The Epigenetic Regulation Involves In The Peripheral Mechaniam Of Chemotherapy Induced Pain:DNA Demethylase TET1 Regulates Potassium Channels In Primary Sensory Neurons

[Background]Chemotherapy-induced neuropathic pain is one of the common complications of chemotherapeutic drugs,with an occurrence rate of 71%to 96%;The clinical features are mainly manifested as a series of neurological disorder symptoms and signs caused by peripheral nerve or autonomic nerve injury.The pathological features were persistent spontaneous pain or intermittent burning pain,severe pain hypersensitivity and abnormal pain response to normal harmless stimuli.It is one of the common types of neuropathic pain.At present,there is limited understanding of the pathogenesis of chemotherapy induced pain,and there is no effective means to treat it.Therefore,it is of great practical significance to explore the pathogenesis of chemotherapy pain.The dorsal root ganglion(DRG),as the primary sensory neurons,plays a critical role in the pathogenesis of neuropathic pain,and the ion channel is closely related.DRG tissues are rich in capillaries and lack of blood-brain barrier.Chemotherapy drugs are easy to accumulate in DRG and can have toxic and side effects on neurons.Ion channel is the molecular basic of neuronal electric activities.Tandem pore domain potassium(K 2P 1.1)is one of the potassium channels,driving by the asymmetric K+gradient across the membrane and is responsible for maintenance of rest membrane potential.Relevant studies have found that the mRNA expression of K 2p 1.1 in DRG was significantly reduced after the intraperitoneal injection of chemotherapy drug paclitaxel.K 2P 1.1 is encoded by gene Kcnkl.Therefore,further study on the relationship between Kcnkl gene and chemotherapy induced neuropathic pain in DRG may reveal the molecular mechanism of chemotherapy induced neuropathic pain.Epigenetic regulation refers to that genetic material other than the genetic information has changed under the environmental stimuli,while the sequence of DNA does not change,and this change can be stable transmitted in the process of growth and cell proliferation;it mainly includes that DNA methylation and demethylation,histone modification.DNA methylation is that the CpG(cytosine-phosphate-guanine)phosphate double cytosine in the nucleotide sequence of 5 carbon atoms on the covalent bonding a methyl group was catalyzed to the reaction of 5-methyl cytosine by DNA methylation transferase catalysis and an S-adenosine methionine was served as a methyl donor.DNA methyltransferase family(DNMTs)has 3 types:DNMT1,DNMT3a,DNMT3b.Overexpression of DNA methylation is usually associated with gene silencing.The TET enzyme family can induce 5-methyl cytosine(5-mC)to oxidize to 5-hydroxymethyl cytosine(5-hmC),leading to DNA demethylation.TETs includes TET1,TET2,and TET3.Related research found that the occurrence of cancer is related to the apparent genetic changes(including DNA methylation,etc.).In previous studies,dorsal root ganglion microinjection HSV-TET1,and overexpression TET1 in SNL model.Overexpression TET1 can promote the DNA methylation in DRG,but after knockout the TET1 in spinal cord,the excitability of spinal dorsal horn was decreased.It is speculated that TET1 plays an important role in DNA methylation and may be an important target for the treatment of chemotherapy pain.Therefore,This research set up chemotherapy pain model,firstly,we detected the changes of DRG Kcnkl gene methylation in chemotherapy pain rats,and to verify the relationships between chemotherapy pain and Kcnkl gene methylation and demethylation changes;Then,Dorsal root ganglion microinjection HSV-TET1,overexpression TET1,and to verify the effect of TET1 on the Kcnkl gene methylation in the chemotherapy pain.To further studying the relationships between TET1 and epigenetic DNA methylation,it is expected to reveal the mechanism of chemotherapeutic pain and provide new thinking and new strategies for clinical treatment of chemotherapy pain.