| BackgroundAccording to the data provided by International Diabetes Faderation (IDF), the number of the diabetic patients is over 250 million, and the number will become 380 million by 2025. China will be the second biggest diabetic country in the world. The first cause of death in diabetic patients is cardiovascular disease. Multivessel coronary artery disease is always seen in diabetic coronary artery disease patients. The incidence of no-reflow after PCI treatment is much higher in diabetic patients compared with those patients without diabetes. The therapy and prevention of no-reflow penomenon have become a new challenge in the field of cardiovascular disease.The possible mechanism of no-reflow penomenon in PCI treatment are the following points:①Microvessel vasodialation dysfunction and neutrophilic granulocyte blocking in microvessles;②Thrombocyte occludes in microvessles;③Endothelial cells swelling;④Change of the stagnation of the blood viscosity. It seems that no-reflow phenomenon is associated with microvascular injury, but the accurate mechanism is not clear. Diabetes is famous of causing mulp-organ microvessle injury. The no-reflow phenomenon seen in diabetic patients may be associated with cardia microvessle injury.Many data reported that the insulin receptor pathway was hurt and the expression of eNOS and secretion of NO were decreased in diabetic patients. These changes were associated with endothelium-dependent vasodialation dysfuncrion and thrombocyte aggragation which were causes of no-reflow phenomenon. But another pathway associated with insulin named MAPK pathway was actived which resulted in endothelial cells apoptosis. This phenomenon was called selective insulin resistance. I/R is another important stimulus for MAPK pathway. What will happen to endothelial cells when stimulated by I/R in diabetic patients? It is not clear now.Objectives1. To determine whether cardia microvessle injury in diabetic rats was more severe than in normal rats following the treatment of I/R.2. If so, to investigate the effect of I/R on PI3K pathway and MAPK pathway, and the possible relationship between the two pathways.3. If the relationship between the two pathways was exsited, to see the role of it in the cardia microvessle injury caused by I/R in diabetic rats.4. To elucidate whether inhibition of the relationship between the two pathways was helpful to improve cardia microvessle injury caused by I/R in diabetic rats.Methods1. Male Sprague-Dawley rat, 200 g, dieted with high glucose and high cholesterol for 12 weeks, diabetes was induced with a single intraperitoneal injection of streptozotocin (50 mg/kg, Sigma). Random serum glucose greater than 16.7 mmol/L were considered success.2. Rats were anesthetized and myocardial ischemia was produced by exteriorizing the heart through a left thoracic incision and placing a 6-0 silk and making a slipknot around the left anterior descending coronary artery. After 30 minutes of ischemia, the slipknot was released and the myocardium was reperfused for 3 hours. Hemodynamic data were continuously monitored on a polygraph (RM-6200C) and simultaneously digitized by using a computer interfaced with an analog-to-digital converter. Even's blue and TTC staining was used to determine the infarction area, and Even's blue and Thioflavin-S staining was used to determine the no-reflow area after 30 min ischemia and 3 hours after reperfusion. Immunofluorescence assay of CD31 was used to determine the number of endothelial cells. Echocardiography was performed at 24 hours after reperfusion.3. Followed by removal of the endocardial endothelium and the epicardial coronaries, the left ventricles were cut into small pieces and incubated in 2 ml 0.2% collagens, isolated and purified cardiac microvascular endothelium cells(CMECs). The isolated primary CMECs were put into hypoxia incubator (95% N2 / 5% CO2 , 37℃) for 1 h and then returned to normal incubator for 3 h. Nitrate reduction kit was used to analyse the secretion of NO. Flow cytometry assay was used to detect the apoptosis of cells, and the expression of PI3K, P-JNK, JNK, MEKK1,IRS-1 were determined by western blot at the end of 3 h reoxygenation.Results1. The No-reflow size in diabetic rats significantly larger than that in normal rats after I/R (1.08±0.07 % vs 3.03±0.11%,P < 0.01). The infarct size (IS)/AAR ratio in diabetic rats was significantly larger than in normal rats (57.49±1.25% vs 68.39±2.31%, P < 0.05)too. I/R decreased the -dp/dtmax in both diabetic rats and normal rats(-4326.75±108.90 vs. -3613±88.67, P < 0.01 and -4342.57±87.49 vs. -4065.09±99.31, P < 0.01), and -dp/dtmax was signaificantly lower in diabetic rats compared with normal rats after I/R treatment. The EF and FS value was attenuated in diabetic rats after I/R treatment (32.5±10.0% vs 57.3±8.5%,P < 0.05). The density of CD31 postive cells was lower in in diabetic rats after I/R campared with that in normal rats (1600±82/mm vs 2653±41/mm, P < 0.05).2. H/R resulted in a significant increased CMECs apoptosis index in both normal and diabetic rats (2.33±0.16% vs. 38.17±1.97%, P < 0.01 and 2.23±0.07% vs. 31±2.32%, P < 0.01). The expression of Caspase-3 showed the same trend. Further more, the expression of Caspase-3 in diabetic CMECs was significantly higher than in normal CMECs (P < 0.05). H/R reduced the secretion of NO in diabetic CMECs compared with normal CMECs after 3 h reoxygenation (74.67±0.96% vs. 56.67±1.28%, P < 0.01).3. The phosphorylation of IRS-1 and the expression of PI3K were significantly attenuated in diabetic CMECs compared with normal CMECs (P < 0.05). Following 3 h reoxygenation, the expression of PI3K was diminished in both diabetic CMECs and normal CMECs (P < 0.05). H/R increased the expression of MEKK1 and phosphorylation of JNK in both diabetic CMECs and normal CMECs (P < 0.01). The phosphorylation of JNK seems increased more in diabetic CMECs following H/R stimulus compared with in normal CMECs (148.47±12.27% vs. 399.67±7.77%, P < 0.01).4. PI3K inhibitor LY294002 (10μM) increased the H/R induced apoptosis in diabetic CMECs (39.07±5.48% vs. 47.55±3.46%, P < 0.01) and increased expression of Caspase-3 (338.00±29.53% vs. 450.00±15.51%, P < 0.05). LY294002 attenuated the reduced secretion of NO caused by H/R in diabetic CMECs (56.67±3.34% vs. 41.00±5.72%, P < 0.05). The phosphorylation of JNK was increased by LY294002 followed by the treatment of H/R in diabetic CMECs (260.75±8.76% vs. 373.5±10.74%, P < 0.01).5. JNK inhibitor SP600125 (10μM) increased the reduced secretion of NO caused by H/R in diabetic CMECs (56.67±3.34% vs. 68.75±8.58%, P < 0.05). SP600125 reduced the H/R increased expression of Caspase-3 (338.00±29.53% vs. 260.67±26.91%, P < 0.05). The reduced expression of PI3K induced by H/R was increased partly by SP600125 in diabetic CMECs (35.00±5.35% vs. 62.00±4.54%,P < 0.05).Conclusions1. Exaggerated no-reflow areas and infarction areas accompanied by severe impairment of cardiac function and endothelial cell density presented in diabetic rat model after I/R.2. H/R induced increased apoptosis and decreased NO secretion, accompanied by increased phosphorylation of JNK and attenuated expression of PI3K.3. JNK inhibitor improved the NO secretion ability of CMECs through increasing the expression of PI3K in diabetic CMECs.4. PI3K inhibitor increased the H/R induced apoptosis through activating JNK in diabetic CMECs. |
