Establishment and evaluation of rat heart ischemia-reperfusion injury model in vivo

AIM: To establish and evaluate a rat model of heart ischemia-reperfusion injury in vivo. METHODS: Seventy-two male Sprague-Dawley rats weighing(250±50)g were randomly divided into sham operation group(sham), ischemia-reperfusion group(I/R) and normal group. The animals were anesthetized and heparinized. Myocardial ischemia-reperfusion was induced by ligating the left anterior descending coronary artery with "U-shape tube" for 35 min followed by 120 min or 240 min reperfusion in vivo. The heart infarct size was measured by triphenyltetrazolium chloride(TTC) staining. The myocardial cell apoptotic index was determined by the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling(TUNEL). Immunohistochemical method was used to detect the expression of Bcl-2 and Bax in rat ischemia myocardium. The blood level of MB isoenzyme of creatine kinase(CK-MB),cardiac troponin I(cTnI),nitric oxide(NO),malondialdehyde(MDA), total superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px) were detected after reperfusion for 2 h and 4 h. RESULTS: Compared with normal group and sham group, there were obvious changes of ST-T segment and Q wave in the electrocardiogram of I/R group. The blood level of CK-MB, cTnI, NO, MDA and GSH-Px in I/R group increased(P<0.05,P<0.01) after reperfusion for 2 h and 4 h, and the blood level of T-SOD in I/R group after reperfusion for 2 h and 4 h also increased(P<0.05). The heart infarct size in I/R group was the largest as compared to other groups. Among these groups, the apoptotic index of I/R group was the highest and the Bcl-2/Bax ratio in I/R group decreased(P<0.01).CONCLUSION: The rat model of heart ischemia-reperfusion injury in vivo can be successfully established with the "U-shape tube". There are obviously changes of heart infarct size, blood level of CK-MB, cTnI, NO, MDA, T-SOD and GSH-Px, myocardial apoptotic index and Bcl-2/Bax ratio between I/R rats and control animals.     

Effects of simvastatin on expression of Bcl-2 and Bax in myocardium of rats with renal ischemia-reperfusion injury

AIM: To observe the effect of simvastatin on myocardial tissue after renal ischemia-reperfusion injury and its mechanism. METHODS: A rat model of renal ischemia-reperfusion injury was prepared by clamping the bilateral renal arteries for 45 min. The rats (n=36) were randomly divided into sham operation group, renal ischemia-reperfusion (I/R) group and simvastatin group with 12 rats in each group. The content of serum creatinine (SCr), blood urea nitrogen (BUN) and myocardial tissue malondialdehyde (MDA), the myocardial activity of lactate dehydrogenase (LDH), creatine kinase (CK) and superoxide dismutase (SOD), and the myocardial protein expression of Bcl-2 and Bax were detected. RESULTS: Compared with sham operation group, the content of SCr, BUN and myocardial MDA, and the myocardial activity of LDH and CK in I/R group were significantly increased (P<0.05), and the activity of SOD was significantly decreased (P<0.05). Compared with I/R group, the content of SCr, BUN and myocardial MDA, and the myocardial activity of LDH and CK in simvastatin group were significantly decreased (P<0.05), while SOD activity was enhanced (P<0.05). The protein expression of Bcl-2 and Bax in sham operation group was less than that in I/R group (P<0.05), and the protein level of Bax in simvastatin group was significantly lower than that in I/R group (P<0.05), while the protein level of Bcl-2 was increased (P<0.05). CONCLUSION: Simvastatin has a protective effect on the myocardium of the rats with renal ischemia-reperfusion injury, and the protective mechanism may be related to the elimination of free radicals by simvastatin, increase in the protein expression of Bcl-2 and decrease in the protein expression of Bax.     

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.     

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.     

