| BackgroundNeuropathic pain(NP)is a chronic disease that plagues approximately 7%of the population and often brings great suffering to patients.Although increased studies have improved the understanding of its pathological mechanisms,optimal therapy still remains a challenge for physicians.Transient receptor potential vanilloid 1(TRPV1),as a nonselective cation channel,integrates multiple injury stimuli in DRG nociceptive neurons and regulates downstream function of multiple signaling pathways.And its own activity has been continuously restored and enhanced,leading to sustained and abnormal activation of neurons,which contributes to the generation and maintenance of chronic pain.However,the mechanism of TRPV1 expression is still lack of further study.Our previous study has found that myeloid zinc finger 1(MZF1)is involved in the expression of Kv gene in CCI rats and thus mediates the generation of neuropathic pain.TRPV1 is similar to the Kv channel,however,whether MZF1 can also regulate TRPV1 expression in DRG contributing to the generation and maintenance of neuropathic pain needs to be further studied.ObjectiveIn this study,the expression of MZF1 and TRPV1 in L4/5 DRG was observed by using chronic constriction injury(CCI)model in rats.And the role of MZF1 and TRPV1 was verified in NP.Finally,the effect of MZF1 on the expression of TRPV1 was investigated by using DRG microinjection of rAAV5-MZF1 or MZF1 siRNA,and the binding of MZF1 to TRPV1 promoter regions was proved to verify the molecular mechanism by which MZF1 regulates the expression of TRPV1.Methods1 Expression of MZF1 and TRPV1 in DRG of rats after CCIMale Sprague-Dawley(SD)rats were randomly divided into 2 groups:experimental group(CCI group)and control group(Sham group).The paw mechanical withdrawal threshold(PWT)and paw thermal withdrawal latency(PWL)were measured to 14th day.The transcription level and protein expressions of MZF1 and TRPV1 in L4/5 DRG were detected by RT-PCR and Western Blot respectively.2 The role of MZF1 on the generation and maintenance of neuropathic pain in DRG of rats(1)The role of overexpressed MZF1 in neuropathic pain in DRG of ratsMale SD rats were randomly divided into 2 groups:rAAV5-MZFl group and rAAV5-EGFP group.The PWT and PWL were measured after DRG microinjection.(2)The role of MZF1 siRNA in neuropathic pain in DRG of ratsMale SD rats were randomly divided into 4 groups:Sham + Vehicle group,CCI +Vehicle group,CCI + MZF1 siRNA group and CCI + MZF1 scrRNA group.The rats were treated with pre-microinjection,then the PWT and PWL were measured.Another group of male SD rats were randomly divided into 4 groups:Sham + Vehicle group,CCI + Vehicle group,CCI + MZF1 siRNA group and CCI + MZF1 scrRNA group.DRG microinjection was used at 7th day following CCI in rats and the PWT and PWL were measured.3 The molecular mechanism of MZF1 regulating the expression of TRPV1 in DRG of CCI rats(1)The effect of overexpression or gene silencing of MZF1 on the expression of TRPV1 in DRG of ratsMale SD rats were randomly divided into 2 groups:rAAV5-MZF1 group and rAAV5-EGFP group.The rAAV5-MZF1 or rAAV5-EGFP was microinjected into DRG in rats.The expressions of MZF1 and TRPV1 were detected by RT-PCR and Western Blot in the L4/5 DRG.Another group of male SD rats were randomly divided into 4 groups:Sham + Vehicle group,CCI + Vehicle group,CCI + MZF1 siRNA group and CCI + MZF1 scrRNA group.The rats were treated with pre-microinjection.The expressions of MZF1 and TRPV1 were detected by RT-PCR and Western Blot in the L4/5 DRG of rats in 7th day after CCI.(2)Verifying the binding of MZF1 to TRPV1 promoter region in DRG of CCI ratsThree male SD rats were used for CCI model operation.The binding of MZF1 to TRPV1 promoter region was detected by chromatin immunoprecipitation in L4/5 DRG.(3)The inhibition of TRPV1 on the generation and maintenance of neuropathic pain in DRG of ratsMale SD rats were randomly divided into 4 groups:Sham + Vehicle group,CCI+ Vehicle group,CCI + TRPV1 siRNA group and CCI + TRPV1 scrRNA group.The rats were treated with pre-microinjection,then the PWT and PWL were measured.Another group of male SD rats were randomly divided into 4 groups:Sham + Vehicle group,CCI + Vehicle group,CCI + TRPV1 siRNA group and CCI + TRPV1 scrRNA group.DRG microinjection was used at 7th day following CCI in rats and the PWT and PWL were measured.Results1 CCI induced pain-related hypersensitivity and upregulation of MZF1 and TRPV1 in DRG of ratsCCI produced mechanical allodynia and thermal hyperalgesia.Pain-related behavioral tests revealed a clear reduction of PWT and PWL,which started at 3rd day after CCI and persistent to 14th day.RT-PCR results showed that the expressions of MZF1 mRNA and TRPV1 mRNA in DRG of CCI rats were significantly increased.Western Blot data confirmed that the expression of MZF1 and TRPV1 protein in DRG of CCI rats also upregulated significantly.2 The role of MZF1 on the generation and maintenance of neuropathic pain in DRG of ratsDRG microinjection of rAAV5-MZF1 induced normal rats pain-related hyperalgesia.Compared with rAAV5-EGFP group,PWT and PWL decreased significantly in rAAV5-MZF1 group.Pain behavior data also confirmed that overexpression of MZF1 in DRG aggravated hyperalgesia in CCI rats.Compared with rAAV5-EGFP group,PWT and PWL were further decreased in rAAV5-MZF1 group.Behavioral tests confirmed that compared with CCI + Vehicle group,pre-DRG microinjection of MZF1 siRNA(3 days before CCI)significantly increased PWT and PWL in CCI rats.To further verify the role of MZF1 in the maintenance of neuropathic pain,the pain behavior data showed that the PWT and PWL of MZF1 siRNA group(CCI + MZF1 siRNA,DRG microinjection at 7th day after CCI)were significantly increased.3 The molecular mechanism of MZF1 regulating the expression of TRPV1 in DRG of CCI ratsThe results of RT-PCR and Western Blot showed that microinjection of rAAV5-MZF1 into DRG induced the upregulation of TRPV1 mRNA and TRPV1 protein in rats.Moreover,microinjection of MZF1 siRNA into DRG significantly inhibited the upregulation of TRPV1 mRNA and TRPV1 protein induced by CCI.Chromatin immunoprecipitation assay(ChIP)confirmed that MZF1 could directly bind to the TRPV1 gene promoter region.Behavior tests data showed that compared with CCI + Vehicle group,pre-DRG microinjection of TRPV1 siRNA(CCI 3 days before surgery)significantly increased the PWT and PWL in CCI rats.To further confirm the role of TRPV1 in the maintenance of NP,behavioral test results showed that the PWT and PWL of TRPV1 siRNA group(CCI + TRPV1 siRNA,DRG microinjection at 7th day after CCI)were significantly increased.ConclusionOur results suggest that chronic constriction injury of sciatic nerve can induce the upregulation of transcription factor MZF1 in L4/5 DRG The increased MZF1 via binding to TRPV1 gene promoter region to regulate the expression of TRPV1 contributes to the generation and development of neuropathic pain. |
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