The Euchromatic Histone-lysine N-methyltransferase 2 In Mediating Diabetic Neuropathic Pain Through Modulating KCNA2/4

Diabetic neuropathic pain(DNP)is associated with irrelievable pain,reduced quality of life,and increased risk of mortality.Despite its substantial impact on diabetic patients,DNP remains undertreated.Moreover,the evidence supporting a benefit for causal treatment is weak,and current pharmacotherapy is largely limited to symptomatic treatment options.Thus,a better understanding of the underlying pathophysiology is mandatory for translation into new treatment approaches.Improved knowledge about pathogenic mechanisms implicated in the development of DNP could lead to novel treatment strategies.Recently,histone lysine N-methyltransferase 2(euchromatic histone-lysine N-methyltransferase 2,EHMT2,also known as G9a)has been found to be involved in the development of neuropathic pain.Potassium channels KCNA2/4 are closely related to excitability of neurons.In this study,streptozocin-induced diabetic rat model in vivo and hyperglycemic dorsal root ganglion DRG neurons in vitro were used to investigate the role of potassium channel KCNA2/4 in diabetic neuropathic pain and its related mechanisms.The relationship between G9a and KCNA2/4 in diabetic neuropathic pain was further investigated.This study may provides a new technique for relieving diabetic neuropathic pain.Part Ⅰ G9a modulates KCNA2/4 expression of DRG neurons with high glucose Induced neurotoxicityG9a,as a kind of histone modification enzyme,has been widely recognized for its role in gene transcription inhibition and can inhibit the expression of a variety of genes.However,the role of G9a in neurotoxicity induced by high glucose is still unclear.Whether G9a participates in the activity and expression of potassium channel protein in high glucose-induced hyperexcitability of DRG neurons needs to be further examined.According to this research background,Firstly,DRG neurons were routinely cultured in the medium for 48 h.The following experiments of DRG neuron culture were designed:(1)Control group:DRG neurons were cultured in medium alone;(2)Inhibitor group:DRG neurons were incubated in cultured midum containing 150 nm/L UNC0638(G9a inhibitor)for 48 h after cultured in ordinary meidum for 24 h;(3)High-glucose group:DRG neurons were cultured in medium containing 45 mmol/L glucose for 24 h;(4)High-glucose + Inhibitor group:DRG neurons were incubated in cultured midum containing 150 nm/L UNC0638 for 48h after cultured in meidum containing 45 mmol/L glucose for 24 h.After the stimulation,the levels of G9a transcription and translation,the expression of KCNA2/4 were detected.The percentage of G9a and kcna2/4 positive neurons were detected.The results showed that high glucose induced G9a upregulatuion and KCNA2/4 downregulation in DRG neurons.Inhibition of G9a could reverse the downregulation of KCNA2/4.These data imply that there is a close relationship between KCNA2/4 expression and G9a in DRG neurons at high glucose condition and provide new experimental evidence for further study the mechanisms of high glucose-induced neurotoxicity.Part Ⅱ G9a mediates diabetic neuropathic pain through modulating KCNA2/4 expressionThe general analgesic drugs used in diabetic neuropathic pain are not effective or have relatively large adverse reactions,which offers a broad prospect for improving the excitability of neurons by changing the expression of potassium channel protein in DRG neurons.G9a is rapidly elevated in the presence of many kinds of nerve injuries.Investigating whether G9a can change expression of potassium channel KCNA2/4 of DRG neurons and participate in the occurrence and development of diabetic neuropathic pain may provide a new analgesic means for diabetic neuropathic pain.Based on the existing research progress,we used STZ to induce diabetic rat model,and designed the following experiments:(1)Control group:normal rats intrathecally injected with normal saline lOul;(2)Inhibitor group:normal rats intrathecally injected with UNC0638(lOug/10ul);(3)Diabetic group:diabetic rats intrathecally injected with normal saline 10ul;(4)Diabetes + Inhibitor group:diabetic rats intrathecally injected with UNC0638 10ul.The changes of pain threshold of mechanical stimulation,the distribution of G9a,KCNA2/4 in DRG neurons and the proportion of positive neurons were detected.The results showed that G9a upregulatuion and KCNA2/4 downregulation in DRG neurons were observed in DRG neurons in STZ-induced DNP rats.Inhibition of G9a could reverse the downregulation of KCNA2/4 in DRG neurons in STZ-induced DNP rats.These data imply that the development and progression of DNP may correlate with G9a-induced KCNA2/4 expression alterations.These data not only provide novel interpretation about the mechanisms of DNP,but also shed a new direction for relieving DNP by targeting G9a and KCNA2/4.