TET1 Participates In Oxaliplatin-induced Neuropathic Pain By Regulating The Expression Of Kv1.2

BackgroundOxaliplatin is a third-generation platinum-based chemotherapeutic drug,which has an effect on a variety of solid tumors such as colorectal cancer,pancreatic cancer,and lung cancer.However,85%?95%of patients who have been used oxaliplatin treatment will develop related peripheral neuropathy.The clinical manifestations were mainly peripheral numbness,hyperalgesia or hypersensitivity,and "stocking-like"paresthesia.Cold stimulation can aggravate its neurological symptoms,and it has a significant dose-and time-dependence.Moreover,the treatment of oxaliplatin was probably interrupted due to severe neuropathy,which affected the treatment effect of patients and shortened survival time.Therefore,exploring the molecular mechanism of oxaliplatin-induced peripheral neuropathy has an important significance in reducing or avoiding the neurotoxic effects of oxaliplatin treatment.Previous studies have shown that oxaliplatin damages peripheral nerves system more severely than the center nerves system.Therefore,exploring the molecular mechanism of ion channel changes and damage in dorsal root ganglia was essential to reveal excitability changes after peripheral nerve injury.Voltage-gated potassium channels were one of the important ion channels involved in neuronal excitability.They participated in the regulation of neuronal excitability by maintaining resting membrane potential and controlling action potential.In oxaliplatin-induced peripheral nerve injury,voltage-gated potassium channel 1.2(Kv1.2)protein expression was reduced,causing neuronal excitability was increased.However,the molecular regulation mechanism of kv1.2 targeting molecules was still unknown.Epigenetics refers to changes in gene expression levels based on non-gene sequence changes,which can regulate gene expression and participate in the regulation of pain.DNA methylation is one of the important regulation.DNA methylation transferases(DNMTs)are involved in DNA methylation and dioxygenases(TETs)are involved in the regulation of DNA demethylation to inhibit gene transcription through different mechanisms.Dioxygenase 1(TET1)converts 5mC to 5hmC by reducing the expression of TETs family proteins,inhibiting DNA demethylation and gene transcription.Our previous results showed that the demethylase 1(TET1)expressed in dorsal root ganglion was reduced in the oxaliplatin-induced neuropathic pain model,but whether TET1 targets the expression of kv1.2 and participates oxaliplatin-induced neuropathic pain remains unclear.Therefore,our study established a model of oxaliplatin-induced chemotherapy pain,and explored the molecular mechanism of the demethylated transferases TET1 and Kv 1.2 to provide new ideas and directions for the treatment of oxaliplatin-induced peripheral neuropathy.ObjectiveThis study explored the changes in potassium channels in neuropathic pain caused by oxaliplatin and related epigenetic regulatory mechanisms,which providing a theoretical basis for reducing oxaliplatin-induced peripheral neurotoxicity.Methods1.We intraperitoneally injected oxaliplatin(6mg/kg)into rats every two days for a total of 4 injections to establish a model of peripheral neuropathic pain caused by chemotherapy.The von-Frey silk was used to observe the threshold of mechanical stimulation in rats.The cold plate test was used to observe the latency of cold stimulation,and the acetone test was used to record the pain score.The qPCR technique was used to screen 8 different subtypes of potassium channels,and the expression levels of DRG and anterior cingulate gyrus in the 14th day after oxaliplatin treatment were detected.2.The immunofluorescence technique was used to detect the colocalization status of kv 1.2 with CGRP,IB4 and NF200 in DRG neuron,respectively,And the mean fluorescence intensity of kv1.2 in DRG neurons on days 7,14,and 21 after oxaliplatin treatment.3.Western-blot and qPCR were used to detect gene and protein expression levels of kv1.2 in DRG neurons on days 7,14,and 21 after oxaliplatin treatment.4.The immunofluorescence was used to detect the co-standards of TET1 and CGRP,IB4 and NF200 in DRG neurons,and the the mean fluorescence intensity of TET1 in DRG neurons on days 7,14,and 21 after oxaliplatin treatment.5.Western-blot and qPCR were used to detect gene and protein expression levels of TET1 on days 7,14,and 21 after oxaliplatin treatment.6.We used TET1-loxp transgenic mice,and injected cre-recombinase through DRG microinjection to conditionally knock out the TET1 gene in DRG neurons and observed the behavioral changes.qPCR was used to detect the gene expression levelsof kv1.2 after TET1 knockout.7.The immunofluorescence was used to detect the co-localization of TET1 and kv1.2 in DRG neurons and their co-standard