The Study Of Cardioprotective Effects And Mechanisms Of Hydroxysafflor Yellow A And Nitric Oxide Against Ischemia Reperfusion Injury

IntroductionThe first concerns about myocardial reperfusion were expressed by Jennings et al. in 1960 when they suggested that reperfusion may accelerate the development of necrosis in irreversible injured myocytes. Reperfusion through thrombolysis, percutaneous coronary angioplasty or bypass surgery is the standard treatment in impending acute myocardial infarction, meaning that reperfusion injury will be a major clinical problem in the near future. The underlying pathophysiological mechanisms of IR have not been fully elucidated. It has been suggested that an overproduction of oxygen-derived free radicals and intracellular calcium overload or redistribution during the first minutes of reflow might be involved. NO is an important cardioprotective molecule via attenuating calcium overload and cleaning free radicals. However, they are not the only candidates responsible for the cardioprotective effect of NO in reperfusion injury.The mitochondrial permeability transition pore (MPTP) is a non-selective pore that penetrates the inner and outer mitochondrial membranes. Opening of the MPTP may result in mitochondrial swelling, collapse of mitochondrial membrane potential,uncoupling of mitochondrial oxidative phosphorylation and cytochrome c release, leading to both necrosis and apoptosis. Conditions associated with postischaemic reperfusion, such as reactive oxygen species (ROS) accumulation,pH normalization and increases in [Ca²⁺]i, create an ideal scenario for mitochondrial permeability transition pore (MPTP) opening. It was demonstrated that preventing MPTP opening may be protective in isolated cardiomyocytes and perfused hearts. In addition, modulation of MPTP opening has been observed in cardioprotection by both preconditioning and post-conditioning of the heart.The flower of the safflower plant Carthamus tinctorius L. has been used extensively in traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases. Hydroxysafflor yellow A, the main chemical component of the safflower yellow pigments, has been demonstrated to attenuate pressure overload hypertrophy in rats. Hydroxysafflor yellow A is also able to reduce myocardial infarct size in rats in vivo and protects the ischaemic myocardium by regulating nitric oxide (NO) and NO synthase (NOS) activity. Moreover, HSYA may provide neuroprotection against cerebral ischaemia/reperfusion (I/R) injury by its anti-oxidant action. However, whether HSYA is able to protect the heart from I/R injury and its mechanism of action remain poorly understood.The reperfusion injury salvage kinase( RISK) is the pathway of cardioprotection during I/R, concluding phosphatidylinositol-3-kinase (PI3K)/ serine/threonine kinase (Akt) signal pathway and (the mitogen-activated protein kinase p42/44extra-cellular signal-regulated kinases (ERK1/2) signal pathway. The RISK pathway, activated during the early period of reperfusion, may act against I/R through attenuating myocardial apoptosis. The mechanism of cardioprotection of precondition and postcondition is tightly connected with that.The purpose of the present study was to determine whether nitric oxide(NO) exerts in the effect of MPTP opening and whether HSYA protects the heart at reperfusion. through a mitochondrial pathway, specifically via inhibition of MPTP opening. The role of Akt and ERK signal pathway in the cardioprotective effect of HSYA was also ivestigated.Method1. The effect of NO to MPTP openingHearts isolated from male Sprague-Dawley rats were perfused on a Langendorff apparatus and subjected to 30 min global ischaemia, followed by 120 min reperfusion. Infarct size and the level of lactate dehydrogenase (LDH) in the coronary effluent were determined. In mitochondria from isolated perfused hearts, Ca²⁺-induced swelling was observed. In isolated ventricular myocytes, depolarization of the mitochondrial membrane was determined by tetramethyl-rhodamine ethyl ester (TMRE) fluorescence.2. The MPTP mechanism of HSYA against I/R injuryHearts isolated from male Sprague-Dawley rats were perfused on a Langendorff apparatus and subjected to 30 min global ischaemia, followed by 120 min reperfusion. Infarct size and the level of lactate dehydrogenase (LDH) in the coronary effluent were determined. In mitochondria from isolated perfused hearts, Ca²⁺-induced swelling was observed. In isolated ventricular myocytes, depolarization of the mitochondrial membrane was determined by tetramethyl-rhodamine ethyl ester (TMRE) fluorescence. Furthermore, levels of phosphorylated endothelial nitric oxide synthase (eNOS) protein were measured by western blot.3. Effect of HSYA on PI3K/Akt and ERK1/2 signal pathway in I/RHearts isolated from male Sprague-Dawley rats were perfused on a Langendorff apparatus and subjected to 30 min global ischaemia, followed by 120 min reperfusion.Levels of Akt,p-Akt,ERK1/2,p-ERK1/2 protein at different time course of reperfusion were measured by western blot. Also, the effect of different doses of HSYA to the levels of Akt,p-Akt were measured.Result1. The effect of NO to MPTP openingPretreatment with 0.2 umol/L cyclosporine A (CsA) for 20 min before ischaemia reduced infarct size and the release of LDH. Administration of 10 mmol/L NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, attenuated the protective effects of CsA. In mitochondria isolated from hearts pretreated with 1 umol/L CsA, a significant inhibition of Ca²⁺-induced swelling was observed and this inhibition was attenuated by L-NAME. In isolated ventricular myocytes, pretreatment with CsA prevented ischaemia-induced cell death and depolarization of the mitochondrial membrane, whereas L-NAME attenuated the effects of CsA.2. The MPTP mechanism of HSYA against I/R injuryPretreatment with HSYA for 5 min before ischaemia reduced infarct size and the release of LDH. Administration of 20 mmol/L atractyloside, an opener of the mitochondrial permeability transition pore, and 10 mmol/L NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, attenuated the protective effects of HSYA. In mitochondria isolated from hearts pretreated with 0.1 mmol/L HSYA for 5 min, a significant inhibition of Ca²⁺-induced swelling was observed and this inhibition was attenuated by L-NAME. In isolated ventricular myocytes, pretreatment with HSYA prevented ischaemia-induced cell death and depolarization of the mitochondrial membrane, whereas atractyloside or L-NAME attenuated the effects of HSYA. Levels of phosphorylated eNOS protein were significantly enhanced in the HSYA-treated group.3. Effect of HSYA on PI3K/Akt and ERK1/2 signal pathway in I/RLevels of p-Akt protein were significantly enhanced in the HSYA-treated group while the levels of Akt protein keep intact. The expression of p-Akt was induced 1.9fold, 2.5fold and 4.3fold higher respectively in 0, 5,10min of reperfusion in HSYA-treated animals than in control (P<0.05). While whether ERK or p-ERK have no significantly change in the levels of protein. HSYA at 0.1 mmol/L significantly enhanced the levels of p-Akt protein in reperfused rat hearts.Conclusion1,The findings of the present study indicate that nitric oxide take a part in the inhibition of mitochondrial permeability transition pores opening.2,The findings of the present study indicate that HSYA protects the myocardium against ischaemia-reperfusion injury by inhibiting mitochondrial permeability transition pore opening.3,The effect of HSYA on mitochondrial permeability transition pore opening may be mediated through enhanced nitric oxide production by eNOS activation.4,HSYA may protect the heart against reperfusion-induced injury by the increase of phosphorylating Akt while ERK1/2 does not take a part in that.