Impacts And Molecular Mechanism Of Low Frequency Vibration On Calcium Influx And Autophagy Influx Of Human Umbilical Vein Endothelial Cells

Vestibular dysfunction,which causes vertigo,disturbs the work ability and reduces life quality of patients.There is a high incidence of peripheral vertigo that is caused by vestibular function disorder or dysfunction,such as common Meniere’s disease.Patients with Meniere’s disease have as the cochlear symptoms of aural fullness,tinnitus and hearing loss in addition to the vestibular symptom.The managements of vertigo including medicine,,surgical treatment,Meniett pulse generator,and various kinds of vestibular rehabilitation treatment have been widely carried out.Low-frequency vibration as a kind of novel treatments in the outpatient department has improved the overall feelings of patients,controlled the dizziness,relieved symptoms such as tinnitus,aural fullness and additionally made the patient fell comfortable on the head.This study observed that the effect of low-frequency vibration in promoting the rehabilitation process of vertigo in patients with unilateral vestibular hypofunction.The low-frequency delivered to the mastoid process obviously promoted the rehabilitation process of vertigo.In order to elucidate the therapeutic mechanism of low-frequency vibration on the vertigo symptom,we explored the mechanism of controlling and modifying the cellular function in the vestibule at the levels of cell biology and molecular biology associated with the low-frequency vibration treatment.We established a cellular model of autophagy interference by mitochondrial dysfunction using mitochondrial toxicin in human umbilical vein endothelial cell(HUVEC)that is a a well-recognized model cell on research of vascular system,which were transfected by mRFP-GFP-LC3 adenovirus.The impact of low-frequency vibration on calcium influx and autophagy influx of HUVECs exposed to mitochondrial toxicin,3-nitropropionic acid(3-NP),was compared with flunarizine that was used as a positive control.A broad-spectrum screening antibody chip for protein phosphorylation was used to analyze the impact of low-frequency vibration on protein phosphorylation level of signaling pathways in HUVECs that were disrupted by mitochondrial toxicin,and to further explore the molecular mechanism of low-frequency vibration interventon.Part I Beneficial effects of low frequency vibration delivered to the mastoid process in promoting vestibular rehabilitation of patients with unilateral vestibular hypofunction.Objective To study the effect of low-frequency vibration on vestibular rehabilitation of patients with unilateral vestibular hypofunction.Methods 25 patients with unilateral vestibular hypofunction were recruited in the outpatient department of Shanghai Changhai Hospital.The patients were diagnosed as the vestibular neuritis.They were randomly divided into two groups as low-frequency vibration intervention group and conventional vestibular rehabilitation group.The patients in low-frequency vibration intervention group received low-frequency vibration delivered to ipsilateral mastoid process.Low-frequency vibration at 100 Hz was delivered for 30 minutes,twice a week,and lasted for 2 weeks.The patients in the conventional vestibular rehabilitationgroup were received TECNOBODY balance test and training in the Rehabilitation Department under the guidance of rehabilitation therapists.The program including sensory integration training,vestibular function training,center of gravity control training was used.The patients in the both groups were evaluated using DHI(Dizziness Handicap Inventory)scores and VVAS(Visual Vertigo Analogue Scale)score before and after intervention.The mean scores between two groups were compared using chi-square with the assistance of SPSS software.A significance was accepted when p < 0.05.Results An effective outcome was granted when the total DHI score reached 18 points.Ten of thirteen patients in the low-frequency vibration intervention group showed effective result,and ten of twelve patients in the conventional vestibular rehabilitation group displayed effective outcome.However,no statistically significant difference was obtained between the two groups.Conclusion The effective rates between the two groups are equivalent in using low-frequency vibration delivered to mastoid process and the conventional vestibular rehabilitation program.T Compared to the conventional vestibular rehabilitation program with professional rehabilitation technician and large place.However,low frequency vibration is simple without special requirements and easy to be mastered by the technician.This present results showed that the low-frequency vibration is effective for the unilateral vestibular hypofunction at least during short-term.However,the long-term effect needs further observation in a large sample size investigation.Part II Impacts of low-frequency vibration on calcium influx of human umbilical vein endothelial cellsObjective To study possible involvement impacts of low-frequency vibration on calcium influx of human umbilical vein endothelial cells.Methods The human umbilical vein endothelial cell(HUVECs)were selected as model cells.We observed real-time changes on calcium influx of HUVECs through using expressing the fluorescently tagged,FLUO-3,which is a calcium ion tracer,by