| Part I Protective effect of nicorandil preconditioning on rat myocardial ischemia reperfusion injury-in vivo studyObjective:To observe the effect of nicorandil preconditioning on rat in vivo myocardial ischemia reperfusion(I/R) injury and the expression of the key factors in endoplasmic reticulum stress signal pathway.Methods:Forty Sprague-Dawley(SD) rats were randomly divided into five groups:S group, I/R group, L-Nic group, M-Nic group and H-Nic group. We established rat in vivo myocardial ischemia reperfusion injury model, gave different doses of nicorandil through tail vein injection before the ligation of left anterior descending coronary. We used HE staining and light microscopy to evaluate pathological changes of the myocardium; Lactate dehydrogenase (LDH) detection kit to test the activity of serum LDH; TUNEL method to detect myocardial apoptosis index; Real-time PCR to detect mRNA expression of GRP78 (ERS marker) and CHOP (ERS related apoptosis marker) in myocardium; Western blot to detect protein expression of GRP78,CHOP, Bcl2 and Bax in myocardium.Results:Compared with S group, the serum LDH activity and myocardial apoptosis index of the I/R group were significantly elevated, the protein expression of Bcl2 in myocardium were significantly decreased, while the Bax protein expression were increased significantly, the mRNA and protein expression of GRP78and CHOP were significantly elevated in I/R group. Compared with I/R group, the serum LDH activity of nicorandil preconditioning groups were significantly lower; Nicorandil preconditioning significantly reduced the myocardial apoptosis index, inhibited the expression of Bax protein and promoted the expression of Bcl2 protein, decreased the mRNA and protein expression of GRP78and CHOP significantly. These effects of nicorandil were dose dependent.Conclusion:Nicorandil preconditioning could alleviate myocardial ischemia reperfusion injury, reduce myocardial cell apoptosis, and inhibit endoplasmic reticulum stress induced by myocardial ischemia reperfusion in a dose-dependent manner in vivo.Part II:Protective effect of nicorandil preconditioning on rat myocardial ischemia reperfusion injury-in vitro studyObjective:To observe the effect of nicorandil preconditioning on rat in vitro myocardial ischemia reperfusion(I/R) injury and the expression of the key factors in endoplasmic reticulum stress signal pathway.Methods:Forty-eight SD rats were randomly divided into six groups:balance group, balance+100μM nicorandil preconditioning group, I/R control group,30μM nicorandil preconditioning group, 100μM nicorandil preconditioning group and 300μM nicorandil preconditioning group. Hearts from each group were excised and subjected to 30min global ischemia followed by 120min reperfusion in a Langendorff apparatus. Homodynamic parameters of left ventricules (LV) were recorded and evaluated with Powerlab system, including LV developed pressure (LVDP), LV end-diastolic pressure (LVEDP), and maximum rates of pressure development or decay over time (+dP/dtmax and-dP/dtmax). Cell death was reflected by lactate dehydrogenase (LDH) activity assay in coronary effluent. Cardiac infarct size was also measured after nicorandil treatment by Triphenyl Tetrazolium Chloride (TTC) staining. I/R induced cell apoptosis was examined by TUNEL staining in myocardium cells. mRNA level of ER stress marker, including GRP78 and CHOP was examined by real time-PCR. Protein expression of GRP78 and CHOP was detected by western blot.Results:Nicorandil preconditioning improved post-ischemic cardiac function of isolated rat heart in a concentration dependent manner, and reduced LDH activity in coronary effluent. Consistent with the above observation, TTC staining shown that nicorandil preconditioning significantly reduced the infarct size after ischemia reperfusion. Nicorandil preconditioning also reduced I/R induced myocardial cell apoptosis. As expected, we found Bcl2 was deceased and BAX was elevated in I/R hearts compared to the balance group. On the contrary, nicorandil treatment suppressed the increase of BAX level and meanwhile enhanced the expression of the anti-apoptotic protein Bcl2 in a dose-dependent manner. Western blot and real-time PCR showed that GRP78 and CHOP were upregulated in I/R heart. When the I/R hearts were treated with nicorandil, both CHOP and GRP78 level were significantly attenuated, which emerged in an dose-dependent way.Conclusions:Nicorandil preconditioning could alleviate myocardial I/R injury, reduce myocardial cell apoptosis, ameliorate post-ischemic contractile function recovery, and inhibit endoplasmic reticulum stress induced by myocardial I/R in a dose-dependent