Effects of ligustrazine ferulate on myocardial apoptosis and apoptosis-related protein expression in rats with myocardial ischemia-reperfusion injury

AIM:To observe the effects of ligustrazine ferulate on the apoptosis of myocardial cells in rats with myocardial ischemia-reperfusion injury, and to explore its possible mechanism. METHODS:Sixty male SD rats were randomly divided into five groups: sham-operation group, ischemia-reperfusion group, ligustrazine (4 mg/kg) group, low-dose (4 mg/kg) ligustrazine ferulate group and high-dose (8 mg/kg) ligustrazine ferulate group. The rat myocardial ischemia-reperfusion model was established by 30 min of myocardial ischemia followed by 120 min of reperfusion. Drugs were administered to the rats by jugular vein injection 10 min before reperfusion. After the reperfusion was finished, the biochemical indicators in serum and the histological indexes in myocardium were detected. RESULTS: Compared with ischemia-reperfusion group, ligustrazine ferulate lowered the serum levels of creatine kinase MB form, lactate dehydrogenase, cardiac troponin I and malondialdehyde, elevated the activity of total superoxide dismutase in serum and the expression of Bcl-2 protein in myocardium, decreased the expression of Bax protein and myocardial apoptotic index, and increased the Bcl-2/Bax ratio (all P<0.01). All the indicators in ligustrazine ferulate groups were dose-dependently superior to those in ligustrazine group (P<0.05 or P<0.01). CONCLUSION: Ligustrazine ferulate protects rats against myocardial ischemia-reperfusion injury. Its anti-apoptotic effect may be related to up-regulation of Bcl-2 and down-regulation of Bax.     

Apoptosis and Bcl-2/Bax expression in the early follicles of reproductive women

AIM: To investigate the apoptosis and Bcl-2/Bax expression in the early follicles of women at reproductive age. METHODS: 12 ovarian specimens were collected from reproductive women (aged 23-38 years) undergoing gynaecological operation. Histopathological examination of these specimens was performed to confirm its' morphological normalities. Using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay and immunohistochemistry method, cell apoptosis and Bcl-2/Bax expression were examined in the early follicles including mainly primordial, intermediary and primary follicles. RESULTS: 18.75% of the oocytes were found TUNEL positive in the early follicles, but no granulosa cells in these follicles were found TUNEL positive. Bax expression was detected in 76.07% of the oocytes in the early follicles, but Bcl-2 expression was negative in these oocytes. In addition, Bcl-2/Bax expression were not present in the granulosa cells in early follicles. CONCLUSION: The oocyte apoptosis occurs in the early follicles of reproductive woman, and pro-apoptotic protein Bax may play a role in regulating this process. It suggests that Bax mediated oocyte apoptosis may be the molecular mechanism of the early follicle atresia in the ovaries of reproductive woman.     

Effects of norepinephrine preconditioning and ischemic preconditioning on apoptosis and Bcl-2, Bax expression in rat myocardial cells during myocardial ischemic reperfusion

AIM: To study the effects of norepinephrine preconditioning(NE-P) and ischemic preconditioning (IP)on apoptosis and Bcl-2, Bax expression in rat myocardial cells in myocardial ischemic reperfusion (I/R). METHODS: The model of rat ischemic-reperfusion was used to conduct NE-preconditioning. Apoptotic myocytes were detected with TUNEL. Bcl-2, Bax expression were detected with immunohistochemistry. RESULTS: The rate of apoptosis cells in I/R group was higher, the rate of apoptosis cells in NE-P group and IP was lower significantly than that in I/R group(P＜0.01). The expression of Bcl-2 in I/R group was lower, but the expression of Bax was higher, the expression of Bcl-2 in NE-P group was higher significantly than that in I/R group(P＜0.01), the expression of Bax in NE-P group was lower than that in I/R group(P＜0.01). There was no significantly difference between NE-P and IP group in the above parameters (P＞0.05). CONCLUSION: NE-P reduced myocyte apoptosis by I/R in rats; The expression of Bcl-2 ,Bax genes played an important role in myocardial apoptosis.     

