| Painful diabetic neuropathy（PDN）is a special type of neuropathic pain.It is an intractable complication of diabetes that causes suffering and severely affects the quality of life of patients.The current clinical treatment is extremely poor.Recently chemokine CXCL13 and its receptor CXCR5 have been shown to play a key role in chronic pain induced by peripheral tissue inflammation or nerve damage.However,it is still unclear whether CXCL13 and its receptor CXCR5 are involved in diabetic neuropathy and how they participate in PDN.In this study,we investigated whether CXCL13/CXCR5 mediates PDN and its possible molecular mechanisms.In this study,db/db mice were used for the type 2 diabetes mouse model and type I mice streptozotocin（STZ）was used to induce the type 2 diabetes mouse model.Db/db mice showed significant hyperglycemia and obesity,increased long-term mechanical hyperalgesia,and increased expression of CXCL13,CXCR5,and pro-inflammatory cytokines TNF-αand IL-6 in the spinal cord.Streptozotocin（STZ）induced hyperglycemia and emaciation,early mechanical allodynia,increased thermal hyperalgesia and mechanical allodynia in the late stage,and increased CXCL13,CXCR5 and proinflammatory cytokines in the spinal cord.The expression of TNF-α,IL-1βand IL-6 was also significantly increased.Furthermore,in the spinal cord of both model mice,there was a significant increase in glial expression and up-regulation of phosphorylated cellular signaling kinases,including pERK,pAKT and pSTAT3.Mechanical allodynia,upregulation of pERK,pAKT and pSTAT3 as well as production of TNF-αand IL-6 can be significantly inhibited by MK-801（a non-competitive NMDA receptor antagonist）.U0126（selective MEK inhibitor）or AG490（JAK/STAT inhibitor）can significantly inhibit mechanical allodynia in db/db mice,and up-regulation of spinal cord pERK（via U0126）or pSTAT3（via AG490）.C57BL/6J mice showed significant thermal hyperalgesia and mechanical allodynia after intrathecal injection of CXCL13,up-regulation of pERK,pAKT and pSTAT3,and increased pro-inflammatory cytokines（TNF-α,IL-6 and IL-1β,）,which were all blockaded by the Cxcr5 gene knockout.In all,our study showed that the chemokine CXCL13 and its receptor CXCR5 play an important role in the development of PDN,and developing novel approaches targeting CXCL13/CXCR5 may contribute to the clinical treatment of PDN.Research purposes:PDN seriously affects the quality of life of patients with diabetes,but the molecular mechanisms remain unclear and treatment options are limited.In this study,a spontaneous db/db mouse model of type II diabetes and streptozotocin（STZ）induced a type I diabetes model were investigated,aiming to study the mechanism of PDN associated with diabetic patients and seek novels molecular targets for treating the PDN.Research methods:1.Both db/db and their wildtype littermates were used,and the changes of fasting blood glucose,body weight and mechanical pain threshold were measured at different time points（starting from 4 weeks）.In the seventh week after birth,their spinal cord lumbar enlargements were dissected and the chemokines were screened by RT-PCR.2.Both db/db and their wildtype littermates were used to stain the astrocytes and microglia of mouse spinal cord and measure the Cxcl13,Cxcr5 expression and pro-inflammatory cytokine levels in the spinal cord.3.Intrathecal injection of MK-801（NMDA receptor antagonist）,U0126（selective MEK inhibitor）or AG490（JAK/STAT inhibitor）into db/db mice,measuring mechanical pain changes,taking most of the spinal cord swelling After homogenization,the cells were centrifuged,and the supernatant was used for Western blot analysis of pERK,pAKT and pSTAT3.4.Intrathecal injection of CXCL13 into Cxcr5–/–mice and their C57 control mice.The spinal cord lumbar enlargements were harvested and homogenized,and then centrifuged.The supernatant was used for Western blot analysis