Angiotensin Ⅱ stimulates the expression of NADPH oxidase subunit p47phox mRNA in kidney in a rat model of hyperoxaluria

AIM: To investigate the roles of angiotensin Ⅱ and NADPH oxidase in the development of renal oxidative stress (OS) in a rat model of hyperoxaluria. METHODS: Animal model of hyperoxaluria was established in adult male Sprague-Dawley rats by administration of 0.8% ethylene glycol (EG) in drinking water for 4 weeks. Simultaneous treatment with apocynin (0.2 g·kg-1·d-1) or losartan (30 mg·kg-1·d-1) by intragastric administration were performed in rats, respectively. At the end of the study, markers for the state of oxidative stress (OS), urinary 8-IP and the enzymatic activity of superoxide dismutase (SOD) in kidney homogenates were assessed. The concentration of angiotensin Ⅱ in kidney homogenates was determined using radioimmunoassay method. Expression of NADPH oxidase subunit p47phox in kidney was localized and evaluated by immunohistochemistry and real time-PCR, respectively.RESULTS: p47phox expressed widely in the kidneys of this rat model, including renal cortex, inner medulla and outer medulla. Compared with the control, OS developed significantly in rats received EG, with increased expression of p47phox mRNA in kidneys. Renal angiotensin II also increased significantly. Treatment with apocynin or losartan significantly reduced the excretion of urinary 8-IP, restored the SOD activity, with decrease in the expression of p47phox mRNA in kidney, but the levels of those OS markers in apocynin or losartan treated rats were still higher than those in normal controls. CONCLUSION: Results suggest that renal Ang II and its stimulation of NADPH oxidase may partially account for the development of OS in kidney in this rat model of hyperoxaluria.     

Effect of erythropoietin on expression of myocardial NADPH oxidase in pressure overload rats

AIM:To explore the effect of erythropoietin （EPO） on the expression of myocardial NADPH oxidase （Nox） in the pressure overload rats. METHODS:Male SD rats (n=36) were used to establish a pressure overload myocardial hypertrophy model by abdominal aorta ligation. The animals were divided into model group, control group (sham, without narrowing abdominal aorta, the rest of the operation was the same as the model) and recombinant human erythropoietin (rhEPO) treatment group (intraperitoneal injection of rhEPO postoperatively, 4 000 U/kg, twice a week). After 8 weeks, the cardiac ultrasound imaging and hemodynamic evaluation were conducted to determine the cardiac functions. Masson staining was used to observe the degree of myocardial fibrosis. The expression of Nox2 and Nox4 at mRNA and protein levels was detected by real-time quantitative PCR and Western blotting. The protein levels of myocardial inflammatory factors CD45, F4/80 and TGF-β were determined by Western blotting. RESULTS:Compared with model group, the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic pressure (LVESP) and left ventricular pressure maximum rising and falling rates (±dp/dtmax) increased significantly in EPO treatment group (P<0.01). At the same time, left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD) and left ventricular end-diastolic pressure (LVEDP) were decreased in EPO treatment group (P<0.01). EPO reduced the degree of myocardial fibrosis caused by pressure overload (P<0.01) and decreased the expression of Nox2 and Nox4 at mRNA and protein levels in the myocardium (P<0.05 or P<0.01), and reduced the protein expression of myocardial inflammatory factors CD45, F4/80 and TGF-β. CONCLUSION: EPO inhibits rat myocar-dial fibrosis induced by pressure overload, improves heart functions by decreasing NADPH oxidase activity and inhibiting myocardial oxidative stress levels and myocardial inflammatory reaction.     

