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Objective@#To investigate the effect of NACHT-LRR-PYD- containing proteins 3 (NLRP3) mediated pyroptosis in myocardial cells undergoing hypoxia/deoxygenation (H/R) injury.@*Methods@#In order to observe whether H/R-treatment could cause pyroptosis, H9c2 cells were divided into 2 groups randomly using the lottery method: control group(without H/R-treatment) and H/R group (in which the H9c2 cells were underwent H/R-treatment). In order to clarify the role of pyroptosis in H/R-injury, H9c2 cells were divided into 4 groups randomly using the lottery method: control group(in which the H9c2 cells were cultivated with normal medium); YVAD group(in which the H9c2 cells were pretreated with z-Val-Ala-Asp(Ome)-fluoromethylketone (Z-YVAD-FMK) 20 μm for 4 hours, then replaced with normal medium); H/R group(H9c2 cells underwent H/R-treatment); YVAD+H/R group (in which the H9c2 cells were pretreated with 20 μm Z-YVAD-FMK for 4 hours before H/R-treatment). To determine whether H/R-induced cell pyroptosis is associated with NLRP3, H9c2 cells were divided into 4 groups randomly using the lottery method: control group (in which cells were transfected with a control nonspecific siRNA); si-NLRP3 group (in which cells were transfected with NLRP3-targeting siRNA); H/R group(in which cells were transfected with a control nonspecific siRNA before H/R-treatment); si-NLRP3+H/R group(in which the H9c2 cells were transfected with NLRP3-targeting siRNA before H/R-treatment). Pore formation on cell membrane was detected by propidium iodide (PI) staining. Cell viability was detected by CCK8 reagent. The protein expression of Caspase-1, interleukin-1β (IL-1β) and NLRP3 was detected by Western blot.@*Results@#(1) The positive rate of PI staining ((26.46±5.15)% vs. (1.69±0.73)%,P<0.01), expression of NLRP3 (0.57±0.16 vs. 0.23±0.06,P<0.01), expression of Caspase-1 (1.07±0.13 vs. 0.37±0.08,P<0.01), and expression of IL-1β (0.38±0.08 vs. 0.16±0.05,P<0.01) were significantly higher in H/R group than in control group. (2)The cell vitality was significantly higher in YVAD+H/R group than in H/R group ((87.31±9.05)% vs. (73.30±7.19)%, P<0.05).The positive rate of PI staining was significantly decreased in YVAD+H/R group than in H/R group ((18.12±4.36)% vs. (26.45±4.60)%, P<0.05). The expression of Caspase-1 (0.72±0.12 vs. 1.07±0.15, P<0.05) and IL-1β(0.29±0.07 vs. 0.39±0.06, P<0.05) were significantly lower in YVAD+H/R group than in H/R group. (3) The cell vitality was significantly increased in si-NLRP3+H/R group than in H/R group ((85.46±7.71)% vs. (72.41±5.53)%, P<0.05). The positive rate of PI staining was significantly lower in si-NLRP3+H/R group than in H/R group ((18.22±4.20)% vs. (26.73±3.26)%, P<0.05). The expression of Caspase-1(0.87±0.07 vs. 1.15±0.15, P<0.05) and IL-1β(0.41±0.07 vs. 0.58±0.10, P<0.05) were significantly decreased in si-NLRP3+H/R group than in H/R group.@*Conclusion@#NLRP3 mediated pyroptosis is involved in H/R injury of myocardial cells.