of pERK,pAKT and pSTAT3.5.Male C57BL/6 mice were administered a 60 mg/kg dose of streptozotocin（STZ）intraperitoneally to induce a type 1 diabetes model.The mechanical pain of STZ-induced diabetic mice was measured,and spinal cord lumbar enlargements were dissected for RT-PCR to screen for chemokines expression.6.C57 group and C57+STZ group were used and their spinal cord tissues were homogenized and centrifuged.The supernatant was used for Western blot analysis of CXCR5,CXCL13,as well as phosphorylated protein expression and cytokine levels.Research results:1.db/db type II diabetic mice showed significant hyperglycemia and obesity,increased long-term mechanical allodynia,and increased expression of chemokines Cxcl13 and Cxcr5 mRNA in the spinal cord.2.There is a significant increase in gliosis in the spinal cord of db/db mice.The levels of pro-inflammatory cytokines TNF-αand IL-6 are increased in the serum of db/db mice.3.In db/db mice,mechanical allodynia and up-regulated pERK,pAKT and pSTAT3,and production of TNF-αand IL-6 were all attenuated by MK-801（the non-competitive NMDA receptor antagonist）.If U0126（selective MEK inhibitor）or AG490（Janus kinase（JAK）STAT inhibitor）were administered to the spinal cord of db/db mice,they both reduced mechanical allodynia,and inhibited pERK（via U0126）or pSTAT3（via AG490）.4.CXCL13 induced C57 mice to produce significant mechanical hyperalgesia for more than 6 hours,but failed in Cxcr5–/–mice.One hour after the injection,CXCL13 significantly increased TNF-α,IL-6 and IL-1βmRNA levels in the spinal cord of C57 control mice,but did not change in Cxcr5–/–mice.Intrathecal injection of CXCL13 enhanced TNF-α,IL-6 and IL-1βlevels in serum of C57 mice,but was ineffective in Cxcr5–/–mice.Finally,the expression of phosphorylated ERK（pERK）,AKT（pAKT）and STAT3（pSTAT3）in the spinal cord after intrathecal injection.CXCL13 induced rapid up-regulation of pERK,pAKT and pSTAT3 in the spinal cord of C57 mice,which was absent in Cxcr5–/–mice.5.STZ-induced type I diabetic mice showed significant hyperglycemia and emaciation,long-term mechanical allodynia,and increased expression of chemokines Cxcl13 and Cxcr5 mRNA in the spinal cord.6.In the spinal cord of STZ-induced type I diabetic mice,there was a significant increase in gliosis and upregulation of phosphorylation of cellular signaling kinases,including pERK,pAKT and pSTAT3.7.The autoimmune and inflammatory pathological processes of diabetes lead to an increase in the expression of CXCL13 and its membrane receptor CXCR5 in the spinal cord.Binding of CXCL13 to CXCR5 activates intracellular signaling pathways such as ERK,STAT3 and AKT.Phosphorylated ERK,STAT3 and AKT lead to increased release of pro-inflammatory cytokines such as TNF-α,IL-6 and IL-1β,regulating various cellular events involving neuro-inflammation,and the development and progression of pain.Research conclusions:In the present study,we provide evidence that the chemokine CXCL13 and its receptor CXCR5 in the spinal cord contribute to the pathogenesis of PDN.We demonstrate that CXCL13/CXCR5 signaling is an important upstream mediator of the pERK,pAKT and pSTAT3 cell signaling pathways and promotes TNF-alpha and IL-6 production.Therefore,treatment for CXCL13/CXCR5 can provide an opportunity for patients with PDN.Research highlights:1.For the first time,our work clearly stated that CXCL13/CXCR5 is involved in the development of diabetic PDN.2.Our work clearly elucidates the cellular and molecular signaling mechanisms at the level of the spinal cord,how CXCL13/CXCR5 works through pERK,pAKT and pSTAT3,and promotes the TNF-αand IL-6 production.3.Our work showed the effects of interventions at different levels of CXCL13/CXCR5,cellular molecular kinases,and inflammatory factors in diabetic mice would provide valuable data for further clinical trials. |
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