Expression of endostatin in ischemic myocardium of myocardial infarction rats

AIM: To study the expression of endostatin in ischemic myocardium of myocardial infarction (MI) rats in various periods and the correlation with VEGF expression and microvascular density (MVD).METHODS: Thirty-two male Sprague-Dawley rats after myocardial infarction were randomly divided into 7, 14, 21 and 28 days group.The sham group was normal control group (eight rats in each group).The expression of endostatin, VEGF and MVD in ischemic myocardium were observed by immunohistochemistry.RESULTS: The expression of endostatin significantly increased in the ischemic myocardium after MI, peaked at 7 days, then gradually decreased at 14, 21 and 28 days.The endostatin level at 28 days was the same as the shams.The changing trends of expression of endostatin in ischemic myocardium after MI were similar to that of VEGF and were significantly correlated with the MVD.CONCLUSION: The expression of endostatin increased in ischemic myocardium of myocardial infarction rats.The changing trends of endostatin were similar to that of VEGF and positively correlated with the MVD.These data suggest that endostatin may modulate ischemic myocardium angiogenesis after myocardial infarction.     

Effect of angelica on myocardial fibrosis post myocardial infarction in rats

AIM: To investigate 1) the role of transforming growth factor-β₁ (TGF-β₁) and macrophage infiltration during the development of myocardial fibrosis (MF) in rats after myocardial infarction (MI);and 2) mechanisms of MF post-MI and the inhibitory effect of angelica.METHODS: Sprague-Dawley (SD) rats were subjected to MI by ligating the left anterior descending coronary artery.The animals were randomly divided into three groups: sham, MI and MI+angelica.After 24 hours of ligation, rats received angelica (20 mL·kg^-1·d^-1, ip) or saline.Left ventricular hemodynamics were measured and rats were killed at week 1, week 2 and week 4, respectively.Collagen content, macrophage infiltration and TGF-β₁ expression were examined in the non-infarcted area.RESULTS: ① In MI group, the numbers of macrophage and TGF-β₁ expression were significantly upregulated compared to sham at week 1 post-MI and remained elevated at week 4 (P<0.01).Angelica significantly decreased macrophage infiltration and TGF-β₁ expression (P<0.01 vs MI).② Collagen content was increased significantly in MI group compared to sham at week 2 and week 4 (P<0.01), and decreased in MI+angelica group (P<0.05 vs MI).③ Cardiac function was markedly decreased post-MI in MI group (P<0.01), and improved at week 4 in MI+angelica group (P<0.05).CONCLUSION: In MF post-MI, angelica may have an antifibrotic effect by decreasing macrophage infiltration and TGF-β₁ expression, by which reactive myocardial fibrosis is reduced, and cardiac function is improved.     

Autoimmune reaction mediates myocardial injury after acute myocardial infarction in rats

AIM: The study was designed to explore the autoimmune mechanism of myocardial injury and ventricular remodeling after acute myocardial infarction (AMI). METHODS: An experimental animal model of AMI was adopted in Wistar rats. After 6 weeks, splenocytes were freshly transferred to syngeneic inbred rats. Four weeks later, these recipient rats were anesthetized for hemodynamics analysis by catheter technique. Serum antibody against cardiac myosin heavy chanin (MHC) was screened by ELISA. Histopathological studies were performed on all hearts. The phenotypes of T lymphocytes in myocardium were analyzed by histocytochemistry stain. RESULTS: Histopathological studies showed the lymphocytes infiltration in non-infarction myocardium in AMI rats and the organ specific inflammation of myocardium in all succedent recipient (AMI-T) rats. Histocytochemistry stain revealed the predominant CD4+T cells infiltration in myocardium. The antibody against MHC was examined in 8/22 cases of AMI rats and AMI-T rats, but none in sham-T rats. The left ventricular dysfunction was found in AMI-T rats, which was characterized by slight decline of +dp/dtmax. CONCLUSIONS: The study showed inflammatory response of non-infarction myocardium in AMI rats and demonstrated the lymphocytes-mediated myocardial injury and cardiac dysfunction by adoptive transfer of splenocytes of AMI rats. The autoimmune-mediated myocardial injury might be a novel mechanism of ventricular remodeling after AMI.     

