The Role And Mechanism Of MZF1 In Dorsal Root Ganglion In CFA-induced Chronic Inflammatory Pain In Rats

BackgroundInflammatory pain,as one of the common clinical diseases,seriously affects patient’s work and daily life.Although the research on the pathogenesis and treatment methods of inflammatory pain has made great progress in recent years,the best treatment method is still a huge challenge for clinicians.The neurons in the dorsal root ganglion(DRG),as the primary neurons of pain transmission,play a key bridge role in the transmission of peripheral sensitization and central sensitization.However,the abnormal continuous discharge of these neurons is related to changes in the expressions of pain-related receptors,voltage-dependent ion channels,enzymes and other molecules at the level of gene transcription and translation in DRG.Myeloid zinc finger 1(MZF1),a member of the Kruppel family of C2H2 zinc finger transcription factors,as a bifunctional transcription factor,plays a key role in the process of transcription and translation.The previous research of this research group found that MZF1 in DRG can enhance the excitability of DRG neurons and the occurrence and maintenance of ectopic discharge frequency-mediated neuropathic pain in rats by regulating the expression of voltage-gated potassium channel(Kv).However,whether MZF1 in DRG is involved in the occurrence and maintenance of inflammatory pain has not been reported.The objective of this study is to evaluate the effect and molecular mechanism of MZF1 of inflammatory pain in rats,and hopefully provide new ideas and theoretical basis for the treatment of inflammatory pain.ObjectiveA model of chronic inflammatory pain induced by subcutaneous injection of plantar of Complete Freund’s Adjuvant(CFA)was used in this study.First,changes in the expression of MZF1 in L4/5 DRG were observed.Then,MZF1 siRNA was microinjected into DRG,and its effect on the occurrence and maintenance of chronic inflammatory pain was demonstrated.Moreover,the molecular mechanism of MZF1-mediated chronic inflammatory pain was further explored.Experimental methods1.Changes in the expression of MZF1 in DRG in rats after the plantar injection of CFA and its cell distribution typeTwenty male SD rats were divided into 2 groups(n=10):the experimental group(CFA group)and the control group(NS group)by using the random number table method.From 3 days before the plantar injection of CFA or normal saline to 14 days after the injection of CFA,mechanical withdrawal threshold and thermal withdrawal latency of the hindfoot were measured.qRT-PCR and Western Blot technology were used to detect changes in transcription levels and protein expression levels of MZF1 in L4/5 DRG.Immunofluorescence technology was used to detect changes in the expression of MZF1 in L4/5 DRG and cell distribution type.2.The effect of MZF1 in DRG in the occurrence and maintenance of CFA-mediated chronic inflammatory pain in ratsThirty male SD rats were divided into 5 groups(n=6):the Naive group,Naive+MZF1 siRNA group,CFA+MZF1 siRNA group,CFA+Vehicle group and CFA+Scramble siRNA group by using the random number table method.One day before modeling,rats in each group underwent L5 DRG microinjection with MZF1 siRNA,normal saline or Scramble siRNA.PWT and PWL values were measured 1,2,3,7,14,21 days after modeling,and the effect of MZF1 in DRG in the occurrence of chronic inflammatory pain was demonstrated.Twenty-four male SD rats were divided into 4 groups(n=6):the Naive+MZF1 siRNA group,CFA+MZF1 siRNA group,CFA+Vehicle group and CFA+Scramble siRNA group by using the random number table method.Three days after modeling,rats in each group underwent L5 DRG microinjection with MZF1 siRNA,normal saline or Scramble siRNA.PWT and PWL values were measured 1,3,4,6,8,10,14 days after modeling,and the effect of MZF1 in DRG in the maintenance of chronic inflammatory pain was demonstrated.3.Molecular mechanism of chronic inflammatory pain in rats mediated by MZF1 in DRGqRT-PCR and Western Blot technologies were used to detect changes in the expressions of MMP-2 and MMP-9 at transcription levels and protein levels in DRG of inflammatory