Effect of L-arginine on expression of bcl-2 and bax mRNA in pulmonary injury induced by ischemia-reperfusion in rabbits

AIM:To investigate the effect of L-arginine (L-Arg) on expression of bcl-2, bax mRNA during pulmonary ischemia and reperfusion injury (PIRI) in rabbits.METHODS:Single lung ischemia and reperfusion animal model was used in vivo. The rabbits were randomly divided into three groups: sham operated group (sham, n=12), ischemia-reperfusion group (I/R, n=12) and I/R+ L-arginine group (L-Arg, n=12). Changes of several parameters, which included apoptotic index (AI), wet to dry ratio of lung tissue weight (W/D) and index of quantitative assessment of histologic lung injury (IQA), were measured at 300 min after reperfusion in lung tissue. Meanwhile the location and expression of bcl-2, bax mRNA as well as the ratio of bcl-2 mRNA/bax mRNA were observed. The lung tissue was prepared for light microscopic and electron microscopic observation at 60, 180 and 300 min after reperfusion. RESULTS:As compared with I/R group, in intima and extima of small pulmonary artery, alveoli, and bronchiole epithelia, the expression of bcl-2 mRNA and the ratio of bcl-2 mRNA/bax mRNA were increased, and the expression of bax mRNA was decreased in L-Arg treatment group. The values of AI, W/D and IQA showed significantly lower than that in I/R group at 180 minutes after reperfusion in lung tissue (P<0.01 and P<0.05). Meanwhile, abnormal changes of the lung tissue in morphologically were markedly lessened in L-Arg treatment group.CONCLUSION:L-arginine produces a notable protective effect on PIRI in rabbits by up-regulating bcl-2 mRNA expression, down-regulating bax mRNA expression in lung tissue and regulating the balance of bcl-2 mRNA and bax mRNA to decrease apoptosis.     

Protective effect of Huangqi injection combined with puerarin injection on myocardium of type 2 diabetes mice

AIM:To investigate the effect of Huangqi injection combined with puerarin injection on the myocardium of the mice with type 2 diabetes. METHODS:Diabetic KKAy mice were randomly divided into model group and treatment group (Huangqi injection combined with puerarin injection). The male KKAy mice of the same age were used as control group. All mice were sacrificed at 21, 24 and 28 weeks. Morphological changes of the myocardium were observed by HE staining. Apoptosis of the cardiomyocytes was measured by TUNEL staining. The mRNA levels of glucose-regulated protein 78(GRP78), C/EBP hoinologous protein (CHOP) and p53 up-regulated modulator of apoptosis (PUMA) were detected by real-time PCR, and the protein levels of GRP78, CHOP, PUMA, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, poly(ADP-ribose) polymerase (PARP) and cleaved PARP were determined by Western blot. RESULTS:Cardiomyocyte hypertrophy, partly dissolved sarcoplasm and necrosis were observed in model group, and these lesion were alleviated in treatment group. Obvious increased apoptosis in model group and significantly decreased apoptosis of cardiomyocytes in treatment group was observed (P<0.05). At 21, 24 and 28 weeks, the mRNA and protein levels of GRP78, CHOP and PUMA and the protein levels of cleaved caspase-3, cleaved caspase-9 and cleaved PARP in model group were increased significantly as compared with control group (P<0.01), and these in treated group were decreased compared with model group. CONCLUSION:Huangqi injection combined with puerarin injection has cardioprotective effects on type 2 diabetes mice and its mechanism of the action was implemented via inhibiting the activation of endoplasmic reticulum stress and caspase pathway, thus resulting in suppressed apoptosis.     

In vitro enhancement of radiation-induced apoptosis in human hepatoma cell line HepG2 by a selective inhibitor of cyclooxygenase-2 enzyme NS-398

