The Effect And Mechanism Of AT1R/ROS Signaling Pathway In Rostral Ventrolateral Medulla On Sympathetic Activity After Traumatic Brain Injury And Its Mechanism

Traumatic brain injury(TBI)is the leading cause of death for all trauma patients during peacetime and wartime,accounting for 1/3 of the deaths due to trauma.Among them,more than half of patients with severe TBI have a poor prognosis,and the incidence of sympathetic overexcitation can reach 30%.Overexcitation of the sympathetic nerve after TBI can lead to intracranial hypertension,damage to the blood brain barrier,and secondary dysfunction of multiple organs and systems such as the heart,lungs,and immune system,which is one of the main reasons for high mortality and disability in TBI patients.However,the pathophysiological basis and molecular biological mechanism of sympathetic excitation after TBI are still unclear,and further research is urgently needed.The previous research by the research team found that the increased level of oxidative stress response in the rostral ventrolateral medulla(RVLM)after TBI is an important pathophysiological basis for sympathetic excitation after TBI.An important product of oxidative stress response is reactive oxygen species(ROS),but the specific molecular biological mechanism of the increased ROS level in RVLM after TBI is unclear.Studies have shown that the activation of the central renin angiotensin system(RAS)is one of the important reasons for inducing sympathetic excitation under various pathological conditions,and an important pathway for its activation is the activation of angiotensin type 1 receptor(AT1R),which increases the level of intracellular oxidative stress reactions.Therefore,activation of AT1R in RVLM may induce oxidative stress responses that induce ROS production,thereby affecting sympathetic nerve activation after TBI.The mechanism by which ATIR activation enhances sympathetic activity in hypertension,heart failure,and other disease models has been reported to involve NADPH oxidase(NOX).NADPH oxidase is a multi subunit oxidoreductase that can transfer electrons from intracellular NADPH to oxygen molecules,generating ROS.Therefore,we speculate that the NOx system in the RVLM region may play a key role in AT1R induced ROS production after TBI.Based on the above research background,this topic proposes the following hypothesis:After traumatic brain injury,AT1R is activated in the rostral ventrolateral medulla,which induces continuous ROS production through the NOX system,ultimately leading to peripheral sympathetic nerve excitation.Part I Changes of sympathetic activity with time and AT1R/ROS signal pathway in rostral ventrolateral medulla after craniocerebral traumaObjective:We used the diffuse axonal injury model as an animal model to study the sympathetic activity after traumatic brain injury,to clarify the change rule of sympathetic activity with time after traumatic brain injury,and to detect the expression level of related indicators of AT1R/ROS signal pathway in RVLM after diffuse axonal injury in rats,and preliminarily determine the effect of AT1R/ROS signal pathway in RVLM on sympathetic activity after traumatic brain injury.Methods:240 Sprague Dawley(SD)rats were randomly selected and randomly divided into experimental group(132 rats)and control group(108 rats).The experimental group made a diffuse axonal injury model(DAI),while the control group only put the animals on the model machine,and did not attack them.108 model rats and 108 control rats that survived after modeling were randomly divided into 1 day group(1D),2 days group(2D),3 days group(3D),5 days group(5D),7 days group(7D),14 days group(14D),21 days group(21D),28 days group(28D)and 35 days group(35D)according to the time point.After modeling,the mean arterial pressure and heart rate variability of rats in each group were monitored according to the time point,and blood and brain tissue samples were collected.The damage of brain tissue in rats was observed by HE staining,silver glycine immersion nerve staining and immunohistochemistry.The femoral artery intubation method was used to monitor the average arterial pressure of rats,the heart rate variability analysis system was used to monitor the heart rate variability of rats,the content of norepinephrine in rat plasma was measured by enzyme-linked immunosorbent assay(ELISA),the expression level of AT1R in rat RVLM was measured by the methods of western-blot,fluorescence quantitative PCR and immunofluorescence,and the NOX2 in rat RVLM was measured by western-blot and fluorescence quantitative PCR The expression