Effects of pioglitazone on myocardial energy metabolism and hemodynamics in rats with myocardial infarction

AIM: To observe the effects of pioglitazone on myocardial energy metabolism and hemodynamics in rats with heart failure after myocardial infarction (MI). METHODS: The model of MI was established by ligation of left anterior descending artery. The 20 surviving rats were randomly divided into MI group (n=10) and pioglitazone intervention group (P group,n=10, pioglitazone 3 mg·kg^-1·d^-1 orally). The sham-operated rats (SH, n=10) served as controls. Hemodynamic parameters were measured. The ratio of left ventricular weight to body weight (LVW/BW) and the ratio of right ventricular weight to body weight (RVW/BW) were calculated after 8-week treatment. The expression of PPARγ was examined by Western blotting. Mitochondrial respiratory function was determined by Clark oxygen electrodes. The size of adenine acid pool (ATP, ADP and AMP) in mitochondria was measured by HPLC. The adenine nucleotide translocator(ANT) activity was detected by the atractyloside-inhibitor stop technique. RESULTS: Compared with SH group, the protein expression of PPARγ was significantly decreased in MI group (P<0.01). The mitochondrial respiratory activity, the transport activity of ANT and the high-energy phosphate content were decreased in MI group (P<0.01), and the hemodynamic parameters were in disorder (P<0.01). Compared with MI group, the protein expression of PPARγ in P group was significantly increased. The mitochondrial respiratory activity, the high-energy phosphate content, the transport activity of ANT were improved (P<0.01). However, the hemodynamic parameters were not significantly changed.CONCLUSION: Pioglitazone increases the protein expression of PPARγ and improves myocardial energy metabolism in the development of heart failure in the rat model of myocardial infarction, but dose not change the hemodynamic parameters significantly.     

Changes of cytokine and collagen in the myocardial remodeling after acute myocardial infarction in rats

AIM:To clarify the relationship between the cytokine and collagen in myocardial remodeling after acute myocardial infarction (MI) in rats. METHODS:In MI group, Wistar rats were undergone acute myocardial infarction by ligation of the anterior descending coronary artery. Sham operation was made in rats as control. The mRNA expression of collagen and cytokines such as TNF-α and TGF-β1 in infract and non-infarct region of left myocardium were detected by RT-PCR at different time point (3 d, 1 and 4 weeks). RESULTS:Collagen type Ⅰ and Ⅲ elevated as well as the TNF-α and TGF-β1 in the MI group at 3th day. Expression of collagen type Ⅰ and Ⅲ were higher in the infarct region than that in the non-infarct region even at 4 weeks. TNF-α and TGF-β1 peaked at 1 week and declined gradually to the baseline, which was still higher than those in control group (P<0.01). Correlation analysis revealed that expressions of TNF-α and TGF-β1 were positively correlated with the collagen type Ⅰand Ⅲ (P<0.01). CONCLUSION:Cytokines participate in the myocardial remodeling after MI. Interfering with expression of cytokines may be the potentially preventative method in the myocardial remodeling.     

MicroRNA profile analysis of ischemic myocardial tissues from rats with acute myocardial infarction

AIM：To identify differentially expressed microRNAs (miRNAs) in ischemic myocardial tissues from the rats with acute myocardial infarction (AMI) by miRNA array technique, and to predict their targets and analyze their functions using bioinformatics. METHODS：The rat models of AMI (n=3) were prepared by ligaturing the left anterior descending coronary artery (LAD) of Wistar rats. Electrocardiogram and blood pressure were detected during the operation, and the myocardial infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Ischemic myocardial tissues were isolated from the infarct area 4 h after ischemia. The same procedure in sham group (n=3) was performed except for ligaturing LAD. Total RNA was extracted from ischemic and normal myocardial tissues. miRNA was isolated from total RNA, labeled with Cy3 and hybridized on miRNA array. Real-time PCR was applied to verify the reliability of miRNA array results. The targets of differentially expressed miRNAs were predicted and their functions were analyzed by bioinformatics. RESULTS：Rat model of AMI was successfully prepared and verified by electrocardiogram detection, blood pressure measurement and pathological observation. Compared with sham group, microarray screening showed that total 11 AMI-related miRNAs were selected, including 6 up-regulated and 5 down-regulated. Three of them (rno-miR-181c, rno-miR-146b and rno-miR-208) were related to the cardiovascular functions, while the functions of the others (rno-miR-672*, rno-miR-743b, rno-miR-128, rno-miR-138-1*, rno-miR-336, rno-miR-138-2*, rno-miR-325-3p and rno-miR-3572) were unknown and might be novel AMI-related biomarkers. Parts of the miRNA targets were also related to the cardiovascular functions. CONCLUSION：Differentially expressed miRNAs in AMI rats may serve as novel biomarkers for diagnosis of AMI and potential targets for treatment of AMI.     

