MiR-30b-5p Alleviates Trigeminal Neuralgia In Rats Through Down Regulated Voltage-gated Sodium Channel Nav1.3 Expression

Objective Trigeminal neuralgia（TN）is a neurological pain attracting much attention.It is currently believed that the parenchyma of TN may be a hyperexcitability,manifested by abnormal discharge or ectopic action potentials in the demyelinated trigeminal nerve after vascular compression.Nav1.3 is a subform of a voltage-gated sodium channel（VGSC）whose abnormal expression plays an important role in the generation of ectopic discharge.Micro RNA（miRNA）is a non-coding RNA with a length of 18 – 25 nucleotides,and certain miRNA can be involved in the development of neuropathic pain by regulating VGSC expression and neuronal excitability.Nav1.3 and miR-30b-5p were highly correlated by bioinformatics analysis.This experiment further validated the target relationship between miR-30b-5p and Nav1.3 by a double-luciferase reporter assay.The rat TN model was constructed by infraorbital nerve-chronic constriction injury（ION-CCI）,and the rat TN model construction was verified by measuring the expression of activating transcription factor 3（ATF3,a marker of nerve damage）in the trigeminal ganglia（TG）.The expression changes of miR-30b-5p and Nav1.3 in postoperative TG after ION-CCI were examined by q RT-PCR and Western blot,and the molecular biological mechanism of miR-30b-5p regulating Nav1.3 in the development of TN were further explored to provide new directions for the treatment of TN.Methods 1.Validate the target relationship between miR-30b-5p and SCN3A（1）Bioinformatics predicted target action on the associated miRNA of the gene SCN3 A encoding for rat Nav1.3.（2）The target relationship of miR-30b-5p and SCN3 A encoding Nav1.3 by double luciferase reporter.2.Rat TN was constructed by the ION-CCI model and verified whether the model construction was successfulThe 180-200 g adult male Sprange-Dawley（SD）rats were selected to construct ION-CCI under general anesthesia,and the mechanical pain threshold of the preoperative and postoperative surgical tentacle pad area was detected by Von Frey hair brush.Changes of ATF3 expression within the operative TG at the lowest point of the rat mechanical pain threshold were determined by q RT-PCR and Western blot.Whether the rat TN model was successfully constructed was identified by the rat mechanical pain threshold and the difference of ATF3 expression within the TG.3.Nav1.3 and miR-30b-5p expression in TG were analyzed by qRT-PCR and Western blotIn the first stage of the experiment,rats were randomly divided into two groups:ION-CCI group and sham surgery group（Sham group）.The same surgical procedures in the Sham group were followed in the ION-CCI group except for non-ligation of the infraorbital nerve.Exchanges of miR-30b-5p and Nav1.3 in TG of ION-CCI and Sham rats were determined by q RT-PCR and Western blot to explore the expression of miR-30b-5p and Nav1.3 in the occurrence and development of TN.4.To explore the mechanism by which miR-30b-5p regulates Nav1.3 in trigeminal neuralgiaIn the second phase of the experiment,miR-30b-5p Inhibitors（antagomir）and antagomir NC were injected into the Sham group TG by the infraorbital foramen injection to inhibit the expression of miR-30b-5p.miR-30b-5p analogues（agomir）and agomir NC were injected into the ION-CCI group TG by the infraorbital foramen injection to advance the expression of miR-30b-5p.Rat mechanical pain threshold and changes in miR-30b-5p and Nav1.3 expression after the intervention were examined to explore the mechanism by which miR-30b-5p regulates Nav1.3 in trigeminal neuralgia.Results 1.The bioinformatics analysis results showed that Nav1.3 was highly associated with miR-30b-5p.The results of the double-luciferase reporter assay showed a target relationship between miR-30b-5p and Nav1.3,and that miR-30b-5p was able to regulate Nav1.3 expression by binding to the SCN3 A 3’UTR.2.Compared with the Sham group,rats in the ION-CCI group showed a significant reduction in the mechanical pain threshold between the postoperative days 2 and 28,reaching the lowest value at the postoperative day 12.The q RT-PCR and Western blot results showed that the ATF3 expression was increased in the TG of rats in the postoperative ION-CCI group compared with the Sham group,and the above results indicated the success of the TN model construction.3.The q RT-PCR results showed that the miR-30b-5p expression was reduced in the postoperative TG and the Nav1.3 m RNA expression was increased compared with the Sham group.The Western blot results showed an increased Nav1.3 protein expression in the TG of the ION-CCI group compared with the Sham group.4.After inhibition of miR-30-5p expression in Sham group,the mechanical pain threshold decreased and the expression of Nav1.3 m RNA and protein increased in TG.After overexpression of miR-30b-5p in the ION-CCI group,the mechanical pain threshold increased and the expression of Nav1.3 m RNA and protein decreased in TG.Conclusion The miR-30b-5p is able to regulate Nav1.3 expression by binding to the SCN3 A 3’UTR.Less miR-30b-5p expression and increased Nav1.3 expression in postoperative TG in the TN model constructed with ION-CCI.Overexpression of miR-30b-5p inhibited Nav1.3 expression in the TG and alleviated the TN induced by ION-CCI.Nav1.3 and miR-30b-5p are involved in the development and development of TN and are a potential therapeutic targets for TN.This experiment explored the mechanism by which miR-30b-5p regulates Nav1.3 involvement in TN,providing new directions for the clinical treatment of TN as well as the selection of regulatory targets.