| BackgroundNeuropathic pain,which is mainly due to nerve injury or inflammation,is one of the most general chronic pains.Its prominent feature is that the painful symptoms still exist after the wound heals,and the pain is strong and stubborn.The mechanism remains unclear.At present,there is no good treatment and poorly curative effect,so neuropathic pain has become a challenging research and distinguished spot in the medical field.Recently,the epigenetic regulation of ion channels,receptors and inflammatory factors,which play an important role in the process of pain,has been widely observed.Some studies have proved that histone demethylation plays an important role in cell function regulation through producing chromatin structural changes to promote the binding of transcription factors and target gene promoters.H3K27me3 is often involved in the occurrence of important epigenetic events.Histone demethylase KDM6B(also known as JMJD3)can specifically demethylate lysine at position 27 of histone H3K27me3 and promote gene expression.Previous studies have found that KDM6B participates in the generation of some inflammatory diseases by promoting the expression of proinflammatory cytokines in vivo.Recently,it has been reported that KDM6B regulates the abnormal expression of cytokines in the brain,which is related to the occurrence of alcohol dependence,drug addiction and depression.Previous studies have proved that neuroinflammation mediated by inflammatory cytokines plays an important role in the generation and maintenance of chronic pain.However,up to now,the expression of KDM6B in dorsal root ganglia(DRG)and spinal dorsal horn,and its function in neuropathic pain,has been still covered.In this current study,we choose the lumbar 5 spinal nerve ligation operation of rats(L5 SNL)to establish the neuropathic pain model,and the role and mechanism of KDM6B in the generation and maintenance of pain were systematically observed.ObjectiveThe purpose of this study is to observe the expression change of KDM6B in DRG and spinal dorsal horn after L5 SNL,to prove the role of KDM6B the emerging and preservation of neuropathic pain induced by L5 SNLin DRG and spinal cord,and to preliminarily reveal the mechanism of KDM6B involved in neuropathic pain after SNL.MethodIn this experiment,the pain behavior of adult male SD rats was observed by detecting paw withdrawal threshold(PWT)and paw withdrawal latency(PWL),which were adopted after the pain model of L5 SNL.The pain behavior of rats was observed by detecting paw withdrawal threshold(PWT)and paw withdrawal latency(PWL).At different time point after SNL,Western blot and qPCR were used to seek the expression change of KDM6B and the methylation level of H3K27me3 of the L4/5 DRGs and the spinal dorsal horn.The cell types of KDM6B in DRG and spinal dorsal horn were further observed by immunofluorescence double staining.The role of KDM6B in the formation and maintenance of neuropathic pain after SNL was searched by intrathecal injection of GSK-J4 and KDM6B siRNA,and the regulation of KDM6B on the binding of transcription factor STAT3 and IL-6 gene promoter was observed by Chromatin Immunoprecipitation PCR(ChIP-PCR).The means ± SD was used to presented the all data,and one-way and two-way ANOVA were selected to analyze all the data from the results.A value of P < 0.05 was considered significant.Results1.Expression changes of KDM6B in Neuropathic pain rats induced by L5 SNL.(1)Establishment of L5 SNL model.There are two groups of experimental Male SD rats which were L5 SNL and Sham group(n = 9,n = 9).And the PWT and PWL at 1,3,5,7,10,and 14 days after surgery were found to be large from 1 day after operation.The PWT and PWL of the hind limbs started to decline remarkablely after the L5 SNL(compared with the Sham group,***P < 0.001),but there was no conspicuous change in the contralateral side.(2)Expression changes of KDM6B in DRG and spinal dorsal horn after L5 SNL in rats.The L4-5 DRG and L4-5 spinal cord of the surgical side and contralateral side were used by Western blot and qPCR to detect changes in KDM6B content.Results of Western blot assay displayed a significant increase in the production of KDM6B protein in the ipsilateral,but not contralateral L4/5 DRGs and spinal dorsal horn after SNL.Compared to Sham group,the significant difference occurred on day 1 or day 3,reached peak on day 7,and persisted to day 14 after SNL,as well as the qPCR result.The above results indicate that L5 SNL can cause up-regulation of KDM6B in the spinal L4-5 DRGs and spinal dorsal horn of rats.(3)Cell types of KDM6B expression in DRG and spinal dorsal horn after L5 SNL in rats.Using immunofluorescence histochemical technique to stain and photograph L4-5 DRG and spinal dorsal cord after L5 SNL and Sham rats,we found L5 SNL led to an augment of KDM6B expression in DRG and spinal dorsal horn.In addition,immunofluorescence double staining images illustrated that KDM6B was made a trail to in neurons in DRG,and in neurons,astrocytes and microglias in spinal dorsal horn.2.The role of KDM6B in the Neuropathic pain caused by SNL.