Epigenetic Regulation Mechanism Of TET1 On Postoperative Incision Pain Delay Caused By Perioperative Stress

Background:Chronic postoperative pain(chronic post-surgical pain syndrome,CPSP)is a common clinical problem and it's mechanisms are unknown.Survey shows that approximately 10%-50% of patients suffered postoperative pain,in which of 10%patients suffered chronic pain badly even the primary disease was healed.Because of the CPSP mechanism is unclear,no suitable treatment for CPSP,so there is a research challenging in this field.In clinical reports,peri-operative patients may be interfere with stress,anxiety,environmental noise on sleep,which can affect the central nervous system of patients that lead to endocrine disorders.The pathological changes may induce CPSP.But the specific molecular mechanism of CPSP is still unknown,especially in epigenetic mechanisms of particular central and peripheral neurons.This research used peri-operative stress animal model,found that peri-operative stress could cause chronic postoperative pain,and DNA demethylase TET1 expression in rat dorsal root ganglion and Spinal Cord was decreasd.So we focused on if or how TET1 participated in CPSP which caused by the peri-operative stress.The aim of this research is to detect the underling mechanisms of TET1 involved in development of CPSP,to provide new ideas to treat CPSP or other neuropathic pain based on this experiment.Research purposesThis project will address behavior characteristics of postoperative pain due to lack of perioperative sleep disturbance;reveal the molecular machanism of increasing neuron excitability induced postoperative pain due to lack of sleep;and explore the epigenetic mechanisms which regulate the development of postoperative pain and extend the postoperative pain due sleep disturbance.This reseach aimed to reveal the underling mechanism of development and maintaince of CPSP through the molecular-cellular-whole body,provide new targets for clinical treatment.Research methods(1)animal modelsRats were divided into 4 groups: N Group(Sham);IN group: Rats were anesthetized with 2% isoflurane delivered via a nose cone.The plantar aspect of left hindpaw was prepared in a sterile manner with a 10% povidone-iodine solution.A1-cm longitudinal incision was made with a number 11 blade,through skin and fascia of the plantar aspect of the foot,starting 0.5 cm from the proximal edge of the heel and extending toward the toes.The plantaris muscle was elevated and incised longitudinally.After hemostasis with gentle pressure,the skin was sutured with 5-0nylon.After surgery,the animals were allowed to recover in their cages;S group: The rats were immobilized with metal mesh packaging and restricting the motion of the head and body or put on the platform aroud the water in a tank,Stress was performed at the same time of the day(between 8 AM and 2 pm)without food and water,animals were subjected to a daily 6-h stress for 3 days;IN+S Group : the rats were suffered perioperative stress before or after incision.Determine model success by detecting behavior changes of mechanical ? thermal ? cold threshold,and pain recovery time;Determine activity of neurons,microglia,astrocytes by Western-blot and immunofluorescence;electrophysiology in DRG and spinal cord.(2)Explore the changes of Methyltransferase and Demethyltransferase enzymes after the chronic pain model in DRG and spinal cord by Western-blot;Detected behavior changes through downexpression TET1 or overexpression TET1 by Si-TET1 or herpes simplex virus carry TET1 CDS,to test effects of TET1 on regulating CPSP.We used Western-blot ? qPCR to test the expression of methylation enzyme DNMT1?DNMT3a?DNMT3b and demethylation enzymes TET1? TET2? TET3;micro-injected siTET1 separately in DRG or Spinal cord to detect if it can induce pain behavior,micro-injected HSV-TET1 separately in DRG and Spinal cord to observe if it can reverse CPSP behavior changes of mechanical ? thermal ? cold threshold,and pain recovery time;From this experient we could know if TET1 is important and necessary elements involved in CPSP.(3)Regulation of glucocorticoid on TET1In order to find which factor caused the changes of TET1,we first detected theserum glucocorticoid levels after rat stress or incision;then we cultured DRG neurons,detected TET1 expression changes in DRG neurons after given corticosteroid or glucocorticoid receptor antagonist Ru486;we still intraperitoneal injected glucocorticoid to observe if glucocorticoid intraperitoneal injection can induced CPSP,and used the glucocorticoid receptor antagonist Ru486 could reverse the CPSP;finally we used Chromatin immunoprecipitation(CHIP)and the dual-luciferase reporter gene(luciferase)to detect the regulation of glucocorticoid receptor GR on TET1 promoter.