Protection And Mechanism Of COX-2 Inhibitor Aganist Oxidative Stress Injury In Heart

BackgroundCyclooxygenase (COX) is the rate-limiting enzyme in the formation of prostanoids by catalyzing the conversion of arachidonic acid to prostaglandin H₂. Two forms of COX have been characterized: a constitutive form (COX-1) and a second form (COX-2) inducible by various factors including mitogens, hormones, serum and cytokines. Although COX-2 is not detectable in normal heart, it is overexpressed in many cardiovascular diseases, such as ischemia/reperfusion, myocardial infarction and congestive heart failure. Increase in ROS is recognized as the key factor during vascular or cardiac injury associated with various heart diseases such as acute myocardial infarction and coronary heart disease. In different kinds of cells, including cardiomyocytes, many factors (such as LPS and TNF-alpha) induce ROS release and significantly enhance COX-2 expression, which is attenuated by antioxidants. All of these studies suggest that COX-2 can be induced by many stimuli, but ROS may be the common factor that mediates this process. Most selective COX inhibitors (such as aspirin and DFU) can reduce left ventricular end diastolic pressure (LVEDP) and infarct size, and improve contractility in ischemic myocardium. Whether COX inhibitors can also provide the protection against ROS is not clear.Objectives(1) To study whether the selective COX-1 piroxicam and the selective COX-2 inhibitor nimesulide could protect myocardial function against oxidative stress injury in rat hearts whichwere exposed to H₂O₂, superoxide anion or hydroxyl free radical.(2) To investigate the role of nitric oxide and coronary endothelial function in the nimesulide-induced cardioprotection in the isolated rat hearts(3) To explore the effect of the selective COX-1 and COX-2 inhibitors on the oxidative stress injury in the PIEC (an endothelial cell line), and to investigate its underlying mechanism.Methods(1) Cardiac contractility was measured by the Langendorff method in isolated SD rat hearts. The concentration of lactate dehydrogenase (LDH) in the coronary effluent was determined. 6-keto PGF_1a (a marker of COX activity) and NO contents in rat hearts were also measured. (2) In the isolated rat hearts, coronary arteries were precontracted with U-46619, and the responses to the endothelium-dependent vasodilator serotonin (5-HT) and the endothelium-independent vasodilator sodium nitroprusside (SNP) were evaluated. (3) The pig endothelial cell line PIEC was cultured in 1640 medium. The cell viability and LDH leakage of cultured PIECs were measured. Western blot was used in the study to detect the expression of GRP78. The concentration of caspase-12 was analyzed by the immune fluorescence method.ResultsPart I Effect of nimesulide and piroxicam on the oxidative stress injury induced by H₂O₂, superoxide anion or hydroxyl free radicalIsolated rat hearts were exposed to hydrogen peroxide (H₂O₂), pyrogallol (which produce superoxide anion) or VitC+Fe²⁺ (which produce hydroxyl free radical) for 10min followed by reperfusion for 30min. (1) H₂O₂ decreased cardiac contractility (LVDP and ±dP/dt_max) and increased LVEDP and LDH release, which was inhibited by nimesulide (3mg/kg) (LVDP 72%±10% vs 61%±11%, LDH 5.5±2.5U/L vs 8.0±2.1U/L, P<0.05). piroxicam (3mg/kg) increased systolic function (LVDP 73%±10% vs 61%±11%, P<0.05), but exacerbated diastolic function (LVEDP 29.00±5.61mmHg vs 23.16±3.57 mmHg, P<0.01) in H₂O₂ treated rat hearts. (2)Nimesulide also protected rat hearts against superoxide anion and hydroxyl free radical injury.Part II Effect of different pretreatment of nimesulide on recovery of contractile performance(1) In isolated rat hearts exposed to H₂O₂ (140 μmol/L) for 20 min, the LVDP, ±dP/dt_max and HR were reduced, while LVEDP was higher than in the control group. Pretreatment with the selective COX-2 inhibitor nimesulide (5 μmol/L) for 20 min antagonized the H₂O₂-induced decrease in LVDP and +dP/dt_max, but aggravated the rise in LVEDP. (2) Nimesulide at a high concentration (25 μmol/L) had no effect on LVDP, ±dP/dt_max and HR in isolated hearts exposed to H₂O₂, while LVEDP was further raised. (3) In isolated hearts pre-perfused with nimesulide (5 μmol/L) for a shorter time (10 min), LVDP and ±dP/dt_max were enhanced compared with that of the H₂O₂ group, while LVEDP did not differ from it.Part III Role of NO and coronary endothelial function in the nimesulide-induced cardioprotection(1) The concentration of 6-keto PGF_1a measured in nimesulide (5μmol//L) pretreated hearts did not differ from that of hearts treated with H₂O₂ alone. (2) Infusion of U-46619 induced a significant vasoconstriction in the arteries (coronary resistances of 10.84 ± 0.81 and 9.37 ± 2.41 mmHg·min/ml in control and H₂O₂ groups, respectively (P>0.05). Perfusion with 5-HT or SNP respectively induced a diminution in coronary resistance of -74.1 ± 9.1 and -45.28 ± 11.8% in the control group. The vasodilatation induced by 5-HT and SNP in the H₂O₂ group was significantly less than in that of control. Pretreatment with nimesulide antagonized the decrease in endothelium-dependent vasodilatation, but had no effect on the decline in endothelium-independent vasodilatation. (3) In isolated hearts pre-perfused with nimesulide (5 μmol/L) for a shorter time (10 min), LVDP and ±dP/dt_max were enhanced compared with that of the H₂O₂ group, and the effect of was abolished by the NOS inhibitor L-NAME. Pretreatment with nimesulide also increased the nitrite/nitrate concentration in the hearts, and this was abolished by L-NAME.Part IV Effect and mechanism of COX inhibitors on the oxidative stress injury in PIECs(1) After incubated with 250 μmol/L of H₂O₂ for 12h, the cell viability decreased and LDH release from cells increased, which were antagonized by nimesulide (25 μmol/L), piroxicam (25 μmol/L) or aspirin (300 μmol/L). (2) The expression of GRP78 enhanced in PIECs after exposed to H₂O₂ for 12h. The expression of GRP78 was inhibited by nimesulide and aspirin, but not by piroxicam. (3) There are no significant differences in caspase-12 content among all groups.Conclusion(1) The selective COX-2 inhibitor nimesulide can improve myocardial function in isolated rat hearts suffering from oxidative stress (induced by H₂O₂, superoxide anion or hydroxyl free radical.). (2) The mechanisms of nimesulide-induced cardioprotection may be through an improvement in endothelium-dependent arterial relaxation and an increase of NO in the heart, but not via the classical pathway that inhibits COX-2 activity. (3) The selective COX-2 inhibitor nimesulide and the selective COX-1 inhibitor aspirin could protect PIECs against H₂O₂-induced injury. The mechanism might through alleviation of the endoplasmic reticulum stress, but not via the caspase-12 related patheway.