miRNA-7-mediated Bax/Bcl-2 expression promotes apoptosis of human nasopharyngeal carcinoma CNE-1 cells

AIM: To investigate the relationship of microRNA-7 (miRNA-7) over-expression and Bax/Bcl-2 expression in human nasopharyngeal carcinoma CNE-1 cells.METHODS: The CNE-1 cells were transfected with miRNA-7 mimics using Lipofectamine 2000. The expression of miRNA-7 was detected by real-time PCR. CCK-8 assay and Hoechst 33258 staining were used to detect the cell activity and apoptosis. The expression of Bax/Bcl-2 at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: The expression of miRNA-7 was increased significantly in the CNE-1 cells compared with negative control group and mock group (P<0.01). The activity of CNE-1 cells were extremely decreased after tansfected with miRNA-7 mimics (P<0.01). The typical apoptotic nuclear morphological changes were observed in the CNE-1 cells under the fluorescence microscope with Hoechst 33258 staining. The expression of Bax at mRNA and protein levels was significantly increased compared with the other 2 groups (P<0.01), while the Bcl-2 expression at mRNA and protein levels was significantly down-regulated (P<0.01).CONCLUSION: Over-expression of miRNA-7 significantly inhibits the growth and promotes the apoptosis of nasopharyngeal carcinoma CNE-1 cells by increasing the expression of Bax and down-regulating Bcl-2.     

Anti-cardiac myosin heavy chain antibodies isolated from patients with acute myocardial infarction induce rat cardiomyocyte apoptosis

AIM: To study the effect of human anti-cardiac myosin heavy chain antibodies (AMHCA) on rat cardiomyocyte apoptosis. METHODS: Rat cardiomyocytes were isolated by the method of enzymolysis. Apoptosis of the cardiomyocytes was observed and measured by DNA end labelling and Annexin-V/PI double-staining assay. The proteins levels of apoptosis related P53 and Bcl-2 and the second messenger calcium were measured by Western blotting, patch clamp and confocal calcium imaging, respectively. RESULTS: AMHCA was able to induce cardiomyocyte apoptosis in a dose dependent manner. In the presence of AMHCA, apoptosis-accelerating nucleoprotein P53 promoted myocardial apoptosis, while apoptosis-inhibiting cytoplasmic protein Bcl-2 inhibited myocardial apoptosis. Meanwhile, the concentration of cytoplasmic calcium was elevated. No effect of AMHCA on L-type calcium currents was observed. CONCLUSION: As a novel triggering factor, AMHCA isolated from the patients with AMI induces cardiomyocyte apoptosis.     

SPK1 interferes with LLC cell apoptosis by regulating Bcl-2/Bax pathway

AIM: To investigate whether sphingosine kinase 1 (SPK1) interferes with apoptosis of Lewis lung cancer (LLC) cells by regulating the Bcl-2/Bax pathway. METHODS: The SPK1 gene siRNA eukaryotic expression vector was constructed, and transfected into the LLC cells. The transfected LLC cells was observed under a fluorescence microscope. The apoptotic rate of LLC cells after transfection was analyzed by flow cytometry. The expression levels of SPK1, Bcl-2 and Bax in LLC cells after transfection were detected by Western blot. The protein levels of Bax and Bcl-2 were measured by ELISA. RESULTS: Transfected LLC cells emitted green fluorescence under a fluorescence microscope. Apoptosis in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group (P<0.01). Western blot analysis showed that the expression of Bax in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group, and the expression of Bcl-2 was lower than that in siRNA-SPK1-Neg group. The ELISA results showed that the protein level of Bax in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group (P<0.01), and the protein level of Bcl-2 in siRNA-SPK1 group was significantly lower than that in siRNA-SPK1-Neg group (P<0.01). CONCLUSION: The expression of SPK1 in LLC cells is related to the apoptotic rate. SPK1 may interfere with the apoptosis of LLC cells via Bcl-2/Bax pathway.     

