The Mechanisms Of CRH In Dorsal Root Ganglion Underlying Neuropathic Pain

Objective Neuropathic pain is one of the chronic pain,which is defined as the pain caused by the damage or disease of the somatosensory system.It is divided into two types,peripheral and central.The different types of pain have similar or common pathogenesis.Long-term pain will not only affect patients’ sleep and work,but also increase the occurrence of anxiety,depression and other emotional disorders,and will cause great harm to the physical and mental health of mankind.At present,research progress in basic research and clinical research is limited,and analgesic drugs with good clinical treatment effect are scarce.Therefore,the development of new targets and analgesic drugs urgently need to be solved.Previous studies have found that corticotropin-releasing hormone(CRH)can regulate the sensory transmission by regulating the excitability of spinal neurons.CRH is present in the dorsal root ganglion(DRG)of mice and the primary afferent fibers in the spinal dorsal horn,but the role of CRH in the pain transmission pathway has not been clarified.We screened the genes with up-regulated expression in DRG after 7d peripheral nerve injury from E-MTAB-3326 data in the Array Express database under EBI,and analyzed the data,and found that the expression of CRH after injury was significantly increased by about 208 times.Therefore,we mainly explored the expression and distribution of CRH in DRG and spinal cord and the mechanism of underlying in neuropathic pain.Methods A model of neuropathic pain induced by spared nerve injury(SNI);The real-time RT-PCR assay was used to detect the expression of CRH m RNA in DRG and spinal cord after the establishment of the mouse SNI model.The expression and distribution of CRH in DRG and spinal cord were detected by immunofluorescence staining.To study the CRH input in spinal dorsal horn,the dorsal root of the spinal nerve was ligated after SNI.The effect of CRH on pain behavior was detected by ethology,and the pain behavior at different time points was detected after intrathecal injection of CRH si RNA(5μg/10μl).According to the bioinformatics data,SMAD1 was selected as the transcription factor,and the luciferase reporting experiment was conducted to verify the transcriptional regulation of CRH by SMAD1.The real-time RT-PCR assay was used to detect the m RNA level of SMAD1,and the expression of SMAD1 m RNA DRG and spinal cord was detected after the SNI model.The effect of SMAD1 on pain behavior was detected by ethology,and the pain induced by mechanical touch at different time points was detected after intrathecal injection of SMAD1 si RNA(5μg/10μl).Behavioral test was performed to detect the effect of intrathecal injection of CRHR antagonists of CRHR1 and CRHR2 on pain behavior of mice after SNI,and the effect of intrathecal injection of CRHR2 antagonist on the expression of c-Fos,IBA1 and GFAP.The behavioral effects of intrathecal injection of CRHR2 agonist in normal mice and its effects on the expression of p ERK in the spinal cord and DRG of mice were examined.The real-time RT-PCR assay was used to detect the m RNA expressions of CRHR1 and CRHR2,as well as the DRG and spinal cord expressions after the SNI model.The expression and distribution of CRHR2 were detected by immunofluorescence staining and western blot.Behavioral test: SNI model was established after 100 nl(AAV-Syn-sh CRHR2-EGFP)of knockdown virus CRHR2 was injected into the spinal dorsal horn of mice,and mechanical touch-induced pain at different time points was detected.The type of CRHR2 neurons was detected by immunofluorescence doublelabeling staining between the transgenic mouse-marked neurons and CRHR2.Results 1.After establishment of the SNI model,the m RNA expression and immunofluorescence indensity of CRH in DRG and spinal cord were significantly increased.CRH-positive neurons in DRG were mainly expressed in small diameter neurons,and CRH-positive nerve endings in spinal dorsal horn were mainly distributed in the superficial amina.2.The dorsal root of the spinal nerve on ipsilateral side was ligated following SNI.The expression of CRH in the spinal dorsal horn was decreased in mice with ligation of spinal dorsal root.CRH gathered at the distal end of the ligation of the dorsal root of the spinal nerve,indicating that the increased CRH in the spinal dorsal horn after SNI was input from DRG;3.Intrathecal injection of CRH si RNA in mice alleviated the mechanical pain induced by SNI;4.SMAD1 was selected as a transcription factor according to data analysis,and luciferase reporting experiment verified the transcriptional regulation of CRH by SMAD1.5.SMAD1 is co-located with CRH in DRG,and the m RNA expression of SMAD1 m RNA in DRG and spinal cord increased after SNI.6.Intrathecal injection of SMAD1 si RNA alleviated the mechanical pain induced by SNI.Intrathecal injection of SMAD1 si RNA in mice interfered with the expression of SMAD1 and CRH in DRG,and SMAD1 regulated the expression of CRH in DRG.7.Intrathecal injection of CRHR2 antagonist in mice alleviated the mechanical pain induced by SNI,and inhibited the expression of c-Fos、IBA1 and GFAP induced by hair brush on the plantar of SNI mice;8.Intrathecal injection of CRHR2 agonist induces mechanical pain behavior in normal mice and increases the expression of p ERK in the spinal cord,but has no significant effect on p ERK in DRG;9.After SNI modeling,the expression levels of CRHR2 m RNA and protein in the spinal cord of mice were increased.The results of immunofluorescence double labeling showed that CRHR2 was mainly expressed in neurons and mainly in excitatory neurons.10.The injection of CRHR2 knockdown virus(AAV-Syn-sh CRHR2-EGFP)in the spinal dorsal horn alleviated the mechanical pain induced by the SNI model.Conclusions In this study,neuropathic pain is induced by a model of SNI.In the SNI mice,CRH expression is increased in the DRG and spinal cord,and the increased CRH in the spinal dorsal horn is input from DRG.CRH is mainly increased in small diameter neurons of DRG.SMAD1 as an upstream transcription factor,regulates the expression of CRH.CRH receptor CRHR2 in the spinal cord is involved in neuropathic pain,and CRH can be used as a potential target for the treatment of neuropathic pain.