AIM:To investigate the possible role of NS-398, a selective inhibitor of cyclooxygenase-2 enzyme, in radiation-induced apoptosis of human hepatoma cell line HepG2 in vitro. METHODS:Hepatoma cell line HepG2 was treated with various concentrations (25, 50, 100, 200 μmol/L) of NS-398 before MTT assay was used to evaluate the cytotoxicity of NS-398. Transmission electron microscopy (TEM) was used to observe the changes of apoptosis in morphology. FCM was performed to quantify the apoptotic percentage. Real-time PCR was used to detect the expression of bcl-2, bax and caspase-3 mRNA, Western blotting was used to measure the expression of Bcl-2 and bax protein, and colorimetric method was provided to analyze the change of caspase-3 activity. RESULTS:The cytotoxicity of NS-398 increased in time-dependent and dose-dependent manners. NS-398 significantly enhanced radiation-induced apoptosis (P<0.01), increased the expression of bax mRNA, Bax protein, caspase-3 mRNA and enhanced caspase-3 activity, whereas no significant change in Bcl-2 expression was found (P>0.05). CONCLUSION:NS-398 enhances radiation-induced apoptosis in hepatoma cell line HepG2. The mechanism may be associated with the up-regulation of the expression of Bax, caspase-3 and enhancement of the activity of caspase-3, which ultimately induce apoptosis in HepG2.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Effect of ginsenoside Rb1 on liver cell pyroptosis in hyperlipidemia rats and its possible mechanism

AIM:To discuss the mechanism of ginsenoside Rb1 against liver lipid deposition by observing the effect of ginsenoside Rb1 on liver cell pyroptosis in hyperlipidemia rats. METHODS:Totally 32 healthy SPF rats were randomly divided into control group, model group, ginsenoside Rb1 group and simvastatin group. The rats in control group was given the basic feed, while the others were given high-fat diet. The rats in ginsenoside Rb1 group and simvastatin group were given corresponding drugs. The rats in control group and model group were intraperitoneal injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by the automatic biochemistry analyzer. The pathological changes of the liver tissues were observed with HE staining. The protein and mRNA expression levels of pyroptosis-related factors NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were detected by Western blot and RT-qPCR. RESULTS:Compared with control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.01), and the HDL-C content was decreased significantly (P<0.05). The steatotic liver cells covered the visual field. The mRNA and protein expression levels of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were increased significantly (P<0.01). Ginsenoside Rb1 significantly decreased the serum levels of TC, TG and LDL-C (P<0.05), and significantly increased the content of HDL-C (P<0.01). Ginsenoside Rb1 also significantly decreased the degree of steatosis, and the number and size of lipid droplets. The mRNA and protein expression levels of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were decreased significantly (P<0.05 or P<0.01). CONCLUSION:Ginsenoside Rb1 atte-nuates liver injury and inhibits liver lipid deposition in hyperlipidemia rats by reducing the expression of hepatic pyroptosis-related factors.     

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.     

Effect of epigallocatechin gallate on cardiomyocyte apoptosis induced by ischemia-reperfusion in rats

AIM: To observe the effects of epigallocatechin gallate (EGCG) on cardiomyocyte apoptosis induced by ischemia-reperfusion (IR) in rats. METHODS: The left anterior descending branch of coronary artery was ligated for 30 min and reperfused for 60 min to make a the myocardial ischemia-reperfusion model in rats. The experiment was divided into five groups: sham, ischemia/reperfusion (IR), EGCG (10 mg/kg and 20 mg/kg) and salvia miltiorrhizae (SM, 100 mg/kg) group. The apoptotic cardiomyocytes were detected by in situ end labeling method, and the expressions of Bcl-2 and Bax were shown through immunohistochemistry method. RESULTS: There was no apoptosis myocardial cell in sham operation group. The apoptosis index and expression of bax significantly increased, and bcl-2/bax reduced in IR group (P<0.01). In EGCG-treated group, however, the changes above were obviously alleviated (P<0.01). CONCLUSION: EGCG significantly inhibits cardiomyocyte apoptosis in ischemia-reperfusion rat hearts. The possible mechanism is to raise the ratio of Bcl-2/Bax proteins by increasing in the expression of bcl-2 gene and decreasing in the expression of bax gene.     