MicroRNA-24 regulates apoptosis of cardiomyocytes after myocardial infarction

AIM：To evaluate the expression of miR-24 in infarcted myocardial tissues and to investigate the function of miR-24 during cardiomyocyte apoptosis in vitro and in vivo. METHODS：The mouse model of myocardial infarcton (MI) was established. The expression of miR-24 in the sections of infarcted myocardial tissues was measured by qRT-PCR. The expression of miR-24 was modified by transfecting oligonucleotide mimic and inhibitor of miR-24 into cardiomyocytes, or injecting lentiviral vectors intramyocardially. The apoptosis of cardiomyocytes was detected by Caspase-Glo 3/7 Assay System. The heart functions were determined by echocardiography and the scar size in MI model was observed with Masson trichrome staining. The apoptosis of infarcted myocardial tissues was detected by TUNEL method. Microarray and bioinformatic analysis were also used to predict the targets of miR-24. RESULTS：The expression of miR-24 in the infarct and border areas was down-regulated after MI. Overexpression of miR-24 in cardiomyocytes reduced the apoptosis induced by hypoxia. miR-24 transfection resulted in reduction of the scar size, and improved left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) of the mice in treatment group after 2 weeks. Furthermore, miR-24 reduced cell apoptosis in the infarct region. BCL2L11, ANK3 and SGPL1 may be the targets of miR-24 during the process of anti-apoptosis. CONCLUSION：miR-24 is down-regulated in the infarcted myocardial tissues. In vitro, miR-24 reduces apoptosis of cardiomyocytes induced by hypoxia. In vivo, miR-24 attenuates cell apoptosis in the infarct and border areas of the heart 2 weeks after MI, and ultimately improves heart functions.     

Effect of Fas/FasL on late reperfusion of acute myocardial infarction and the potential oxidative stress mechanism in canine

AIM: To discuss the effect of Fas/FasL on the late reperfusion of acute myocardial infarction (AMI) and the potential oxidative stress mechanism. METHODS:Eighteen anesthetized dogs were randomly divided into three groups: late reperfusion group (n=6): ligated the coronary for 6 h, followed by reperfusion for 6 h; permanent ischemia group (n=6): after pericardium were opened for 6 h, ligated the coronary for 6 h, and did not reperfuse; control group (n=6): did not ligate the coronary but operation last for 12 h. Infarction brim myocardial Fas/FasL was detected by immunohistochemistry. Apoptosis index (AI) was detected by TUNEL. SOD and GR activity and MDA content were detected by colorimetry. RESULTS:The expression of Fas/FasL and apoptosis index were significantly higher in permanent ischemia group and late reperfusion group than those in control group (P<0.05, P<0.01), and the difference between them was also significant (P<0.05). SOD and GR activities were lower in permanent ischemia group and late reperfusion group than those in control group (P<0.05, P<0.01). The MDA contents in permanent ischemia group and late reperfusion group were higher than that in control group (P<0.05). CONCLUSION:The late reperfusion of AMI promotes the expression of Fas/FasL and myocardial apoptosis, and it may be due to oxidative stress mechanism.     

Effects of celecoxib on cardiac myocyte apoptosis after myocardial infarction

AIM: To investigate the effects of celecoxib, a selective cyclooxygenase-2 inhibitor, on antioxidative capability and apoptosis of cardiac myocytes after myocardial infarction. METHODS: 24 New Zealand rabbits were divided into three groups randomly (8 in each group): sham-operated group (sham group), myocardial infarction group (MI group), celecoxib group (Cele group, 10 mg kg^-1·d^-1, qd, with the drugs gastric gavage for six weeks). The NO concentration, total antioxidative capability (T-AOC), the activity of constitutive nitric oxide synthase (cNOS) and inducible NOS (iNOS) in cardiac tissue homogenate, adjacent to the infracted area, were detected. The pathological changes were observed by light microscope and electron microscopy. The expressions of Bcl-2 and Bax protein in myocytes were observed using immunohistochemistry, and the degree of apoptosis were examined by TUNEL. RESULTS: Cardiac tissue in MI group presented interstitial edema, fibroplastic proliferation, inflammatory cellular infiltration, and vacuolar degeneration in cardiac myocytes. The results of electron microscopy showed that myocytes presented more changes caused by ischemic injury: widened interspace of myofibril, disordered myofibrillae, focal lysis of myofilament, ectasia of sarcoplasmic reticulum. In Cele group, the pathological changes were light, the NO^-₂/ NO^-₃ concentration, the activity of iNOS were lower (P<0.05), while the activity of cNOS and T-AOC were higher (P<0.05) than those in MI group. The expression rate of Bcl-2 protein in Cele group was higher than that in MI group, while the Bax was lower (P<0.01). The number of apoptotic myocytes was lower than that in MI group (P<0.01).CONCLUSION: Celecoxib decreases the number of apoptotic cardiomyocytes and increases the antioxidative capability after myocardial infarction.     

