Pathophysiological Mechanism Of The Effect Of Panic Attacks On Vascular Endothelial Function In Rats

Objective:Panic disorder（PD）is a sub-type of anxiety disorder,and it is also a chronic and disabling mental disorder with high prevalence.Panic attack（PA）is the core symptom of panic disorder,manifested as a sudden outbreak of intense fear or tension,accompanied by a series of autonomic nervous symptoms such as sweating,chest tightness,dyspnea,tachycardia,arrhythmia,headache,and a strong sense of near-death and loss of control,and these symptoms are similar to those of angina.Therefore,most PD patients often go to the cardiovascular department repeatedly and the relationship between PD and cardiovascular disease has been attracted much attention.Studies have shown that PD can increase the prevalence of cardiovascular diseases（such as coronary heart disease,arrhythmia and hypertension）,but the relevant mechanisms are still unclear.Vascular endothelial function plays an important role in the pathogenesis of cardiovascular diseases such as atherosclerosis and hypertension.Vascular endothelial function includes regulating vasodilation and contraction,secreting vasoactive substances,controlling angiogenesis and other physiological functions,among which vascular endothelium-dependent vasodilation function is an important index to measure vascular endothelial function.The decrease of endothelium-dependent vasodilation will lead to vascular stenosis and induce series of processes such as platelet aggregation and monocyte adhesion,which will lead to atherosclerosis and increase the risk of cardiovascular disease.At present,studies have found that psychosocial stress,acute mental stress and anxiety in patients with panic disorder are related to decreased vascular endothelial function.However,it is unclear that whether panic attacks will lead to vascular endothelial dysfunction and also increasing risk of cardiovasular disease within PD patients.Fluoxetine,a representative drug of serotonin reuptake inhibitor,is the first-line drug for clinical treatment of PD,which can effectively control panic attacks.Whether fluoxetine has a protective effect on vascular endothelial function is still unclear.There are studies that report sympathetic excitation during panic attacks.Sympathetic excitation promotes vasoconstriction,an increase in inflammatory factors（such as TNF-α,IL-6,hs-CRP）,and oxidative stress（such as reactive oxygen species）.These substances can activate the RhoA/ROCK signaling pathway,which will lead to decreased expression and activity of nitric oxide synthase（e NOS）,which will result in the reduction of the production or bioavailability of nitric oxide（NO）,and the increasing production of contractile factors（such as endothelin-1）,ultimately leading to vascular endothelial dysfunction.Whether panic attacks affect vascular endothelial function through this pathophysiological mechanism is also unclear.Therefore,this study intends to use animal models to observe the effects of panic attacks on vascular endothelium-dependent relaxation function,observe whether fluoxetine has a protective effect on vascular endothelial function,and explore whether panic attacks affect vascular endothelial function by activating RhoA/ROCK signaling pathway.It provides a theoretical basis for the pathophysiological mechanism of the effect of panic attacks on vascular endothelial function and the protective effect of fluoxetine on vascular endothelial function.Methods:Animal model preparation:Animal models were prepared based on previous reports in the studies,a commonly used and well-theorized method of escape behavior induced by electrical stimulation of dorsolateral periaqueductal gray（d PAG）in rats.Previous literature has shown that the physiological and behavioral changes shown in this model rats meet symptomatic homology with human panic disorder,and anti-panic drugs and panic promoting drugs meet the the pharmacological homology in this model,so electrical stimulation d PAG of rat can be used to simulate panic attacks.The specific methods are as follows:After anesthesia,the rats were placed on a stereo