The Regulative Mechanism Of VGSCs Subtype Nav1.6 On The Stress-induced Hypertension In The Rostral Ventrolatral Medullar

Background: Hypertension,which results from genetics,living habits and environmental factors,is a common cardiovascular disease and the major aggravating cause of social and psychological stress among adults.Hypertension is characterized by elevated arterial blood pressure.There is no obvious symptom in the early stage of the disease,then the pathological features such as arteriosclerosis appear in the middle stage.At the late stage,hypertension could cause excessive damage to vital organs and induce the multiple complications.Furthermore,the hypertension even lead the people to die following the disease worsens.Stress is one of the dangerous factors,along with the modern life rhythm speeding up,social and psychological stress aggravated,the incidence rate and mortality rate of stress-induced hypertension(SIH)occupies a large proportion in the high blood pressure.Stress can activate a series of pathological molecular and cellular alterations,leading to activation of sympathetic neurons.Interestingly,several lines of experimental evidence have indicated a key role for the sympathetic nervous system in stress-mediated cardiovascular disease.The rostral ventrolateral medulla(RVLM)is integral for vasomotion,and controls arterial pressure and activation of the basal sympathetic nerve.Presympathetic neurons in the RVLM are responsible for generating sympathetic drive to the cardiovascular system,eventually regulating both cardiac output and vascular resistance.Nav1.6,a voltage-gated sodium channels(VGSCs)subtype located within the axonal initial segment and nodes of Ranvier,plays a key role of generation and propagation of neuronal action potential.There is no lack of central regulation research on hypertension,but the research of the excessive activation of Nav1.6 on SIH in the RVLM was poorly reported.Therefore,our present studies aim to reveal the regulative mechanism of SIH by activation of sympathetic neurons,which result from over-activation Nav1.6 in neurons in RVLM.Objective: 1)The changes of Nav1.6 expression in RVLM in stress-induced hypertension rats;2)Nav1.6 is mainly expressed on neuronal cells in RVLM in stress-induced hypertension rats;3)The relationship between the Nav1.6 expression and the level of cardiovascular index;4)The level of cardiovascular index including SBP,HR and RSNA is alleviated after silencing expression of Nav1.6 in RVLM in stress-induced hypertension rats.Methods: Adult Sprague-Dawley rats were used in all experiments.Rats in SIH group were treated with plantar electric shock plus noisy stimulation for 15 days to prepare stable stress hypertensive rats.All rats were randomly divided into four groups: normotensive(control),stress,stress with GFP(Lv-CRISPR-eGFP)and stress with gRNA(Lv-CRISPR-eGFP-Scn8a-gRNA).Systolic blood pressure(SBP),heart rate(HR)and weight were monitored under conscious and anesthetic conditions.Renal sympathetic nerve activity(RSNA)was recorded in vivo under anesthesia.Western blot and immunofluorescence assays were used to monitor the expression of Nav1.6.Results: 1)Compared with the control group,the SIHR group exhibited increased anxiety in performance tests and significantly increased SBP and HR,but the animals in the SIHR group weighed less.On the tenth day,SBP exhibited its peak increase after stress.The weight of rats in the SIHR group was increased after the 12 th day of stress compared with the control group.Level of RSNA began to be significantly increased 10 days of daily stress induction.2)Expression of Nav1.6 protein was significantly higher in the SIHR group on the tenth and fifteenth days compared with the control group.Numbers of Nav1.6-immunoreactive(ir)cells in the RVLM of the SIHR group on the tenth day of stress was significantly higher than observed for control group rats.3)The expression of Nav1.6-positive neurons in the RVLM was significantly increased compared with the control group.Activation of microglia and astrocytes was not observed in the RVLM at the tenth day.4)In the RVLM,increased co-localization of Nav1.6 with NeuN and increased protein expression of Nav1.6 both positively correlated with increased days of stress and changes in SPB.5)The level of cardiovascular index including SBP,HR and RSNA is alleviated after silencing expression of Nav1.6 in RVLM in stress-induced hypertension rats.Conclusion: The present study shows that Nav1.6 expression at protein level was significantly increased in RVLM neurons after stress induction compared to the control group,a dynamic,prominent increase of Nav1.6 immunoreactivity in neurons was observed in RVLM of SIHR.Furthermore,the upregulation of Nav1.6 expression in RVLM neurons was strongly correlated with the SBP after stress induction.Furthermore,compared with the control group,the level of SBP and RSNA was decreased by knocking down the Nav1.6 in RVLM in SIHR group.These findings suggest that Nav1.6 expression in RVLM neurons may contribute to the development of SIH.Further investigation will be performed to clarify the role of Nav1.6 expression in different types of neurons in RVLM in response of SIH.