Protective Mechanism Of Melatonin On Chronic Stress Induced Cardiac Injury In Rats By Regulating ROS/CA²⁺ Signal Pathway

Chronic stress,as an important inducement of a variety of diseases,seriously affects animal husbandry and animal health.Strong and constant stress will lead to the damage of multiple systems and organs of animal body,among which the heart is one of the most vulnerable organs in stress.Chronic stress often results in functional and organic changes in the heart,and even life-threatening.Therefore,clarifying the molecular mechanism of chronic stress-induced cardiac injury and exploring effective prevention and treatment drugs are hot and difficult issues in veterinary clinical research.Previous studies have found that melatonin?Mel?existed anti-stress and cardioprotection effects,the research group intends to establish a chronic stress model in rats and intervention with melatonin to explore the protective mechanism of Mel on chronic stress-induced myocardial injury from the perspective of inhibiting ROS/Ca²⁺signaling pathway and alleviating myocardial cell apoptosis.Sixty-four healthy male Wistar rats were randomly divided into 8 groups?n=8?.The modeling period is 21 days,during the last 7 days of the experiment,the rats in group C,CF,CN and CM were respectively administered by gavage normal saline,50 mg/kg glucocorticoid antagonist,100 mg/kg reactive oxygen species scavenger,10 mg/kg Mel;the rats in group C,CSF,CSN and CSM were restrainted for 6 h every day for 21 consecutive days,and at the last 7 days of the experiment,30minutes before restraint,they were given intragastric normal saline,50 mg/kg MIF,100 mg/kg NAC,10 mg/kg Mel respectively.The body weight change,open field test and serum CORT concentration were detected to verify the success of the chronic stress model.Blood and myocardial tissue were obtained,cardiac function was evaluated by measuring CK,LDH activity and CK-MB,c-Tn I concentrations in serum;the myocardial histopathological changes were observed by H.E staining;cardiomyocyte ultrastructural change were observed by transmission electron microscopy;the oxidative stress indicators were detected by colorimetry;the Calpain activity was detected by fluorescence;the gene level related to Ca²⁺regulation was detected by q RT-PCR;the protein expression related to Ca²⁺regulation and apoptosis was detected by western blotting and cardiomyocyte apoptosis was detected by TUNEL.So as to clarify the protective mechanism of Mel on cardiomyocyte apoptosis induced by chronic stress in rats.The results showed that the chronic stress model in rats was successfully established.The results of cardiac biochemical indicators tests showed that the activity of CK and LDH,as well as the concentration of CK-MB and c-Tn I in the CS group were significantly increased?P<0.01?,which were significantly reduced after Mel intervention?p<0.01?.Histopathological results showed that the morphology and structure of myocardium in the C group was normal,the arrangement of myocardial fibers in the CS group were disordered,the myocardial cells were swollen and vacuolated,the myocardial damage in the CSF,CSN and CSM groups was significant mitigated.Ultrastructural test results showed that the myocardial fibers of the C group were orderly arranged in sarcomere,with clear mitochondrial structure and complete cristae,in the CS group,mitochondrial crista was fractured or disappeared,matrix density decreased and vacuoles appeared,the ultrastructural damage of myocardial tissue in the CSF,CSN and CSM groups was significantly reduced.The detection results of oxidative stress related indexes showed that the ROS level and MDA content of the CS group were significantly increased?p<0.01?,and the GSH content and T-SOD activity weresignificantly reduced?p<0.05,p<0.01?,while Mel intervention significantly reversed the changes in the above indicators?p<0.01?.The detection results of Ca²⁺regulation-related gene expression showed that the relative m RNA expression of Ry R2 was not significantly different in each group?p>0.05?,CACNA1C,IP3R,Calpain-1 and Calpain-2 in CS group were significantly increased?p<0.01?,and NCX,SERCA2 were significantly decreased?p<0.01?,while Mel intervention significantly reversed the change of the relative m RNA expression?p<0.01?.The trend of Ca²⁺regulatory relative protein expression was consistent with that of m RNA.The detection results of[Ca²⁺]_i and Calpain activity showed that the[Ca²⁺]_i and Calpain activity in the CS group were significantly increased?p<0.01?,which were significantly reduced after Mel intervention?p<0.01,p<0.05?.The statistical results of the percentage of TUNEL positive cells showed that the percentage of TUNEL positive cells in the CS group was significantly increased?p<0.01?,which was significantly reduced after Mel intervention?p<0.01?.Apoptosis-related protein expression results showed that the relative expression levels of Bax/Bcl-2,Cyt C,Cleaved Caspase 9/Caspase 9 and Cleaved Caspase 3/Caspase 3 in the CS group were significantly increased,which were significantly reduced after Mel intervention.In the experiment,MIF and NAC intervention had similar effect to Mel.In conclusion,Mel plays a protective role in chronic stress-induced myocardial injury in rats by decreasing CORT content,reducing ROS production,enhancing antioxidant capacity,restoring Ca²⁺homeostasis,down-regulating[Ca²⁺]_i,inhibiting Calpain-1 and Calpain-2 activity,and reducing apoptosis.