The Role And Mechanism Of Dorsal Root Ganglion Syncrip In Neuropathic Pain

Neuropathic pain(NP)is a chronic disease caused by peripheral and central nervous system injury and dysfunction.Its characteristic clinical manifestations are spontaneous pain,abnormal pain and hyperalgesia.At present,NP has become a global problem,affecting6.9% ~ 10% of the world population.It not only seriously affects the quality of life and work of patients,but also brings a heavy economic burden to society and families.At present,the treatment of NP is mainly drugs.The long-term use of opioids can cause increased risk of drug resistance and various adverse reactions.Due to the lack of understanding of the pathogenesis of NP,there is a lack of effective clinical treatment.Syncrip is a special sequence of RNA binding proteins(RBPs),which is widely expressed in a variety of organisms and tissues.It belongs to the family of cellular heterogeneous ribonucleoprotein(hn RNP).This family is considered to be involved in all processes involving m RNA maturation,among which Syncrip is considered to be a potentially important regulator of neuronal homeostasis.Studies have shown that Syncrip can mediate the post transcriptional regulation of the pathogenesis of many neurological diseases,but the mechanism of Syncrip in NP is still poorly understood.Therefore,this project intends to prepare a mouse peripheral nerve injury model on this basis,in order to explore the key role and related mechanism of Syncrip in NP,and provide a theoretical basis for clinical prevention and treatment.Part one: Peripheral nerve injury can cause changes in the expression and distribution of SyncripObjective: To observe the changes of Syncrip expression and distribution in mouse dorsal root ganglion(DRG)after peripheral nerve injury.Methods: Male CD1 mice aged 7-8 weeks were randomly divided into three groups with 12 mice in each group.Sham group: sham operation group,only the skin behind the ipsilateral lower limb of mice was cut,and then the skin was nailed;CCI group: the mouse model of chronic constriction injury of sciatic nerve was established;SNL group:L4 spinal nerve ligation mouse model was established.Von Frey cilia,thermal radiation stimulator and 0℃ aluminum plate were used to detect the behavioral changes of mice at different time points after operation;The co-labeling of Syncrip with different types of neuronal marker(Neu N,NF200,IB4,CGRP)and satellite glial marker(GS)in normal mouse DRG was observed by immunofluorescence staining,and the proportion of cell distribution was calculated;q RT-PCR and Western blot were used to detect the expression of Syncrip in DRG and spinal dorsal horn at different time points after operation;the number of Syncrip positive cells in impaired DRG of CCI mice was observed by immunofluorescence staining and calculate the distribution proportion.Results: Compared with sham group,the paw mechanical withdraw frequency of ipsilateral hindlimb in CCI and SNL group increased significantly on the 3rd,5th,7th and 14 th day after operation(P < 0.01),and the paw withdraw latency to thermal and cold stimuli decreased significantly(P < 0.01).The expression of Syncrip in ipsilateral DRG in CCI group and SNL group increased significantly on the 3rd,7th and14 th day after operation(P < 0.05),and there was no significant change in the ipsilateral spinal dorsal horn.Syncrip was specifically co-expressed with Neu N but not GS in normal mouse DRG,approximately 57.4% of DRG neurons were positive for Syncrip,about 35.6% of Syncrip-labelled neurons were small(< 300 μm2 in area),23.6% were medium(300–600μm2 in area),and 40.8% were large(> 600 μm2 in area),about 24.1%were positive for CGRP,35.4% for IB4 and 35.9% for NF200.Compared with sham group,the number of Syncrip positive cells in ipsilateral DRG in CCI group increased significantly on the 7th day after operation,including 30.7% small neurons,43.9% medium neurons and 25.5% large neurons.Conclusion: After nerve injury,the expression and distribution of Syncrip in mouse DRG neurons changed significantly,and Syncrip may play an important role in the occurrence and development of NP.The specific role of syncrip in NP and its possible mechanism need to be further studied.Part two: The role of dorsal root ganglion Syncrip in neuropathic painObjective: To explore the specific role of DRG Syncrip in the early development and maintenance of NP,and to evaluate its significance as an analgesic target.Methods: The above animals were randomly divided into four groups: scramble + sham,scramble + CCI,si RNA + CCI and si RNA +sham,with 12 animals in each group.The animals in each group were DRG microinjected with scramble si RNA or Syncrip si RNA before operation,and then underwent sham operation or CCI operation,5 days after operation animals were sacrificed.Then the above animals were randomly divided into another CCI + scramble and CCI + si RNA groups,with 12 animals in each group.On the 7th day after CCI,all mices were microinjected with scramble si RNA or syncrip si RNA in DRG,and sacrificed 14 days after operation.Finally,the above animals were randomly divided into two groups: AAV5-GFP and AAV5-Syncrip,with12 rats in each group.Mice were microinjected with AAV5-GFP or AAV5-syncrip virus by DRG microinjection respectively,the animals were sacrificed 8 weeks after injection.Von Frey cilia,thermal radiation stimulator and 0℃ aluminum plate were used to detect the behavioral changes of the above groups at different time points;q RT-PCR and Western blot were used to detect the