The Mechanism Of TRESK Two-pore Domain Potassium Channel In Inflammatory Pain

Objective:To investigate the expression and effect of TRESK(TWIK-related spinal cord K+channel,TRESK)in the trigeminal ganglion(TG)of inflammatory pain.To clarify upstream stimulatory factor2(USF2)participating in hyperalgesia of inflammatory pain by regulating the expression of TRESK.Methods:1)An injection of complete freund's adjuvant(CFA)into the left whisker pad area of rat was used to construct a facial inflammatory pain model.Behavioral changes of mechanical pain threshold in rats with von frey filaments.2)The expression of TRESK in the trigeminal ganglia of rats was measured by western blot and RT-qPCR methods.Immunofluorescence was used to determine the cellular distribution of TRESK in TG.3)The whole cell patch clamp method was applied to study the current density of TRESK in TG medium-small neurons.4)Molecular cloning,dual luciferase reporter gene method and the bioinformatics software of Alibaba2.1 were used to screen transcription factor that regulate TRESK expression.5)Using lentivirus expressing TRESK to up-regulate the expression of TRESK in TG,knockdown of TRESK channels using TRESK small interfering RNA(siRNA).And the regulation of USF2 in TG was detected by USF2 siRNA.6)Dural luciferase reporter gene assay was used to detect the binding of USF2 to the promoter region of TRESK gene,and to verify the inhibitory effect of USF2 on TRESK gene transcription.Results:1)The mechanical pain of threshold of CFA rats decreased significantly in the 1d after CFA,and the rats in the 10 d continued to suffer from hyperalgesia.2)CFA induced a decrease of TRESK expression in TG,and the TRESK current density decreased significantly.In addition,the fraction of lamotrigine-sensitive currents was reduced in CFA rats.3)TRESK was mainly distributed in the medium-small diameter neurons with CGRP and IB4 markers,no expression detected in glial cells.4)When the role of TRESK was inhibited by TRESK siRNA in TG of normal rats,TRESK expression was decreased,causing a significant reduction in mechanical pain threshold.5)Over-expression of TRESK increased relieve the hyperalgesia of CFA rats.6)A series of truncated TRESK 5'-promoter luciferase constructs and Dural luciferase reporter gene assay suggest that there is a transcription factor that can negatively regulate TRESK expression.7)Bioinformatics software prediction and RT-qPCR results suggest that USF2 can regulate the expression of TRESK.8)USF2 and TRESK were co-expressed in TG,and the expression of USF2 was increased in CFA rats9)Dural luciferase reporter gene assay results showed that USF2 bind to the promoter region of TRESK,and USF2 negatively regulates TRESK expression.10)Down-regulate the expression of USF2 in TG of CFA rats increased TRESK expression and alleviated the hyperalgesia.Conclusion:The expression of TRESK was decreased in TG of CFA rats,which was involved in inflammatory pain.Transcription factor USF2 negatively regulates TRESK expression and participates in pain regulation.