[Methods]1.Establish the chemotherapeutic pain model.SD(Sprague-Dawley)male rats were divided into Vehicle groups and Paclitaxel(Paclitaxel,Pac)groups(each group>5).Paclitaxel was injected intraperitoneally into rats every other day(d 0,2,4,6).Control solution was injected intraperitoneally into Vehicle group rats,the injection methods is the same as Paclitaxel group.2.Observe the behavioral changes of rats with chemotherapy pain model.After chemotherapy pain models were established,Vehicle groups and Paclitaxel groups in 4,7,10 days to observe rats' induced pain behaviors,record the two groups of rats mechanical,heat and cold stimulation of behavioral responses,and statistical analysis was carried out as on the record.3.Immunofluorescence double standard observation K 2p 1.1 expressed common localization between different cells in dorsal root ganglion.After the rat models of chemotherapy pain was established,and L4-5 dorsal root ganglion tissues were obtained.In this study,the expression in the dorsal root ganglion distribution of neurons and other cells were detected by immunofluorescence bidirectional method.4.HSV-TET1 was injected into the dorsal root ganglion of rats with chemotherapy pain model,and the behavioral changes of rats were observed respectively.After the chemotherapy pain model rats were microinjectioned HSV-TET1 on dorsal root ganglion,the rats were divided into two groups:the groups,at 4th day,HSV-TET1 was micro-injectioned into Pac + HSV + TET1 groups,Vehicle +HSV + TET1 groups in dorsal root ganglion,were observed behavioral changes between groups of mechanical stimulation,heat stimulation and cold stimulation,and statistical analysis of each record;The other groups,at 7th day,TET1 was micro-injectioned into Pac + HSV + TET1 groups,observe rats respectively behavior change of mechanical stimulation,heat stimulation and cold stimulation,and statistical analysis of each record.5.Expression of K 2p 1.1 in the dorsal root ganglion of rats with chemotherapy pain model.After the rats chemotherapy pain models were established,the pain model rats L4-5 dorsal root ganglions were observed,using Western blot test technology,the detecting different points in time L4-5 dorsal root ganglion tissues about expression changes of K 2 p 1.1.The immuno-fluorescence double standard staining technique was used to detect the change of K 2p 1.1 expression and the expression of localization between different cells in rats with the chemotherapy pain model L4-5 dorsal root ganglion tissue.After microinjection HSV-TET1 in L4-5 dorsal root ganglion,to observe the chemotherapy pain model rats L4-5 dorsal root ganglion after overexpressing TET1,TET1 was overexpressed in pain model rats,the influence of experimental results.6.Chemotherapy pain model rat dorsal root ganglion after microinjection HSV-TET1,TETI tested expression in rat dorsal root ganglion changes in DNA methylation level and expression changes of K2 p 1.1 channel protein.Build the herpes simplex virus vector of HSV-TET1.In establishing the rat pain model 4th and 7th day after chemotherapy,pain model rats were given chemotherapy L4-5 dorsal root ganglion microinjection HSV-TET1,according to the expected time,chemotherapy pain model rats were taken of L4-5 dorsal root ganglion tissue microinjection HSV-TET1 dorsal root ganglion tissue,and then to each dorsal root ganglion tissue,using Western blot technique,testing different time points of dorsal root ganglion Kcnkl gene expression changes in organization;Taking each dorsal root ganglion organization,the use of immunofluorescence double marking technology,TETI and Kcnkl in dorsal root ganglion cells within the positioning,the relationship between overexpression of TET1 and potassium channel K 2 p 1.1 protein expression changes in dorsal root ganglion.7.After microinjection of HSV-TET1 in the dorsal root ganglion of the rats with chemotherapeutic pain model,it was verified that whether TET1 was involved in chemotherapeutic pain by regulating the methylation of Kcnkl gene.After rat chemotherapy pain models was established,the HSV-TET1 was microinjectioned into dorsal root ganglion,taking the root ganglion tissue,using Western blot technique,Chemotherapy pain models in rat dorsal root ganglion expressing TET1 and Kcnkl gene promoter wereobserved;Taking the dorsal root ganglion tissue,using the chromosome(ChIP)-PCR immune coprecipitation method,observe the level change of chemotherapy pain model Kcnkl promoter region methylation in rat dorsal root ganglion,validation Kcnkl gene promoter region of 5-mC and 5-hmC.