Effect of senegenin on H2O2-induced damage in hippocampal neurons of SD rats

AIM: To observe the effect of senegenin (Sen) on hippocampal neuron injuries induced by H₂O₂.METHODS: Hippocampal neurons were isolated from neonatal SD rats. The primarily cultured neurons were divided into control group, H₂O₂ group, Sen group and Sen+H₂O₂ group. The cell viability, the content of malondialdehyde(MDA) and the activity of superoxide dismutase(SOD) in the neurons were detected after treated with Sen. The morphological changes of nucleus of the neurons were observed by Hoechst 33258 staining. The mRNA expression of bcl-2 and bax was quantified by real-time PCR. The protein levels of Bcl-2 and bax were measured by Western blotting. The activity of caspase-3 was also assayed.RESULTS: Compared with H₂O₂ group, the levels of antioxidative enzyme were increased in Sen+H₂O₂ group (P<0.05). In addition, mRNA expression of bcl-2 increased and that of bax decreased (P<0.05) in Sen+H₂O₂ group. Moreover, Sen increased the protein level of Bcl-2, and reduced the protein level of Bax and the activity of caspase-3 in the neurons exposed to H₂O₂ (P<0.05).CONCLUSION: The protective effect of Sen on hippocampal neurons with H₂O₂ -induced injury may be involved in the mechanisms of     

The anticancer activity of genistein on implanted tumor of human primary gastric carcinoma cells in nude mice

AIM: To investigate the apoptosis of implanted tumor of primary human gastric cancer cells in nude mice induced by genistein and the relation between this apoptosis and expression of bcl-2 and bax.METHODS: Establishing a transplanted tumor model by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice.The different doses of genistein (0.5mg/kg,1mg/kg and 1.5 mg/kg ) were directly injected beside tumor body respectively,for six times at an interval of two days.Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated.We observed the morphologic alteration by electron microscope,measured the apoptotic rate by TUNEL staining method,detected the expression of apoptosis-regulated gene bcl-2 and bax by immunohistochemical staining and RT-PCR.RESULTS: Genistein could significantly inhibit carcinoma growth when it was injected near the carcinoma.Genistein induced implanted tumors cells to undergo apoptosis with apoptotic characteristics by transmission electron microscope.The apoptosis index of above three groups was increased progressively.Positive rate of Bcl-2 protein of above three groups was decreased progressively and positive rate of Bax protein of above three groups was increased progressively by immunohistochemical staining.The density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR.CONCLUSION: Genistein is able to induce the apoptosis of transplanted tumor cells.This apoptosis may be mediated by down-regulating bcl-2 and up-regulating bax mRNA and its protein.     

Protective effect of human insulin-like growth factor 1 gene transfection on rat skeletal myoblasts with ischemic/reperfusion injury

AIM: To observe the protective effect of human insulin-like growth factor 1 (hIGF-1) on rat skeletal myoblasts with ischemic/reperfusion injury. METHODS: Myoblasts were isolated from SD rats, cultured, purified, and transfected with plasmid pLghIGF-1SN or pLgGFPSN. The myoblasts were divided into insulin-like growth factor (IGF) group (myoblasts transfected with pLghIGF-1SN), green fluorescent protein (GFP) group (myoblasts transfected with pLgGFPSN), and control group (untransfected myoblasts). The expression of hIGF-1 in myoblasts was investigated by immunocytochemistry, RT-PCR and ELISA. The proliferation rate of myoblasts 14 days after transfection was detected. To observe the protective effect of IGF-1 gene on skeletal myoblasts with ischemic/reperfusion injury 7 days after transfection, the apoptotic myoblasts were detected by the method of in situ TdT-mediated dUTP nick end labeling (TUNEL). The expression of bax and bcl-2 mRNA was detected by RT-PCR, and the expression of caspase-3 was determined by Western blotting. RESULTS: The expression of hIGF-1 in myoblasts transfected with pLghIGF-1SN was detected by immunocytochemistry, RT-PCR and ELISA, but not in myoblasts transfected by pLgGFPSN and untransfected myoblasts. The proliferation rate of myoblasts in IGF group was higher than that in other groups (P<0.05). The results of RT-PCR showed that the expression of bax mRNA significantly decreased and bcl-2 mRNA significantly increased in IGF group compared with GFP group (P<0.05). The results of Western blotting showed that the expression of caspase-3 significantly decreased in IGF group compared with GFP group (P<0.05). CONCLUSION: The transfection of hIGF-1 gene mediated by a retroviral vector produces a protective effect in rat skeletal myoblasts with ischemic/reperfusion injury. The mechanisms may be associated with down-regulating the expression of Bax and caspase-3 and up-regulating Bcl-2 expression.     

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.     

