| Granolocyte-macrophage colony stimulating factor(GM-CSF),originally identified as a colony stimulating factor because of its ability to induce granulocyte and macrophage populations from precursor cells,is one of the most common growth factors in the blood system.GM-CSF is widely used for the treatment of myelodysplastic syndromes,aplastic anemia,tumor radiotherapy and chemotherapy-induced neutropenia.During these trestments,the most severe adverse reaction for GM-CSF is bone pain,with an incidence up to 90%.A recent study has,for the first time,functionally linked GM-CSF secreted by tumor cells in bone metastases to sensitization of pain-sensing nerves(nociceptors),tumorevoked pain and remodelling of peripheral nerves.The level of GM-CSF in the bone marrow of the bone metastatic pain model is significantly increased.Indeed,blocking GM-CSFR signaling by receptor neutralising antibodies leads to an abrogation of bone tumor-induced pain hypersensitivity.GM-CSF receptor is abundantly expressed in the dorsal root ganglion.Three main cellular signaling pathways:Janus kinase-signal transducerandactivatoroftranscription(Jak-Stat)pathway,the mitogen-activated protein kinase(MAPK)pathway and the phosphoinositide3-kinase(PI3K)pathway are activated when GM-CSF exert their functions in hematopoietic cells,all of which have been directly or indirectly implicated in pain modulation.However,it is unknown what consequences of these GM-CSF-mediated cell signaling have on ion channels which are important for pain transduction,and the targeted ion channels by GM-CSF inpain-sensing neurons remain unknown.Dorsal root ganglion(DRG)neurons are the primary somatosensory neurons,and are crucial for the pain signal initiation and propagation.In pathological conditions,nociceptors in dorsal root ganglia can transduce noxious heat,mechanical,and chemical signals into electrical signals which are then sent to the central nervous system.At least five different voltage-gated sodium channels are expressed in DRG,including the TTX-sensitive Nav1.1,Nav1.6 and Nav1.7 and the TTX-resistant Nav1.8 and Nav1.9.Nav1.7,Nav1.8 and Nav1.9 channels are mainly distributed in small diameter DRG neurons,and are related to pain initiation.Nav1.7 channel mutations lead to inherited erythromelalgia(IEM),paroxysmal extreme pain disorder(PEPD),congenital insensitivity to pain(CIP)and other pain related diseases.Mutation of Nav1.8 gene is fund in patients with peripheral neuropathy,and Nav1.9 channel mutations cause familial episodic pain syndrome.We first screened ion channels and measured their mRNA levels in DRG which are treated with GM-CSF.The results show that mRNA levels of Nav1.7,Nav1.8,Nav1.9 channel were significantly increased with treatmet of GM-CSF.Based on these results,Further experiments were carried out and the results were demonstrated in this thesis,which are described in two parts:in the first part,we established a method for targeted delivery of drugs into DRG,and then investigated the effects of DRG targeted delivery of GM-CSF on the expression level of Nav1.7,Nav1.8 and Nav1.9;in the second part,we investigated the mechanism for GM-CSF induced upregulation of Nav1.7,Nav1.8,Nav1.9.Part 1 Targeted delivery of GM-CSF to DRG lead to up-regulation Nav1.7,Nav1.8 and Nav1.9 mRNA and painful behavior in ratsObjective:To investigate effects of targeted delivery of GM-CSF into DRG on the expression level of Nav1.7,Nav1.8 and Nav1.9 mRNA and on the pain behavior of rats.Methods:(1)A cannula was inserted and fixed upon to the DRG of rats,to facilitate the targeted delivery of GM-CSF into DRG.The mechanical pain threshold of rats was measured using the von Frey fibers,the heat pain threshold of rats was measured using a heat radiation apparatus.(2)DRG neurons were acutely disassociated and were incubated with GM-CSF for 4 h or 24 h;after incubation with GM-CSF,the mRNA expression level of Nav1.7,Nav1.8 and Nav1.9 channels in DRG neurons were measured by qPCR.