The effect of postconditioning on apoptosis-related gene expression of lung tissues in rat intestinal ischemia-reperfusion

AIM: To investigate the effect of postconditioning on apoptosis-related gene expression of lung tissues in rat intestinal ischemia-reperfusion(I/R). METHODS: Male Sprague-Dawley rats were divided into 3 groups which were sham operation group (sham), I/R group and ischemic postconditioning (IPOST) group. The experimental animals were sacrificed after reperfusion for 6 h. The tissue sections were stained with hematoxylin and eosin and examined under light microscope to observe lung histopathological changes. Lung W/D ratios were measured. Lung tissue samples were taken to detect Bcl-2, Bax and caspase-3 mRNA and protein expression using RT-PCR and Western blotting analysis, respectively. RESULTS: Compared with I/R group, the morphological changes of lung were alleviated and the lung W/D ratios significantly decreased in IPOST group (P<0.05). The mRNA and protein expression of Bcl-2 in IPOST group was obviously higher than that in I/R group (P<0.05), while the mRNA and protein expression of Bax and caspase-3 in IPOST group was obviously higher than that in I/R group (P<0.05). CONCLUSION: Ischemic postconditioning attenuated intestinal ischemia-reperfusion-induced acute lung injury partly by increasing Bcl-2 mRNA and protein expression, reducing Bax mRNA and protein expression.     

Cardiomyocyte apoptosis and expression of Bcl-2, Bax protein after acute myocardial infarction and late reperfusion in dogs

AIM: To study the effect of late reperfusion on apoptotic cardiomyocytes in the risk area of acute myocardial infarctin in dogs. METHODS: The experiment was divided into three groups: sham operation group, acute myocardial infarction (AMI) group, and late reperfusion (LR) group. Apart from sham operation group, the other two groups were subjected to left anterior descending branch of coronary artery ligation. The acute myocardial infarction group was only subjected to ligation for 12 hours, late reperfusion group was subjected to ligation for 6 hours following by 6 hours of reperfusion. The cardiomyocyte apoptosis was measured by TUNEL assay. Immunohistochemistry and Western blotting analysis were used to detect the expression of Bcl-2 and Bax protein. RESULTS: The number of apoptotic cardiomyocytes in late reperfusion group was much less than acute myocardial infarction group (P<0.05), and increased significantily as compared with sham operation group (P<0.01). The expression of Bcl-2 protein was enhanced gently in late reperfusion group in contrast to acute myocardial infarction group, but no significant difference in the two groups (P>0.05) was observed, although it was much more in the two groups than that in sham operation group (P<0.01). The expression of Bax protein in late reperfusion group was much higher than that in sham operation group (P<0.01), and was lower than that in acute myocardial infarction group (P<0.05). CONCLUSION: Late reperfusion reduces cardiomyocyte apoptosis in the risk area of acute myocardial infarction. The mechanism may be that late reperfusion can decrease the expression of Bax protein.     

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 intestinal lymphatic pathway on apoptosis of lung tissue in rats by two-hit

AIM: To observe the effects of mesenteric lymph duct ligation on apoptosis of lung tissue,correlated gene expression with apoptosis and TNF-α,IL-6 contents in rats by two-hit.METHODS: 45 Wistar rats were divided into three groups: the ligation group,the non-ligation group and sham group,and the two-hit model was established by means of hemorrhage and LPS treatments.Ligating mesenteric lymph duct was conducted in ligation group.After 24 hours,the pathological sections of lung tissue were prepared for determining the apoptosis rate by TUNEL method and expressions of Bcl-2 and Bax were observed by immunohistochemical test.The lung homogenate was also prepared for determining the contents of TNF-α and IL-6 by ELISA.RESULTS: After two-hit,the apoptosis rate,Bax expression in lung tissue and contents of TNF-α and IL-6 in serum and lung homogenate in non-ligation group were increased and Bcl-2 expression was lower than that in sham group and ligation group significantly (P<0.01,P<0.05).Apoptosis rate in ligation group was no statistics difference with sham group (P>0.05),and the expression of Bcl-2 protein was increased and Bax was lower than that of sham group (P<0.01,P<0.05).CONCLUSION: Blockage of intestinal lymphatic pathway reduces the apoptosis of lung in two-hit rats,and its mechanism might relate to reduced the levels of TNF-α and IL-6 and improved the expression of Bcl-2 protein in lung by the ligation of mesenteric lymph duct.The mesenteric lymph of two-hit might play an important role in the pathogenesis of acute lung injury (ALI).     