(4)Explore the regulatory mechanism of TET1 on downstream proteins.Check the pain related genes from DRG and Spinal cord by Western-blot ?qPCR,to find the difference expression between IN+S group and the other groups,especially check MOR?KOR?Kv1.2 expression changes;Overexpressed TET1 by using HSV-TET1 or down-expressed by using si-TET1 transfection in DRG cell culture culture and in vivo micro-injection in DRG and spinal cord to observe the changes of pain-related genes,especially check MOR?KOR? Kv1.2 expression changes.Then we focus on MOR,through bioinformatics software,we designed 7primers of MOR promoter,we used TET1 antibody to check if TET1 and OPRM1 promoter is binding together and the binding degree changes under stress;we still used immunofluorescence to detect TET1 expression in spinal cord and ganglion and the coexpression in one cell with MOR and KOR;Gave MOR,KOR agonists or antagonists to observe if IN+S behavioral could change.Results1.Animal models are builded successfully.Compared to IN groups,IN+S groups appeared more sensitive pain behavior from 7d to 9d of mechanical pain,thermal pain,cold pain.Compared to IN group,the incision pain disappeared at postoperative 9d,but in IN+S group pain threshold of machanical ?thermal?cold didn't back to normal untill postoperative 13 d.Immunofluorescence showed microglia marker Iba1 and astrocytes marker GFAP expression increased in ipsilateral side of L5 spinal cord at postoperative 9d;Western-blot showed pERK1,pERK2,GFAP expression increased in ipsilateral side of L5 spinal cord in IN+S Group;Electrophysiology showed excitability of dorsal root ganglion neurons increased inIN+S Group at 9d compared to the IN Group.2.Western-blot?qPCR results displayed methylase enzyme DNMT1?DNMT3a?and DNMT3 b,demethylase enzyme TET2?TET3 expression in the spinal cord and the DRG had no changes,but demethylase TET1 expression decreased in the spinal cord and the DRG of IN+S group compare to IN group.We microinjected si-TET1 to normal rat DRG and Spinal cord respectively and found the threshold of mechanical?thermal?cold decreased compared to negative microinjection.Though microinjected HSV-TET1 to DRG and spinal cord,We found overexpression TET1 can reversed the decrease of the mechanical threshold ?thermal threshold?cold threshold in IN+S Group and shorten the duration of postoperative pain.3.The cortisone lever of blood were increased more than thousand folds in IN+S group or S group compared to N group or IN group.Sucrose preference results showed that rats of IN+S group and S group intake less sucrouse than N group and IN group;Forced swimming test showed the immobilization time in water extended of IN+S and S group compared to N group and IN group;Western blot and qPCR result all showed TET1 expression decreased in DRG or spinal cord after intraperitoneal injection cortisone continuous 3 days and then incision rats,as well as the same phenomenon in DRG cell culture after cortisones stimulating;this decrease of TET1 stimulated by cortisone could be reverse after GR receptor block agent Ru486 offering no matter in cultured cell or intrathecal Ru486 in IN+S rats.We still found GR antibody could pulldown TET1 promtor by chromatin immune precipitation(CHIP),and activate GR could decreased the luciferase by double fluorescent enzyme report gene carried TET1 promotor after transfected into HEK293 cell.4.Compared to IN group,Western-blot?qPCR result showed that MOR or KOR expression decreased in DRG or spinal cord;the function of MOR and KOR were weaken detected by conditioned place preference experiments of IN+S compared to IN group.Western-blot?qPCR showed MOR,KOR expression decreased in cultured DRG cell after Si-TET1 tranfection;Western-blot,qPCR showed MOR,KOR expression increased after transfection of HSV-TET1 in DRG cells.As well as in vivo experiment,Western-blot?qPCR showed MOR,KOR expression decreased after DRG or spinal cord injection of si-TET1;Western-blot?qPCR showed MOR,KORexpression increased after DRG or spinal cord injection of HSV-TET1 in IN+S group,as well as pain behavior reversed compared to microinjection of negative control in IN+S group.We still found TET1 antibody could pulldown MOR promtor by chromatin immune precipitation(CHIP),and TET1 could increased the luciferase by double fluorescent enzyme report gene carried MOR promoter after transfected into PC12 cell.DNA-blot displayed although total methyl of level didn't variable,but the high-through sequencing results show that MOR promoter methylation increased in IN+S Group compared to IN group.Research conclusionsPerioperative stress caused glucocorticoid receptor activation that inhibited TET1,the decrease of TET1 expression affected the function and expression of MOR?KOR because of 5mc/5hmc increased.TET1 is the important and necessary factor involved in the development and progression of chronic postoperative pain.It is very important epigenetic molecules of TET1 to fully clarify the mechanisms of CPSP to provide a theoretical basis for opening up new ideas for treatment of CPSP.So TET1 might be a potential targets for the prevention and treatment of CPSP.