rates on day 14 after oxaliplatin treatment.8.Before establishing chemotherapy model in rats,we overexpressed TET1 through DRG microinjecting lentivirus.SD rats were divided into three groups:Naive,TET1-NC+OXA,TET1-Lv+OXA.And we observe the behavioral changes of rats and the expression levels of TET1 and kv1.2 proteins among the groups.After establishing the chemotherapy model in rats,we overexpressed TET1 through DRG microinjecting lentivirus.SD rats were divided into three groups:Naive,OXA+TET1-NC,OXA+TET1-Lv.And we observed the behavior changes of rats among the groups.9.Before establishing a chemotherapy model in mice,we overexpressed TET1 through DRG microinjecting lentivirus.C57/BL mice were divided into three groups:Naive,TET1-NC+OXA,TET1-Lv+OXA.We observed the behavioral changes of mechanical pain and the expression levels of TET1 and kv1.2 genes in mice among the group.Results1.After we established a rat oxaliplatin chemotherapy pain model,compared with the Naive group,the bilateral mechanical pain threshold and cold pain threshold in the OXA group were significantly reduced from 7 to 21 days after oxaliplatin treatment(P<0.001),which indicated that the animal models were successfully established.qPCR screening results showed that kv1.1(P<0.001),kv1.2(P<0.01),kv2.1(P<0.01),and kv3.4(P<0.05)mRNA expression in DRG were decreased.The mRNA expression of kv3.4(P<0.05),kv4.3(P<0.01),and kv7.5(P<0.01)were decreased in the anterior cingulate gyrus of the brain.However,the mRNA expressions of kv 1.4 and kv4.2 were unchanged in these two parts.2.Immunofluorescence found that kv1.2 was mainly expressed on the cytoplasm and membrane of neurons,and co-standardized with large,medium and small diameter neurons and peptide energy neurons,and hardly co-standardized with satellite glial cells.The mean fluorescence intensity of kv1.2-positive cells of DRG neurons was decreased significantly at 7(P<0.001),14(P<0.01),and 21 days(P<0.01)after oxaliplatin treatment.3.Western blot showed that the expression of kv1.2 protein in DRG neurons was significantly downregulated on days 7,14,and 21 after oxaliplatin treatment in rats(P<0.05).qPCR showed that the mRNA expression of DRG neurons kv1.2 was decreased at 7(P<0.05),14(P<0.05),and 21(P<0.01)days after oxaliplatin treatment.4.Immunofluorescence found that TET1 was mainly expressed in the nucleus,and co-standarded with large,medium and small diameter neurons and peptide energy neurons.On the 7(P<0.001),14(P<0.01),and 21(P<0.01)days after oxaliplatin treatment,the average fluorescence intensity of TET1-positive cells in DRG neurons was significantly reduced.5.Western blot and qPCR showed that the expressions of TET1 protein(P<0.001)and mRNA(P<0.01),not DNMT3A were reduced on the 14th day after oxaliplatin treatment.The expression of TET1 protein in DRG neurons was significantly reduced on days 7,14,and 21 after oxaliplatin treatment in rats(P<0.001).And the mRNA expression of TET1 in DRG neurons was also reduced on days 7(P<0.05),14(P<0.05),and 21(P<0.01).6.In the TET1-loxp transgenic mice,we found that a mechanical hyperalgesia on the 21 day(P<0.05)after DRG micro injection of cre-recombinaseand lasted to 28 days(P<0.001).qPCR showed that the expression levels of mRNA of TET1 and kv1.2 were significantly reduced(P<0.01).7.The immunofluorescence double standard found that kv1.2 and TET1 were co-standard,and the co-standard rate of both was reduced on the 14th day after oxaliplatin treatment in rats(P<0.05).8.TET1-Lv/NC was given 7 days before oxaliplatin treatment in rats.Compared with the TET1-NC+OXA group,the rats that the mechanical pain threshold of the TET]-Lv+OXA group on the 7th day after oxaliplatin treatment(P<0.05)and cold pain threshold(P<0.05)were alleviated and it lasted until 21 days.Western blot showed that the protein expressions of kvl.2 and TET1 were reversed on the 14th day after oxaliplatin treatment in rats(P<0.05).TET1-Lv/NC was given on the 7th day after oxaliplatin treatment.Compared with the OXA+TET1-NC group,behavior showedthat the mechanical pain threshold(P<0.05)and cold pain threshold(P<0.05)of the OXA+TET1-Lv group were alleviated significantly on the 14th day after oxaliplatin treatment,and it lasted until 28 days.9.We established a mouse model of oxaliplatin chemotherapy pain.TET1-Lv/NC was administrated 14 days before oxaliplatin treatment,and the results showed that compared with the TET1-NC+OXA group,the frequency of withdrawal was alleviated on the 14th day after oxaliplatin treatment whether it was 0.07g or 0.4g(P<0.05).qPCR showed that the mRNA expression of kv1.2 was reversed in mice(P<0.05).ConclusionIn the model of oxaliplatin-induced pain,dorsal root ganglion TET1 participates in the neuropathic pain induced by oxaliplatin by down-regulating the gene and protein expression of kv1.2.