Effect And Mechanism Of Icariside Ⅱ On Attenuating Cardiomyocyte Apoptosis In Spontaneously Hypertensive Rats

Objective:To observe the interventional effect of icariside II（ICSII）on left ventricular cardiomyocytes apoptosis in spontaneously hypertensive rats（SHR）,and to investigate its impact on the related apoptotic signal molecules of oxidative stress,mitochondrial apoptosis and endoplasmic reticulum stress（ERS）.Methods:13-week SHRs were randomly divided into four groups:model group（SHR group）,ICSII low dose group（4 mg/kg,ICSII-L）,ICSII medium dose group（8mg/kg,ICSII-M）and ICSII high dose group（16 mg/kg,ICSII-H）.Wistar-Kyoto（WKY）rats served as control group.After a week of adaptive feeding,ICSII groups were given ICSII oral administration once a day to 26 weeks of age,meanwhile,WKY and SHR groups were given equal volume of double distilled water.In this process,the rat tail blood pressure was measured weekly.The left ventricular function was detected by ultrasonography after administration.After sacrifice,the left ventricular was separated and weighed to calculate the left ventricula mass index.The left ventricular pathological change was observed by H&E staining,the apoptosis of left cardiomyocytes was observed by TUNEL staining.The contents of H₂O₂,·OH,MDA and the activity of SOD were detected by ELISA in serum.The levels of ASK1,JNK,p38,p53,Bcl-2,Bax,GRP78,CHOP,PERK,ATF-4 and ATF-6 m RNA were detected by RT-PCR in left ventricular tissue.The expressions of p-ASK1,p-JNK,p-p38,p53,Bcl-2,Bax,cleaved-caspase3,GRP78,CHOP,p-PERK,ATF-4,ATF-6,cleaved-caspase12 and cleaved-caspase9 protein were detected by western blot in left ventricular tissue.Results:Compared with WKY,the arterial blood pressure was increased significantly in SHR.Specifically,systolic and diastolic blood pressure were 1.8 times and 1.5 times that of WKY at 26 weeks,respectively.The left ventricular diameter（LVID）,left ventricular anterior wall thickness（LVAW）and left ventricular posterior wall thickness（LVPW）were increased in SHR group（P<0.05）,while ejection fraction（EF）and fractional shortening（FS）were decreased（P<0.05）,and the left ventricular mass index was increased（P<0.05）.H&E staining showed that myocardial cells were hypertrophy and disorganized,with widening of cell gap and partial rupture of muscle fibers in SHR group.TUNEL staining showed that apoptosis of left cardiomyocytes was significantly increased in SHR group（P<0.05）.The contents of H₂O₂,·OH and MDA were increased and the activity of SOD was decreased in SHR group（P<0.05）.The levels of ASK1,p38,Bax,GRP78,CHOP,PERK,ATF-4 and ATF-6 mRNA were significantly increased（P<0.05）and the level of Bcl-2 mRNA was decreased（P<0.05）,while the levels of JNK and p53 mRNA did not change significantly in the left ventricular tissue of SHR.In the left ventricle of SHR,the phosphorylation of ASK1,JNK,p38 and PERK were increased significantly（P<0.05）,and the activation of caspase12,caspase9 and caspase3 were increased（P<0.05）.Meanwhile,the expressions of ATF-6 was upregulated（P<0.05）and the ratio of Bax/Bcl-2 was increased（P<0.05）,while the expression of Bcl-2 was downregulated（P<0.05）.After the treatment of ICSII,however,the arterial blood pressure was decreased in different degrees compared with the SHR group,especially in the middle and high dose groups.In ICSII middle and high dose groups,the LVID was narrowed,the LVAW and LVPW were decreased,the EF and FS were increased（P<0.05）,meanwhile,the left ventricular mass index was decreased（P<0.05）.H&E staining showed that cardiomyocyte hypertrophy was improved and the cells tended to be neat arrangement and the interstitial space was narrowed without myofilament rupture,especially in the medium and high ICSII dose groups.TUNEL staining showed that the number of apoptotic cells was decreased after ICSII treatment（P<0.05）.Moreover,the percentages of myocardial apoptosis in ICSII-M and ICSII-H groups were respectively decreased by 9%and 30%（P<0.05）.Compared with SHR group,the levels of H₂O₂,OH and MDA were decreased（P<0.05）,while the activity of SOD was increased in ICSII groups（P<0.05）.The levels of ASK1,Bax,GRP78,CHOP,PERK and ATF-4 mRNA were significantly decreased in left ventricular tissue of ICSII groups（P<0.05）,and the level of Bcl-2 mRNA was increased（P<0.05）,while the levels of JNK,p38,p53 and ATF-6 m RNA did not change significantly.In ICSII groups,the phosphorylation of ASK1,JNK,p38 and PERK were significantly decreased in the left ventricle（P<0.05）,and the activation of caspase12,caspase9 and caspase3 were decreased（P<0.05）.Meanwhile,the expression of ATF-6 was down-regulated（P<0.05）,the expression of Bcl-2 was up-regulated（P<0.05）,and the ratio of Bax/Bcl-2 was decresed（P<0.05）.Conclusion:SHRs have been associated with arterial hypertension,ventricular remodeling and left ventricular dysfunction at 26 weeks of age,and left ventricular cardiomyocyte apoptosis increased significantly.ICSII could reduce SHR blood pressure,improve ventricular remodeling and left ventricular function and reduce left ventricular cardiomyocytes apoptosis.The mechanism may be related to lowering blood pressure,inhibiting oxidative stress,mitochondrial apoptosis signaling pathway and endoplasmic reticulum stress.