the demyelination of the sural nerve aggravated with the prolongation of time,at 6 weeks the arrangement of the lamellar was disorder,some nerve myelin sheaths were dissolved and absorbed,there were lots of necrosis in the axon.Conclusion:Using single STZ(60 mg/kg,intraperitoneal injection)was a reliable method for establishing diabetic neuropathic pain model.This method was low in mortality,high in modulus,stable in model and simple in operation.Part Two:Effects of exogenous NRG1 on mechanical pain threshold in diabetic ratsObjective:To investigate whether exogenous NRG1 could allevate diabetic neuropathic pain in rats.Methods:20 rats,successful diabetic model of the first part,were randomly divided into diabetic control group(group D,n=10)and NRG1 intravenous injection group(group N,n=10),another 10 rats with the same month were used as normal control group(group C,n=10).Group N were received 10 ?g·kg-1d·-1 rhNRG1 intravenous injection for 7 consecutive days,while group C and D were received vehicle(saline).Von Frey test was used to determine the mechanical sensitivity threshold for nociception every week.Meanwhile,the changes of blood glucose were monitored every week.Results:1.Changes of pain threshold:compared with the diabetic group,administration of NRG1 significantly attenuated the development of mechanical allodynia in diabetic rats(P<0.05).Quantitative analysis showed that NRG1 increased the mechanical pain threshold with the extension of time.2.Changes in blood glucose:no significant changes in blood glucose in group C;blood glucose levels in group D and group N were elevated,and continued during the whole experimental process.Compared with group N and group D,the increase of blood glucose in group N was lower than that in group D(P<0.05).Conclusion:NRG1 exerted positive effects on the behavioral changes of rats with STZ-induced diabetic neuropathic pain,and it could improve hyperglycemia in rats.Part Three:The mechanism of exogenous NRG1 in alleviating neuropathic pain in diabetic ratsObjective:To analyze the possible underlying mechanisms of exogenous NRG1 in alleviating diabetic neuropathic pain in rats.Methods:After consecutive giving rhNRG1 7 days,four weeks later,all of the rats were sacrificed,the expression of NRG1,NGF and the levels of IL-1?,TNF-? in the lumbar enlargement of spinal cord were determined,meanwhile the morphological changes of the dorsal root ganglion and sural nerve were observed.Results:1.Compared with group C,both in group D and group N,the expressions of NRG1 and NGF were significantly decreased,the protein expression of TNF-? and IL-?increased(P<0.05).Compared with group D,in group N the expressions of NGF were increased,while the protein expression of TNF-? and IL-? dicreased(P<0.05),although there was an increase of NRG 1 expression in group N,it did not reach significance.2.Pathological results:the structure of dorsal root ganglion and the sural nerve were normal in group C;in group D,the nuclear membrane was shrink,there were large vacuolar degeneration in cytoplasm,there was significant swelling of rough endoplasmic reticulum,the myelinated axon was serious demyelination,the myelin sheath was arranged in disorder,broken and swollen;in group N,the nuclear membrane was basically integral,slight vesiculation was observed in the nucleus and cytoplasm,the myelinated axon was moderate segmental demylination,the myelin sheath was arranged disorder in local areas,and a small amount of vacuolization could be seen.Conclusion:NRG1 exerted positive effects on the pathological changes of rats with STZ-induced diabetic neuropathic pain,the underlying mechanism might be related to the promotion of NGF excretion and the inhibition of inflammatory pathway. |
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