Salvia extract promotes angiogenesis of myocardium in rats with myocardial infarction

AIM: To determine the effect of salvia extract on angiogenesis of the myocardium in the rats with myocardial infarction (MI) and to analyze its possible mechanism. METHODS: Left coronary artery of Sprague-Dawley rats was ligated to establish a MI model. The rats were randomly divided into MI model group, 3 different dose groups of salvia (10, 20 and 40 mg·kg^-1·d^-1), and sham operation group. Each group consisted of 8 rats. The rats in all treatment groups were orally administered with the salvia extract, and the rats in MI group and sham operation group were fed with the same volume of saline. The rats were sacrificed 4 weeks later. The hemodynamic changes of the rats were determined, and the segmental heart samples were used for morphological observation by hematoxylin and eosin staining, Masson staining, or electron microscopic analysis. The expression of vascular endothelial growth factor (VEGF) and cluster of differentiation 34 (CD34) was analyzed according to immunohistochemistry. RESULTS: Compared with sham operation group, the morphological changes of the myocardium in MI group were disordered, part of myocardial cell outline disappeared, and obvious fibrosis in the necrosis myocardial tissue and fuzzy or disappearing microvascular ultrastructure were also observed. Compared with MI group, the number of new microvessels in all the treatment groups increased obviously, and the morphological changes of the endothelial cells were relatively complete according to electron microscopy. Compared with sham operation group, the protein expression of VEGF and CD34 in the cytoplasm of the myocardial tissues in MI group increased only a little. Compared with MI group, the protein expression of VEGF and CD34 in the cytoplasm of the myocardial tissues in all treatment groups increased significantly (P<0.01). CONCLUSION: Salvia extract obviously promotes angiogenesis of the myocardial tissues in the rats after myocardial infarction.     

Short-term cardioprotective effects of trimetazidine on acute myocardial infarction in rats

AIM: To observe the myocardial protective effects of trimetazidine on myocardial infarction (MI) in Sprague-Dawley (SD) rats. METHODS: Ninety SD rats were randomly assigned to 3 groups (n=30 each): myocardial infarction group (MI group), MI+trimetazidine group (MT group) and sham group (S group). By permanently ligating the left anterior descending artery, the MI model was set up in the rats in MI group and MT group. Before and after setting up the MI model, normal saline was given to the rats in MI and S group by gavage. On the other hand, trimetazidine (3 mg/kg,twice per day) was given to the rats in MT group by gavage. At 8 h, 24 h and 48 h after applying trimetazidine, the serum level of cardiac troponin I (cTnI) was measured. At the 1st week, 2nd week and 4th week after treated with trimetazidine, the size of myocardial infarction, the maximum rising rate of the left ventricular systolic pressure (+dp/dt_max) and the maximum descending rate of the left ventricular diastolic pressure (-dp/dt_max) were measured. Also at the 1st week after applying trimetazidine, the cardiomyocyte apoptotic index was detected. RESULTS: Compared with MI group 2 weeks after applying trimetazidine, +dp/dt_max significantly increased in MT group , and -dp/dt_max also significantly increased in MT group . Four weeks after applying trimetazidine, +dp/dt_max significantly increased in MT group , and -dp/dt_max also significantly increased in MT group . At 8 h and 48 h after applying trimetazidine, no statistically significant difference (P＞0.05) of serum cTnI between MI group and MT group was observed. However, at 24 h after applying trimetazidine, the serum level of cTnI decreased in MT group as compared with MI group . Aditionally, trimetazidine significantly decreased the infarction size of myocardium in MT group (0.248±0.052) as compared with MI group (0.362±0.082, P<0.01). CONCLUSION: Trimetazidine has short-term cardioprotective effects on the rats with acute MI by improving myocardial systolic and diastolic functions, reducing infarct size and inhibiting apoptosis.     

