Stress-based Effect And Mechanism Of Microwave Radiation-induced Cardiac Injury In Rats

Objective:Microwave radiation of certain intensity could cause damage to multiple systems and organs of the living organism.Currently,it has been divided into the thermal and non-thermal effect injuries,while in the study of electromagnetic injury mechanism was mostly based on the non-thermal effect,and the thermal effect study was rare,especially the injury study based on cardiovascular system has been not reported.Therefore,this study intended to start from the cardiovascular system,which was the most abundant in electrical signals of living organisms,and explored the injury mechanism innovatively based on the clarification of electromagnetic radiation-induced injury effect.Studies have shown that the heart was one of the sensitive target organs for microwave radiation,that the electrical activity of the heart was directly related to the sympathetic-adrenal medulla and parasympathetic nerves,and that the hormone of the endocrine system were important factors in triggering cardiac diseases.The neuroendocrine disorders could not only lead to cardiac injury,but also were the physiological and material basis for the occurrence of stress,which suggested that cardiac injury and stress occurrence were closely related.There was a lack of research in the field of electromagnetic bioeffects to track the mechanisms of stress-injury effects in depth,and there was no relevant study to evaluate stress-injury using the heart as an entry point.Therefore,in this study,we proposed to use a single and long-term S-wave radiation in rats and classified the detection indexes according to physiological-stress and psychological-stress,and evaluated physiological-stress damage by indexes that could reflect the function and structure of the hear,and used body weight and the Open Field Experiment as the gold standard to verify the successful establishment of psychological-stress model,in order to clarify the successful construction of stress-model at the physiological and psychological levels.Further,in terms of mechanistic studies,it was proposed to examine the changes of Hsp72,an important marker of heat-stress injury,and to examine its downstream key molecules in the JNK pathway in post-irradiation cardiac injury,aiming to broaden the direction for the study of the effect mechanism of radiation-induced cardiac injury.Methods:1.Animal model establishment:In this study,50 Wistar male rats（200±20）g were randomly divided into control group（C）and radiation group（R）,with R group receiving microwave radiation at 2.856 GHz,30 m W/cm²,for 35 min,and C group receiving the same treatment except not receiving radiation.The anal temperature of six rats from each of C group and R group were measured with a digital thermometer before and immediately after radiation.2.Evaluation of physiological-stress injury in rats’heart:on the 1^st d,7^th d,14^th d and 28^th d after radiation,the structure of myocardial tissue was observed by Optical Microscope and Transmission Electron Microscope;peripheral serum was measured by Automatic Biochemical Analyzer,the activity of myocardial enzyme（AST,LDH,CK,CK-MB and HBDH）,ion(K⁺,Na⁺,Ca²⁺and Cl^-)content were calculated;the myocardial injury marker（c-Tn T,H-FABP and NT-pro BNP）was analyzed by Elisa;mitochondrial function（m PTP and MMP）was measured by Flow Cytometry;the marker of oxidative stress（MDA content and SOD activity）was measured by Microplate Reader;the changes in heart rate,P-wave,T-wave and R-wave were observed by Multi-channel Physiological Recorder;and the hormone in serum（ANP and ET）was measured by Radioimmunoassay.3.Validation of a psychological-stress model in rats:stress-hormone in serum（CRH,ACTH,GC,COR,NE and DA）was measured by Radioimmunoassay on the 1^st d,7^th d,14^th d and 28^th d after radiation;HRV（Mean HR,Mean RR,HRV tri index,RMSSD,SDNN,LF-ms²,HF-ms²,LF-nu,LF/HF radio and LF-nu）was analyzed by Multi-channel Physiological Recorder;rats were weighed on the 14^th d,7^th d,and 1^st d before radiation,1^st d,3^th d,5^th d,7^th d,14^th d,21^th d,and 28^th d after radiation;the Open Field Experiment was performed on the 6^th h,1^st d,7^th d,and 14^th d after radiation（total distance traveled,average speed,active time,number of trips into the central zone,time spent in the central zone,distance traveled in the central zone,number and time spent upright,and number and time spent grooming）.4.Mechanistic study of radiation-induced heat stress injury:the expression of Hsp72in rats’myocardial tissue was detected by Western blot on the 1^st d,7^th d,14^th d and 28^thd after radiation;the distribution of Hsp72 in rats’myocardial tissue was analyzed by Immunofluorescence on the 1^st d and 7^th d after radiation;the expression of JNK,p-JNK,HSF1,NFATc4 in rats’myocardial tissue was detected by Western blot on the 1^st d and7^th d after radiation;the distribution of JNK,p-JNK,HSF1,and NFATc4 in rats’myocardial tissue was analyzed by Immunofluorescence on the 1^st d after radiation.Results:1.Physiological-stress injury in rats after radiation was manifested by increased anal temperature,structural damage to myocardium,decrease of ion concentrations in serum,disturbance in activity of myocardial enzyme,increased myocardial injury-marker levels in serum,increase of ANP and ET concentration in serum,m PTP and MMP damage in myocardial tissue,increased MDA content and decreased SOD activity in myocardial tissue,and abnormal ECG.（1）An immediate increase in anal temperature>2.3°C was seen（P<0.01）.