(3)Anti-senseoligodeoxynucleotide(ASODN)against Nav1.7,Nav1.8and Nav1.9 were injected in DRG(L5)via DRG cannula to lower down the expression Nav1.7,Nav1.8 and Nav1.9 and the effect of these ASODNs on pain behavior was tested.Results:(1)Inbehavioral experiments and compared with the vehicle-treated rats,GM-CSF induced significantdose-dependent thermal and mechanical hyperalgesia,respectively.Focal injection of GM-CSF(20 ng or 200 ng,respectively)via the DRG cannula significantly increased sensitivity of rats to thermal and mechanical stimuli as measured with the Hargreaves and Von Frey tests(*P<0.05);however,the nociceptive responses of rats were not affected when 2 ng of GM-CSF was injected.(2)The mRNA expression level of Nav1.7(1.45±0.18 folds,n=11,*P<0.05),Nav1.8(3.8±0.4 folds,n=11,*P<0.05)and Nav1.9(1.56±0.15folds,n=11,*P<0.05)in DRG cells were significantly increased after incubated with GM-CSF for 4 h.The mRNA expression levels of Nav1.7(1.75±0.23 folds,n=6,*P<0.05),Nav1.8(2.25±0.3 folds,n=6,*P<0.05)and Nav1.9(1.6±0.23 folds,n=6,*P<0.05)in DRG cells were also significantly increased after incubated with GM-CSF for 24 h.(3)Injection of antisense oligonucleotides against Nav1.7,Nav1.8 and Nav1.9 separately in DRG(L5)via DRG cannula reduced the expression Nav1.7,Nav1.8 and Nav1.9,and at the same time reduced the nociceptive responses of the rats to GM-CSF which was also delivered via the DRG cannula.Conclusions:GM-CSF induces pain behaviors possibly through increased mRNA expression level of Nav1.7,Nav1.8 and Nav1.9 channels in DRG.Part 2 Jak2-Stat3 signaling pathway is involved in GM-CSF induced upregulation of Nav1.7,Nav1.8,Nav1.9 mRNAObjective:To explore the cellular signaling mechanism involved in GM-CSF induced upregulation of Nav1.7,Nav1.8,Nav1.9 mRNA.Methods:(1)The acutely disassociated DRG neurons were incubated with GM-CSF and the blockers of related signaling pathways,and were divided into four groups:control,GM-CSF,AG490+GM-CSF(AG490 is a Jak2blcoker),and Stattic+GM-CSF(Stattic is a Stat3 blocker).Both blockers were added to cells separately 15 min or 30 min prior to and concurrently present with GM-CSF treatment.The mRNA expression levels of Nav1.7,Nav1.8 and Nav1.9 in DRG neurons were measured with qPCR.(2)Immunofluorescence study was used to detect the protein expression of Nav1.7,Nav1.8 and Nav1.9 in DRG neurons before and after administration of Jak2 inhibitor.(3)ASODN against Jak2,Stat3 and Stat5a were injected in DRG(L5)via DRG cannula to reduce the expression Jak2,Stat3 and Stat5a and then the pain behavior induced by GM-CSF in rats was observed.Results:(1)Same as shown above,when DRG neurons were incubated with GM-CSF,the mRNA expression levels of all three Na~+channels were significantly increased compared with the control:Nav1.7(1.45±0.18 folds,n=11,*P<0.05),Nav1.8(3.8±0.4 folds,n=11,*P<0.05),Nav1.9(1.56±0.15 folds,n=11,*P<0.05).These effects of GM-CSF were prevented when the Jak2 inhibitor AG490 was present;compared with the GM-CSF alone treated neurons,the mRNA levels in the presence of both GM-CSF and AG490 were:Nav1.7(0.63±0.13 folds,n=6,~#P<0.05),Nav1.8(0.52±0.19 folds,n=6,~#P<0.05),Nav1.9(0.72±0.15 folds,n=6,~#P<0.05).Similarly Stat3 inhibitor Stattic also prevented the effect of GM-CSF:Nav1.7(0.4±0.22 folds,n=6,~#P<0.05),Nav1.8(0.68±0.17 folds,n=10,~#P<0.05),Nav1.9(0.26±0.12 folds,n=6,~#P<0.05).(2)In protein expression level study,measured with immunofluorescence,when DRG neurons were incubated with GM-CSF,the protein expression levels of all three Na~+channels were significantly increased compared with the control.These effects of GM-CSF were prevented when the Jak2 inhibitor AG490 was present;compared with the GM-CSF alone treated neurons,the protein levels of Nav1.7,Nav1.8 and Nav1.9 in the presence of both GM-CSF and AG490 were reduced.(3)Antisense oligonucleotides against Jak2,Stat3 and Stat5a administrated via DRG cannula in DRG(L5)reduced the expression level of Jak2,Stat3 and Stat5a mRNA.With the pronounced reduction of Jak2 and Stat3 mRNA levels a reduced pain behavior induced by GM-CSF was observed(~#P<0.05 with respect to the corresponding GM-CSF alone),however,the pain behavior induced by GM-CSF were not affected when Stat5a mRNA levels was reduced by its antisense oligonucleotides.Conclusions:Jak2-Stat3 signaling pathway is a key target signaling pathway involved in GM-CSF induced upregulation of Nav1.7,Nav1.8 and Nav1.9 channels. |
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