Correlation between myocardial interstitial collagen remodeling and changes in hemodynamics in rats with myocardial infarction

AIM: To study the relationship between cardiac extracellular matrix remodeling and cardiac function after myocardial infarction. METHODS: We observed sequential changes in collagen contents and collagen Ⅰ/Ⅲ ratios in infarct zone (IZ) and non-infarct zone (NIZ) and their relationship to the parameters of left ventricular systolic and diastolic function in the rat model of myocardial infarction induced by ligation of left main coronary artery. RESULTS: Collagen conteants in IZ and NIZ after 3d of myocardial infarction were significantly higher than those in sham group at corresponding time (P<0.05, P<0.01). Collagen Ⅰ/Ⅲ ratio in IZ decreased on day 3, significantly increased after 7 d (P<0.01). Collagen Ⅰ/Ⅲ ratio in NIZ increased significantly afte14 d. Correlated analysis between collagen contents in IZ or NIZ and collagen type Ⅰ/Ⅲ ratio and maximal ascending velocity (+p'_max) or maximal descending velocity of the left ventricular pressure (-p'_max) was performed and the negative correlation between collagen contents in NIZ and +P'_max (r=-0.589, P>0.05) and -P'_max (r=-0.788, P<0.01) was found. Collagen content in IZ positively correlated to the +P'_max (r=0.70, P<0.50), but not to -P'_max (r=-0.29, P>0.05). Collagen type Ⅰ/Ⅲ ratios in NIZ correlated negatively to the +P'_max (r=-0.504, P>0.05) and -P'_max (r=-0.545, P>0.05), but there were no relationship between collagen type Ⅰ/Ⅲ ratios in IZ and +P'_max or -P'_max in IZ. CONCLUSION: Collagen deposition in IZ after myocardial infarction was of benefit to improvement of systolic function. Collagen deposition in NIZ was harmful to systolic and diastolic function.     

Expression of angiogenic factors in myocardial tissue of diabetic rats

AIM：To observe the expression of angiogenesis factors in the myocardial tissue of streptozotocin-induced diabetic rats. METHODS：The diabetic rat model was induced by intraperitoneal injection of streptozotocin. After 12 weeks, the cardiac function was measured by MPA cardiac function analysis system. The myocardial collagen volume fraction (CVF) was assessed by Masson staining. The capillary vessels was quantified as the ratio of capillary to myocyte (C/M) using CD31 immunostaining. The expression levels of vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1, endostatin and Ang-2 were observed by Western blotting. RESULTS：Compared with normal control group, the left ventricular end-diastolic pressure (LVEDP) was evidently increased (P<0.01), but left ventricular pressure rise maximum rate (+dp／dtmax), left ventricular pressure decrease maximum rate (-dp／dtmax) and the ratio of capillary/myocyte (C/M) were significantly decreased (P<0.05). The CVF and the expression level of endostatin were significantly increased, whereas the expression levels of VEGF and Ang-1 evidently decreased (both P<0.05) in diabetic rats. However, no marked difference in the expression of Ang-2 between the 2 groups was observed (P>0.05). CONCLUSION：Imbalances between the angiogenic factors (VEGF and Ang-1) and anti-angiogenic factors (endostatin) may play an important role in the pathogenesis of diabetic cardiomyopathy.     

