| Objective:The vestibular system is an important component of maintaining body balance and an important feature of maintaining human upright biological attributes.At the same time,the vestibular system is also involved in spatial positioning,emotion regulation,memory and other higher sensory functions.The vestibular system is composed of semicircular canals,utricle,saccule,vestibular nerves,vestibular nuclei,cerebellum,thalamus,cerebral cortex and the conduction pathways formed between them.Any part in the system occurs pathological changes,can cause vestibular dysfunction.Vestibular dysfunction is mainly manifested as vertigo,dizziness,posture imbalance,vestibular vision symptoms,accompanied by nausea and vomiting,palpitations,sweating,defecation,fear,etc.,seriously affecting the patient’s quality of life.Vertigo is a kind of motion illusion.According to statistics,vertigo as the chief complaint accounts for about 5%～10%in neurology outpatient department,about 6～8%in inpatient cases,and about 7%in otorhinolaryngology outpatient department.Different according to be involved region,vertigo can be divided for peripheral and central vertigo,peripheral vertigo basically involves labyrinth,vestibular nerve.Acute peripheral vertigo refers to vertigo caused by acute lesion of peripheral vestibular receptors or vestibular nerves.Acute peripheral vertigo common disease has vestibular neuritis,labyrinthitis,sudden deafness with vertigo.The clinical manifestations of vestibular dysfunction can be alleviated and improved over time,is known as vestibular compensation.Vestibular compensation is divided into"static compensation" and "dynamic compensation".Vestibular compensation mainly involves vestibular nucleus,cerebellum and other vestibular central structures.Vestibular compensation can be realized in three ways:restoration,habutiation and adaptation.Vestibular compensation is a complex process involving a variety of biochemical changes.Current studies have confirmed that the biochemical mechanisms of vestibular compensation may include protein phosphorylation and synthesis,gene regulation,neuronutrient expression,stress and glucocorticoid effects,nitric oxide,etc.Protein kinase C and Ca2+/Calmodulin-dependent kinase Ⅱ are mainly involved in protein phosphorylation and synthesis.In terms of gene regulation,c-FOS,e-Jun,Krox-24 and micrornas have been confirmed,such as miR-218a-5p,miR-219a-5p,miR-2213p.However,there is no domestic study on the relationship between protein phosphorylation,synthesis and gene regulation in vestibular compensation.This study preliminarily explored the expression changes and regulatory pathways of miR-219a-5p,CaMKⅡγ and PKC in vestibular compensation process of acute peripheral vertigo.Methods:1.Construct a rat model of Unilateral vestibular deafferentation by Unilateral labyrinthectomy to simulate acute peripheral vertigo.The UVD model of rats with overexpression and silenced miR-219a-5p and overexpression of CaMKⅡγ was established by lateral ventricle micropumping technique.Vestibular dysfunction score(postural asymmetry,head tilt,nystagmus)and rotarod score were used to detect vestibular compensation.The expression of miR-219a-5p in MVN was detected by qRT-PCR.The expression of CaMKⅡγ,P-CaMKⅡγ and PKC in MVN was detected by Westernblot.BrdU detected cell proliferation in MVN when miR-219a-5p was overexpressed and silenced.Immunohistochemistry was used to detect the expression of PKC in MVN.Pearson correlation model was used to analyze the correlation of Mir-219a-5p,CaMKⅡγand PKC expression levels.2.Trans-retinoic acid was used to induce the differentiation of P19 embryonic cancer cells into neural cells,double luciferase reporter gene assay was used to determine the targeted regulation of miR-219a-5p on CaMKⅡγ and fluorescence in situ hybridization was used to detect the subcellular localization of miR-219a-5p.miR219a-5p and CaMKⅡγ were transfected into C57BL/6 mouse cortical cells,and the effects of overexpression and silenced miR-219a-5p on CaMKⅡγ expression and overexpression of CaMKⅡγ on PKC expression were detected by Western blot.Rat UVD models with overexpression and silenced of miR-219a-5p and overexpression of CaMKⅡγ were established by lateral ventricular micropumping technique.Western blot was used to detect the expression of CaMKⅡγ in MVN when miR-219a-5p was overexpressed and silenced,and the expression of PKC in MVN when miR-219a-5p was overexpressed.Immunohistochemistry was used to detect the immunoactivity of PKC.Results:1.The vestibular compensation is fastest 1～3 days after UL operation,and basically returned to normal one week later.2.Western blot showed that during vestibular compensation in UVD rats,the expression of miR-219a-5p and PKC inoperation ipsolateral MVN was significantly higher than that in sham operation group(p<0.05),and the expression of CaMKⅡγ was significantly lower than that in sham operation group(p<0.05).Immunohistochemistry showed that the immunohistochemistry score of PKC in operation ipsilateral MVN of UVD rats was significantly higher than that of sham operation(p<0.05).3.UL was performed 10 h after injection of miR-219a-5p agomir or antagomir by lateral ventricular micropump,and ipsilateral MVN was taken 3d after surgery.qRT-PCR results showed that the expression of miR-219a-5p in agomir group