pain model rats.Twenty-eight male SD rats were randomly divided into 4 groups(n=7):the Saline+Vehicle group,CFA+Vehicle group,CFA+MZF1 siRNA group,CFA+Scramble siRNA group.Rats in each group underwent L5 DRG microinjection with MZF1 siRNA,normal saline or Scramble siRNA 3 days after plantar injection of CFA or normal saline.After 3 days,samples were taken for qRT-PCR(n=3)and Western Blot {n=4)to detect changes in MZF1,MMP-2,and MMP-9 at transcription and protein levels.ChIP-PCR technology was used to detect the binding of MZF1 and the MMP-2/9 promoter region.Immunofluorescence technology was used to detect the co-localization of MZF1 and MMP-2/9.Results1.CFA-induced hypersensitivity and up-regulation of MZF1 expression in L4/5 DRG in ratsPain behavior data showed that compared with the NS group,PWT values of rats of CFA group began to decline 6 hours after modeling,and continued to 14 days after modeling;However,PWL values began to decline 6 h after modeling and continued to 10 days after modeling.The above results suggested that plantar injection of CFA can induce abnormal pain and hyperalgesia in rats.Results of qRT-PCR showed that compared with 0 h,the expression of MZF1 mRNA in the L4/5 DRG was up-regulated on the same side of rats 6 h after modeling,and continued to 7 days.Among them,the expression level was the highest at 6 h.Western Blot results showed that compared with 0 d,the expression of MZF1 in L4/5 DRG was up-regulated on the same side of rats 6 h after modeling,and continued to 7 days.Among them,the expression level was the highest at 1 day.The above results suggested that plantar injection of CFA can induce up-regulation of the expression of MZF1 of L4/5 DRG at the transcription and protein levels on the same side of rats.Immunofluorescence single-staining results showed that compared with 0 d,the number of positive cells of MZF1 in L4/5 DRG was increased on the same side of rats 1 day after modeling,and continued to 7 days.Immunofluorescence double staining results showed that MZF1 can be co-marked with NF200(class A neuron marker),IB4(class C non-peptide neuron marker),GFAP(satellite glial cell marker),the results suggested that MZF1 in DRG can be expressed in type A,type C neurons and satellite glial cells.2.The effect of MZF1 in CFA-induced chronic inflammatory painPain behavior data showed that 1 day before modeling,the microinjection of L5 DRG with MZF1 siRNA can alleviate the decrease of CFA-induced PWT and PWL compared with DRG microinjection solvent.The microinjection of L5 DRG with MZF1 siRNA can alleviate the decrease of CFA-induced PWT and PWL 3 days after modeling.That is,MZF1 siRNA can alleviate CFA-induced abnormal pain and hyperalgesia during the occurrence and maintenance of pain.In addition,the microinjection of L5 DRG with MZF1 siRNA can not affect the basal pain threshold of rats.3.MZF1 participates in chronic inflammatory pain by regulating the expression of MMP-2/9Western Blot and qRT-PCR results showed that the protein and mRNA expression levels of MMP-2/9 in L4/5 DRG on the same side after the injection of CFA were significantly up-regulated.Compared with 0 d,the expressions of MMP-2 and MMP-9 in the same L4/5 DRG was up-regulated 6 h after modeling,and continued to 7 days,of which the expression level was highest at 1 d and continued to 7d.Compared with the L5 DRG injection solvent group,the injection of DRG with MZF1 siRNA can reverse the up-regulation of expressions of MZF1,MMP-2/9 mRNA and protein.Immunofluorescence double staining showed that MZF1 and MMP-2/9 co-localized in DRG neurons.ChIP-PCR results showed that MZF1 can bind to the promoter regions of Mmp2 and Mmp9,indicating that the transcription factor MZF1 can directly participate in chronic inflammatory pain by regulating the expression of MMP-2/9.ConclusionIn the chronic inflammatory pain model established by plantar injection of CFA,CFA can cause up-regulation of the expressions of MZF1,MMP-2 and MMP-9 in L4/5 DRG in rats,and the transcription factor MZF1 can regulate the occurrence and maintenance of chronic inflammatory pain by directly binding to the promoter regions of Mmp2 and Mmp9.