Effect of Shenshuguanxin granula on coronary circulation in rats with myocardial infarction

AIM：To explore the effect of traditional Chinese medicine Shenshuguanxin granula on coronary circulation in a rat model of myocardial infarction (MI). METHODS：SD rats (n=50, SPF grade) were randomly divided into 5 groups (n=10):sham group, MI group, and high-dose, middle-dose and low-dose Shenshuguanxin granula treatment groups. The rat MI model was established by ligation of the coronary artery. The cardiac markers, small and medium-sized blood vessels ［microvessel count (MVC) value］ in the infarct zone, and platelet endothelial cell adhesion mo-lecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) expression in the infarct border zone were measured. RESULTS：After 4 weeks of coronary artery ligation, the significant increases in MVC in the infarct zone, and the expression of PECAM-1 and VEGF in the infarct border zone were detected compared with sham group (P<0.05). The differences of cardiac markers between MI group and other groups were insignificant (P>0.05). CONCLUSION:Shenshuguanxin granula improves coronary circulation in the rats with myocardial infarction by increasing the expression of PECAM-1 and VEGF, and promoting small and medium-sized angiogenesis.     

Effects of granulocyte colony-stimulating factor on the myocardial infarction in experimental rats

AIM: To investigate the effects of granulocyte colony-stimulating factor (G-CSF) on the myocardial infarction in experimental rats. METHODS: Rats were randomly divided into GT group and saline control group (SC).The rats of GT group were treated with G-CSF (10 μg/kg) once a day subcutaneously for 5 days and those of SC group were received saline.On the third day, both groups were injected with isoprenaline (ISO) interaperitoneally to develop acute ischemic model. The hearts were harvested from 2 weeks to 4 weeks after administration of ISO for histopathological examination. RESULTS: Compared with saline control group, G-CSF treatment group significantly reduced the scar size (P<0.05). We also found the regeneration of myocytes, smooth muscle and endothelial cells. CONCLUSION: G-CSF treatment could be benefical to the regeneration of infarcted myocardium and significantly reduce scar size and it could be used for therapeutic intervention of the acute myocardial infarction.     

Combinational effects of trimetazidin and parecoxib on acute myocardial infarction in rats

AIM: To investigate the protective effects and mechanisms of combinational use of trimetazidine(TMZ) and parecoxib sodium on acute myocardial infarction (AMI) in rats. METHODS: Sixty-six Sprague-Dawley rats were randomly divided into 5 groups: sham group; AMI group; AMI+TMZ group; AMI+parecoxib group; AMI+TMZ+parecoxib group. All rats were sacrificed and cardiac functions (HR, LVSP, LVEDP, +dp/dt_max,-dp/dt_max) were measured with a Pclab-3804 biological signal processing system on the 8th day. The infarct size in each group was checked up by TTC staining method. RT-PCR was employed to detect the bax mRNA and bcl-2 mRNA. The protein levels of COX-2, Bax, Bcl-2 and cleaved caspase-3 in myocardium were determined by Western blotting. The activity of caspase-3 in each group was measured by colorimetric assay kit, and the apoptotic rates were detected with DNA ladder kit.RESULTS: Compared with sham group, increased expression of COX-2 protein (P<0.01) was observed in AMI group. The expression of COX-2 protein in parecoxib group was lower than that in AMI group (P<0.01). Compared with AMI group, the combinational use of trimetazidin and parecoxib improved contractile functions (LVSP and +dp/dt_max), reduced the infarct size and lowered the apoptotic rates remarkably. Specifically, the combinational use of trimetazidin and parecoxib showed better effects than use of trimetazidin or parecoxib alone. Reduced expression of Bax/Bcl-2 mRNA and protein, the reduced caspase-3 activity and cleaved caspase-3 expression were also found in combinational group as compared with other groups (P<0.05).CONCLUSION: The combinational use of trimetazidin and parecoxib effectively improves cardiac functions and reduces infarct size. The mechanism of the protective effect is probably associated with inhibiting apoptosis of cardiac myocytes.     