was significantly higher than that in antagomir and UL group(p<0.05),suggesting that the modeling was successful.The vestibular dysfunction score showed that the scores of posture asymmetry,head tilt and nystagmus in agomir group were significantly lower than those in antagomir group and UL group at 2-7d after UL(p<0.05),and the scores of rotation test were significantly higher than those in antagomir group and UL group(p<0.05).BrdU staining showed that the number of BrdU positive cells in agomir group was significantly higher than that in antagomir group and UL group(p<0.05).4.After UL,overexpressing CaMKⅡγ lentivirus was injected into the lateral ventricle of rats with micropump,and the ipsilateral MVN was extracted 3d after surgery.Western blot showed that the expression of CaMKⅡγ and p-CamKⅡγ in oe-CaMKⅡγ group was significantly increased compared with that in oe-NC group and sham group(p<0.05),indicating that molding is successful.The vestibular dysfunction deficit score showed that oe-CaMKⅡγ group posture asymmetry score,head tilt score and nystagmus score were significantly higher than those of oe-NC group(p<0.05),the scores of rotation test was significantly lower than that of oe-NC group(p<0.05).5.Pearson correlation analysis showed that CaMKⅡγ was negatively correlated with miR219a-5p(r=-0.890,p<0.05),PKC was positively correlated with miR-219a-5p(r=0.779,p<0.05),PKC was negatively correlated with CaMKⅡγ(r=-0.907,p<0.05).6.Dual-luciferase reporter gene assay showed that there was no significantly change in the luciferase activity of CaMKⅡγ-Mut group when miR-219a-5p was overexpressed or silenced.In CaMKⅡγ-Wt group,overexpression of miR-219a-5p inhibited luciferase activity,while silenced of miR-219a-5p promoted luciferase activity,with statistical significance(p<0.05).7.Fluorescence in situ hybridization showed that miR-219a-5p was expressed in the cytoplasm of neural cells.8.C57BL/6 mouse cortex cells were transfected with miR-219a-5p mimics or inhibitor and negative control,and the transfection efficiency was detected by qRT-PCR.The results showed that the expression level of miR-219a-5p in mimics group was significantly higher than that in inhibitor group(p<0.05),indicating that the transfection was successful.Western blot results showed that overexpression of miR-219a-5p inhibited the expression of CaMKⅡγ,and silenced of miR-219a-5p promoted the expression of CaMKⅡγ,with statistically significant differences compared with the negative control group(p<0.05).9.Cortical cells were transfected with miR-219a-5p mimic,oe-CaMKⅡγ and negative control.Western blot results showed that the expression of PKC in miR-219a-5p mimic group was significantly higher than that in the negative control group(p<0.05).The expression of PKC in oe-CaMKⅡγ group was significantly decreased compared with negative control(p<0.05),and the expression of PKC in miR-219a-5p mimic+oeCaMKⅡγ group was significantly increased compared with oe-CaMKⅡγ group(p<0.05).10.After UL,the oe-CaMKⅡγ model was established by injecting overexpressing CaMKⅡγ lentivirus into the lateral ventricle of rats with micropump,and the ipsilateral MVN was taken for Western Bolt and immunohistochemistry 3d after operation.The results showed that the expression of PKC in oe-CaMKⅡγ group was significantly lower than that in oe-NC group(p<0.05),and the immunohistochemical score of PKC in oeCaMKⅡγ group was significantly lower than that in oe-NC group(p<0.05).11.Agomir or antagomir of miR-219a-5p was pumped into the lateral ventricle micropump to establish miR-219a-5p overexpression or silencing model.UL was performed 10h after administration,and 3d after surgery,ipsilateral MVN was extracted.The expression of CaMKⅡγ in agomir group was significantly decreased compared with antagomir group and UL group(p<0.05),and the expression of PKC in agomir group was significantly increased compared with antagomir group and UL group(p<0.05).Immunohistochemistry showed that the immunohistochemistry score of PKC in agomir group was significantly higher than that in antagomir group and UL group(p<0.05).Conclusion:1.The vestibular compensation is fastest 1～3 days after UL operation,and basically returned to normal one week later.2.During vestibular compensation in UVD model rats,the expression of miR-219a-5p and PKC increased in ipsilateral MVN,while the expression of CaMKⅡγ decreased.3.Overexpression of miR-219a-5p promoted vestibular compensation and silent suppression in UVD rats.Overexpression of CaMKⅡγ inhibited vestibular compensation and silencing promotedin UVD rats.4.miR-219a-5p can negatively regulate the expression of CaMKⅡγ by targeting regulation,and miR-219a-5p is expressed in the cytoplasm of nerve cells.5.Overexpression of miR-219a-5p inhibits the expression of CaMKⅡγ,silencing miR219a-5p promotes the expression of CaMKⅡγ,overexpression of miR-219a-5p promotes the expression of PKC,and overexpression of CaMKⅡγ inhibits the expression of PKC.miR-219a-5p and CaMKⅡγ were overexpressed,and PKC expression was recovered.6.During vestibular compensation in UVD rats,overexpression of CaMKⅡγ inhibited PKC expression.Overexpression of miR-219a-5p inhibited CaMKⅡγ and promoted PKC expression,while silencing miR-219a-5p promoted CaMKⅡγ and inhibited PKC expression. |
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