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Objective@#To investigate the possible mechanism related to the protective effects of salvianolate in H9c2 cells underwent hypoxia/reoxygenation (H/R)-injury.@*Methods@#H9c2 cells were divided into four groups: control group, salvianolate group (S group), H/R group, and salvianolate+ H/R group(S+ H/R group), in which the H9c2 cells were pretreated with salvianolate before H/R-treatment.Apoptotic cells were detected by Tunel assays and AnnexinⅤ-FITC apoptosis detection kit.The intracellular ATP level, the change of mitochondrial membrane potential and the mitochondrial DNA oxidative damage were also determined in these groups.@*Results@#(1) The apoptosis rate of H/R group(26.36±5.14)% was significantly higher compared to control group(2.71±1.66)%(P=0.000 4), which could be significantly reduced in S+ H/R group(17.28±4.75)%(P=0.012 8 vs. H/R group , P=0.003 9 vs. control group). The ratio of AnnexinⅤ and PI double positive cells in H/R group(28.23±6.73)% was significantly higher compared to control group(3.53±2.83)%(P=0.001 1), which was significantly reduced in S+ H/R group(18.10±4.56)%(P=0.037 2 vs. H/R group, P=0.038 3 vs. control group). (2)The ATP level of H9c2 cells in H/R group(49.05±10.12)% was significantly lower than in control group 100%(P=0.000 5), which was significantly increased in S+ H/R group(68.67±13.32)%(P=0.019 9 vs. H/R group). Confocal microscope showed that red fluorescence was dominant in the control group, red fluorescence was significantly reduced, while green fluorescence was significantly increased in H9c2 cells of H/R group and the fluorescence ratio of red to green in H/R group((37.13±8.47)%) was significantly decreased compared to control group (100%, P=0.000 1), fluorescence ratio of red to green was significantly increased in S+ H/R group((63.77±12.32)% vs. H/R group, P=0.007 3). (3)The mitochondrial DNA oxidative damage in different groups: there was only few 8-hydroxyguanine (8-OHdG) expression, which marked as green, in control group, and 8-OHdG expression was significantly upregulated in H/R group, moreover, the 8-OHdG was co-localized with mitochondria.The expression of 8-OHdG was significantly lower in S+ H/R group compared to H/R group.@*Conclusion@#Salvianolate can reduce mitochondrial DNA oxidative damage, and protect mitochondrial function, thus inhibit myocardial cell apoptosis and eventually reduce the myocardial H/R-injury in H9c2 cells.
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Objective@#To investigate the effect and related mechanism of salvianolate on myocardial ischemia-reperfusion (I/R) injury in rats.@*Methods@#Thirty-six adult Sprague-Dawley rats were divided into three groups (n=12 each) using random number table method: control group (coronary artery was not ligated) , I/R group (myocardial I/R model was established by ligation and opening of left anterior descending coronary artery) , and salvianolate+I/R group (5 mg/kg of salvianolate was injected through the tail vein at the time of reperfusion) .Triphenyltetrazolium chloride (TTC) stain was utilized to measure the myocardial infarct size. The ELISA method was used to detect myocardial necrotic markers, including cardiac troponin T (TnT) , creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) . Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to analyses the levels of apoptosis. The levels of cleaved Caspase-3 protein were analyzed with Western blot.Cold luminescence method was used to detect the ATP level of myocardial tissue. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in myocardial tissue were detected by immunofluorescence.@*Results@#(1) The infarct size in control group, I/R group and salvianolate+I/R group were 0, (23.90±5.66) %, and (12.06±5.97) %, respectively (P<0.01 or 0.05) . (2) The TnT level was (0.04±0.03) , (16.96±2.80) , and (6.95±2.31) ng/ml, the CK-MB level was (43.6±23.5) , (1 135.8±180.6) ,and (390.3±121.5) U/L, the LDH level was (119.0±58.6) , (1 838.6±543.8) , and (631.6±228.3) U/L in control group, I/R group and salvianolate+I/R group, all significantly lower in salvianolate+I/R group than in I/R group (all P<0.01) . (3) The rate of TUNEL positive myocardial cells were (1.07±1.16) %, (21.36±4.11) %,and (13.30±3.67) % in control group, I/R group,and salvianolate+I/R group (all P<0.01) . (4) The cleaved Caspase-3 expression was 0.11±0.05, 0.84±0.20,and 0.43±0.09 in control group, I/R group, and salvianolate+I/R group (all P<0.01) . (5) The ATP level of myocardial tissue was (100.0±0.0) %, (34.2±9.2) %,and (62.1±18.0) % respectively in control group, I/R group, and salvianolate+I/R group (all P<0.01) . (6) There was almost no 8-OHdG expression in the myocardial tissue of control group. The expression of 8-OHdG in the myocardial tissue of I/R group was greater than that of the control group. The expression of 8-OHdG in the myocardial tissue of salvianolate+I/R group was less than that of the I/R group.@*Conclusion@#Salvianolate may alleviate myocardial I/R injury of rat through reducing the mitochondrial DNA oxidative damage, protecting mitochondrial function and inhibiting the apoptosis of myocardial cells.