a fluorescence microscope We established 3-NP treatment of cell damage model and then,applied low frequency vibration on its treatment intervention,at the same time,flunarizine was used as positive control.On morphology,we observed real-time changes on calcium influx of 3-NP treatment of cell damage model and low frequency vibration on its treatment intervention through image analysis and semi-quantitative tests.Results Compared with normal cells,the green fluorescence intensity obviously strengthened in HUVECs damage model treated with 3-NP.There was calcium overload in HUVECs.In 3-NP treatment group,we respectively join flunarizine,or apply low frequency vibration.Respectively,in 3-NP + low frequency vibration treatment group and 3-NP + flunarizine group,at 1 minute,5 minutes,15 minutes,20 minutes,30 minutes,compared with 3-NP treatment of HUVECs damage model at different time points,we observed that green fluorescence intensity obviously decreased and level of calcium overload gradually reduced.There is obvious difference in statistical analysis by the semi-quantitative fluorescence.Conclusion Low-frequency vibration significantly reduced the level of calcium influx in HUVECs that were impaired by mitochondrial dysfunction.Low-frequency vibration may be slow or reduce the occurrence of apoptosis through reducing the level of calcium influx in HUVECs,and thus relieve the pathological process in inner ear disease.Part Ⅲ Impacts of low-frequency vibration on autophagy Influx of human umbilical vein endothelial cellsObjective To study possible involvement of autophagy in regulating vascular function of the inner ear by low-frequency vibration.Methods The human umbilical vein endothelial cell(HUVECs)were selected as model cells.HUVECs were transfected using adenovirus expressing the fluorescently tagged microtubule-associated protein 1 light chain 3 with mRFP-GFP-LC3,which is a marker protein of autophagy.The mitochondrial dysfunction cell model was established by exposing the transfected cells to 3-NP.A low-frequency vibration was delivered to the culture vessel in which the transfected cells grow for 30 min.In another group,flunarizine was added to the medium as positive control.The influence of 3-NP on autophagy Influx was followed real-time under laser confocal microscope.Then,a quantitative analysis on autophagy Influx was performed on the same cell type using automatic flow cytometry.Results Compared with normal cells,the number of the autophagosome(green)reduced in cells exposed to 3-NP,most of which showed fusion with lysosomes(yellow and red).Both low-frequency vibration and flunarizine significantly increased autophagy in the cells exposed to 3-NP.Conclusion Low-frequency vibration significantly raised the level of autophagy in HUVECs that were impaired by mitochondrial dysfunction.Low-frequency vibration may inhibit the excessive inflammatory response through enhancing autophagy,thus relieve the pathological process in inner ear.Part IV Impacts and molecular mechanism of low-frequency vibration on human umbilical vein endothelial cellsObjective To explore impacts of low frequency vibration on protein phosphorylation level of signaling pathways in HUVECs induced for mitochondrial dysfunction,and further investigate molecular mechanisms of the pathological process of HUVECs associated with mitochondrial dysfunction followed by low-frequency vibration intervention.Methods HUVECs were exposed to either 3-NP,or low-frequency vibration following 3-NP exposure,or flunarizine following 3-NP exposure.Whole cellular protein of normal HUVECs,3-NP exposed HUVECs,low-frequency vibration treated HUVECs following 3-NP exposure,and flunarizine treated HUVECs following 3-NP exposure.The signaling pathways phosphorylation broad-spectrum screening antibody microarrays(PEX100)was used to detect protein phosphorylation,A total of 1318 kinds of highly specific antibodies were attached to the high-density film using a patented three-dimensional polymer film technology.679 phosphorylation sites of 432 signal proteins in the four groups were analyzed.A large number of experimental data were analyzed and the phosphorylation ratio was calculated with a formula and compared between defined groups.Results Compared with 3-NP exposure group without intervention,low-frequency vibration group increased by more than 6 times the phosphorylation level of the following main protein signaling pathways : NMDAR2B(Phospho-Tyr1472),MEK1(Phospho-Ser298),Lamin A/C(Phospho-Ser392),HDAC1(Phospho-Ser421),PECAM-1(Phospho-Tyr713),P90RSK(Phospho-Thr359/Ser363),P90RSK(Phospho-Thr573),LAT(Phospho-Tyr171),PP2A-alpha(Phospho-Tyr307),Merlin(Phospho-Ser10),MKK4/SEK1(Phospho-Ser80),BLNK(Phospho-Tyr96),14-3-3 zeta/delta(Phospho-Thr232).3-NP + flunarizine group increased phosphorylation level more than 6 times of the following signaling pathways : MEK1(Phospho-Ser298),Lamin A/C(Phospho-Ser392),PAK1/2/3(Phospho-Thr423/402/421),PECAM-1(Phospho-Tyr713),PPAR-gamma(Phospho-Ser112),MKK4/SEK1(Phospho-Ser80),P90RSK(Phospho-Thr573),MDM2(Phospho-Ser166),PP2A-alpha(Phospho-Tyr307),14-3-3 zeta/delta(Phospho-Thr232).Conclusion: In HUVECs,3-NP reduced most of the protein phosphorylation,the low-frequency vibration and flunarizine enhanced the molecular phosphorylation level associated with autophagy and cellular viability and against apoptosis.