Mechanisms Of Peripheral Neuropathy Mediated By Neuroinflammation And Microcirculatory Disturbance Participating In Chemotherapy-induced Pain

Objective: Chemotherapy induced peripheral neuropathy(CIPN)is one of the serious side effects caused by chemotherapy,which brings endless pain and seriously declining the quality of life of patients.However,the pathogenesis of CIPN has not been elucidated and effective treatment is urgently needed in clinical.Matrix metalloproteinase-9(MMP-9)and matrix metalloproteinase-2(MMP-2)play an important role in the development of inflammation.Considering that the peripheral sensitization in CIPN is closely related to inflammation,MMP-9/2 may play an important role in the pathogenesis of CIPN.As an important source of MMP-9,macrophages mediate inflammatory response,are recruited to activate and release MMP-9 to promote neuronal injury and apoptosis.When neurons are stimulated or injured,they also secrete a large amount of MMP-9,which leads to local inflammation.The interaction between them may play an important role in the occurrence and development of CIPN.High mobility group box-1 protein(HMGB-1)can activate toll like receptor 4(TLR4)signal transduction to trigger and amplify the inflammatory process.After TLR4 activation,Nuclear factor ?B(NF-?B)was further activated,and NF-?B promoted the expression of MMP-9.It has been shown that the expression of MMP-9 can also be increased through the phosphatidylinositol 3-kinase(PI3K)/ protein kinase B(Akt)-NF-?B signaling pathway.Therefore,HMGB-1 may mediate the activation of MMP-9 in CIPN through TLR4-PI3K/Akt-NF-?B signaling pathway,and participate in the occurrence of CIPN.Here,we aimed to illustrate the crosstalk between macrophage and neuron mediated by the HMGB-1-TLR4-PI3K/Akt-MMP-9 signaling pathway in the process of CIPN and explored whether N-acetyl-L-cysteine(NAC),as a powerful antioxidant,can alleviate CIPN.Methods: Oxaliplatin(L-OHP)was administrated to mice to establish CIPN model.Western blot,gelatin zymography and immunohistochemistry were used to measure the expression or activity of protein MMP9/2 in DRG and spinal cord.Immunofluorescence was used to detect the expression of calcitonin gene-related peptide(CGRP)and microglia marker(Iba-1).The nociception in mice was detected by Von Frey Hairs.RAW264.7 cells and SH-SY5 Y cells were cultured to investigate separately the signal pathway of macrophages and neurons in vitro.N-Acetyl-L-cysteine(NAC)was administered intragastrically to measure the activity of MMP-9/2 in mice.Results: L-OHP induced mechanical pain and activation of MMP-9/2 in dorsal root ganglion(DRG).MMP-9 gene knockout alleviated the occurrence of CIPN.Macrophage scavenger clodronate liposomes attenuated mechanical pain and decreased MMP-9/2 expression in CIPN mice.In vitro,L-OHP increased the release of MMP-9/2 from neuron and HMGB-1 from both macrophage and neuron.Upregulation of MMP-9 activity induced by recombinant HMGB-1 was abolished by TLR4,PI3 K or Akt inhibitor respectively in macrophage(Raw 264.7 cells)and neuron(SH-SY5 Y cells)in vitro.Finally,we found that NAC inhibited the expression of CGRP and IBA-1 in spinal cord of CIPN mice,reduced MMP-9/2 activity,and improved CIPN.Conclusions: HMGB-1-TLR4-PI3K/Akt-MMP-9 axis is involved in the crosstalk between macrophage and neuron in the pathogenesis of CIPN in mice.NAC may be a potential therapeutic regimen and strategy for CIPN treatment.Objective: Chemotherapeutic drugs can induce the release of inflammatory factors from macrophages,and also induce macrophage expression of tissue factor(TF)which participate in thrombosis.Considering that tissue factor(TF)is an endogenous coagulation factor involved in ischemia and hypoxia,its high expression may induce microthrombus formation around the peripheral nerves,especially in the microenvironment around the sciatic nerve,causing hypoxia-related molecular events,such as an increase in hypoxia-inducible factor-1?(HIF-1?).Studies have shown that chemotherapeutic drugs can significantly increase the expression of TF,and TLR4-induced p38 activation is an important upstream signal of TF expression in macrophages.Similar to HMGB1,heat shock protein 70(HSP70)is one of the important endogenous ligands of TLR4,which can be rapidly released into the extracellular environment under cellular stress,activating TLR4 signaling to trigger and amplify the inflammatory process.Therefore,we hypothesized that TF-induced microcirculatory disorders may be involved in the development of CIPN.Here,we aimed to investigate the important role of hypoxia-related molecular signaling in CIPN,especially the relationship between the TF-HIF-1? signaling pathway axis and MMP-9/2 in the sciatic microenvironment on the formation and development of CIPN.At the same time,we aimed to explore the upstream signal of chemotherapy-induced high expression of TF,that is,to investigate the effect of HSP70-mediated activation of TLR4-p38 signaling pathway on TF expression and the possibility of using the anticoagulant hirudin to interfere with the TF-HIF-1?-MMPs axis and alleviate CIPN.Methods: L-OHP was injected intraperitoneally to establish a mouse CIPN model.Von Frey Hairs was used to determine the mechanical pain threshold of mice.The mouse macrophage cell line RAW 264.7 was used for cell experiments.Immunoblotting(Western blot)was used to detect the expression of HIF-1?,p-p38,p38,TF and HSP70 release in mouse sciatic nerve,blood and cell.Gelatin zymography was used to detect changes in the activity of MMP-9/2 in plasma and sciatic nerves before and after chemotherapy in mice and after hirudin treatment.Immunofluorescence assay was used to detect the effect of cellular level p38 inhibitor on TF expression before and after L-OHP treatment.Results: At the overall animal level,L-OHP significantly reduced the mechanical pain threshold in mice and significantly increased the expression of TF and MMP-9/2 in the sciatic nerve and blood of mice.In addition,L-OHP promoted the release of HSP70 to plasma.At the cellular level,L-OHP promoted the release of HSP70 by macrophages and increased p-p38 and HIF-1? protein levels in vivo and in vitro.Hirudin significantly inhibitd L-OHP-induced high expression of p-p38,HIF-1? and MMP-9/2 in sciatic nerve and reduced L-OHP-induced mechanical pain threshold reduction in mice.Immunofluorescence results showed that the p38 inhibitor SB203580 significantly inhibited L-OHP-induced high expression of TF in RAW264.7 cells.Conclusions: This study suggests that a novel HSP70-TLR4-p38-TF-HIF-1?axis-mediated microcirculatory disorders may play a pivotal role in the pathological process of CIPN.It is also shown that the use of anticoagulant Hirudin can inhibit the above mechanisms and improve CIPN.