| Objective:Neuropathic pain?NPP?is the most intractable chronic pain in clinical.The pathogenesis and mechanisms of NPP remain unknown,which leads to the lack of effective theraputic strategies,and make a heavy burden for the society and patients.Postherpetic neuralgia?PHN?is known as the most common type of NPP with the severe pain intensity.Therefore,protein chip technique was used to screen the differentially expressed proteins in PHN patients,and verified the target differentially expressed proteins by expanded clinical samples.Then,animal model of NPP was established to explore the potential mechanisms of target differentially expressed proteins in NPP,thus providing a meaningful molecular basis for in-depth exploration of the pathogenesis of NPP.Methods:1.Forty-three PHN patients who came from Hospital?T.C.M?affiliated to Southwest medical university from June to October2019 were selected as the case group?PHN group?.Forty-three healthy individuals of similar gender and age were included as the control group?CON group?.The gender,age,body mass index?BMI?and other general information of the subjects in the two groups were recorded.Visual Analogue Scale?VAS?was used to evaluate pain intensity of PHN patients.Venous blood was extracted from the two groups of subjects to harvest the serum.Then,serums of all subjects were stored in-80?refrigerator.3 serum samples were randomly selected from PHN group and CON group respectively.TMT protein chip analysis was used to screen out the differentially expressed proteins of PHN patients.2.Enzyme-linked immunosorbent assay?ELISA?was used to validate the expression of target differentially expressed protein?DPP4?in serums of subjects in the two groups based on the results of protein microarray screening.3.Ninety adult Sprague-Dawley?SD?male rats?200-250g,6-8weeks old?were selected as experimental animals and randomly divided into three groups:sham group?S group?,model group?M group?,DPP4inhibitor group?D group?,n=30.The rats in M group were performed with Spared nerve injury?SNI?model;The rats in S group only exposed the sciatic nerve without injury.After the establishment of SNI model,rats in D group were intrathecally injected with DPP4 inhibitor?Vildagliptin 10mg/ml?10ul once a day.Rats in M group and S group were treated with intrathecal injection of 10ul saline as an intervention control.At the time points of preoperative day 1?T0?,postoperative day 3?T1?,postoperative day 7?T2?,postoperative day 14?T3?,behavioral tests of three groups were detected by Thermal withdrawl threshold?TWL?and Mechanical withdrawl threshold?MWT?.After the behavioral tests,the L4-5 spinal cord segment and L5 Dorsal root ganglion?DRG?of all rats were extracted;The expression of DPP4 in L4-5 spinal cord and L5 DRG and the release of inflammatory cytokines TNF-?and IFN-?in L4-5were detected by ELISA.Expression of CGRP and microglia marker Iba-1 in L5 DRG and L4-5 spinal cord were detected by Immunofluorescence and Western blot..Results:1.There was no significant difference in age,gender and BMI between the two groups?P<0.05?.The VAS score of PHN group was 6.0±0.8,while subjects in CON group without pain.After protein microarray analysis,127 differentially expressed proteins were found in the serum of PHN patients,including 79 up-regulated proteins and 48down-regulated proteins.2.Among the up-regulated differentially expressed proteins,DPP4 was selected as the target protein.It was found that the serum DPP4 concentration in PHN group was4093.02±858.30pg/ml,and that in CON group was 1978.10±579.01pg/ml.3.SNI rats began to exhibited the symptoms of NPP at T1.MWT and TWL of M group and D group were significantly lower than those in S group at T13?P<0.05?.However,intrathecal injection of DPP4inhibitor improved the pain intensity of SNI rats,and MWT and TWL of group D were significantly increased at T13 compared with M group?P<0.05?.In addition,the expression level of DPP4 in L4-5 and L5 DRG in M group and D group increased significantly from T1 to T3 compared with that of S group?P<0.05?.However,compared with M group,the expression level of DPP4 in L4-5 and L5 DRG in D group significantly decreased from T1 to T3?P<0.05?.The microglia of M group and D group were activated at T133 by SNI surgery.However,compared with M group,the activation of microglia was inhibited in D group by the DPP4inhibitor,and the expression level of Iba-1 in L4-5 of D group was significantly decreased at T3 compared with M group?P<0.05?.Moreover,the release of TNF-?and IFN-?in L4-5 of rats was increased after SNI.TNF-?and IFN-?in L4-5 of rats in M group and D group were significantly up-regulated at T13 compared with S group?P<0.05?,but the up-regulated TNF-?and IFN-?was significantly inhibited by DPP4inhibitor in D group?P<0.05?.The overexpression of CGRP in L5 DRG in D group and M group were detected compared with S group at T13,but the expression of CGRP in D group was significantly inhibited compared with M group,and the expression of CGRP in L5 DRG of D group was significantly decreased at T3 compared with M group?P<0.05?.Conclusion:1.The overexpression of DPP4 in peripheral blood,L5DRG and L4-5 might contribute to the development of NPP.2.DPP4 might participate in the peripheral and central sensitization of NPP by promoting the excitability of traumatic sensory neurons in DRG and the activation of microglia in spinal dorsal horn. |
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