fixator,and electrodes were implanted into the d PAG of the rats（lateral to lambda 1.9mm,5.2mm below the skull surface）according to the coordinates of Paxinos atlas and Watson atlas.After 5-7 days of recovery,d PAG was stimulated by electric stimulation with the lowest current intensity（less than 200μA）that caused running or jumping behavior of rats.After the experiment,brain tissue was collected and frozen to verify the electrode position with Nissl stain,and only the rats with the electrode tip present at the d PAG site were statistically analyzed.Experiment 1:Effect of panic attacks on endothelium-dependent vasodilation in ratsBased on the duration of panic attack（5-20min）in the population,27 rats were randomly divided into 3 groups with 9 rats in each group.The experiment started after 5-7 days of postoperative recovery:（1）Electric stimulation d PAG 5min group（ES5）:electric stimulation d PAG will be excuited 10 times,in which 30s sine-wave pulse and 30s interval are included.That means the total duration of electric stimulation d PAG was 5min.（2）Electric stimulation d PAG 10min group（ES10）:electric stimulation d PAG will be excuited 20 times,in which 30s sine-wave pulse and 30s interval are included.That means the total duration of electric stimulation d PAG was 10min.（3）Electric stimulation d PAG 15min group（ES15）:electric stimulation d PAG will be excuited 30 times,in which 30s sine-wave pulse and 30s interval are included.That means the total duration of electric stimulation d PAG was 15min.The thoracic aorta was taken immediately after the end of electrical stimulation.Acetylcholine（Ach）was used to induce vascular endothalium-dependent relaxation response,and the endothalium-dependent relaxation function of the thoracic aorta was detected by vascular tonometer.Experiment 2:Changes of endothelium-dependent vasodilation in rats after fluoxetine treatmentThirty rats were randomly divided into 3 groups with 10 rats in each group.（1）Fluoxetine+electrical stimulation group（FLX+ES15）:Basal escape threshold was measured 5-7 days later after electrode implantation recovery and fluoxetine was continuously injected intraperitoneally with 5mg/kg（≈55.5mg calculated by human body weight 70kg）for 21 days.After intraperitoneal injection of fluoxetine for 30min on the21st day,the escape threshold of rats was measured again,and the d PAG of rats was electrically stimulated with the basic escape threshold for 15min（the shortest electrical stimulation time that caused vascular endothelial injury in the experiment 1）.（2）Electric stimulation group（ES15）:intraperitoneally injected physiological saline with equal volume of fluoxetine and the remaining treatment were the same as the FLX+ES15 group.（3）SHAM operation group（SHAM）:all same as ES15 group,except no current given.The thoracic aorta was taken immediately after the end of electrical stimulation.Acetylcholine（Ach）was used to induce vascular endothalium-dependent relaxation response,and the endothalium-dependent relaxation function of the thoracic aorta was detected by vascular tonometer.Experiment 3:To investigate the pathophysiological mechanism of vascular endothelial dysfunction induced by panic attacks in rats45 rats were randomly divided into 5 groups with 9 rats in each group.After 5-7 days of postoperative recovery,the d PAG of rats was electrically stimulated with the escape threshold for 15min（the shortest electrical stimulation time that caused vascular endothelial injury in the experiment 1）:（1）Propranolol group（Pro+ES15）:10mg/kg propranolol（βblocker）was intraperitoneally injected 30min before electrical stimulation,and physiological saline equivalent to fasudil（ROCK inhibitor）was intraperitoneally injected 10min before electrical stimulation.（2）Fasudil group（Fa+ES15）:intraperitoneally injected with propranolol equivalent physiological saline 30min before electrical stimulation,and intraperitoneally injected with fasudil 10mg/kg 10min before electrical stimulation.（3）Electric stimulation group（ES15）:injected with equal volume physiological saline at 30min and 10min before electrical stimulation and the remaining treatments were same as Pro+ES15 and Fa+ES15.