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Objective To study the influence of ultralow‐frequency transcranial magnetic stimulus(TMS) on the cognitive a‐bility of rats with cerebral ischemia and its mechanism .Methods 60 healthy rats were divided into 4 groups(15 cases each):A (sham‐operation) ,B(model) ,C(TMS) and D(TMS+ H89) .The escape latency time ,times of passing through platform ,expression level of VEGF ,BDNF and nestin protein were compared among 4 groups .Results In the group A ,the escape latency time was (16 .31 ± 2 .33)s ,times passing through platform were (8 .02 ± 1 .76) times ;in group B ,which were (57 .14 ± 2 .89)s and (3 .15 ± 0 .88) times;in group C ,which were (29 .18 ± 1 .95)s and (5 .44 ± 0 .75) times ;in group D ,which were (45 .87 ± 2 .06)s and (4 .16 ± 1 .02) times .Compared with the group A ,the escape latency time in the group B ,C and D was significantly extended ,more‐over that in the group B was longer than that in the group D and C ,the differences were statistically significant (P<0 .05);the times of passing through platform decreased ,which in the group B was less than that in the group D and C ,the differences had sta‐tistical significance(P<0 .05) .The expression levels of VEGF ,BDNF and nestin had statistical differences among various groups (P<0 .05) .Conclusion Low‐frequency TMS can significantly improve the cognitive ability of rats with cerebral ischemia ,its effect is related to the expression of cAMP‐response element binding protein and its following genes(VEGF and BDNF) .
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Objective To investigate the possible mechanism of metoprolol on protecting against ischemia‐reperfusion in‐duced injury .Methods A total of 32 healthy 3-4 months male C57BL/6 mice were divided into four groups(n=8)as following :Sham‐operating group(control group);metoprolol group;ischemia‐reperfusion group(I/R group);metoprolol + I/R group .Myo‐cardial injury ,apoptosis ,cytochrome c release ,Caspase‐3 activity and calpain activity were determined in these groups .Results Al‐though there was no obvious changes in the regions at risk between I/R group and metoprolol + I/R group ,metoprolol pretreat‐ment significantly reduced the ratio of the infarct to risk regions(P<0 .05) .In the I/R group ,the rate of cardiomyocyte apoptosis , cytochrome c release ,as well as the activity of Caspase‐3 and calpain significantly increased compared to the control group(P<0 .01) .However ,these effects of I/R injury were alleviated by pretreatment with metoprolol .Conclusion metoprolol might protect against ischemia‐reperfusion induced injury by preventing calpain activation .
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BACKGROUND:Myocardial fibrosis following myocardial infarction is an important mechanism of ventricle reconstitution. However, there are few reports concerning effects of myocardial transplantation related to stern cells on this process. OBJECTIVE: To investigate the effects of auto-skeletal muscle satellite cells implanted into ischemic myocardium on myocardial fibrosis in rats with myocardial infarction and their mechanisms.DESIGN, TIME AND SETTING: The randomized controlled animal study was performed at the Third Research Room, Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA from July to September 2007. MATERIALS: A total of 45 Wistar rats, of both genders, weighing 150-200 g, were used in this study. Of them, 30 rats were used to establish models of myocardial infarction.METHODS: A total of 45 rats were assigned to 3 groups (n=15). Rats in the myocardial infarction group received ligation of the left anterior descending coronary artery to induce myocardial infarction. 2 weeks later, 0.2 mL serum-free M199 medium was infused into the juncture between infarct region and normal myocardium through multiple points. In the transplantation group, following model induction, 0.2 mL auto-skeletal muscle satellite cells in rats after 2-weeks in vitro culture were transplanted into the surrounding of infarct region. Rats in the sham operation group were not induced to create models, only injected with 0.2 mL saline in the heart anterior wall surrounding the left anterior descending branch through multiple points. MAIN OUTCOME MEASURES: Four weeks after injection, vascular endothelial growth factor mRNA and vascular endothelial growth factor protein expression in the ischemic myocardium was demonstrated. Capillary density changes in the ischemic myocardium were detected. Growth and proliferation of myocardial cells in the infarct region were observed using hematoxylin-eosin staining.RESULTS: Vascular endothelial growth factor mRNA and vascular endothelial growth factor protein expression was significantly decreased in the sham operation and myocardial infarction groups compared with the transplantation group at 4 weeks following satellite cell transplantation (P<0.01). Capillary density was greater in the myocardial infarction group compared with the sham operation group (P<0.05). Capillary density was significantly higher in the rat ischemic myocardium in the transplantation group compared with the sham operation and myocardial infarction groups (P<0.01). Hematoxylin-eosin staining demonstrated that myocardial morphology was normal in rats of the sham operation group, with clear structure, orderly myocardial fibrosis. There were no fibroblastaggregation and hyperplasia among myocardial fibrosis. Fibroblast hyperplasia and collagent formation were found in the rat myocardium in the myocardial infarction group, with disorderly myocardial structure. Myocardial cells with transverse striation and many nuclei were observed in the rat infarct region of the transplantation group, with orderly arrangement. Fibrous tissue was significantly less in the transplantation group compared with the myocardial infarction group.CONCLUSION: Satellite cells can proliferate and differentiate into striated muscle-like cells with flexible and systolic functions in the infarct region. Satellite cells secrete vascular endothelial growth factor and promote blood capillary hyperplasia in ischemic myocardium by autocrine and paracrine, which finally effectively inhibits fibrosis progress in the ischomic myocardium.
