| Background and ObjectiveNitric oxide (NO) is one of gaseous signaling molecules, which play a central role in cell apoptosis/survival. NO is a double edged sword to the myocardium. The Biological effect that NO serves as depends on its origin,concentration and the redox state at the local microenvironment. The NO derived from eNOS can activates the mitochondrial KATP (Mito KATP) channels and inhibit mitochondrial permeability transition pore (MPTP) opening that promotes both apoptotic and cell death. However, the NO derived from iNOS yet increases the myocardial infarct size and has pro-inflammatory action; NO donors increase cardiomyocyte cell death and switch the form of cell death from apoptosis to necrosis as a function of their concentration; when the reactive oxidative species (ROS), such as O2- is over-produced, NO will reacts with O2- to form peroxynitrite (ONOO-), a particularly destructive molecule within biological systems. Therefore to elucidate the effect of NO on different Physiological and pathological state will provide us some new therapeutic targets to remedy the ischemia injury resulting from different causesCardiovascular disease is a major complication of diabetes and the leading cause of early death among people with diabetes. About 65 percent of people with diabetes die from heart disease. Strong evidence exists that diabetics suffer from increased incidence and severity of myocardial infarction (MI) and are much more likely to suffer from heart failure following MI compared to non-diabetics. These results indicate that the diabetic heart is more sensitive to ischemia injury compared to that of non-diabetics. Recent study reports that overexpression of eNOS protects liver ischemia/reperfusion injury in control animal but worsens the injury in diabetic animals. These studies indicate that diabetes can alter NO's biologic activity in ischemic/reperfused liver. However whether diabetes may also alter NO's biologic activity in ischemic/reperfused heart remains completely unknown.Oxidative/nitrative stress has been implicated as a modulator in the MI/R injury. NO will reacts with O2- to form peroxynitrite (ONOO-), a particularly destructive molecule within biological systems. Both I/R injury and diabetes increased the production of ONOO-, which not only damages the DNA but also modify posttranslationally some key proteins related to gene expression, signal transduction, and antioxidant defense, altering the response of hearts to I/R injury. Thioredoxin is a small protein expressed in living cells. Trx not only exerts cytoprotective functions against oxidative stress, but also regulates cell survival signaling pathways. It has been demonstrated that in addition to upregulation or downregulation of Trx expression at the gene level, Trx activity is regulated by posttranslational modification. Trx can be nitratively modified, resulting in the irreversible inhibition of activity during the MI/R condition. Recent study demonstrates that increased oxidative/ nitrative stress and nitrative inactivation of thioredoxin enhanced the vulnerability of diabetic hearts to I/R injury .However, whether diabetes may alter NO's biologic activity in ischemic/reperfused heart by promoting of nitrative inactivation of thioredoxin remains completely unknownTherefore, the aim of the present study were (1) to determine the opposite effects of NO on the diabetic and non-diabetic mice, if so (2) to identify the mechanism by which diabetes alters NO's cardioprotective action a in ischemic/reperfused heart. MethodsPart One: The establishment of diabetes and MI/R model in mice. Mice were rendered diabetic by 5 days of daily intraperitoneal injection with 40 mg/kg STZ (Sigma) in 0.05 M sodium citrate, pH 4.5. Blood glucose was measured 2 days after the final injection, and diabetic condition was confirmed by markedly elevated fasting-blood glucose levels (>11.1μmol/L). Myocardial ischemia/reperfusion was produced by temporarily exteriorizing the heart via left thoracic incision and placing a 6-0 silk suture slipknot at the distal 1/3 of the left anterior descending coronary artery. After 30 min of ischemia, the slipknot was released.PartTwo: The effect of diabetes on the level of oxidative stress and MI/R injury in diabetic mice.The diabetic and age-matched non-diabetic mice were subjected to MI/R injury 7 days after the establishment of diabetes. After 30 min of ischemia, the slipknot was released, and the myocardium was reperfused for 3 h (for apoptosis, the level of oxidative stress) or 24 h (infarct size assay). Sham-operated mice underwent the same surgical procedures except that the suture placed under the left coronary artery was not tied. Part three: The effect of GSNO MI/R injury in diabetic and non-diabetic mice and possible mechanism7 days after the establishment of diabetes, the diabetic mice were subjected to MI/R. 10 min before MI/R 1, the diabetic and non-diabetic mice were received GSNO or GSNO in combination with Mn (III) TBAP by intraperitoneal injection. After 30 min of ischemia, the slipknot was released, and the myocardium was reperfused for 3 h (for apoptosis, the level of oxidative stress, Trx activity, Trx nitration) or 24 h (infarct size assay). ResultsPart one: The fasting-blood glucose level was significantly elevated (20±3.9 mmol/L in STZ-treated mice VS 6.9±2.3 mmol/L in control mice). The hearts suffering from ischemia/reperfusion present infarcted and ischemia area by TTC. These results indicate that the diabetes condition and myocardial ischemia/reperfusion model were established successfully.Part two: In Control+Sham group, the superoxide generation was increased compared with that in DM+Sham group. After I/R injury, in DM+MI/R group the infarct size, apoptosis and caspase-3 activity were significantly increased , compared with that in Control+MI/R group. The increase of superoxide generation was further amplified between DM+MI/R group and Control+MI/R group.Part three:1) GSNO increased the MI/R injury in diabetic mice, but decreased the MI/R injury in non-diabetic mice: GSNO increased the infarct size, apoptosis and caspase-3 activity, compared to the vehicle in diabetic mice; GSNO also increased the superoxide generation, nitrotyrosine content, and Trx nitration and decreased the Trx activity, compared to the vehicle in diabetic mice. However, GSNO decreased the infarct size, apoptosis and caspase-3 activity, compared to the vehicle in diabetic mice.2) Mn (III) TBAP attenuated the increase of GSNO-induced MI/R injury: Mn (III) TBAP in combination with decreased the infarct size, apoptosis , caspase-3 activity, nitrotyrosine content, Trx nitration and increased the Trx activity, compared to GSNO.Conclusions1) NO donor GSNO increased the MI/R injury in diabetic mice, but it has the opposite effect on that in no-diabetic mice;2) The mechanism by which GSNO increased the MI/R injury in diabetic mice may partly attributed to the enhanced oxidative stress, which can promote the ONOO- formation, resulting in the nitrative thioredoxin inactivation in diabetic mice. |
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