levels of NOX4 and superoxide dismutase(SOD)were detected by using the method of ROS staining in frozen sections.Result:1.The survival rate of rats in the experimental group after modeling was 84.09%.HE staining,glycine silver immersion nerve staining β-APP immunohistochemical showed that there was diffuse axonal injury in the brain tissue of rats in the experimental group.2.Compared with the control group,the plasma norepinephrine content(11.34±0.44 vs 24.46±1.20,P<0.05),the mean arterial pressure(105.2±3.56 vs 131.4+5.32,P<0.05)and the heart rate variability(3.86±0.50 vs 2.01±0.74,P<0.05)of the rats in the experimental group on the third day after the injury reached the maximum difference.3.There was co-localization of PMNT(location C1 neurons)and AT1R in the RVLM of rats in the experimental group,and the protein expression and mRNA content of AT1R was significantly higher than that in the control group(P<0.05).4.Compared with the control group,the protein expression and mRNA content of NOX2 and NOX4 in RVLM of rats in the experimental group were significantly higher(P<0.05).5.Compared with the control group,the protein expression and mRNA content of SOD in RVLM of rats in the experimental group was significantly lower(P<0.05),and the expression level of ROS was significantly higher(P<0.05).Conclusion:1.The model of diffuse axonal injury was successfully made,which is simple to operate and has a high success rate.2.After craniocerebral injury,the sympathetic activity of rats increased and reached a peak on the 3rd day after injury,followed by a decrease in sympathetic activity.3.AT1R expression exists in the presympathetic neurons of rat RVLM,and the expression of AT1R and NOX systems in rat RVLM is significantly increased after craniocerebral trauma,and the level of oxidative stress response is significantly increased.4.The increased expression of AT1R and NOX systems in RVLM of rats after craniocerebral injury may be related to the increase of oxidative stress reaction after craniocerebral injury,which may cause the increase of sympathetic activity.Part Ⅱ AT1R increases the level of oxidative stress in PC12 cells by regulating the NOX systemObjective:We use highly differentiated PC12 cells as a cell model,excluding other interfering factors in vivo,to clarify that highly differentiated PC 12 cells stimulated by Ang ⅡI can be used to simulate the state after craniocerebral trauma,and that AT1R can affect the level of oxidative stress in PC 12 cells through the NOX system.Methods:Using highly differentiated PC12 cells as an experimental neuronal cell model in vitro,the highly differentiated PC 12 cells were stimulated with Ang Ⅱ.The expression levels of AT1R,NOX2,NOX4,and SOD were measured using Western Blot and fluorescence quantitative PCR methods.The expression levels of ROS were measured using reactive oxygen species fluorescence probes and flow cytometry.The expression of AT1R in PC 12 cells was reduced by lentivirus transfection.After 24 hours of Ang Ⅱ stimulation,the expression levels of NOX2,NOX4,and SOD were detected using Western Blot and fluorescence quantitative PCR,and the expression levels of ROS were detected using reactive oxygen species fluorescence probes and flow cytometry.After 24 hours of Ang Ⅱ stimulation by inhibiting the NOX system in PC 12 cells,the expression level of SOD was detected using Western Blot and fluorescence quantitative PCR,and the expression level of ROS was detected using reactive oxygen species fluorescence probes and flow cytometry.Result:1.Compared with the control group,the protein expression and mRNA content of AT1R,NOX4 and NOX2 in PC 12 cells of the experimental group were significantly increased(P<0.05),the protein expression and mRNA content of SOD were significantly decreased(P<0.05),and the expression level of ROS was significantly increased(P<0.05)after 24 hours of AngⅡ stimulation.2.Compared with the control group,the expression of AT1R in PC12 cells was reduced.After 24 hours of AngⅡ stimulation of PC 12 cells,the protein expression and mRNA content of NOX2 and NOX4 in PC12 cells in the experimental group were significantly decreased(P<0.05),the protein expression and mRNA content of SOD were significantly increased(P<0.05),and the expression level of ROS was significantly decreased(P<0.05).3.Compared with the control group,inhibiting the NOX system in PC12 cells,the protein expression and mRNA content of SOD were significantly increased(P<0.05),and the expression level of ROS was significantly decreased(P<0.05).Conclusion:1.The highly differentiated PC 12 cells stimulated by Ang