manner in vivo.Part Ⅲ:The effects of nicorandil preconditioning on endoplasmic reticulum stress during myocardial ischemia reperfusionObjective:The aim of this study is to explore whether endoplasmic reticulum stress was involved in the cardioprotective mechanism of nicorandil preconditioning.Methods:Forty SD rats were randomly divided into five groups:balance group, I/R control group, 100μM nicorandil preconditioning group,2.5g/ml tunicamycin preconditioning group,2.5g/ml tunicamycin and 100μM nicorandil preconditioning group. Hearts from each group were excised and subjected to 30min global ischemia followed by 120min reperfusion in a Langendorff apparatus. Homodynamic parameters of left ventricules were recorded and evaluated with Powerlab system, including LVDP, LVEDP,+dP/dtmax and-dP/dtmax. Cell death was reflected by LDH activity assay in coronary effluent. Cardiac infarct size was also measured after nicorandil treatment by TTC staining. I/R induced cell apoptosis was examined by TUNEL staining in myocardium cells. mRNA level of GRP78 and CHOP was examined by real time-PCR. Protein expression of GRP78 and CHOP was detected by western blot.Results:Nicorandil preconditioning improved post-ischemic cardiac function of isolated rat hearts. The protective effects of nicorandil on the myocardial contractile function against I/R were abolished by the exposure to tunicamycin. Furthermore, LDH activity in coronary effluent and the infarct size was significant increased after treatment of endoplasmic reticulum stress inducer tunicamycin. TUNEL staining assay showed that tunicamycin abolished the protective effect of nicorandil preconditioning on the myocytes death. We found that tunicamycin preconditioning resulted in a restoration of the level of CHOP and GRP78 in the nicorandil treament group.Conclusions:The protective effects of nicorandil on ischemia reperfusion myocardium were abolished by the endoplasmic reticulum stress inducer, tunicamycin, suggested that endoplasmic reticulum stress might be one of the important targets of nicorandil to protect the heart from ischemia reperfusion injury.Part Ⅳ:Nicorandil protects the heart from ischemia reperfusion injury by attenuating endoplasmic reticulum stress through PI3K/Akt signaling pathwayObjective:To explore whether PI3K/Akt signaling pathway was involved in the nicorandil preconditioning mediated cardioprotection and its effect on endoplasmic reticulum stress.Methods:Forty-eight SD rats were randomly divided into six groups:balance group, balanced-100μM nicorandil group, I/R control group, 100μM nicorandil preconditioning group, wortmannin preconditioning group, wortmannin and 100μM nicorandil preconditioning group. Hearts from each group were excised and subjected to 30min global ischemia followed by 120min reperfusion in a Langendorff apparatus. Homodynamic parameters of left ventricules were recorded and evaluated with Powerlab system, including LVDP, LVEDP,+dP/dtmax and-dP/dtmax. Cell death was reflected by LDH activity assay in coronary effluent. Cardiac infarct size was also measured after nicorandil treatment by TTC staining. I/R induced cell apoptosis was examined by TUNEL staining in myocardium cells. Protein expression of GRP78, CHOP, Akt, p-Akt, Bcl2 and Bax was detected by western blot.Results:Nicorandil preconditioning improved post-ischemic cardiac function of isolated rat heart. The protective effects of nicorandil on the myocardial contractile function against I/R were abolished by the exposure to wortmannin. Furthermore, LDH activity in coronary effluent and the infarct size was significantly increased after treatment with PI3K/Akt specific inhibitor wortmannin. Furthermore, the infarct size was significantly increased after treatment with wortmannin. TUNEL staining assay showed that wortmannin abolished the protective effect of nicorandil preconditioning on the myocytes death. Wortmannin abolished the effect of nicorandil on decreasing the expression of BAX level and increaseing the anti-apoptotic protein Bcl2 level. We examined the activity of PI3K-Akt pathway in rat I/R heart and found that compared with I/R control heart, there was a marked increase of Akt phosphorylation in I/R heart treated with nicorandil, as determined by western blot. The level of CHOP and GRP78 was comparable to that in the I/R group without nicorandil treatment.Conclusions:The cardioprotective effects of nicorandil and its effect on endoplasmic reticulum stress were abolished by PI3K/Akt specific inhibitor wortmannin, suggested that PI3K/Akt signaling pathway might play an important role in these effects of nicorandil. |
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