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Objective To investigate the effect of auto-skeletal muscle satellite cell implantation into ischemic myocardium on cardiac function and the mechanisms.Methods Approximately 10 7 to 10 9 muscle satellite cells(SCs)were cultivated in vitro.The left anterior descending(LAD)artery was ligated in Wistar rats to create myocardial infarction(MI).Some rats only underwent sham operation served as control.Two weeks after MI,autologous SCs,serum-free culture medium and sodium chloride injection were injected into ischemic myocardium of implantation rats(n=15),control rats(n=15)and myocardium around LAD of sham operation rats(SO,n=15),respectively.Four weeks after injection,hemodynamic parameters and cardiac function in all groups were evaluated by polygraph system,capillary density in the ischemic myocardium was demonstrated by immunohistochemical method,serum VEGF concentration was examined by ELISA,and the differentiated myofibers from SCs in the infarcted site were observed by pathologic examination and immunohistochemical method.Results Four weeks after injection,the SCs had progressively differentiated into striated muscle fibers in the myocardial infarction site,and immunohistochemical analysis confirmed their skeletal muscle origin.Compared with the SO rats,systolic blood pressure(SBP),diastolic blood pressure(DBP),mean ar-tery pressure(MAP),left ventricular systolic pressure(LVSP)and dp/dtmaxwere markedly decreased(P
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Objective To evaluate the role of transfected angiotensinⅡ(Ang Ⅱ) receptor AT1 anti-sense nucleotide (AT1A) in the expression of subtypes of AngⅡ receptor mRNA, synthesis of protein and nucleic acid in cardiomyocytes. Methods AT1 cDNA sequence (476 bp) was cloned with RT-PCR and reversely inserted into PcDNA3.1 (5.4 kb) to construct an intact plasmid containing AT1A (PAT1A). The plasmid was then transfected into the cultured cardiomyocytes and identified with RT-PCR and Western blot. The synthesis of protein and nucleic acid identified by 3H-Leu and 3H-TdR incorporation, and expressions of AT1 and AT2 mRNA by RT-PCR, were compared between transfected and nontransfected cardiomyocytes after being stimulated with 10-7 mol/L AngⅡ for 24 h. Results The plasmid PAT1A were successfully constructed. The AT1 mRNA and its protein were expressed significantly less in the transfected cardiomyocytes than in the control (P<0.01). In the transfected cardiomyocytes, AT1 mRNA expression was markedly decreased, but that of AT2 mRNA obviously increased (P<0.01) when compared with the nontransfected cardiomyocytes after stimulation for 24 h with AngⅡ 10-7 mol/L; no significant difference was found in 3H-Leu and 3H-TdR incorporation between them. Conclusion After the cardiomyocytes was tranfected with AT1A, the expression of AT1 mRNA was markedly suppressed，while AT2 mRNA up-regulated at the same time. Our results indicate that AT1A blocking can not effectively interrupt the Ang Ⅱ-induced synthesis of the protein and nucleic acid in cardiomyocytes.
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AIM: To investigate the involvement of calcineruin (CaN) signal pathway mediated by angiotension Ⅱ (Ang Ⅱ) in the myocardial remodeling mechanism in patients with congestive heart failure (CHF). METHODS: 39 patients of mitral valve disease with CHF were randomly selected and 30 cases of healthy persons were included as controls. Cardiac function parameters were measured by echocardiography. Concentration of Ang Ⅱ in plasma and myocardial tissues were determined by radioimmunoassay. Immunoprecitipation was used to assay the protein expression and phosphorylation CaN, nuclear factor of activated T cells (NFAT_3),zinc finger transcription factor (GATA_4) in myocardial tissues. The mRNA expression of ?-myosin heavy chain (?-MHC) was measured by RT-PCR. RESULTS: The Ang Ⅱ concentrations in patients with CHF were positively correlated with the parameters of the cardiac dialtion respectively, but negatively correlated with the paremeters of cardiac function. Compared to the control group, protein of CaN and GATA_4, phosphorylation of CaN, and ?-MHC mRNA expression in myocardial tissues in CHF groups were highly expressed and their expression were positively correlated to the levels of CHF, but the phosphorylation of NFAT_3 was negatively correlated with the levels of CHF. CONCLUSION: CaN signal pathway may play important roles in the myocardial remodeling in CHF.