Beclin-1 expression is upregulated by spermine in rat myocardial ische-mia-reperfusion injury

AIM: To observe the effects of spermine (SP) on myocardial ischemia-reperfusion (IR) injury in rats. METHODS: SD rats (weighing 220~250 g) were equally randomized to 3 groups:sham control group, in which the rats were only treated with thoracotomy; IR group, in which the rats were treated with ischemia for 30 min and reperfusion for 60 min; and IR+SP group, in which 0.5 mmol/L SP (2 mL/kg) was intravenously injected just 15 min before reperfusion. The morphological changes of myocardial tissues were assessed by HE staining. The levels of cardiac troponin I (cTnI) and creatine kinase isoenzyme MB (CK-MB) in plasma were determined. Myocardial infarct size and no-reflow range of the myocardium were measured by Evans blue and thioflavin S staining. Inflammatory responses in the myocardial tissues were detected by myeloperoxidase (MPO) assay. The autophagy function was detected by measuring the protein expression of beclin-1 by Western blot. RESULTS: The myocardial injury and inflammatory infiltration in IR+SP group were reduced under light microscope. Treatment with SP decreased the plasma levels of cTnI and CK-MB, and reduced the IR-induced infarct size and no-reflow range size of the left ventricle (P<0.05). Tissue MPO assay showed that myocardial inflammatory responses were attenuated in IR+SP group compared with IR group. Beclin-1 was upregulated in IR+SP group compared with IR group (P<0.05). CONCLUSION: Exogenous SP attenuates myocardial ischemia-reperfusion injury by upregulating the expression of beclin-1.     

Effect of Qiliqiangxin granule on apoptosis of renal tissues in rats with cardiorenal syndrome

AIM: To investigate the effect of Qiliqiangxin granule on the apoptosis of renal tissues in rats with cardiorenal syndrome (CRS) and its possible mechanism. METHODS: A rat model of CRS was established by ligation of the left anterior descending coronary artery and acute renal ischemia/reperfusion injury. After operation, the rats were divided into 6 groups:2-week sham operation (2w sham) group, 2-week model (2w CRS) group, 2-week drug (2w CRS-Q) group, 4-week sham operation (4w sham) group, 4-week model (4w CRS) group and 4-week drug (4w CRS-Q) group. The rats in 2w CRS-Q group and 4w CRS-Q group were given Qiliqiangxin granule (4 g·kg^-1·d^-1) by gavage for 2 weeks and 4 weeks, respectively. The levels of serum cystatin C (Cys-C), plasma angiotensin Ⅱ (Ang Ⅱ), urine neutrophil gelatinase-associated lipocalin (NGAL) and urine microalbumin (UMA) were measured by ELISA. The serum level of creatinine (Cre) was detected by sarcosine oxidase method. The renal histopathological changes were observed by HE staining. The mRNA and protein expression levels of Ang Ⅱ, Bcl-2 and Bax were evaluated by RT-qPCR and Western blot, respectively. The apoptosis rate of renal cells was assessed by TUNEL staining. RESULTS: The levels of serum Cys-C, serum Cre, plasma Ang Ⅱ, urine NGAL and UMA were significantly increased in 2w CRS group and 4w CRS group compared with 2w sham group and 4w sham group after modeling (P<0.05). The mRNA and protein expression levels of Bax and Ang Ⅱ in the renal tissues of CRS rats were significantly up-regulated (P<0.05), while Bcl-2 was significantly down-regulated (P<0.05) compared with 2w sham group and 4w sham group. Compared with 2w sham group and 4w sham group, the damage of renal tissues in 2w CRS and 4w CRS group was severe, and the apoptotic rates of renal cells were significantly increased. Compared with 2w CRS group and 4w CRS group, Qiliqiangxin granule greatly decreased the levels of Cys-C, Cre, Ang Ⅱ, NGAL and UMA, down-regulated the mRNA and protein expression levels of Bax and Ang Ⅱ in the renal tissues, and up-regulated the expression of Bcl-2 at mRNA and protein levels at 2 and 4 weeks. In addition, Qiliqiangxin granule also greatly attenuated the damage and apoptosis of the renal tissues. CONCLUSION: Qiliqiangxin granule significantly inhibits the apoptosis of renal tissues and improves the renal function of CRS rats, and its mechanism may be related to the inhibition of Ang Ⅱ expression.     