Up-regulation of mRNA expressions of fas,bcl-2, bim,bax, caspase-3, caspase-9, and bcl2l12 in K562 treated with bortezomib

AIM: To detect the treatment of K562 leukemia cells with bortezomib altering the expression of genes fas, bcl-2, bcl2l12, bim, bax, caspase-9 and caspase-3.METHODS: MTT assay was used to detect the inhibition of proliferation. Apoptosis was detected by Annexin-V staining and mitochondrial transmembrane potential (Δψm). RT-PCR was used to analyze the mRNA expressions of fas, bcl-2, bcl2l12, bim, bax, caspase-3 and caspase-9.RESULTS: Bortezomib caused a time- and dose-dependent inhibition of cell proliferation and IC50 of 24 h and 48 h were 161.41 nmol/L and 96.33 nmol/L, respectively. At the concentration of 104 nmol/L, bortezomib induced apoptosis in a time-dependent manner, including increasing annexin-V positivity and decreasing the Δψm. RT-PCR showed that bortezomib up-regulated the mRNA expression of fas, bcl2l12, caspase-9 and caspase-3, but mRNA expressions of bcl-2, bim and bax did not changed obviously.CONCLUSION: Bortezomib inhibits the proliferation of K562 and induces apoptosis, in which fas, bcl2l12, caspase-9 or caspase-3 gene is one of the main genes taking part in.     

Potential mechanism of myocardium apoptosis of right ventricle in rat under chronic hypoxia

AIM:We used an animal model of chronic hypoxia to mimic right ventricular hypertrophy and try to study the potential mechanism of myocardium apoptosis of right heart in rat under chronic hypoxia. METHODS: Rat hypoxia models were established by exposing the rats to normobaric chronic hypoxia (oxygen levels were maintained at 9.5%-10.5%). Sixty rats were separated into two groups: one exposed to hypoxia and the other serving as control. Ten rats, randomly selected from each group were killed at 14, 21, 28 d after hypoxia. The apoptosis was determined. The changes of RV weight to left ventricle and interventricular septum weight ratio［RV/(LV+S)］, the RV weight to body weight ratio (RV/BW) were also observed. The β-MHC, bcl-2 and bad mRNA levels in right ventricle were detected by semi-quantitative RT-PCR assays and expression of β-MHC, Bcl-2 and Bad protein levels were detected by Western blotting.RESULTS: The RV/(LV+S), RV/BW and apoptosis index in chronic hypoxia group were higher than those in normal control group (P<0.01). The results of RT-PCR and Western blotting showed that β-MHC mRNA levels and protein levels in chronic hypoxia group were higher than those in normal control group (P<0.01). The rate of apoptosis, the RV/(LV+S), RV/BW and the expression of β-MHC in hypoxia group all increased with time. The bcl-2 mRNA and Bcl-2 protein expressions in chronic hypoxia group were lower compared with control group at 14, 21 and 28 d (P<0.05). In contrast, no significant change of bad mRNA and Bad protein expressions in chronic hypoxia group were observed compared with control group (P>0.05). Finally, a decreased bcl-2/bad〖STBZ〗 ratio in chronic hypoxia group was found compared with control group (P<0.05). Both the expression of bcl-2 and the bcl-2/bad ratio decreased with time (P<0.05).CONCLUSION:These data demonstrate that chronic hypoxia may induce right ventricular hypertrophy, as well as cardiomyocytes apoptosis. Furthermore, apoptosis in hypertrophic cardiomyocytes induced by hypoxia is mainly due to the inhibition of bcl-2 expression and decrease of bcl-2/bad ratio.     

Dexmedetomidine reduces renal injury induced by lung ischemia/reperfusion in mice through inhibiting endoplasmic reticulum stress response