（4）SHAM operation group（SHAM）:in addition to no current given,the same as ES 15 group.（5）Control group:No intervention.Blood,the thoracic aorta and brain tissues were collected immediately after the experiment:（1）Determination of sympathetic nerve excitability:The changes of blood pressure and heart rate before and after electrical stimulation were measured,and the serum noradrenalin（NA）concentration of rats after electrical stimulation was detected by ELISA.（2）The levels of inflammatory factors:serum hs-CRP,IL-6 and TNF-αwere detected by ELISA.（3）Determination of oxidative stress level:Serum malondialdehyde（MDA）concentration was determined by thiobarbituric acid（TBA）method and serum superoxide dismutase（SOD）activity was determined by hydroxylamine method and the expression of reactive oxygen species（ROS）in thoracic aorta were detected by immunofluorescence assay.（4）Determination of RhoA/ROCK signaling pathway activation state:The proteins expression of RhoA,ROCK,MYPT and p-MYPT in the thoracic aorta of rats was detected by Western blot.The m RNA expression of RhoA and ROCK in thoracic aorta was detected by PCR.（5）Expression of diastolic and contractile factors related to vascular endothelial function:The proteins expressions of e NOS and p-e NOS in the thoracic aorta of rat were detected by Western blot.The m RNA expression of e NOS in thoracic aorta of rat was detected by PCR.The expression of endothelia-1（ET-1）in the thoracic aorta of rats has been detected by immunohistochemical method.（6）Determination of endothelium-dependent vasodilation function:The endothelium-dependent relaxation function of thoracic aorta was measured by vascular tonometer.Results:Experiment 1:Effect of panic attacks on endothelium-dependent vasodilation in ratsAt Ach10^-7,10^-6and 10^-5mol/l,the thoracic aortic diastole of the ES15 group decreased to（25.52±6.46）%,（51.31±8.21）%and（72.66±3.69）%,respectively.Compared with ES5 group and ES10 group,the diastolic degree of thoracic aorta in ES15 group was significantly lower（P<0.05）.Experiment 2:Changes of endothelium-dependent vasodilation in rats after fluoxetine treatmentThe escape threshold measured 21 days after fluoxetine 5mg/kg injection was 10-20μA higher than the baseline escape threshold measured before administration.It is suggested that fluoxetine plays an anti-panic attack role.At Ach10^-6,10^-5mol/l,the diastolic degree of thoracic aorta in the FLX+ES15 group increased to（79.08±11.11）%and（96.57±5.03）%.Compared with the ES15 group,the diastolic degree of thoracic aorta in the FLX+ES15 group was significantly higher（P<0.05）.Experiment 3:To investigate the pathophysiological mechanism of vascular endothelial dysfunction induced by panic attacks in rats1.Effects of panic attacks on blood pressure,heart rate and serum noradrenalin in rats（1）Compared with the blood pressure before electrical stimulation,the blood pressure of rats in the ES15 group increased after electrical stimulation（135.20±9.52 vs120.20±10.11mm Hg,P<0.05）,the difference was statistically significant;but there was no significant change in blood pressure measured in the Pro+ES15 group and in the Fa+ES15 group.（2）Compared with the heart rate before electrical stimulation,the heart rate of rats in the ES15 group and in the Fa+ES15 group increased after electrical stimulation（468.70±53.01 vs 411.70±49.38bpm,P<0.05;408.00±50.48 vs 362.50±35.01bpm,P<0.05）.After the application of propranolol,the heart rate of rats in the Pro+ES15group decreased after the electrical stimulation of d PAG（343.80±46.01 vs 383.50±42.66bpm,P<0.05）,the difference was statistically significant.