HDAC2 Regulates Kv1.2 Expression To Mediate Neuropathic Pain

BackgroundNeuropathic pain is caused by injury or disease of the somatosensory nervous system.It can cause prolonged pain and diminish the patient's quality of life.Its clinical features are spontaneous pain,hyperalgesia,and allodynia.However,the molecular mechanism of the neuropathic pain is not very clear.There is still a lack of effective treatment,Therefore,an indepth study of the mechanism of pain will increase our awareness of pain itself,and it also has important theoretical and clinical meanings for the development of novel analgesics.Potassium channels selectively allow the passage of potassium ions across the membrane and are the most various and most complex class of ion channels,which play an important role in the regulation of membrane potential and excitability of nerve cells.The ion channels directly affect the excitability of DRG neurons.In neuropathic pain model induced by peripheral nerve injury?such as CCI model?,the expression of Kv1.2 protein expression is down-regulated,potassium current is decreased and the excitability of neurons is increased in DRG,and pain occurs.Recent studies have focused that gene expression is also epigenetically regulated through chromatin remodeling and altered transcriptional regulation.Histone acetylation,one type of epigenetic modifications,has been implicated in synaptic plasticity and chronic NPP,which is mainly regulated by histone acetyltransferase and histone deacetylase,affecting the transcriptional activity of genes.HATs promotes depolymerization of chromosomes and activates transcription while HDACs block DNA and inhibit transcription.Research shows that histone deacetylase overexpression in neurons reduces dendritic spine density,synapse number,synaptic plasticity and memory formation.The non-selective HDAC inhibitor,suberoylanilide hydroxamic acid,relieves the inflammatory pain and neuropathic pain in rats.However,the underlying mechanisms of HDAC and HDAC inhibitor in the NPP remain unclear.Therefore,in this study,we established chronic constrictive injury model to investigate the relationship between HDAC and Kv1.2 and provided a new idea for clinically more targeted drug development.PurposesIn this study,we established chronic constriction injury to investigate the hypersensitivity and the expression of HDAC1 and HDAC2 at 0,3,7 and 14 days.By intrathecal injection with deacetylase inhibitor SAHA,we detected the behavior and the expression of Kv1.2.Then we meaned to evaluate whether HDAC1 and HDAC2 were double-labeled with Kv1.2 in the spinal cord and DRG respectively.HDAC1 siRNA and HDAC2 siRNA were intrathecally injected into the rats to detect behavioral changes and Kv1.2 expression.On the other hand,si-HDAC1 and si-HDAC2 were transfected in PC12 cells,and we detected the expression of Kv1.2.Methods1.We established of CCI model and detected the expression of HDAC1 and HDAC2.Rats were anesthetized with 10%chloral hydrate?0.35mL/100g?by intraperitoneal injection.Left hind limbs were disinfected with iodophor and the mid-thigh sciatic nerves were exposed by blunt dissection.Sciatic nerves were then separated using a glass needle and loosely ligated four times with 4-0 surgical wire.The distance between ligatures was approximately 1 mm.Incisions were sutured layer by layer.This surgical manipulation was not performed on the sciatic nerves of the sham control group.The mechanical pain thresholds and thermal pain thresholds were measured at 0,3,5,7 and 14 days after CCI.Western blot and qPCR were used to detect the expression of HDAC1 and HDAC2 at 0,3,7 and 14 days after CCI.The distribution of HDAC1 and HDAC2 in DRG and spinal cord was detected by immunofluorescence.2.We detected behavioral changes and Kv1.2 expression after the administration of SAHA.Male SD rats were divided into 4 groups randomly:CCI+vehicle,CCI+0.05?g/?L SAHA,CCI+0.1?g/?L SAHA and CCI+0.2?g/?L SAHA.We injected SAHA intrathecally 7 days in a row.And we detected PWTs and PWLs at 1 day before