AIM:To investigate the effect of dexmedetomidine (DEX) on renal injury induced by lung ischemia/reperfusion (I/R) in mice and its relationship with endoplasmic reticulum stress response.METHODS:Healthy SPF male C57BL/6J mice,weighing 20~24 g,aged 8~10 weeks,were randomly divided into 5 groups (n=10 each):sham operation group (sham group),I/R group,atipamezole (Atip) group,DEX group,and DEX+Atip group.In vivo lung I/R model was established by occlusion of the left pulmonary artery for 30 min followed by 180 min of reperfusion in the mice.The Atip (250 μg/kg),DEX (20 μg/kg) and DEX+Atip were intraperitoneally infused into the mice before left pulmonary hilus was blocked in Atip group,DEX group and DEX+Atip group,and other operations were the same as I/R group.After experiment,the mice were killed,and the renal tissues were harvested to observe the morphological changes.The enzymatic activity of caspase-3,serum creatinine and blood urea nitrogen,and cell apoptotic index of the renal cells were also analyzed.The expression of c-Jun N-terminal kinase (JNK),caspase-12,CCAAT/enhancer-binding protein homdogous protein (CHOP) and glucose-regulated protein 78(GRP78) at mRNA and protein levels in the renal tissues was determined by RT-PCR and Western blot.RESULTS:Compared with sham group,the enzymatic activity of caspase-3,serum creatinine and blood urea nitrogen,renal cell apoptotic index,and the mRNA and protein levels of JNK,caspase-12,CHOP and GRP78 in I/R group were significantly increased (P<0.01),and the renal tissues had obvious damage under light microscope.Compared with I/R group,Atip group and DEX+Atip group,the enzymatic activity of caspase-3,serum creatinine and blood urea nitrogen,renal cell apoptotic index,and the mRNA and protein levels of JNK,caspase-12 and CHOP in DEX group were significantly decreased,and the expression level of GRP78 significantly increased (P<0.01).Furthermore,the renal tissue damage was obvious reduced.CONCLUSION:DEX effectively relieves the renal injury induced by lung I/R in mice,which may be associated with exciting α₂-adrenergic receptor and inhibiting endoplasmic reticulum stress response.     

C-reactive protein induces apoptosis in human renal tubular epithelial cells

ATM: To explore whether the C-reactive protein (CRP) level in microinflammation state induces the apoptosis of renal tubular epithelial cells. METHODS: HK-2 cells were stimulated with recombinant human CRP. Annexin-FITC-PI staining and flow cytometry were used to detect the percentage of apoptotic cells. Morphology observation of apoptosis was assessed by Hoechst 33258 staining. Caspase-3 activity was measured by a colorimetric assay. The expression of apoptotic gene bax and anti-apoptotic gene bcl-2 at mRNA levels was determined by real-time PCR. RESULTS: CRP induced apoptosis of HK-2 cells in a time- and dose-dependent manner. The maximal apoptotic effect of CRP concentration was 10 mg/L CRP at concentration of 20 mg/L. CRP treatment was associated with the characteristic morphological features of apoptosis such as condensation, fragmentation or margination of nuclear chromatin. CRP exposure increased caspase-3 activity, up-regulated the mRNA expression of Bax and down-regulated the mRNA expression of Bcl-2. CONCLUSION: Slightly increased CRP level has the potential to induce apoptosis of renal tubular cells.     

Role of cAMP signaling in cardioprotective mechanism of ischemic postconditioning

AIM: To assess the role of the cAMP signaling in cardioprotection by brief intermittent ischemia at the time of onset of reperfusion (i.e. postconditioning). METHODS: The model of rat myocardial ischemia/reperfusion (I/R) was used. The left ventricular functions were assessed by measuring the left ventricular developed pressure (LVDP) and the coronary flow (CF). The injury of myocardium was further confirmed by detecting the releases of lactate dehydrogenase(LDH) and creatine kinase(CK) in coronary effluent. The mRNA expression of caspase-3, bcl-2 and bax in myocardium was determined by real-time PCR. RESULTS: I/R treatment led to the decrease in LVDP and CF, and the increase in the releases of CK and LDH in coronary artery effluent. The mRNA expression of caspase-3 and bax/bcl-2 ratio was up-regulated simultaneously. Postconditioning treatment relieved the injury induced by I/R, which was enhanced by the specific phosphodiesterase 4(PDE4) inhibitor rolipram. On the other hand, the specific adenylyl cyclase inhibitor SQ22536 attenuated those protective effects of postconditioning. CONCLUSION: The cAMP signaling participates in the protective effect of postconditioning on heart from I/R injury, and the effect may be associated with the regulation of apoptosis.     