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.     

Effect of PKC activition on cardiac myocyte apoptosis and Bcl- 2 expression during myocardial ischemia/reperfusion in rats

AIM: To explore the effect of PKC activition on cardiac myocyte apoptosis and expression of bcl- 2 during myocardial ischemia/reperfusion(I/R) in rats. METHODS: TUNEL,immunohistochemistry and in situ hybridization were used. RESULTS: The TUNEL data showed that the numbers of positive cardiac myocyte nucleus and the percentage of positive cardiac myocyte nucleus in PMA+IR_{3 h} group decreased significantly(P＜0.05,P＜0.01), compared to those in IR_3h group. The number of Bcl-2 protein positive cardiomyocytes and the percentage of Bcl-2 protein positive cardiomyocytes in PMA+IR_3h group were higher than those in IR_3h group (P＜0.01) bcl- 2 mRNA expression showed the same changes in PMA+IR_0h group compared to IR_1h group.CONCL USIONS:Activation of PKC decreased cardiomyocyte death during I/R.Upregulation of bcl-2 gene expression in cardiomyocytes during I/R may be one of the mechanisms of decreasing cardiomyocyte death by PCK activating during I/R.     

Panax quinquefolium saponin attenuates ventricular remodeling after acute myocardial infarction in rats by inhibiting endoplasmic reticulum stress-related apoptosis

AIM: To investigate the effect of Panax quinquefolium saponin (PQS) on ventricular remodeling after acute myocardial infarction (AMI) in rats and its mechanism. METHODS: Ninety healthy male SD rats were randomly divided into sham group, AMI group, taurine 300 mg·kg-1·d-1 group, PQS 50 mg·kg-1·d-1 group, PQS 100 mg·kg-1·d-1 group and PQS 200 mg·kg-1·d-1 group. AMI models were produced by ligating the left coronary arteries in SD rats. The rats in each treatment group were gavaged with drugs dissolved in water (10 mL·kg-1·d-1), and the rats in sham group and AMI group received equal volume of water. Four weeks after MI, the left ventricle fractional shortening, ejection fraction and structure were evaluated by echocardiography. Myocardial infarct size was measured by 2,3,5-triphenyltetrazolium chloride staining. The hydroxyproline level was measured by colorimetric method. Apoptosis of the cardiomyocytes was detected by TUNEL. In addition, the expression of endoplasmic reticulum stress-related molecules in the noninfarcted myocardium was determined by Western blotting. RESULTS: Compared with AMI group, the left ventricular end-systolic dimension in PQS 50 mg·kg-1·d-1 group, PQS 100 mg·kg-1·d-1 group and PQS 200 mg·kg-1·d-1 group decreased by 17.2%, 20.3% and 38.8% respectively,and the left ventricular end-diastolic dimension decreased by 8.91%, 8.95% and 17.20%, respectively.The left ventricular end-systolic volume decreased by 31.4%, 38.5% and 67.0%, respectively, and the left ventricular end-diastolic volume decreased by 18.2%, 18.8% and 34.2%, respectively.The left ventricular ejection fraction increased by 44.9%, 60.1% and 118.0%, respectively,and the fractional shortening increased by 55.4%, 71.0% and 148.0%, respectively.The infarction size decreased by 4.6%, 39.5% and 55.8%, respectively,and the hydroxyproline level in noninfarcted myocardium decreased by 34.5%, 35.9% and 48.7%, respectively. Compared with AMI group, the myocardial apoptotic index in PQS 200 mg·kg-1·d-1 group decreased by 27.3%, the protein expression of Bcl-2 increased by 114.0%, and that of Bax, GRP78, CRT and CHOP decreased by 53.1%, 79.9%, 80.8% and 42.5%, respectively. The above mentioned protective effects in PQS 200 mg·kg-1·d-1 group and taurine group were similar. The Spearman correlation analysis revealed that CHOP expression had significant positive correlation with apoptotic index (r=0.797, P<0.01). CONCLUSION: PQS attenuates ventricular remodeling in rats. The underlying mechanism may be associated with the inhibition of CHOP-mediated endoplasmic reticulum stress-related cardiomyocyte apoptosis.     