Effects of heptanol preconditioning on structure,function and Cx43 content of mitochondria in rabbit model of myocardial ischemia/reperfusion injury

AIM: To investigate the effects of heptanol preconditioning on the changes of structure, function and connexin 43 (Cx43) content in mitochondria in a rabbit model of myocardial isehemia-reperfusion (IR) injury. METHODS: In anesthetized open-chest rabbits, the left anterior descending artery (LAD) was occluded for 30 min and reperfused for 4 h. Sixty-four rabbits were randomly divided into 4 groups (n=16 in each group): sham operation group (sham group), ischemia-reperfusion group (IR group), ischemic preconditioning group (IP group) and heptanol preconditioning group (HT group). All rabbits in the 4 groups were killed 4 h after reperfusion. Myocardial infarct size was determined at the end of the experiment. Mitochondria was isolated by centrifugations. The ultrastructural changes of the mitochondria were observed under electronic microscope. The mitochondrial membrane potential, Ca2+ concentration, MDA content and SOD activity of myocardial mitochondria were also examined. The content of mitochondria Cx43 was detected by Western blotting. RESULTS: Compared to IR group, the myocardial infarct size was significantly reduced in IP (18.97%±2.80%) and HT (19.97%±3.80%) groups, the damage of mitoehondrial ultrastructure was milder (P<0.05), mitochondrial membrane potential was significantly higher and Ca2+ concentration was much lower (P<0.01) in IP group and HT group. No significant difference of MDA content and SOD activity in myocardial mitochondria between IR group and HT group was found. However, MDA content were much lower and SOD activity was significantly higher in IP group as compared to IR group (P<0.01). Compared to sham group, the mitochondria Cx43 expression was distinctly decreased compared to IR group (P<0.05) and no significant difference was found between IP group and HT group (P>0.05). CONCLUSION: Heptanol preconditioning protects myocardium from ischemia-reperfusion injury. The mechanism may be related to increasing in mitochondrial membrane potential, alleviating Ca2+ overload in myocardial mitochondria and attenuating the decrease in mitochondria Cx43 expression induced by isehemia-reperfusion.     

Tetramethylpyrazine combined with bone marrow mesenchymal stem cell transplantation inhibits neuronal apoptosis in rats with cerebral ischemia

