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AIM:To study the effects of noninvasive delayed limb ischemia preconditioning ( NDLIP) on ani-mal cardiac function , myocardial morphology and myocardial apoptosis after myocardial infarction ( MI ) .METHODS:Healthy SD male rats [n=45, weighing (250 ±10) g] were randomly divided into 3 groups:MI group:the animal model of MI was established by surgical ligation of left anterior descending artery ( LAD) after 2 weeks;NDLIP group:after the success of the MI animal model , NDLIP was carried out every other day until the 4th, 6th and 8th weeks;sham group:as the negative control group , the animals were taken heart LAD threading but no ligation .All rats were fed conventionally .At the end of the 4th, 6th and 8th weeks, all rats were made ventricular intubation , and then the hemodynamic parameters were recorded .The blood samples were withdrawn from the abdominal aorta and the serum was separated via centrifugation . The serum contents of Bcl-2 and Bax were measured by ELISA .Left ventricular anterior wall was homogenized .The mito-chondrial respiratory chain complexes Ⅰ,Ⅱ,Ⅲand Ⅳin the myocardial tissues were detected by ELISA .RESULTS:At the end of the 4th, 6th and 8th weeks, compared with MI group, left ventricular systolic pressure in NDLIP group was significantly increased , while left ventricular end-diastolic pressure in NDLIP group was significantly decreased ( both P<0.05).Mitochondrial respiratory chain complexesⅠ, Ⅱ, Ⅲ and Ⅳ in NDLIP group were significantly increased (P<0.05).The serum level of Bcl-2 in NDLIP group was significantly increased and Bax level was reduced remarkably (both P<0.01) .CONCLUSION:NDLIP improves the hemodynamic indexes , promotes the mitochondrial respiratory function and inhibits cell apoptosis , thus improving the prognosis of MI .
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Aim To study the preventative effects of noninvasive delayed limb ischemic preconditioning ( NDLIP) on sudden cardiac death in rats with myocar-dial infarction. Methods Thirty healthy SD male rats weighting ( 250 ± 10 ) g were randomly divided into 3 groups:① myocardial infarction ( MI ) group: animal model of MI was established by making surgical ligation of animal LAD. ② MI plus NDLIP group: after the success of the animal model of MI, NDLIP was carried out every other day until 4th week. ③Sham group:as the negative control group, animals were taken heart LAD threading but no ligation. All rats were fed con-ventionally. At the end of 4 weeks, three groups of rats were administered with metaraminol ( 0. 2 mg · min-1 ) . ECG, drug cumulant of sudden death and death onset time were recorded. After sudden death, blood samples were withdrawn from abdominal aorta and serum was separated via centrifugation. ELISA method was used to measure serum caspase-3 , HSP70 and SOD concentration. Results While metaraminol led animal cardiac sudden death, the rats heart rate ( HR) kept declining with the increase of dosage of metaraminol during the administration period. Rat HR of MI+NDLIP group [ ( 479 ± 8 ) vs ( 416 ± 19 ) beat ·min-1 , ( 446 ± 32 ) vs ( 370 ± 20 ) beat · min-1 , (376 ± 53) vs (305 ± 29) beat·min-1, (307 ± 63) vs (244 ± 33) beat·min-1, (283 ± 45) vs (121 ± 35 ) beat · min-1 , P <0. 01 ] was markedly higher than that of MI group at 0 , 5 , 10 , 30 , 50 min before death. Compared with MI group, drugs cumulant to sudden death and death onset time of MI + NDLIP group [ ( 14. 58 ± 3. 03 ) vs ( 10. 76 ± 2. 73 ) mg, (72. 9 ± 15. 2 ) vs ( 53. 8 ± 13. 6 ) min, P <0. 01 ] were significantly increased. Compared with MI group, serum caspase-3 content of MI+NDLIP group was sig-nificantly reduced [ ( 2. 01 ± 0. 52 ) vs ( 2. 34 ± 0. 38 )μg·L-1 , P<0. 01 ]; HSP70 levels were remarkably increased [ ( 3. 01 ± 0. 58 ) vs ( 2. 70 ± 0. 43 ) μg · L-1 , P <0. 05 ]; SOD levels were significantly im-proved [(1. 99 ± 0. 65) vs (1. 70 ± 0. 58) mg·L-1, P<0. 01 ] . Conclusion NDLIP can prevent sudden cardiac death after myocardial infarction in rats, which may be mediated by reducing the myocardial cell apop-tosis, increasing protective protein expression and en-hancing antioxidant capacity.