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.     

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.     

Effect of cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor gene transferred neonatal cardiomyocytes

AIM:To observe the secretion of vascular endothelial growth factor (VEGF) when adenovirus induced VEGF165 (AdVEGF165) gene transferred into neonatal cardiomyocytes in vitro,and to investigate the impact on heart function after transplanted the transferred cardiomyocytes into infarct myocardium in rats.METHODS:Neonatal cardiomyocytes were cultured in vitro and labeled with BrdU,then transferred by AdVEGF165.ELISA was applied to assay the expression and secretion of VEGF.Wistar rats,in which left descending branch of coronary artery was ligated,were randomly divided into four groups and transplanted into MI area with transferred cardiomyocytes (group Ⅰ),untransferred cardiomyocytes (group Ⅱ),AdVEGF165 (group Ⅲ) and culture medium (group Ⅳ),respectively.The echocardiograph was applied to evaluate the heart function before and after cell transplantation.Then the rats were executed and the hearts were harvested for histological (hematoxylin-eosin) and immunohistological (anti-BrdU dyeing) examinations.The vessels were also counted in injected area.RESULTS:ELISA indicated that the expression and secretion of VEGF in groupⅠwere higher than those in the rest (P<0.01).Echocardiograph revealed that the improvement of ejection fraction (EF) in groupⅠwas greater than that in other groups (P<0.01).Immunohistological study showed that the implanted cardiomyocytes survived in MI area.More blood vessels in groupⅠthan other groups were observed (P<0.01) by vessel-counting.CONCLUSIONS:AdVEGF165 gene transfer increased the VEGF-production,accelerated neovascularization in MI area and increased the blood flow.The method makes favor for the survival of implanted cells and greatly improves the heart function.     

Effect of rhEGF activating extracellular signal-regulated kinase on the expression of Bcl-2 and p53 protein in human amniotic cells

AIM: To explore the effect of recombinant human epidermal growth factor (rhEGF) on the ERK1/2 pathway and its safety in use when the proliferation of cultured FL cells (human amnion epithelial cells) were promoted by rhEGF.METHODS: FL cells were stimulated by rhEGF at various concentrations.The proliferation rate of FL cells was evaluated by MTT assay.Western blotting was used to detect phosphorylation of ERK1/2 (p-ERK1/2) and the change of Bcl-2 and P53 protein.RESULTS: The proliferation rate FL cells reached the maximum when the concentration of rhEGF was 10 μg/L (42.4％，P<0.01).Western blotting showed that the activity of p-ERK1/2 increased significantly when rhEGF ranged in 10 μg/L to 60 μg/L.Level of Bcl-2 was unchanged in each groups.Level of P53 decreased significantly when the concentration of rhEGF was 60 μg/L.CONCLUSION: It is safe when rhEGF presents in optimization concentration of 10 μg/L.The capability of apoptosis may be depressed when rhEGF is in larger dose.     

Inmbition of nodosin on cell proliferation and expression of Bcl-2 and Bax in HepG2 cells

AIM:To investigate the effects of nodosin extracted from Chinese traditional medicine on the proliferation of HepG2 cells cultured in vitroand to detect the protein expression of Bcl-2 and Bax in HepG2 cells. METHODS:HepG2 cells were treated with different concentrations (1.25, 2.5, 5, 10 and 20 μmol/L) of nodosin for 24 h. The morphological changes of HepG2 cells were observed under inverted microscope. The inhibitory rates of HepG2 cell growth were detected by MTT assay. The apoptotic rates and the protein expression of Bcl-2 and Bax were analyzed by flow cytometry. RESULTS:Shrunken and suspended HepG2 cells increased with the increases in the concentrations of nodosin. The apoptotic rates and the expression of Bax increased with the increases in the doses of nodosin, while the expression of Bcl-2 decreased. CONCLUSION: Nodosin inhibits the growth of HepG2 cells in a dose-dependent manner. The inhibition of HepG2 cell growth is induced by decreasing Bcl-2 and increasing Bax, thus promoting cell apoptosis.     