ATM: To investigate the effects of tetramethylpyrazine (TMP) combined with bone marrow mesenchymal stem cells (BMSCs) on neuronal apoptosis, and Bcl-2 and Bax expression in rats with cerebral ischemia. METHODS: The BMSCs were isolated by the whole bone marrow adherent method and cultured, and those in the 3rd passage were used for tail-vein transplantation. The rats were subjected to right middle cerebral artery occlusion (MCAO) using suture method, and the rats except sham group were randomly divided into model group, BMSCs (1×10⁹ cells/L) group, TMP (40 mg/kg) group and combination (TMP+BMSCs) group with 12 rats in each group. Neurological function was evaluated by modified neurological severity scoring (mNSS) on 1 d, 7 d and 14 d after cerebral ischemia. Toluidine blue staining was performed to detect cerebral infarct volume, HE staining was used to observe brain histopathological change, neuronal apoptosis was observed by TUNEL staining, and the mRNA and protein expression of Bcl-2 and Bax was detected by real-time fluorescence quantitative PCR and Western blot at 14 d after cerebral ischemia. RESULTS: Compared with BMSCs group and TMP group, TMP combined with BMSCs significantly reduced the score of mNSS (P<0.01) and the infarct volume (P<0.01), alleviated the pathological damage in the peripheral area of cerebral ischemia, decreased the number of TUNEL positive cells (P<0.01), increased the expression of Bcl-2 and decreased the expression of Bax at mRNA and protein levels (P<0.01).CONCLUSION: Tetramethylpyrazine combined with transplantation of BMSCs improves the functional recovery, reduces the infarct volume, relieves the ischemic injury of the brain tissue, and attenuates neuronal apoptosis in the rats with cerebral ischemia. The mechanism may be related to regulating the expression of Bcl-2 and Bax.     

Minocycline postconditioning protects myocardium from ischemia-reperfusion injury through attenuating poly(ADP-ribose) polymerase excessive activation

AIM: To investigate whether minocycline postconditioning protects rat myocardium from ischemia-reperfusion (I/R) injury through attenuating poly(ADP-ribose)polymerase-1(PARP-1) excessive activation. METHODS: The left anterior descending coronary artery was ligated for 45 min and then reopened for 2 h to establish the rat model of myocardial ischemia-reperfusion injury. The male Wistar rats (n=90) were randomly divided into sham group, I/R group, low-and high-dose minocycline groups, and 3-aminobenzamide (3-AB, PARP inhibitor) group. The myocardial infarct size was measured by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. The morphological changes of the myocardium were observed with HE staining. The cardiomyocyte apoptosis was detected using in situ TDT-mediated dUTP nick end labeling (TUNEL). The level of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in the serum were measured by ELISA. The content of poly(ADP-ribose) (PAR) in the reperfused myocardium and peripheral leukocytes were detected by Western blot. RESULTS: Compared with sham group, PAR expression, TNF-α content and IL-1β concentration increased in all other groups. Compared with I/R group, treatment with low and high doses of minocycline and 3-AB significantly reduced the infarct size and myocardial apoptosis. PAR expression, TNF-α content and IL-1β concentration in low-and high-dose minocycline groups and 3-AB group all decreased. No significant difference of the above parameters between high-dose minocycline group and 3-AB group was observed. CONCLUSION: Minocycline postconditioning may attenuate myocardial ischemia-reperfusion injury by depressing the activation of PARP-1 in cardiomyocytes and peripheral leukocytes in rats.     

Protective effect of ischemic-preconditioning under the mild hypothermia against small intestine ischemia-reperfusion injury in rats

AIM: To investigate the protective effect of ischemic-preconditioning under the mild hypothermia against small intestine ischemia-reperfusion injury in rats and its mechanism. METHODS: Thirty-two rats were randomized into 4 groups (8 rats in each group): sham operated group (Sham), ischemia-reperfusion (I/R) group, ischemic-preconditioning (IP) group, mild hypothermia ischemic-preconditioning (MHIP) group. The wet/dry ratio, Ca²⁺-Mg²⁺-ATPase activity in intestine tissue, the malondialdehyde (MDA) content, activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and total antioxdase (TAX) in blood were determined. Ultrastructure, Bcl-2 and Bax expression in intestinal mucosa tissue were also observed. RESULTS: After I/R, the intestinal tissue wet/dry ratio, the content of MDA, LDH activity, the optic density of Bcl-2 and Bax proteins were significantly higher in I/R group than those in sham group (P<0.01). The activities of Ca²⁺-Mg²⁺-ATPase, SOD, TAX were significantly lower in I/R group than those in sham group (P<0.01). The intestinal tissue wet/dry ratio, the content of MDA, LDH activity and the optic density of Bax protein were significantly lower in IP group than those in I/R group (P<0.01), and also lower in MHIP group than in IP group (P<0.05). The activities of Ca²⁺-Mg²⁺-ATPase, SOD, TAX and the optic density of Bcl-2 protein were significantly higher in IP group than in I/R group (P<0.01). CONCLUSION: MHIP can protect intestine against I/R injury in rats, which may be related to enhancing oxidation-resistance of intestine, inhibiting lipid peroxidation, upregulating the expression of Bcl-2 protein and downregulating the expression of Bax protein.     