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Aim To study the protective effects of sphingosine 1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation in reperfusion injury salvage kinase ( RISK ) signal path-way. Methods The cultured rat H9c2 cells were ran-domly divided into seven groups: ( 1 ) control group;(2) hypoxia/reoxygenation (H/R) group; (3) S1P group;(4) S1P+LY294002 group(S1P+LY); (5) LY group; ( 6 ) S1 P +PD98059 group ( S1 P +PD );(7) PD group. The viability of H9c2 cells was detec-ted using MTT method. The content of MDA in the cultured medium and the activity of T-SOD and Mn-SOD were measured with colorimetry. The concentra-tion of intracellular free calcium ion was detected by confocal microscopy. The rate of cell apoptosis was de-termined by flow cytometric analysis. Western blot was used to assess phosphorylation of Akt and ERK1/2 in H9c2 cells. Results Compared with the H/R group, S1P significantly increased vaibility of cells, lowered the rate of apoptosis, decreased the content of MDA in the culture medium, increased the activity of T-SOD and Mn-SOD, reduced concentration of intracellular calcium and increased the phosphorylation of Akt and ERK1/2 . When added LY294002 or PD98059 , the effects of S1P above were inhibited. Conclusion S1P protects H9 c2 cells against hypoxia/reoxygenation inju-ry. The protection of S1P was inhibited by LY294002, the inhibitor of PI3 K/Akt and PD98059 , the inhibitor of ERK1/2 . S1 P protects H9 c2 cells against hypoxia/reoxygenation injury via RISK pathway.
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Aim To construct a lentiviral low expres-sion of miR-139-5 p vector and validate its expression efficiency in H9c2 cells. Method Target sequence was designed according to the sequence of rat miR-139-5p. Oligonucleotide duplex was synthesized and cloned into the lentiviral vector pGC-LV. The recombi-nant lentiviral vector, pHelper 1. 0, and pHelper 2. 0 were co-transfected into 293T cells, packaging virus. Then H9 c2 cells were infected with the supernatant containing lentiviral particles, and its infection effi-ciency and miR-139-5 p expression were determined by fluorescent microscope and real-time quantitative PCR, respectively. Results A lentiviral low expression of miR-139-5p vector was successfully constructed. The infection efficiency in H9c2 cells reached over 95%, and the relative expression of miR-139-5p was significantly down-regulated. Conclusion The lentiviral low expression of miR-139-5p vector is successfully constructed, and the expression of miR-139-5p in infected H9c2 cells is inhibited effectively.
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Aims To investigate the protective effects of noninvasive limb ischemic preconditioning (LIPC) on myocardial ischemia-reperfusion (I /R)injury,and to explore the mechanism.Methods Healthy male Wistar rats were divided randomly into I /R,I /R +LIPC,I /R +5-Hydroxydecanoate (5-HD)and I /R +LIPC +5-HD groups.The I /R +LIPC and I /R +LIPC-5-HD groups of rats were subjected to three cy-cles of LIPC induction per day with 5 min of reperfu-sion after occlusion for 5 min at the left hind limb for 3 days.All rats were subjected to myocardial I /R injury on the fourth day.The I /R +5-HD and I /R +LIPC-5-HD groups of rats were given the inhibitor of ATP-sen-sitive potassium channel 5-HD before and during myo-cardial I /R injury. Results Compared with I /R group,LIPC reduced myocardial infarct size (P <0.05),lowered cardiocyte apoptosis index and Fas, FasL positive cell number (P <0.01 ),increased the reduced nitric oxide (NO)/endothelin (ET)-1 ratio (P <0.05)in serum in I /R +LIPC group.5-HD a-bolished the protective effects induced by LIPC in I /R+LIPC-5-HD group.Compared with normal myocardi-al tissue,expression of mir-30a-3p was increased in I /R group (P <0.01 )and was decreased in LIPC group (P <0.01 ).Conclusion LIPC alleviates myocardial I /R injury and improves endothelial function. The mechanism may be related with the opening of ATP-sensitive potassium channel,regulating the balance be-tween NO and ET-1 and decreasing the expression of myocardial mir-30a-3p.
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Objective To establish a flow-cytometric method to detect microvesicles (MVs) in rat peripheral blood, and to detect platelets-derived MVs (PMVs) and endothelial cell-derived MVs (EMVs) in blood from ischemic precondition-ing (IPC) treated rats. Methods Blood was withdrawn from rat abdominal aorta and anticoagulated with sodium citrate. Platelets-free plasma (PFP) was isolated through two centrifugations at room temperature. PFP was incubated with FITC-conjugated mouse anti-rat CD61 or PE-conjugated mouse anti-rat CD144. Standard beads in diameter of 1 and 2μm were used for calibration and absolute counting, respectively. Analysis was performed on flow cytometer. Results When 3.5%so-dium citrate was mixed with blood at volume ratio of 1∶4, clear supernatant was collected after centrifugation. Signals of parti-cles smaller than 1μm accounted for more than 99%of overall signals. PMVs and EMVs were CD61 positive and CD144 positive, respectively. Their diameters were both smaller than 1 μm. The concentration of PMVs and EMVs in peripheral blood from IPC treated rats was (4 053±1 987)/μL and (4 870±825)/μL, respectively. Conclusion The method for MVs de-tection by flow cytometry was successfully established and optimized, and verified through detecting PMVs and EMVs in pe-ripheral blood from IPC treated rats.