expression of Syncrip in DRG and the expression of ERK,p-ERK and GFAP in spinal dorsal horn;At the fifth week after virus injection,CPP test detected the non irritating spontaneous pain behavior of mice between AAV5-GFP and AAV5-Syncrip groups.Results: Compared with the scramble + sham group,the paw mechanical withdraw frequency of ipsilateral hind limb in scramble +CCI group increased significantly on the 3rd and 5th day after operation(P < 0.01),the paw withdraw latency to thermal and cold stimuli decreased significantly(P < 0.01),the expression of Syncrip,p-ERK1/2and GFAP increased significantly on the 5th day after operation(P <0.01),and there was no significant change in the above indexes in si RNA+ sham group.Compared with scramble + CCI group,the paw mechanical withdraw frequency of ipsilateral hind limb in si RNA + CCI group decreased significantly on the 3rd and 5th day after operation(P <0.01),the paw withdraw latency to thermal and cold stimuli prolonged significantly(P < 0.01),and the expression of Syncrip,p-ERK1/2 and GFAP decreased significantly on the 5th day after operation(P < 0.05).Compared with CCI + scramble group,the paw mechanical withdraw frequency of ipsilateral hind limb in CCI + si RNA group was significantly reduced(P < 0.01),and the paw withdraw latency to thermal and cold stimuli were significantly prolonged(P < 0.01).Compared with naive group,the expression of Syncrip,p-ERK1/2 and GFAP in CCI +scramble group were significantly up-regulated on the 14 th day after operation(P < 0.01).Compared with CCI + scramble group,the expressions of Syncrip,p-ERK1/2 and GFAP in CCI + si RNA group were significantly down regulated on the 14 th day after operation(P < 0.05).Compared with AAV5-GFP group,the paw mechanical withdraw frequency in AAV5-syncrip group was significantly increased at 4,6 and8 weeks after injection(P < 0.01),and the paw withdraw latency to thermal and cold stimuli were significantly decreased(P < 0.01).In CCP experiment at 5 weeks after injection,the time of mice staying in lidocaine room was significantly prolonged(P < 0.01),The expressions of Syncrip,p-ERK1/2 and GFAP were significantly up-regulated at the8 th week after injection(P < 0.05).Conclusion: Up regulated DRG Syncrip plays an important role in the development and maintenance of NP and participates in the central sensitization of spinal cord caused by nerve injury in mice,which provides a theoretical basis for a new target for pain diagnosis and treatment in the future.Part three: Syncrip participates in neuropathic pain by mediating the stability regulation of CCR2 m RNAObjective: To clarify the mechanism of Syncrip participating in neuropathic pain by mediating the stability regulation of CCR2 m RNA.Methods: The in vivo experiment is the same as the above research,and the experimental groups are carried out.In vitro,DRG primary neurons were divided into scramble + AAV5-GFP group,scramble +AAV5-syncrip group,si RNA + AAV5-syncrip group and si RNA +AAV5-GFP group by si RNA and virus transfection;CAD cells were divided into naive group,GFP + scramble group,Syncrip + scramble group and si RNA + Syncrip group.CAD cells in the above groups were treated with actinomycin D(Act D)6,12 and 24 hours before cell collection.q RT-PCR and Western blot were used to detect the expression of CCR2 in DRG of mice in each group,and the expressions of Syncrip and CCR2 in DRG primary neurons were detected by Western blot.The expression of CCR2 m RNA in CAD cells was detected by q RT-PCR.RIP was used to detect the binding of Syncrip and CCR2 m RNA in mouse DRG.The co-labeling of Syncrip and CCR2 in injury DRG of CCI mice was observed by immunofluorescence.Results: Compared with sham group,the expression of CCR2 m RNA in ipsilateral DRG in CCI group was significantly up-regulated on the 3rd,7th and 14 th day after operation(P < 0.05).Compared with scramble + sham group,the expression of CCR2 in ipsilateral DRG in scramble + CCI group was significantly up-regulated on the 5th day after operation(P < 0.05).Compared with scramble + CCI group,the expression of CCR2 in si RNA + CCI group decreased significantly on the5 th day after operation(P < 0.05).Compared with AAV5-GFP group,the expression of CCR2 in AAV5-Syncrip group was significantly up-regulated at the 8th week after microinjection(P < 0.05).Compared with scramble + AAV5-GFP group,the expression of Syncrip and CCR2 in scramble + AAV5-Syncrip group was significantly up-regulated(P < 0.05),compared with scramble + AAV5-Syncrip group,the expression of Syncrip and CCR2 in si RNA + AAV5-Syncrip group was significantly down regulated(P < 0.05).Compared with naive group, the remaining level of CCR2 m RNA in Syncrip + scramble group was significantly higher after 6,12 and 24 hours of Act D treatment(P < 0.05),compared with Syncrip + scramble group,the remaining level of CCR2 m RNA in si RNA + Syncrip group was significantly lower after 12 and 24 hours of Act D treatment(P < 0.05).Compared with sham group,the binding activity between CCR2 m RNA fragment and Syncrip in impaired DRG on the 5th day after CCI operation was significantly higher(P <0.01).Immunofluorescence staining confirmed that Syncrip and CCR2 could be co-expressed in the same neuronal cells in the impaired DRG of CCI mice.Conclusion: Syncrip can increase the stability of mRNA by combining with CCR2 m RNA,so as to promote the expression of CCR2 and participate in the occurrence and development of NP.Therefore,targeting syncrip may have potential value in the prevention and treatment of neuropathic pain.