Effect of fluvastatin on left ventricular remodeling after myocardial infarction in rats

AIM: To clarify the protective effect of long-term administration of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor fluvastatin on ventricular remodeling after myocardial infarction (MI) in rats and its mechanisms. METHODS: Myocardial infarction were established by ligated left coronary anterior artery in SD rats, 24 hours after the operation, the survival rats were treated by gavage fluvastatin (20 mg·kg^-1·d^-1) or distilled water for 8 weeks. Doppler echocardiography, homodynamic and cardiac histomorphometry were used to assess the ventricular remodeling and cardiac function. The plasma levels of total cholesterol (Tch), creatinine (Cr), glutamic-oxal (o) acetic transaminase (AST), lipid peroxidation (LPO), glutathione perioxidase (GSH-PX), nitrogen monoxide (NO₂^-/NO₃^-) were detected. RESULTS: The Tch, Cr and AST were not significant difference in groups. Left ventricular end-diastole pressure, right relative weight, left ventricular posterior wall thickness, collagen volume fraction and the lung weight were decreased in AMI+fluvastatin group compared to AMI group (P＜0.05, P<0.01); The levels of LPO, NO₂^-+NO₃^- in plasma and LPO in myocardium decreased, but plasma GSH-PX level increased in AMI+fluvastatin group (P＜0.05). CONCLUSION: Fluvastatin ameliorates the ventricular structural remodeling in a rat model of infarction, and delays the development of heart failure. The anti-oxidation mechanism of fluvastatin may take part in this process.     

Effects of atorvastatin on release of endothelial microparticles and myocardial apoptosis in rats with acute myocardial infarction

AIM: To investigate the effect of atorvastatin(AT) on the release of endothelial microparticles(EMP) and myocardial apoptosis in the rats with myocardial infarction. METHODS: SD male rats(n=24) were randomly divided into 3 groups:sham operation(sham) group, myocardial infarction(MI) group and MI+AT group. The rat model of acute myocardial infarction was prepared by coronary artery ligation. At 2 h and 24 h after modeling, the peripheral blood was collected to detect creatine kinase-MB(CK-MB) and cardiac troponin T(cTnT). The circulating levels of EMP were measured by flow cytometry. The myocardial apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay. RESULTS: At 2 h after modeling, the level of CK-MB was significantly increased in MI group compared with sham group, and the level of EMP and the myocardial apoptotic rate were significantly increased in MI group and MI+AT group compared with sham group. At 24 h after modeling, the level of EMP was significantly increased in MI group compared with sham group. The levels of CK-MB, cTnT, EMP and the myocardial apoptotic rate were significantly decreased in MI+AT group compared with MI group. Moreover, the level of CK-MB in MI group was significantly increased at 24 h compared with that at 2 h after modeling. The levels of CK-MB, cTnT and EMP were significantly decreased in MI+AT group at 24 h compared with those at 2 h after modeling. CONCLUSION: Ator-vastatin may reduce the level of EMP and the myocardial apoptotic rate in the rats with acute myocardial infarction, indicating that atorvastatin plays a role in protecting endothelium.     

Cardiomyocyte apoptosis and related gene expression after acute myocardial infarction in rats

