SPOCK2 Modulates Neuropathic Pain By Interacting With MT1-MMP To Regulate Activation Of MMP-2 In Rats With Chronic Constriction Injury

Objective:Neuropathic pain(NP)is an intractable pain caused by a lesion or disease affecting the somatosensory system.Traditional analgesics have poor efficacy and are prone to adverse reactions,such as overdose toxicity and opioid dependence.However,the pathogenesis of NP remains a complicated issue for pain management practitioners.SPARC/osteonectin,cwcv,and kazal-like domains proteoglycan 2(SPOCK2)is a secreted multidomain proteoglycan belonging to the BM-40/SPARC family of extracellular proteins.Currently,few functional studies have focused on SPOCK2,and SPOCK2 may be associated with lung adenocarcinoma,prostate cancer,ovarian cancer,and bronchopulmonary dysplasia.A recent genome-wide association study of back pain(BP)combining data from the UK Biobank and CHARGE Consortium databases identified and replicated three BP-associated loci,including one novel SPOCK2 gene region.BP is usually caused by spinal nerve inflammation,and long-term BP can produce central sensitization and evolve into NP.Therefore,the present study aimed to investigate the role of SPOCK2 in the pathogenesis of NP using a well-characterized rat model of chronic constriction injury(CCI).Methods:Part Ⅰ.Healthy adult male SD rats were used to make sciatic nerve chronic constriction injury model(CCI)for animal experiments.The paw withdrawl threshold(PWT)and paw withdrawl latency(PWL)of the affected hind limb were monitored before and at 1,3,7,14,21 days after CCI operation to determine the changes of pain behavior in rats.After the expression of SPOCK2 in the spinal cord of CCI rats was down-regulated by RNAi mediated,the changes in pain behavior of the rats were observed.The expression and localization of SPOCK2 in the dorsal horn of the affected spinal cord in rats were detected by Western Blot,immunohistochemical staining of paraffin sections and immunofluorescence staining of frozen sections.Part Ⅱ.After RNAi mediated down-regulation of SPOCK2 expression in the spinal cord of CCI rats,Western Blot and immunofluorescence results showed that the phosphorylation of ERK1/2 and the cleavage and release of inflammatory factor IL-1βcould be reduced synchronously with the decrease of RNAi mediated SPOCK2 expression in the spinal cord of rats.The phosphorylation of JNK and the expression of chemokine CCL2 were not decreased.Western Blot showed that the expression level of MMP-2 in the spinal cord of the affected side increased gradually with the postoperative CCI,and reached a peak 7-14 days after the postoperative CCI.After the expression and activation of MMP-2 in the spinal cord of CCI rats were down-regulated by MMP-2specific inhibitors,mechanical and thermal pain sensitivity of rats could be partially relieved.Pearson correlation analysis showed strong positive correlation between SPOCK2 and active-MMP-2 expression at each time point after CCI surgery.In order to further analyze the relationship between SPOCK2 and the activation of MMP-2,we found that the expression of SPOCK2 in the spinal cord of CCI rats was down-regulated by RNAi mediated,and the activation of MMP-2 was decreased simultaneously.However,the expression and activation of MMP-2 in the spinal cord of CCI rats mediated by MMP-2 inhibitor decreased,but the expression level of SPOCK2 in the spinal dorsal horn of rats did not change significantly.Part Ⅲ.The co-localization of SPOCK2 and MT1-MMP in the spinal dorsal horn of rats was observed by immunofluorescence co-localization in frozen tissue sections.Then,through cell experiments,HEK293 T cell lines were selected and full-length plasmids of SPOCK2 were transfected exogenically to determine whether SPOCK2 and MT1-MMP combined into a complex to perform biological functions.Then,in HEK293 T cell lines transfected with SPOCK2 mutant plasmid,the binding domain of SPOCK2 and MT1-MMP was determined by co-immunoprecipitation,and the expression and activation of MMP-2 were detected by Western Blot.Results:Part Ⅰ.Postoperative changes in pain behavior(PWT and PWL)can be observed in the rat CCI model.With the appearance of neuropathic pain,SPOCK2 expression in the spinal cord of the affected side of the rat is up-regulated with time,and reaches its peak7-14 days after CCI surgery,and the high expression level can be maintained until 21 days after surgery.The expression levels of SPOCK2 and active-MMP-2 were positively correlated with each time after CCI operation.Immunofluorescence co-localization showed that SPOCK2 was basically expressed in astrocytes but not in microglia in the spinal dorsal horn of rats.However,when neuropathic pain was induced by CCI,the expression of SPOCK2 in astrocytes increased significantly,while the expression of SPOCK2 in microglia remained unchanged.Part Ⅱ.RNAi mediated down-regulation of SPOCK2 expression in the spinal cord of CCI rats can significantly relieve pain(PWT and PWL)in rats,reduce the activation of MMP-2,and cause down-regulation of the activation of ERK1/2 in astrocytes and reduce the cleavage and release of IL-1β.After downregulating the expression and activation of MMP-2 in CCI rats,MMP-2 inhibitor can also relieve the pain of rats,but the expression level of SPOCK2 in the spinal dorsal horn of rats has no significant change.Part Ⅲ.The colocalization of SPOCK2 and MT1-MMP is obvious in the spinal dorsal horn of rats,which provides the basis for their interaction.Cell experiments confirmed that SPOCK2 can promote the up-regulation of MMP-2 activation by binding MT1-MMP with the SPARC＿EC domain.Conclusion:Our findings highlight a new pathogenesis mode of NP.Part Ⅰ: SPOCK2 is positively correlated with neuropathic pain.SPOCK2 expression increased in spinal dorsal horn when pain occurred.When pain occurs,if the expression of SPOCK2 in the spinal cord is down-regulated,the pain can be partially relieved.In order to further analyze how SPOCK2 affects neuropathic pain,we found that when neuropathic pain occurs,the expression of SPOCK2 in astrocytes will increase significantly,considering that the effect of SPOCK2 on neuropathic pain may be related to the astrocytic activation.Part Ⅱ: SPOCK2 can be used as an upstream molecule to influence the generation of neuropathic pain by regulating the activation of MMP-2 and then affecting the phosphorylation of ERK1/2 and the cleavage and release of IL-1β in astrocytes.Part Ⅲ: In cell experiments,we confirmed that SPOCK2 can interact with MT1-MMP by binding specific SPARC＿EC domain to form a complex,thus promoting the activation of MMP-2.In the future,we can conduct in vivo experiments targeting this mechanism of action,which may provide new therapeutic targets for neuropathic pain.