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Aim To investigate the protective effects of nicorandil, glutamine and metoprolol used in single and combination on the antiapoptosis ability of cardio-cytes and the expression of HSP70 after myocardial is-chemia/reperfusion injury in rats. Methods Male Wistar rats were divided randomly into seven groups:( 1 ) Sham group:in which the coronary artery was not roped;(2) I/R group:in which only 30 min ischemia and 120 min reperfusion of LAD was executed; ( 3 ) Nicorandil group; ( 4 ) Glutamine group; ( 5 ) Meto-prolol group; ( 6 ) Nic + Glu + Met ( NGM ) group;(7) low dose of Nic+Glu+Met ( NGML) group. In the pharmacological precondition groups, correspond-ing drugs were administered 30 min before I/R proto-col, and the initial drug dose in each group was 1 mg ·kg-1 . Myocardial apoptotic rates were measured with TUNEL ( terminal dexynucleotidyl transferase-mediated dUTP nick end labeling) , and the expression of apop-tosis related proteins-Bcl-2/Bax and protective pro-tein-HSP70 was detected by Western blot. ResultsIn I/R group, the apoptotic rate in ischemic region was very high ( 36.9% ± 10.3%) , and Bcl-2/Bax was very low ( 0.14 ±0.08 ) . Compared with I/R group, the apoptotic rate of pharmacological precondition groups was decreased significantly ( P <0.01 ) , and the ratio of Bcl-2 to Bax was raised ( P<0.01 ) . Com-pared with Sham group, the expression of HSP70 in-creased significantly ( P<0.01 ) in I/R group. Com-pared with IR group, the expression of HSP70 of phar-macological precondition groups was elevated ( P <0.01 ) , and the expression in NGM group was higher than in the single drug group ( P<0.01 ) . Conclusion Compared with single therapy after I/R in rat, the combination therapy of nicorandil, glutamine, meto-prolol can decrease the apoptotic rate, and the expres-sion of apoptosis related proteins is developed. In the mean time, the expression of protective protein HSP70 is elevated.
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Aim To investigate the protection of ramipril,BQ-123 and their combination against myocardial ischemia/reperfusion(I/R)injury in vivo in anesthetized rats,and to explore the mechanism of action of drugs on myocardial oxidation-antioxidation system.Methods Healthy male Wistar rats were divided into 5 groups randomly,sham operated(Sham)group,I/R group,ramipril(RAM)group,BQ-123(BQ)group and ramipril and BQ-123(R&B)group.All groups but not sham were subjected to I/R procedure.Twenty four hours before ligation,ramipril(1 mg·kg~(-1))was intragastrically administered to rats in RAM and R&B groups.The equal volume of normal saline was given to rats in other groups.BQ-123(10 μg·kg~(-1)· min-1)was infused intravenously from 10 min before ligation to the end of 30 min ischemia to rats in BQ and R&B groups.The equal volume of normal saline was given to other groups.HR,MAP and the change of ST-segment were observed;ventricular arrhythmias were monitored during ischemia;the infarct size was examined by TTC staining;the activity of myocardial T-SOD,Mn-SOD,CAT and the content of MDA were detected by spectrophotometer.Results Compared with I/R group,the elevation of ST-segment was decreased,onset of VPC and VT was delayed,duration of VPC and VT was shortened,incidence of VPC,VT and VF was decreased,IS and IS/AAR were improved,activity of T-SOD,Mn-SOD and CAT was increased,the content of MDA was decreased in RAM,BQ and R&B groups.Compared with RAM and BQ alone group,onset of VPC and VT,duration of VPC and VT,size,activity of T-SOD and Mn-SOD and content of MDA were changed dramatically in R&B group.Conclusions Ramipril,BQ-123 and the combined use of these two agents protected myocardium from I/R injury in vivo.The protective effects of the combination on delaying onset of VA,shortening duration of VA,decreasing infarct size and content of MDA,and increasing activity of SOD are better than those of using ramipril or BQ-123 alone.
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As a powerful endogenous protection,myocardial ischemic preconditioning and postconditioning have cardioprotection against ischemia reperfusion injury and cardiopretection against reperfusion injury respectively.They perhaps share some mechanisms during reperfusion,such as reduction of the production of oxygen free radical,activation of adenosine receptors,recruit of reperfusion injury salvage kinases,opening of the mitochondrial KATP channel,and inhibition of the mitochondrial permeability transition pore.These common targets may provide new strategy for development of drugs against reperfusion injury.
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The important progress of the relationship between genomics and cardiovascular diseases has elucidated several abnormalities of genes leading to familial cardiomyopathy and ion channel diseases.Investigations of genomics show that the difference including resistance and susceptivity to diseases among human is determined by over 3 million base pairs of all 3,000 million.Single nucleotide polymorphism plays an essential role in elucidating hereditary cardiovascular diseases.