AIM: To investigate cardiomyocyte apoptosis and the expression of caspase-3, Bcl-2 and Bax after acute myocardial infarction (AMI) in rats.METHODS: AMI model was established with the ligation of left coronary artery in 78 randomly selected female SD rats.Twenty-four hours after operation, 43 survivors were randomly divided into 48-hour and 4-week two groups according to the time points: MI 48 h (n=11) and MI4 weeks (n=13) groups, sham-operated rats (S, n=27) were also randomly selected and reassigned to S48 h (n=10) and S4 weeks (n=10) groups.Cardiomyocyte apoptosis was detected with in situ terminal deoxynucleotidyl transferase (TdT)-dUTP nick-end labeling (TUNEL staining) and DNA gel electrophoresis.Caspase-3, Bcl-2 expression and Bax expression were detected with immunohistochemistry and Western blotting analysis.RESULTS: Compared with sham-operated group, after AMI, systolic, diastolic, and mean arterial blood pressures (SBP, DBP, MAP), left ventricular systolic pressure (LVSP) and the maximum change rate of left ventricular pressure rise and fall (±dp/dt) were significantly decreased (P<0.05, P＜0.01), while left ventricular end diastolic pressure (LVEDP) was significantly increased (P<0.05) in MI 48 h group.All the above indices in MI 4 weeks group had the same change as that in MI48h group, with the LVEDP significantly higher (P<0.01), except for a non-significantly change in SBP, DBP and MAP (all P>0.05).In both MI 48 h and MI 4 weeks groups, myocyte apoptotic index was significantly increased in the infracted/scar, border and non-infarcted areas (P<0.05,P＜0.01) with caspase-3 and Bax expressions increased significantly (P<0.05, P＜0.01) in myocytes of the above three areas and Bcl-2 expression increased only in myocytes of the infracted area in MI 48 h group.Western blotting indicated that Bcl-2/Bax ratio was also decreased in MI 48 h subgroup.CONCLUSIONS: After AMI in rats, cardiomyocyte apoptosis happened in the infarction/scar, border and non-infarcted areas, with caspase-3 and Bax expression in myocytes increased, and with Bcl-2 expression increased in myocytes of infracted area and Bcl-2/Bax ratio decreased only early after AMI.     

Changes of calcium handling protein after acute myocardial infarction in rats and effect of carvedilol

AIM: To observe the changes of sarcoplasmic reticulum Ca2+-ATPase (SERCA)， phospholamban (PLB) during heart failure after acute myocardial infarction (AMI) in rats and the effect of carvedilol. METHODS: Rats were randomly assigned to normal control group, sham-operation group, AMI group and carvedilol (CAR) group. 6 weeks later, in vivo hemodynamic, morphometry and SERCA, PLB mRNA and protein expression of myocytes were measured in all animals. RESULTS: In comparison with sham-operation group, LV end diastolic pressure (LVEDP) and weight of ventricles were increased, while maximal rate of rise and fall (±dp/dt) of LV pressure were decreased in AMI group. After treatment with carvedilol, these parameters were all improved. The mRNA and protein expression of SERCA were downregulated (P<0.01). PLB mRNA and protein expression were upregulated (P<0.01) in AMI group relative to sham-operation group. Carvedilol restored the low expression of SERCA mRNA and protein (P<0.05), but was no effect on PLB mRNA and protein expression (P>0.05). CONCLUSIONS: The changes of SERCA and PLB may be the important mechanism of contractile dysfunction in heart failure after AMI. Carvedilol is effective in preventing LV dysfunction after AMI. The molecular mechanism may be related with normalization of SERCA expression.     

瓜类褪绿黄化病毒p22 基因在大肠杆菌中的表达及抗血清的制备

 以被瓜类褪绿黄化病毒（Cucurbit chlorotic yellows virus,CCYV）侵染的甜瓜叶片为供试材 料,采用RT-PCR 方法克隆其P22 蛋白基因,并将其连接到原核表达载体pGex-4T-3 上,PCR 验证及克隆 测序确定开放阅读框的正确性。将重组载体pGexp22 转化大肠杆菌BL21 菌株,诱导表达,SDS-PAGE 分 析表明,经IPTG 诱导,p22 基因在大肠杆菌BL21 中得到了高效表达。以表达的蛋白作为抗原,免疫家 兔,制备了CCYV P22 的特异性抗血清。ACP-ELISA 检测结果表明,血清效价高达1.28 × 10⁵。Western blot 检测甜瓜叶片,结果表明抗血清能够特异性地检测CCYV 侵染的甜瓜叶片中的CCYV P22 蛋白。