[Results]1.Before chemotherapy drugs were injected into the Vehicle groups and paclitaxel groups,the rats lientang foot behavioral based threshold was tested(mechanical stimulation threshold,heat stimulation threshold and cold stimulation threshold),had not found the obvious difference(P>0.05).Statistical analysis was performed on the mean values of the left hind legs of the two groups.Chemotherapy pain model rats were observed 4th,7th,10th day after behavioral response of rats,compared with the Vehicle group,mechanical stimulation shrinkage foot reflex threshold significantly lower(P<0.05),latent heat shrinkage foot threshold significantly decreased(P<0.05),cold stimulation shrinkage foot reflex threshold number increased significantly(P<0.05).2.Compared with the Vehicle group,the expression of K 2p 1.1 protein and mRNA in the dorsal root ganglion,paclitaxel groups were significantly reduced compared with that of the Vehicle groups.3.Chemotherapy pain model rats microinjection TET1 in dorsal root ganglion,which were observed behavioral response of rats found that mechanical stimulation threshold,heat stimulation threshold and cold stimulation threshold have obvioused,that were difference(P<0.05).It was suggested that TET1 could block chemotherapeutic drug induced neuropathic pain behavior change in rats with dorsal root ganglion.4.The changes of DNA methylation level and the changes of K 2p 1.1 channel protein the expression,which was expressed after the DRG microinjection of DRG microinjection in rats with chemotherapy pain model.5.Chromosome immune coprecipitation-PCR experiment,founding in Kcnkl gene promoter R1 and R3 area,5-mC and the change of 5-hmC exists is in contrast,when the chemotherapy DRG Kcnkl higher gene expression within 5-mC,but the 5-hmC is reduced,therefore,the methylation s of DRG Kcnkl gene in chemotherapy pain model rats was rised;It was shown that DRG injection of HSV-TET1,and overexpression TET1 could reverse this change and reduce the methylation level of Kcnk1 gene.6.Westion Blot technique test shows that chemotherapy did not change the expression of TET1,but lowered Kcnk1 expression,overexpression of TET1 can reverse chemotherapy reduced DRG Kcnkl protein expression,the expression of TET1 and Kcnkl reverse change of protein expression,prompt TET1 down-regulate Kcnkl protein expression.7.After Chemotherapy pain model rats microinjection HSV-TET1,and overexpression TET1,using Westion Blot technique,testing shows that chemotherapy pain does not change the expression of TET1,but lowered Kcnkl gene expression;However,the overexpression of TET1 in the dorsal root ganglion can reverse K 2 p 1.1 reduced protein expression at dorsal root ganglion,the change of expression TET1 and K 2 p 1.1 protein expression is reverse,prompting TET1 expression can increase K 2 p 1.1 protein expression.[Conclusions]The behavioral data of rats showed that DRG microinjection of hsv-tetl was able to relieve the chemotherapy pain induced by paclitaxel.Decreased K 2p 1.1 expression in DRG neurons caused increased excitability of neurons and the pathogenesis of chemotherapy pain.The decrease of K 2p 1.1 protein expression was due to the increase of methylation in the promoter region of DRG Kcnkl gene.TET1 induces 5-mc in the promoter region of Kcnkl gene to generate 5-hmc,which increases the methylation level of Kcnkl gene and reduces the methylation of Kcnkl gene.The chemotherapy pain did not change the expression of TET1,but the expression of K 2p 1.1 protein was down-regulated,and overexpression of DRG TET1 in the chemotherapy pain model could increase the expression of Kcnk1 protein and alleviate the chemotherapy pain in rats.Therefore,TET1 may be played an important role in chemotherapy pain occurrence and development,the TET1 may help clear epigenetic mechanisms,and provide reference to reveal the pathogenesis of pain chemotherapy,and help opening up new ideas for clinical treatment chemotherapy pain.In short,TET1 regulates DNA methylation and demethylation,which may be a potential target for the prevention and treatment of chemotherapy pain.