Effect Of Exchange Protein Directly Activated By Cyclic Adenosine Monophosphate 1 On Mechanical Pain Of Diabetic Rats

ObjectiveThe anamorphism of guanine nucleotide transforming factor exchange protein 1(Epac1)is one of the important initiation factors of pain,but wh ether Epac1 is involved in the pathogenesis of diabetic mechanical allodyn ia(DMA)Not sure.In this study,Lentivirus-mediated sh RNA was used to inhibit the expression of Epac1.The hind paw withdrawal threshold and th e expression of Epac1 in dorsal root ganglion were observed in DMA mo del rats to investigate the role of Epac1 in the pathogenesis of DMA.Methods32 male SD rats(180-220 g body weight)were housed in animal hou se without specific pathogens,and were free to eat and water.They were accustomed to the environment for 1 week.The body weight,fasting blo od glucose and hindpaw withdrawal threshold were measured before the e xperiment in all rats.The experimental steps are as follows:(1)Preparation of Diabetic Rat Model A randomized selection of 20 rats for intraperitone al injection of streptozotocin was used to make a diabetic model.Streptoz otocin was dissolved in citric acid-sodium citrate at a concentration of 20mg/ml.In the buffer,the rats were weighed and streptozotocin was injecte d intraperitoneally at a dose of 65 mg/kg.The remaining 12 served as no rmal controls and were injected intraperitoneally with an equal volume of c itric acid-sodium citrate buffer.After 3 days,all rats' tail vein blood glucos e levels were measured to verify the diabetic model.Fasting blood glucos e level ?13.9 mmol/L was a successful model for diabetes.(2)Prepare D MA rat model to detect the withdrawal threshold of rat hind paw before(0d),3 d,7 d and 14 d after intraperitoneal injection.The significant decre ase in the ratio was statistically significant,that is,a successful DMA mod el rat.According to the above method,a total of 11 rats were successfull y prepared as a DMA model.(3)Grouping 12 normal control rats were ra ndomly divided into 2 groups: Group 1: normal rats were intrathecal inject ed with control group(n=6),intrathecal injection of shRNA lentivirus was i neffective,and shRNA lentivirus did not result in any cells The mRNA wa s degraded as a negative control of Epac1 shRNA lentivirus;2 groups: no rmal rats intrathecal injection group(n=6)and intrathecal Epac1 shRNA le ntivirus.11 DMA model rats were randomly divided into 2 groups,3 group s: DMA intrathecal injection control group(n=5),intrathecal injection shRN A lentivirus;4 group: DMA intrathecal injection group(n=6)Intrathecal Epa c1 sh RNA lentivirus.(4)Detection of changes in hind paw withdrawal thre shold and changes in mRNA and protein expression Changes in paw with drawal thresholds before and after intrathecal injection(0 d),1 d,3 d,6d,9 d,12 d,and 14 d were detected.14 days after intrathecal injection,1% sodium pentobarbital(65 mg/kg)was injected intraperitoneally to rats t o take L4-L6 dorsal root ganglion.Real time-PCR and Western-Blot were used to detect the dorsal root of the rats in the 4 groups.Mitochondrial si te Epac1 mRNA and protein expression.Results(1)Compared with the normal control group(group 1,group 2),the h ind paw withdrawal threshold in the DMA group(group 3,group 4)was si gnificantly decreased,and there was a statistically significant difference be tween the 7th and 14 th days after intraperitoneal injection.(P = 0.020,P= 0.000).At the same time,the expression of Epac1 m RNA and protein was up-regulated in the DMA group(group 3,group 4)compared with the normal control group(group 1 and group 2)(P=0.000,P=0.000).(2)On the 14 th day after intrathecal injection,the hind paw withdrawa l threshold in the DMA intrathecal injection group(4 groups)increased sig nificantly,which was significantly higher than that in the DMA intrathecal g roup(group 3)rats.The difference was statistically significant(P < 0.05).= 0.000),indicating that Epac1 sh RNA lentivirus can upregulate mechanica l pain threshold in rats and relieve mechanical pain in rats with diabetes mellitus;analysis of variance analysis and statistics of factorial design sho wed that intrathecal injection of lentivirus group was compared with intrath ecal injection of ineffective lentivirus group.Epac1 mRNA and protein expr ession decreased(P=0.000,P=0.020).ConclusionsThe expression of Epac1 in DMA rats was increased.The inhibition of Epac1 expression by lentivirus-mediated sh RNA upregulates the rat mechanical pain threshold and relieves mechanical pain in diabetic rats.