Changes of heart function and serum cardiac troponin I in early type 2 diabetic rats

AIM: To observe the changes of heart function and the expression of serum cardiac troponin I(cTnI) in early type 2 diabetic rats, and to explore the role of cTnI in the development of type 2 diabetes and early diabetic cardiomyopathy.METHODS: The type 2 diabetes rat model was established by an injection of streptozotocin after high fat diet(5 weeks). The rats were randomly divided into control group, model group of 2 weeks, and model group of 4 weeks. M-mode echocardiography was performed for echocardiographic measurements. Fasting blood glucose(FBG), total cholesterol(TC), triglyceride(TG), high density lipoprotein-cholesterol(HDL-C), low density lipoprotein- cholesterol(LDL-C), fasting insulin(FINS) and cTnI levels were tested. HE staining was used to observe the pathological changes of myocardial structures. The alteration of cTnI in myocardium was determined by Western blot.RESULTS: Compared with normal group, the levels of TC, TG and LDL-C in type 2 diabetic rats were significantly increased, HDL-C levels were significantly reduced. Cardiac histological analysis revealed that type 2 diabetes induced cardiomyocytes degeneration and necrosis. The expression of cTnI increased significantly in diabetic groups compared to control group, and that in model group of 4 weeks increased far more than that in model group of 2 weeks(P<0.05).CONCLUSION: The increased level of cTnI and the change of the heart function may be associated with the development diabetic cardiomyopathy. These changes are valuable for the early clinical diagnosis of myocardial injury in diabetic cardiomyopathy.     

Effect of diet with high fats and cholesterol on PPARα gene expression and body cholesterol level in mice

AIM:To investigate the effects of dietary fats and cholesterol on liver PPARα gene expression and body cholesterol (Chol) level in C57BL/6J mice. METHODS:The animals (n=75) were randomly divided into five groups and respectively received formula mash for 6 weeks. RESULTS:As compared to Chol diet, Chol+PUFA diet produced significantly higher liver cholesterol (P<0.01), serum total cholesterol (TC), focusing on HDL-C. While Chol+MUFA diet resulted in unchanged serum TC and lower liver cholesterol (P<0.01). Chol+SFA diet rsulted in higher liver cholesterol (P<0.01) and serum TC, focusing on LDL-C. Furthermore, Chol+PUFA diet increased the mRNA and protein content of PPARα (P<0.01) in liver, while Chol+MUFA and Chol+SFA diets decreased the mRNA content of PPARα significantly (P<0.01). CONCLUSIONS:These results indicated that addition of fats containing PUFA to diet high in cholesterol increased PPARα mRNA and protein expression, addition of fats containing MUFA or SFA to diet high in cholesterol decreased PPARα mRNA expression. The change of PPARα gene expression may further affect body cholesterol level.     

Liraglutide ameliorates liver injury of type 2 diabetic mice via micro-RNA-33-AMPK pathway

AIM: To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expression of AMP-activated protein kinase (AMPK) and apoptosis-related proteins in mice with type 2 diabetes mellitus (T2DM), and to explore its possible mechanism. METHODS: High-fat diet and intraperitoneal injection of streptozocin were used to establish the type 2 diabetic model in C57BL/6 mice. The mice were randomly divided into 4 groups (n=15):in control group, the normal mice were subcutaneously injected with equivalent volume of saline; in model group, the T2DM mice were subcutaneously injected with equivalent volume of saline; in low-and high-dose liraglutide treatment groups, the T2DM mice were subcutaneously injected with 100 and 200 μg·kg^-1·d^-1, respectively. After 4 weeks of administration, the levels of FBG, TG, TC, HDL-C, LDL-C, ALT and AST were determined. HE staining was used to observe the pathological changes of the liver tissues. The protein level of cleaved caspase-3 in the liver tissue was detected by the technique of immunofluorescence. The protein levels of p-AMPK/AMPK and apoptosis-related proteins were detected by Western blot. The expression of miR-33 in the liver tissues was detected by real-time PCR. RESULTS: Compared with model group, the contents of FBG, TG, TC, LDL-C, ALT and AST were decreased significantly, while the content of HDL-C was increased significantly in low-dose liraglutide group and high-dose liraglutide group (P<0.05). The protein levels of phosphorylated AMPK and Bcl-2 were up-regulated significantly, and the expression of cleaved caspase-3 was down-regulated significantly (P<0.05). The level of miR-33 was decreased significantly (P<0.01). CONCLUSION: Liraglutide alleviates liver injury in type 2 diabetic mice, and the mechanism may be associated with reducing the level of miR-33 and increasing the phosphorylation of AMPK in the liver tissues, thereby inhibiting hepatocyte apoptosis.