| BackgroundOxaliplatin is the third generation of the platinum,which is effective in the treatment of multiple solid tumors such as ovarian cancer,gastric cancer,lung cancer,and pancreatic cancer.However,it can cause peripheral neuropathic pain and there is no good treatment.The molecular mechanisms remain unknown.This research used oxaliplatin animal model,found that Nav1.6 expression in rat dorsal root ganglion was increased.So we concentrated on how Nav1.6 participated in oxaliplatin-induced peripheral neuropathic pain.The aim of this research is to detect upstream mechanisms of Nav1.6 involved in development of oxaliplatin-induced peripheral neuropathic pain,to provide new ideas to treat neuropathic pain.Research purposes:In the present study,we established the peripheral neuropathic pain rat model by intraperitoneal injecting with oxaliplatin.To reveal the molecular machanism of increasing neuron excitability induced postoperative pain due to injected oxaliplatin.This research aimed to reveal the mechanisms of oxaliplatin-induced peripheral neuropathic pain development through the molecular-cellular-whole body,provide new targets for clinical treatment.Research methods1.Establishment of oxaliplatin-induced peripheral neuropathic pain in SD rats. We simulated the clinical medication time giving oxaliplatin in SD male rats(twice a week,a total of nine intraperitoneal injections).we detected the mechanical,coldand and thermal allodynia in the model rats.The m RNA and protein expression of Nav1.6 in DRG tissues were detected by q RT-PCR and Western-blot.To define the localization of Nav1.6 and mi R-30 b,double-labeled immunofluorescence and in FISH were conducted in DRG neurons.2.mi R-30 b actually targeted the 3'UTR sequences of SCN8 A in PC12 cells.To construct a dual luciferase vector containing the sequence of SCN8 A 3'UTR.mi R-30 b agomir and wild-type and mutant-type vectors were transfected into PC12 cells by lipofectamine 2000,and detect the change of fluorescence intensity in each group.To determine whether there is a target relationship between mi R-30 b and SCN8 A.3.To study the regulation of mi R-30 b and Nav1.6 in DRG cells.The primary DRG neurons were stimulated by different concentration of oxaliplatin.The m RNA and protein expression of Nav1.6 in primary DRG cells were detected by q RT-PCR and Western-blot.Transfecting mi R-30 b agomir into primary DRG cells stimulated by oxaliplatin and then determine the change of Nav1.6 m RNA and protein.Meanwhile,we transfected mi R-30 b inhibitor into normal DRG neurons of rats,and detected the expression of Nav 1.6 m RNA and protein.4.Intrathecal injection of mi R-30 b relieves oxaliplatin-induced peripheral neuropathic pain.mi R-30 b was overexpressed to the model rats for 4 days following day 15 after the first oxaliplatin administration,and mechanical,thermal and cold allodynia were observed.q RT-PCR and Western-blot were performed to assess the expression of Nav 1.6 m RNA and protein in DRG in oxaliplatin model rats.We administered mi R-30 inhibitor into normal rats,and mechanical,thermal and cold allodynia were detected.Meanwhile,the exepression of Nav1.6 m RNA and protein were detected.5.Transfecting mi R-30 b agomir into LS-174 t cells.To investigate the effect of mi R-30 b on the proliferation of colon cancer cells.mi R-30 b agomir and antagomir were transfected into LS-174 t cells and detect the proliferation of the tumor cells,and the change of the Caspase-3 Protein was detected in tumor cells.Results1.Establishment of oxaliplatin-induced peripheral neuropathic pain in SD rats.We established the peripheral neuropathic pain rat model by intraperitoneal injecting with different concentration oxaliplatin(2.4mg/kg,3.2mg/kg,and 4.0mg/kg).The significant mechanical and cold allodynia of model rats were decreased from 8 days after the first injection and dose-denpent.Thermal allodynia and weight were not changed in the treatment period in all groups.2.Changes of Nav1.6 expression at different concentrations of oxaliplatin and at different time points.The expression of Nav1.6 m RNA and protein in DRG after injection of oxaliplatin were markedly higher than the control group.It found that the expression of Nav1.6 m RNA and protein increased after injection of oxaliplatin at different time points from 8th to 21 st day,but there was no significant difference between the 15 th and 21 st days.The change of Nav1.6 are dose dependent and time dependent in oxaliplatin-induced peripheral neuropathic pain rats.3.mi R-30 b directly targets the 3'UTR of SCN8 A in PC12 cells.We discovered that SCN8 A was the primary target of mi R-30 b using Target Scan software.The wild-type plasmid vector was transfected into PC12 cells by Lipofectamine 3000.Mi R-30 b mimic decreased relative luciferase activity.But the luciferase activities of others groups were unchanged.4.mi R-30 b agomir inhibit the Expression of Nav1.6 m RNA in oxaliplatin stimulate the primary DRG Neurons.We used to oxaliplatin stimulate the primary DRG neurons.The levels of mi R-30 b and SCN8 A m RNA were measured by q RT-PCR and the changes in Nav1.6 protein expression was determined by western-blot.oxaliplatin caused a significantly increase of Nav1.6 at m RNA and protein level,while a reduction of mi R-30 b was observed.Moreover,mi R-30 b agomir reversed the up-regulation of SCN8 A and Nav1.6 in DRG cells.5.Intrathecal mi R-30 b agomir inhibits the expression of Nav1.6 and attenates oxaliplatin-induced neuropathy.We delivered mi R-30 b agomir to oxaliplatin-induced peripheral neuropathic pain rats for 4 days following the 15 days after the first injection.The mechanical and cold allodynia were relieved by intrathecal injection with mi R-30 b mimics in model rats.The upregulation of Nav1.6 protein was significantly suppression by mi R-30 b agomir in DRG. We administrated mi R-30 b antagomir to normal rats for 4 days.We observed that mechanical and cold allodynia were markedly lower during mi R-30 b antagomir application than normal rats injected with scrambled mi RNAs,showing that down-expression mi R-30 b generated pain in normal rats.Moreover,down-regulation of mi R-30 b increases Nav1.6 m RNA and protein in DRG neurons of normal rats.6.miR-30 b agomir inhibit the proliferation of the LS-174 t cells.We over-expressed mi R-30 b in LS-174 t cells by transfecting mi R-30 b agomir.CCK-8 assay showed that over-expression of mi R-30 b dramatically decreased the growth rate of LS-174 t cells compared with transfecting scramble cells.Down-expression of mi R-30 b significantly increased the growth rate of LS-174 t cells.ConclusionWe found that mi R-30 b can relieve pain by reducing Nav1.6 expression in oxaliplatin induced peripheral neuropathic pain,and mi R-30 b may become a new target for relieving oxaliplatin-induced peripheral neuropathic pain. |
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