Upregulation Of Nav1.6 Channel By P38 MAPK Pathway In Dorsal Root Ganglion Contributes To The Development Of Bone Cancer Pain

Objective:To explore the aberrant expression of m RNA and related signaling pathways by transcriptome sequencing in dorsal root ganglia of rats with bone cancer pain.Methods:Cancer pain was induced by inoculation of MRMT-1 breast carcinosarcoma cells（3×10⁵/10μl）into the right tibia of adult female Sprague Dawley（SD）rats.Mechanical withdrawal threshold（MWT）was tested at day 2 before surgery and 4,7,11,14,17 and21 days after surgery.X-ray imaging and pathology was employed to identify the successful establishment of the rat model.Then,we performed transcriptome sequencing of the ipsilateral dorsal root ganglion（DRG）to explore the aberrant expression of m RNA and performed Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis on the differently expressed m RNAs.Q-PCR was used to validate the significantly up-regulated and down-regulated gene expression.Results:Rat with bone cancer showed an apparent decline in the MWT at day（d）11-21days after MRMT-1 cells inoculation（*P<0.05）.Compared with the Sham group,we found 149 differently expressed m RNAs,including 124 expression up-regulated and 25down-regulated.The data showed that the expression of Nav1.6 was significantly up-regulated by 2^2.4times,ranking in the top ten up-regulated genes.The GO and KEGG analysis showed that the differently expressed m RNAs are mainly involved in cell signal transduction,mmune,inflammation and other physiological and pathological processes.The MAPK pathway,one of the important signal transduction pathways,is concentrated in the top three in the KEGG analysis and was proven to be associated with chronic pain.The q-PCR results showed that the expression of LOC100911356 and Ndst2 genes increased in the BCP group compared with the Sham group,while the expression of Press29,Epyc,and Fcrla genes were down-regulated,which was consistent with the microarray results.Conclusions:The injection of MRMT-1 breast cancer cells in tibia of rats induces bone cancer pain.The up-regulation of Nav1.6 in the DRG of rats with bone cancer pain and the activation of MAPK signaling pathway can be closely related to the mechanism of bone cancer pain.Objective:To determine the role of MAPK signaling pathway on the regulation of Nav1.6 in the development of bone cancer pain in rats.Methods:1.To examined the expression of Nav1.6 in rats with bone cancer pain.12 adult female SD rats were randomly divided into 2 groups: Sham and BCP group,n=6.MRMT-1 tumor cells were inoculated into the right tibia of SD rat in BCP group.Western Blot（WB）,q-PCR,and immunofluorescence were used to compare the expression of Nav1.6 in DRG of rats between two groups.To further confirmed whether the Nav1.6 knockdown in DRG can alleviate the development of bone cancer pain,84 adult female SD rats were divided into 6 groups（n=14）: Sham group,BCP group,LV-NC Group,sh Nav1.6#1 group,sh Nav1.6#2 group,sh Nav1.6#3 group.The Nav1.6sh RNA lentivirus（2×106TU/10μl）targeting three specific sequence and non-targeting lentivirus were delivered to region of L5 DRG via the intrathecal injection catheter in sh Nav1.6#1 group,sh Nav1.6#2 group,sh Nav1.6#3 group and LV-NC group separately.In all groups（n=8）,the MWT of rats was measured on 2 days before surgery and 4,7,11,14,17,21 days after operation.The expression of Nav1.6 in DRG was examined on the 14 day after surgery by WB and q-PCR.2.In order to explore whether the activation of MAPK pathway contributes to bone cancer pain by regulating the expression of Nav1.6,we detected the phosphorylation levels of p38,JNK and ERK in the DRG of rats with bone cancer pain.Then,84 SD rats were randomly divided into 6 groups（n=14）: Sham group,BCP group,BCP+SB203580（p38 inhibitor）group,BCP+SP600125（JNK inhibitor）group,BCP+U0126（ERK upstream MEK inhibitor）group,BCP+Vehicle group.Inhibitors and vehicle were given daily for three consecutive days from 12 to 14 days after surgery.MWT was measured on the 14 day,before administration and at 2,4,6,8h after drugs injection.The expression of p-JNK/JNK,p-ERK/ERK,p-p38/p38,Nav1.6 in DRG was examined at 4hours after administration on the 14 day by WB.Nav1.6 m RNA level,the colocalization of Nav1.6 and p-p38 was detected by q-PCR and immunofluorescence respectively.Results:1.The expression of Nav1.6 m RNA and protein in DRG of rats with bone cancer pain was significantly increased（*P<0.05 vs Sham group）.2.Compared with BCP group,the expressions of Nav1.6 m RNA and protein in the sh Nav1.6#2 group and sh Nav1.6#3 group were significantly down-regulated（#P<0.01 vs BCP group）,and the decrease of MWT induced by bone cancer can be alleviated in sh Nav1.6#2 group and sh Nav1.6#3 group.3.Increased phosphorylation of p38,JNK and ERK in the DRG of rats with bone cancer pain was shown.Intrathecal injection of inhibitors respectively can significantly inhibit phosphorylation of p38,JNK and ERK.However,only SB203580（p38 inhibitor）can decreased the expression of Nav1.6 and attenuated pain behavior induced by bone cancer.Conclusions:Up-regulation of Nav1.6 by p38 MAPK pathway instead of JNK、ERK pathway activation in rat dorsal root ganglia of rat contributes to the development of bone cancer pain.