| OBJECT:To study the possible cardioprotective mechanism of CT-1 in rat cardiomyocytes injured by hypoxia-reoxygenation, assess the effection of Phosphatidylinositol 3-kinase(PI-3K/GSK3β) pathway induced by CT-1.METHODS:1. Culture of neonatal myocyte:Cardiomyocytes were isolated from the ventricles of 1-3 days Sprague-Dawley neonatal rats and primarily cultured for experiment. Ischemia-reperfusion injury was mimicked by hypoxia-reoxygenation model.2. Preparation of hypoxia-reoxygeonation model:The experiment start from cardiomyocytes converging to synchronous pulsation. Before hypoxia, we replaced the medium by hypoxia solution, which having been deoxygenated by bubbling with 95% nitrogen and 5% carbon dioxcide for 1 hours. Then myocytes were transferred into the chamber (filled with 95% nitrogen and 5% carbon dixocide) to induce hypoxia for 3 hours at 37℃. After the hypoxia of 3 hours, we replaced the hypoxia solution with the reoxygenation solution, which was oxygenated by bubbling with the mixture of 95% oxygen and 5% carbon dixocide for 1 hour. Reoxygenation was carried out by transferring cells into the chamber full of the same gas for 3 hours at 37℃.3.Experiment grouping:(1) Control group: Cultivated normally in incubator full of CO2 at 37℃; (2) hypoxia-reoxygeonation group: hypoxia-reoxygeonation for 6h and cultivation with reoxygeonation for 3h;(3) CT-1 group: CT-1 (10ng/ml) was given after 3h hypoxia cultivation with reoxygeonation for 3h; (4) LY294002 group: (PI-3K blocking agent): LY294002 with final concentration 10umol/l was given after 3h hypoxia ,CT-1(10ng/ml) was given 10min later, then processed with reoxygeonation for 3h; (5)DMSO group: (auxiliary solvent-dimethyl sulfide group) DMSO was given after 3h hypoxia , CT-1(10ng/ml) was given 10min later, then processed with reoxygeonation for 3h.RESULTS:1.Rate and rhythm of cardiomyocyte beat : cardiomyocyte beated regularly and the beating frequency was about 148 beats/min in each group before hypoxia, there was no significant difference. The cardiomyocyte beated slowly, weakly and irregularly after hypoxia-reoxygeonation. The cardiomyocyte beats in CT-1 group was similar to those control group as well as those before hypoxia-reoxygeonation . However, it beated slowly , weakly and irregularly after processing with blockers LY294002, while DNSO could not affect the role of CT-1.Therefore, it indicates that LY294002 could particularly block the effect of CT-1 in protecting cardiomyocyte from hypoxia-reoxygeonation injury.2. Cardiomyocyte injury: The survival and apoptosis rates were 96.2 +3.2% and 2.8+0.4% respectively in control group.The survival rate decreased apparently to 76.8+4.6% and apoptosis rate increased obviously to 19.4+2.3% in hypoxia-reoxygeonation group, there was significantly different (p<0.05) between two groups. LDH and CK-MB increased significantly comparing with those in control group.The survival rate in CT-1 group increased apparently comparing with that in hypoxia-reoxygeonation group increased apparently comparing with that in hypoxia-reoxygeoation group (89.8+8.3%VS 78.8+4.6%,P<0.05). Besides,LDH and CK-MB decreased and apoptosis was inhibited obviously in CT-1group. However, those effects of CT-1 could be blocked obviously by signaling pathway blocker LY294002, because it's had fouded that survival rate decreased and apoptosis rate increased after processing with LY294002.3. Cardiomyocyte mitochondrial membrane potential(△ψm): Results of flow cytometry: Cardiomyocyte△ψm in hypoxia-reoxygeonation group was significantly lower than that in control group(86.28±7.15VS40.55±4.25,P<0.01),Cardiomyocyte△ψm of CT-1 group was higher than that in hypoxia-reoxygeonation group (69.13±6.84VS40.55±4.25,P<0.05),while myocyte△ψm was lower than that in CT-1 group after processing with LY294002 blocker (50.13±5.82VS69.13±6.84, 54.51±6.62VS69.13±6.84, 54.51±6.62VS69.13±6.84,P<0.05),but myocyte△ψm in DMSO group was close to that in CT-1 group.4. PI3K protein and phosphorylated PI3K protein in cardiomyocyte: There was no significant difference among PI3K protein of groups. Phosphorylated PI3K protein increased in hypoxia-reoxygeonation group comparing with control group(0.475+0.032,P<0.05). Phosphorylated PI3K protein decreased obviously after processed with PI3K/GSK3βblocker (0.325+0.029 vs 0.986+0.093,P<0.05) ,however,there was no obvious difference between DMSO group and CT-1 group (0.963+0.048 vs 0.986+0.093,P>0.05), which indicate that CT-1 had activated PI3K pathway to increase the Phosphorylated PI3K protein.5. Expression of GSK3βprotein and phsphorylated GSK3βprotein in cardiomyocyte: There was not statistical difference about expression of GSK3βprotein in hypoxia and reoxygeonation. But expression of phosphorylated GSK3βprotein in hypoxia-reoxygeonation group decreased apparently comparing with control group while that in CT-1 group increased obviously comparing with hypoxia-reoxygeonation group. Phosphorylated GSK3βprotein was significant lower than that in CT-1 group after processing with PI3K/GSK3βblockers while there no obvious difference between auxiliary solvent group and CT-1 group, which indicates that CT-1 can induce GSK3βprotein phosphorylation in cardiomyocyte with hyoxia-reoxygeonation. PI3K/GSK3βblocker can block the effect of CT-1of causing GSK3βprotein phosphorylation .6. Expression of Caspases3 in cardiomyocyte: There was no significant expression of protein in each group. But after hypoxia-reoxygeonation ,the expression of Caspases3 in hypoxia-reoxygeonation group increase apparently comparing with control group(0.953±0.015VS0.329±0.017,P<0.05). while that in CT-1 group reduced obviously comparing with hypoxia-reoxygeonation group (0.412±0.034VS0.953±0.015,P<0.05) . After processing with PI3K/GSK3βblockers, the expression of Caspases3 increased obviously(0.746±0.026VS0. 412±0.034, P<0.05). However,there was no obvious difference between DMSO group and CT-1 group(0.433±0.031VS0.412±0.034,P>0.05), which indicated that CT-1 had activated PI3K pathway to inhibit the apoptosis of myocyte and PI3K/GSK3βblocker can block the protection effect of CT-1.Conclusion:1. CT-1 can protect cardiac cells against hypoxia-reoxygeonation injury, which include improving cell survival rate and reducing apotosis as well as release of myocardial damage enzyme. Besides, it can help myocardial cell beat regularly and powerfully.2. CT-1 can increase the mitochondrial membrane potential△ψm of cardiocyte and reduce the expression of Caspases3 to inhibit myocyte from apoptosis.3. CT-1 perform anti-apoptosis effection through PI-3K/GSK3βsignal pathway.
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