surgery,3,5,7,8,9,10,11,12,13,14 days after CCI.Spinal cord and L₄-L₆DRGs were collected from the lumbar region of rats,and the expression of Kv1.2protein were detected by Western blot.We identified the colocalization of Kv1.2with HDAC1 and HDAC2 in DRG by immunofluorescence.3.We detected Kv1.2 expression after the intrathecal injection of HDAC1siRNA,HDAC2 siRNAHDAC1 siRNA and HDAC2 siRNA were transfected into PC12 cells and Kv1.2 expression was detected by Western blot.Subsequently,rats were divided into5 groups:CCI,CCI+HDAC1 NC,CCI+HDAC1 siRNA,CCI+HDAC2 NC,CCI+HDAC2 siRNA.The corresponding siRNA was intrathecally injected into CCI and the PWTs and PWLs were detected at 0,3,5,7,8,9,10,11 and 12 days.The protein expression of Kv1.2 was detected by Western blot.Results1.Nerve injury upregulated the expression of HDAC1 in the DRG and HDAC2in the DRG and spinal cord.PWTs and PWLs in CCI rats were significantly decreased at 3-14d?P<0.001?compared with sham group.But contralateral side have no change,indicating the success of CCI surgery.Afterwards,Western blot revealed the expression of HDAC1in the spinal cord did not change significantly,while the expression of HDAC2 was significantly increased at 7 and 14 days after CCI?P<0.05?.However,the expression of HDAC1 was significantly increased in DRG at 3,7 and 14 days postoperatively?P<0.01?.And HDAC2 expression was also increased at 7 and 14 days postoperatively?P<0.01?.The mRNA expression was detected by qPCR,which was consistent with the protein expression results.By immunofluorescence,HDAC1 was mainly expressed in glial cells in spinal cord,with little expression in neurons.At DRG,HDAC1 was also expressed only in peripheral glial cells and small or medium sized neurons.The HDAC2 mainly colocalization with neurons.In the DRG,HDAC2 was also colocalized with large neurons and a small amount of small and medium-sized neurons.2.The HDAC inhibitor,SAHA,attenuated mechanical and thermal hypersensitivity and reversed decreased expression of Kv1.2 after CCI.The rats were intrathecally injected with SAHA,and SAHA at a concentration of 0.1?g/?L and SAHA 0.2?g/?L were found to be able to relieve the mechanical and thermal hyperalgesia.The expression of Kv1.2 in the spinal cord and DRG was detected by Western blot.The expression of Kv1.2 in both spinal cord and DRG was lower in CCI rats compared with sham group?P<0.001?.After administration of SAHA,Kv1.2 expression was significantly up-regulated?P<0.05?.By immunofluorescence,Kv1.2 is mainly expressed in large neurons in DRGs.Immunofluorescence showed that HDAC1 was not colocalized with Kv1.2,while HDAC2 was found to be completely double-labeled with Kv1.2.3.Treatment with HDAC2 siRNA relieved mechanical and thermal hypersensitivity and upregulated Kv1.2 expression.In vitro experiments,HDAC1 siRNA and HDAC2 siRNA were transfected into PC12 cells and proteins were extracted from the cells to detect Kv1.2 expression.It is HDAC2 siRNA increased the expression of Kv1.2?P<0.05?.After intrathecal injection of HDAC1 si RNA and HDAC2 siRNA to rats,mechanical and thermal thresholds were tested.The results showed that intrathecal injection of HDAC2siRNA significantly increased mechanical and thermal pain thresholds in rats compared with CCI group?P<0.05?.There was no significant difference in contralateral mechanical pain thresholds and thermal pain thresholds.Western blot results showed that the protein levels of Kv1.2 in DRGs and spinal cord increased significantly?P<0.05?.Intrathecal injection of NC?negative control?group had no effect on Kv1.2 levels.There was no significant change in PWTs,PWLs and Kv1.2expression after intrathecal injection of HDAC1 siRNA.ConclusionAfter CCI,the expression of HDAC1 and HDAC2 were increased in DRGs.Intrathecal injection of SAHA can relieve pain and reverse the expression of Kv1.2.Kv1.2 and HDAC2 are colocalized in DRGs.Intrathecal injection of HDAC2 siRNA can ease pain and increase Kv1.2 expression.This indicates that HDAC2,but not HDAC1 regulates Kv1.2 expression to mediate neuropathic pain.