Effects of HES 130/0.4 on no-reflow after myocardial ischemia-reperfusion injury in rats

AIM: To observe the effects and mechanisms of hydroxyethylstarch(HES) 130/0.4 on no-reflow phenomenon after myocardial ischemia-reperfusion in rats. METHODS: SD rats were randomly divided into 4 groups:sham operation group, ischemia-reperfusion(IR, treated with normal saline) group, normal saline ischemia-reperfusion(NS-IR, treated with NS) group and HES ischemia-reperfusion(HES-IR, treated with HES) group. Myocardial infarct size and no-reflow range were determined by staining methods, and the activities of myocardial enzymes(CK-MB, cTnI and MPO) were measured. Meanwhile, cardiac microvascular endothelial cells of the rat were cultured and divided into 4 groups:control group, hypoxia/reoxygenation(H/R) group, NS-H/R group and HES-H/R group. Acute ischemia reperfusion models were simulated, and the concentration of calcium ions was measured. The relative cell activity was evaluated by CCK-8 assay, and the apoptotic rate was detected by flow cytometry. RESULTS: In HES-IR group, the myocardial infarct size, the no-reflow zone, CK-MB, cTnI and MPO activity were all significantly lower than those in IR group(P<0.05). In microvascular endothelial cells, the concentration of calcium ions and the apoptotic rate in HES-H/R group were significantly decreased, while the relative cell activity increased compared with H/R group(P<0.05). CONCLUSION: HES reduces no-reflow in acute myocardial ischemia-reperfusion. The mechanism may be involved in the inhibition of both the infiltration of neutrophils and the calcium overload of endothelial cells.     

X-ray irradiation increases sensitivity of myocardium to ischemia/reperfusion injury in rats

AIM：To observe the sensitivity of myocardium to ischemia/reperfusion (I/R) injury in the rats with chest radiotherapy. METHODS：The radiation-induced heart disease model was established by local 20 Gy of X-ray irradiation in the chest. Male Wistar rats (n=42) were randomly divided into 6 groups:sham trauma group, trauma group, sham trauma+sham operation group, sham trauma +I/R group, trauma+sham operation group and trauma+I/R group. The rats were subjected to 30 min of ischemia and 1 h of reperfusion 2 week after trauma. The left ventricular developed pressure (LVDP) and ±dp/dtmax were recorded by BL-410 biological signal recording and analysis system. The serum cardiac troponin I (cTnI) and creatine kinase isoenzyme (CK-MB) were measured by ELISA. The myocardial infarct size was determined by nitroblue tetrazolium(NBT) staining method and BI2000 image analysis software. RESULTS：Compared with sham trauma+I/R group, significant decreases in LVDP and ±dp/dtmax were observed in trauma+I/R group (P<0.01) with significant increases in the infarct size and the concentrations of cTnI and CK-MB (P<0.01). CONCLUSION:Chest X-ray irradiation increases the sensitivity of myocardium to I/R injury in rats.     