(1)Effect of intrathecal injection of KDM6B specific inhibitor GSK-J4 on the formation and maintenance of neuropathic pain after SNL.In the current study,the intrathecal(i.t.)injections of GSK-J4 at the doses of 5μg,25 μg,and 50 μg were performed to examine the role of KDM6B in the SNL-induced neuropathic pain.The data of behavioral test showed that repeat i.t.injections(half hour before SNL and once daily thereafter for 5 days)of GSK-J4 alleviated the pain-related hypersensitivity after SNL.Compared to SNL plus vehicle(Veh: 0.01 M PBS containing 0.5% DMSO)group,the treatment of GSK-J4 resulted in significant increases in mechanical paw withdrawal threshold(PWT)and thermal paw withdrawal latency(PWL)in the manner of dose-dependently in SNL rats.To explore the influence of KDM6B inhibition to the neuropathic pain,the i.t.injection of GSK-J4 was accomplished on day 7(once daily for 3 consecutive days),a time-point at which the neuropathic pain has been established fully after SNL.The results showed that SNL-induced mechanical allodynia and thermal hyperalgesia were partially reversed following the treatment of i.t.injection of GSK-J4.Compared with SNL plus vehicle group,i.t.injection of GSK-J4 led to significant increase in PWT and PWL in SNL rats.In the comparison of the L5 SNL + Veh group,in the high-dose group(i.t.50μg/10 μL)on the 7th day after surgery,the expression of KDM6B decreased by Western blot.This shows that inhibiting the activity of KDM6B can partially inhibit the occurrence of neuropathic pain.(2)Effect of intrathecal injection of KDM6B siRNA on the formation and maintenance of neuropathic pain after SNL.To further prove the preventive and therapeutic effect of GSK-J4 on neuropathic pain after SNL,small interfering RNA(siRNA)was designed for KDM6B.Firstly,PC12 cells were cultured to screen and verify the effectiveness of siRNA.qPCR and Western blot results showed that siRNA 39 inhibited the expression of KDM6B by65% and 54.3%.In vivo,intrathecal injection of KDM6B siRNA 39 effectively inhibited up-regulation of KDM6B induced by SNL in the spinal dorsal horn of rats,nevertheless the transfection reagent group and the scRNA group restrained highly.Behavioral data showed that prior i.t.injection of KDM6B siRNA attenuated the development of mechanical allodynia and thermal hyperalgesia following SNL.Compared with SNL plus vehicle or SNL plus scRNA group,the KDM6B siRNA treatment resulted in significant increase in PWT and PWL after SNL.The established neuropathic pain was also alleviated by the treatment of i.t.injection of KDM6B siRNA which started on day 6 after SNL.Taken together,the above results indicate that SNL-induced upregulation of KDM6B in the DRG and spinal dorsal horn was involved in the development and maintenance of neuropathic pain.3.Molecular mechanism of KDM6B involved in mediating neuropathic pain after SNL.(1)Effect of KDM6B inhibition on IL-6 expression in DRG and spinal dorsal horn after SNL.Previous studies have reported that IL-6 in DRG and spinal dorsal horn is involved in the development and maintenance of neuropathic pain,KDM6B is involved in the regulation of IL-6 expression in vascular endothelial cells after spinal cord injury.Therefore,this study first detected the changes of IL-6 expression in L4/5DRG and spinal dorsal horn at distrinct time point after SNL.The expression level of proinflammatory cytokines IL-6 were preserved at a high threshold,and peaked on day 7.What’s more,compared with the L5 SNL + Vehicle group,the expression of proinflammatory cytokine IL-6 in the high-dose group(i.t.50 μg/10 μL)on the 7th day after L5 SNL decreased by Western blot.What’s more,the SNL-produced augment of IL-6 mRNA and protein expression in the spinal dorsal horn were observably suppressed by repeat i.t.injections of GSK-J4 when compared to that of SNL plus vehicle(transfection trgent)group through qPCR and Western blotting assay.This shows that inhibiting the activity of KDM6B can partially inhibit the occurrence of neuropathic pain,and also inhibit the up-regulation of IL-6 expression in spinal dorsal horn caused by L5 SNL in rats.It shows that KDM6B may be involved in the regulation of IL-6 expression in DRG and spinal dorsal horn after SNL.(2)Molecular mechanism of KDM6B regulating IL-6 expression.It has been proved that KDM6B can activate the expression of related genes by demethylating H3K27me3.Therefore,the methylation levels of H3K27me3 in L4/5DRGs and spinal dorsal horn were detected at different time points after SNL.H3K27me3,which is the substrate of KDM6B,showed a reduction in both L4/5DRGs and spinal dorsal horn,lowly peaked on day 3 and lasted more than 7 days after SNL surgery.Bioinformatics prediction results showed that KDM6B promoted the expression of IL-6 by promoting the binding of transcription factor STAT3 and IL-6 gene promoter.In our study,the ChIP-PCR was chosen to seek the binding of STAT3 and IL-6 gene promoter after SNL,and the effect of inhibiting KDM6B on the binding of STAT3 and IL-6 gene promoter was further observed by intrathecal injection of GSK-J4.These results indicate that the binding of STAT3 to IL-6 gene promoter is dependent on KDM6B.(3)Cell types of KDM6B,STAT3 and IL-6 in DRG and spinal dorsal horn after SNL.The double immunofluorescence staining was performed to observe the colocalization of STAT3 with IL-6 to verify the regulation of STAT3 to the expression of IL-6.Our results showed that both p-STAT3 and IL-6 were expressed in neurons in DRG,and in neurons,microglia and astrocytes in the spinal dorsal horn.This result is similar to that of KDM6B in DRG and spinal dorsal horn.Therefore,it is suggested that KDM6B,STAT3 and IL-6 have the common space in DRG and spinal dorsal horn for cell action.ConclusionIn summary,our study demonstrated that SNL-induced upregulation of KDM6B via H3K27me3 demethylation facilitated the binding of STAT3 with IL-6 promoter,and subsequently enhanced-IL-6 expression in the DRG and spinal dorsal horn contributed to the development and maintenance of neuropathic pain. |
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