（2）Structural and ultrastructural damage in cardiac:on the 1^st d after radiation,myocardial fibers were disorganized and nuclei were deeply stained under Optical Microscope,myocardial fibers were faintly lysed,Z-line was unclear,mitochondria was cavitated and perinuclear gap was widened under Transmission Electron Microscope;the damage was aggravated on the 7^th d,nuclei were deeply stained under Optical Microscope,and myocardial fibers were segmentally lysed under Transmission Electron Microscope;the above damage was recovered on the 28^th d.（3）Decrease of ion concentration in serum:K⁺,Na⁺,Cl^-and Ca²⁺concentration were significantly decreased on the 1^st d after radiation（P<0.05 or 0.01）,Cl^-concentration was significantly decreased（P<0.05）and the Ca²⁺concentration was significantly increased on the 7^th d and 14^th d（P<0.05 or 0.01）,and the recovery of the above indicators was seen on the 28^th d（P>0.05）.（4）Disturbance in activity of myocardial enzyme in serum:the activity of AST,CK and LDH were increased on the 1^st d after radiation（P<0.05 or 0.01）,the activity of CK-MB andα-HBDH were decreased on the 7^th d（P<0.01）,and each of these indicators were recovered on the 14^th d（P>0.05）.（5）Elevated concentration of myocardial injury-markers in serum:concentration of c-Tn T was significantly increased on the 1^st d,7^th d,14^th d and 28^th d after radiation（P<0.01）,concentration of NT-pro BNP was significantly increased on the 1^st d,7^th d and14^th d（P<0.05 or 0.01）,concentration of H-FABP was significantly increased on the 7^thd（P<0.05）,and the changes of above indicators were not fully recovered on the 28^th d（P<0.05）.（6）Abnormal endocrine-function in the cardiac:concentration of ANP was significantly increased on the 1^st d,7^th d,14^th d and 28^th d after radiation（P<0.05 or0.01）,and concentration of ET was significantly increased on the 14^th d and 28^th d（P<0.05 or 0.01）,and each of these indicators were not fully recovered on the 28^th d（P<0.05）.（7）Damage to mitochondrial function in myocardial tissue:m PTP was significantly decreased on the 1^st d,7^th d,14^th d and 28^th d after radiation（P<0.05 or0.01）,MMP was impaired on the 7^th d and 14^th d（P<0.05 or 0.01）,and each of the above indicators were still not fully recovered on the 28^th d（P<0.05）.（8）Oxidative-stress damage in myocardial tissue:MDA content was significantly increased on the 7^th d and 14^th d after radiation（P<0.05）,SOD activity was significantly decreased on the 1^st d,7^th d and 14^th d（P<0.05）,and all of the above indicators were recovered from damage on the 28^th d（P>0.05）.（9）Dysfunction of ECG:heart rate was significantly increased on the 1^st d,7^th d and 14^th d after radiation（P<0.05 or 0.01）,P-wave amplitude was significantly increased on the 1^th d and 7^th d（P<0.05 or 0.01）,P-wave duration was significantly decreased on the 1^th d and 7^th d（P<0.05）,R-wave amplitude was significantly increased on the 7^th d（P<0.05）,T-wave amplitude was significantly decreased on the 1^th d and 7^th d（P<0.05or 0.01）,and the damage had completely recovered on the 28^th d（P>0.05）.2.Psychological-stress damage in rats after radiation were manifested by elevated stress-hormone level in serum,abnormal heart rate variability,slow weight-gain,and changes in the indices of the Open Field Experiment.（1）Elevated stress-hormone in rats’serum:CRH,COR,NE and DA were significantly increased on the 1^st d,7^th d,14^th d and 28^th d after radiation（P<0.05 or0.01）,ACTH was increased on the 7^th d and 14^th d（P<0.05 or 0.01）,and GC was increased on the 7^th d（P<0.05）,and the above indicators were still not fully recovered on the 28^th d（P<0.05）.（2）Abnormal heart rate variability:Mean HR and LF-ms² were significantly increased on the 1^st d,7^th d and 14^th d after radiation（P<0.05 or 0.01）,Mean RR and HRV tri index were decreased on the 1^st d,7^th d and 14^th d（P<0.05 or 0.01）,RMSSD was decreased on the 1^th d and 7^th d（P<0.05 or 0.01）,and SDNN was decreased on the14^th d（P<0.01）,HF-ms² was decreased on the 7^th d and 14^th d（P<0.05）,LF-nu and LF/HF radio were increased on the 1^st d,7^th d,14^th d and 28^th d（P<0.05 or 0.01）,HF-nu was decreased on the 1^st d,7^th d,14^th d and 28^th d（P<0.05 or 0.01）,and the above indices were still not fully recovered on the 28^th d（P<0.05）.（3）Slow weight-gain:the body weight of R group was significantly lower than C group on the 1^st d,3^th d,5^th d and 7^th d after radiation（P<0.05 or 0.01）,and there is no significant difference between the two groups on the 14^th d（P<0.05）.（4）Decreased motility,lower activity,reduced exploratory and grooming behaviors were most significant at the 6^th h after radiation（P<0.05 or 0.01）,with a trend toward recovery on the 7^th d,and complete recovery on the 14^th d（P>0.05）.3.Radiation could cause the significant increase in the expression of Hsp72,JNK and p-JNK on the 1^th d and 7^th d after radiation（P<0.05 or 0.01）,and the significant increase in the expression of HSF1 and NFATc4 on the 1^st d in rats’myocardial tissue（P<0.05 or 0.01）.Conclusion:1.Certain condition of S-band microwave radiation could cause physiological and psychological stress injury in the cardiac of rats.2.Physiological and psychological stress injury in the cardiac of rats was the result of a combination of the thermal and non-thermal effects.3.Hsp72 was involved in the pathophysiological process of physiological and psychological stress injury in the cardiac of rats.4.Activation of important molecules in the JNK pathway was an important molecular mechanism underlying physiological and psychological stress injury in the cardiac of rats.