（3）Compared with the Control group and the SHAM group,the serum noradrenaline level in the ES15 group increased（69.01±5.69 vs 57.23±2.87ng/L,P<0.05;69.01±5.69 vs 60.83±3.88ng/L,P<0.05）;Compared with the ES15 group,the serum noradrenaline level in the Pro+ES15 group decreased（59.26±4.96 vs 69.01±5.69ng/L,P<0.05）with statistical difference;There was no significant change in serum noradrenaline in the Fa+ES15 group compared with the ES15 group.2.Changes of systemic inflammatory factors in rats with panic attacksCompared with SHAM group,serum hs-CRP level in the ES15 group increased（3.24±0.33 vs 2.51±0.23ng/L,P<0.05）,with statistical difference.Compared with the ES15 group,the serum IL-6 level in the Pro+ES15 group and Fa+ES15 group decreased（69.16±4.26 vs 80.49±4.63μg/L,P<0.05;62.66±6.59 vs80.49±4.63μg/L,P<0.05）,and the difference was statistically significant.There was no significant difference in TNF-αamong all groups.3.Changes of systemic and vascular oxidative stress levels in rats with panic attacksCompared with Control group and SHAM group,ROS fluorescence intensity in the thoracic aorta and serum MDA concentration of rats in the ES15 group significantly increased,while serum SOD activity significantly decreased,with statistical difference（P<0.05）.Compared with the ES15 group,ROS expression was down-regulated,serum MDA concentration decreased and serum SOD activity increased in the Pro+ES15 group and Fa+ES15 group,with statistical difference（P<0.05）.4.Effect of panic attacks on RhoA/ROCK signaling pathway in the thoracic aorta of ratsWestern blot showed that:compared with the Control group and the SHAM group,the proteins expression of RhoA,ROCK,MYPT and p-MYPT in thoracic aorta of rats in the ES15 group increased,and the differences were statistically significant（P<0.05）.PCR showed that compared with the Control group and the SHAM group,m RNA expression of RhoA and ROCK in thoracic aorta of rats in ES15 group increased,and the difference was statistically significant（P<0.05）.Compared with the ES15 group,the protein expression of RhoA,ROCK,MYPT,p-MYPT and m RNA expression of RhoA and ROCK in thoracic aorta of rats in the Pro+ES15 group and Fa+ES15 group decreased,and the differences were statistically significant（P<0.05）.5.Effects of propranolol and fasudil on vascular endothelial function related active factors e NOS and ET-1 in ratsWestern blot showed that:compared with Control group and the SHAM group,the protein expression of e NOS and p-e NOS in thoracic aorta of rats in ES15 group decreased,and the differences were statistically significant（P<0.05）.PCR showed that:compared with the Control group and the SHAM group,the expression of e NOS m RNA in thoracic aorta of rats in ES15 group decreased,and the difference was statistically significant（P<0.05）.Immunohistochemical showed that the expression of ET-1 in thoracic aorta of rats in the ES15 group was higher than that in the Control group and the SHAM group,and the difference was statistically significant（P<0.05）.Compared with the ES15 group,the expression of e NOS,p-e NOS protein and e NOS m RNA in the thoracic aorta of rats in the Pro+ES15 group and Fa+ES15 group increased,while the expression of ET-1 in the thoracic aorta of rats in the Fa+ES15 group decreased,and the differences were statistically significant（P<0.05）.6.Effect of propranolol and fasudil on endothelium-dependent vasodilation in ratsAt Ach 10^-5mol/l,the diastolic degree of thoracic aorta decreased to（72.66±3.69）%in the ES15 group.Compared with the Control group and the SHAM group,the diastolic degree of thoracic aorta in the ES15 group was significantly reduced（P<0.05）.At Ach10^-6and 10^-5mol/l,the diastolic degree of thoracic aorta in the Pro+ES15group increased to（86.67±12.85）%and（94.39±6.12）%.The diastolic degree of thoracic aorta increased to（85.83±13.28）%and（93.08±7.80）%in the Fa+ES15 group.Compared with the ES15 group,the diastolic degree of thoracic aorta in Pro+ES15 group and Fa+ES15 group was significantly increased（P<0.05）.Conclusion:1.Panic attacks induced by electrical stimulation of d PAG can lead to endothelium dependent diastolic dysfunction in rats.2.Fluoxetine can improve endothelium-dependent diastolic dysfunction in rats by increasing the escape threshold of rats with panic attacks.3.Panic attacks induced by electrical stimulation of d PAG can increase the release of inflammatory factors and the level of oxidative stress in rats through sympathetic nerve excitation,and then activate RhoA/ROCK signaling pathway,leading to the imbalance of expression of e NOS and ET-1 in vascular endothelium and vascular endothelium dependent diastolic dysfunction in rats.