Effects of tetrahydroxystilbene-2-O-β-D-glucoside on apoptosis and expression of bcl-2/bax/caspase-3 in HUVECs treated with homocysteine

AIM：To explore the effects of tetrahydroxystilbene-2-O-β-D-glucoside (TSG) from Polygonum multiflorum on the apoptosis and the mRNA expression of bcl-2, bax and caspase-3 in human umbilical vein endothelial cells (HUVECs) treated with homocysteine (Hcy). METHODS：Cultured HUVECs were treated with Hcy (3 mmol/L) to establish a Hcy-damaged model. HUVECs in TSG treated groups were pre-incubated with TSG at concentrations of 1 μmol/L and 10 μmol/L for 2 h before treated with Hcy. Cell nuclear damage was detected by Hoechst 33342 staining. Cell apoptosis was determined by flow cytometry. The mRNA expression of bcl-2, bax and caspase-3 was measured by real-time fluorescence quantitative RT-PCR. RESULTS： After treatment with Hcy at concentration of 3 mmol/L, the nuclear damage and apoptotic rate of HUVECs were higher than that in normal group. The expression of bcl-2 was lower, and the expression of Bax and caspase-3 was higher than that in normal group. On the other hand, pre-incubation with TSG at concentrations of 1 μmol/L and 10 μmol/L decreased the nuclear damage and cell apoptosis, increased the expression of bcl-2, and decreased the expression of bax and caspase-3 as compared with the cells only treated with Hcy. CONCLUSION：TSG reduces the apoptosis of HUVECs induced by Hcy, and the mechanism might be associated with regulating the expression of bcl-2, bax and caspase-3.     

Cardiac protective effect of granulocyte colony-stimulating factor combined with ischemic postconditioning on acute myocardial infarction in rabbits

AIM: To investigate the protective effect of granulocyte colony-stimulating factor (G-CSF) combined with ischemic postconditioning (IP) on acute myocardial infarction (AMI). METHODS: Male New Zealand rabbits were randomly divided into 4 groups (n=15) after 30 min of left ventricular artery (LVA) occlusion: the rabbits in ischemia-reperfusion (IR) group were directly given reperfusion|the rabbits in G-CSF group were subsequently treated with G-CSF (10 μg·kg^-1·d^-1) by subcutaneous injection after direct reperfusion|the rabbits in IP group received 4 episodes of 30 s reperfusion and 30 s occlusion before total reperfusion|the rabbits in IP combined with G-CSF (IP+G-CSF) group were treated with both IP and G-CSF. Electrocardiogram (ECG) monitoring was performed during the operation. Blood was drawn to evaluate white blood cell count (WBC) and cardiac troponin I (cTnI) before operation and 7 d later. Ultrasound cardiography was performed to evaluate left ventricular remodeling and functions 4 weeks after operation. The sizes of infarcted myocardium were determined by triphenyltetrazolium chloride (TTC) staining. Apoptosis of cardiomyocytes was measured by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining. RESULTS: ST-segment resolutions were significantly decreased in IP group and IP+G-CSF group compared with direct reperfusion groups (P<0.05). WBC significantly increased in the groups treated with G-CSF for 1 week. The values of cTnI after operation were significantly lowered in G-CSF group, IP group and IP+G-CSF group as compared with IR group (P<0.05). Left ventricular ejection fraction, the size of infarcted myocardium and apoptosis of cardiomyocytes were better in IP group, G-CSF group and IP+G-CSF group than those in IR group. CONCLUSION: G-CSF combined with IP is a promising strategy against cardiac reperfusion injury and accelerates cardiac repair in AMI.