Ⅱ can increase the expression of AT1R and NOX systems,improve the level of oxidative stress response,and can be used to simulate the state after craniocerebral trauma.2.The expression of NOX system and the level of oxidative stress response in PPC12 cells after Ang Ⅱ stimulation depend on the expression of AT1R.3.The regulation of AT1R on oxidative stress response in PC12 cells after Ang Ⅱstimulation depends on the expression of NOX system.Part Ⅲ AT1R/ROS signal pathway and its effect on sympathetic activity in the ventrolateral medulla oblongata region after experiencing traumatic brain injuryObjective:We used the diffuse axonal injury model as an animal model to study the sympathetic activity after traumatic brain injury,and explored the effect of AT1R/ROS signal pathway and sympathetic activity in RVLM after traumatic brain injury.Methods:①To investigate the effect of AT1R expression in RVLM on sympathetic activity after craniocerebral trauma,adeno-associated virus was injected into RVLM to interfere with AT1R expression,and then animal models were established.The surviving rats were randomly divided into DAI-AAV-EGFP group and DAI-AAV-shRNA group,with 12 rats in each group.Twelve SD rats were randomly selected as a control group(SHAM).Three days after modeling,the mean arterial pressure and heart rate variability of the rats in each group were monitored,and blood and brain tissue samples were collected.The femoral artery intubation method was used to monitor the average arterial pressure of rats,the heart rate variability analysis system was used to monitor the heart rate variability of rats,the enzyme linked immunosorbent assay was used to detect the content of norepinephrine in rat plasma,Western Blot and fluorescence quantitative PCR were used to detect the expression level of NOX2,NOX4,and SOD in rat RVLM,and the expression level of ROS in rat RVLM was detected using frozen section ROS staining method.②In order to investigate the effect of the expression of NOX system in RVLM on the sympathetic activity of rats after injury,SD rats were randomly divided into an experimental group(DAI group)of 30 rats and a sham operation group(sham group)of 12 rats.The experimental group established a DAI model.Twenty four surviving rats were randomly selected and divided into a DAI+APO(inhibitor of the NOX system)group and a DAI+ACSF(artificial cerebrospinal fluid)group,with 12 rats in each group.Three days after modeling,the average arterial pressure and heart rate variability of the rats in each group were monitored,Blood and brain tissue samples were collected.The femoral artery intubation method was used to monitor the average arterial pressure of rats,the heart rate variability analysis system was used to monitor the heart rate variability of rats,the enzyme linked immunosorbent assay was used to detect the content of norepinephrine in rat plasma,Western blot and fluorescence quantitative PCR were used to detect the expression level of SOD in rat RVLM,and the expression level of ROS in rat RVLM was detected using frozen section ROS staining method.Result:1.Compared with the DAI-AAV-EGFP group,the protein expression and mRNA content of NOX2 and NOX4 in the RVLM region of the DAI-AAV-shRNA group significantly decreased(P<0.05),the protein expression and mRNA content of SOD significantly increased(P<0.05),the expression level of ROS significantly decreased(P<0.05),the mean arterial pressure,the content of norepinephrine significantly decreased(P<0.05),and the heart rate variability(SDNN Index)significantly increased(P<0.05).2.Compared with the DAI+ACSF group,the protein expression and mRNA content of SOD in the RVLM region of the DAI+APO group were significantly increased(P<0.05),the expression level of ROS was significantly decreased(P<0.05),the mean arterial pressure and the content of norepinephrine were significantly decreased(P<0.05),and the heart rate variability(SDNN index)was significantly increased(P<0.05).Conclusion:1.The increase of NOX system,oxidative stress response level and sympathetic activity in RVLM of rats after craniocerebral trauma depends on the expression of AT1R in RVLM.2.The regulation of AT1R in RVLM of rats after traumatic brain injury on oxidative stress response and sympathetic activity depends on the expression of NOX system.3.The increased expression of AT1R in RVLM of rats after traumatic brain injury the increased level of oxidative stress response by regulating the NOx system may be an important pathophysiological and molecular mechanism for the increased sympathetic activity after traumatic brain injury.