Effects of maternal limb ischemic preconditioning on fetal hippocampal neuron apoptosis induced by intrauterine distress-reoxygenation in rats

AIM: To evaluate the influence of maternal limb ischemic preconditioning (LIP) on the apoptosis of fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats. METHODS: Intrauterine ischemia was induced by clamping the uterine and uterine branch of the ovarian blood vessels with aneurysm clamps for a period of 15 min followed by removal of the clamps to permit reperfusion. Sprague-Dawley (SD) rats (n=12) were randomly divided into 4 groups on the 19th pregnant day: sham (S) group, LIP group, fetal distress (FD) group and LIP+FD group. The cesarean birth occurred on embryonic day 21 to obtain 12 fetal rats alive in each group. The fetal rats were decapitated and the pyramidal cells in CA1 hippocampus were observed under light microscope. The neuronal apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and the apoptotic rate was calculated. The expression of Bcl-2 and Bax were detected by immunohistochemical method and Western blotting. RESULTS: The rates of neuronal apoptosis in FD group and LIP+FD group were significantly higher than that in S group (P<0.05), while no significant difference was observed between S group and LIP group (P>0.05). The expression of Bcl-2 and Bax in FD group and LIP+FD group was significantly higher than that in S group (P<0.05), while no significant difference was observed between S group and LIP group (P>0.05). The ratio of Bcl-2/Bax was lower in FD group than that in S group. Compared with FD group, the rate of neuronal apoptosis was significantly lower (P<0.05), while the ratio of Bcl-2/Bax was significantly higher (P<0.05) in LIP+FD group. CONCLUSION: Maternal limb ischemic preconditioning attenuates the apoptosis of fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats, which may be associated with the up-regulation of Bcl-2 expression.     

Effect of dexmedetomidine on astrocytes in rats with focal cerebral ischemia-reperfusion

AIM: To investigate the effects of dexmedetomidine on astrocytes in rats with focal cerebral ischemia-reperfusion. METHODS: Sixty female SD rats, weighing 230~250 g, were randomly divided into sham operation group, ischemia-reperfusion group, dexmedetomidine preconditioning group 1 and dexmedetomidine preconditioning group 2. The model of middle cerebral artery occlusion (MCAO) was established by thread embolism of middle cerebral artery. In sham operation group, the carotid arteries were exposed without performing MCAO. In ischemia-reperfusion group, NS was injected intraperitoneally 30 min before focal cerebral ischemia-reperfusion. The rats in dexmedetomidine preconditioning group 1 and dexmedetomidine preconditioning group 2 received intraperitoneal injection of dexmedetomidine at doses of 20 μg/kg and 40 μg/kg, respectively. The neurological scores were studied, and the pathological changes were observed under microscope with HE staining. The expression of glial fibrillary acidic protein (GFAP) and tumor necrosis factor α (TNF-α) in astrocytes was detected by the methods of immunohistochemistry and immunoblotting 24 h after cerebral ischemia-reperfusion. RESULTS: No neurological change was observed in sham operation group. The neurological deficiency scores in ischemia-reperfusion group were markedly higher than those in dexmedetomidine preconditioning group 1 and group 2 (P<0.05). Compared with sham operation group, the expression of GFAP and TNF-α in astrocytes and the level of GFAP increased significantly 24 h after focal cerebral ischemia-reperfusion. Pretreatment with dexmedetomidine significantly attenuated the expression of GFAP and reduced the infarct size and inflammation. CONCLUSION: Dexmedetomidine has a neuroprotective effect on focal cerebral ischemia-reperfusion injury by inhibiting the astrocytes.     

Effects of berberine and yohimbine on splenocyte apoptosis in septic mice

AIM: To observe the effects of berberine and yohimbine on splenocyte apoptosis in septic mice and underlying mechanisms. METHODS: The mice were subjected to cecal ligature and puncture (CLP). The drugs or vehicle were given intragastrically 2 h after the surgery according to the following 5 groups: sham, CLP, CLP+berberine, CLP+yohimbine, and CLP+berberine+yohimbine. The apoptosis of splenocytes stained by TUNEL was observed under laser scanning confocal microscope 20 h after CLP. The splenic lymphocytes were isolated and observed using flow cytometry. The activities of caspase-3, caspase-8 and caspase-9 in splenic lymphocytes were detected, and the expression of Fas, Bim, Bcl-2 and Bax in the splenocytes was also determined by Western blotting. RESULTS: The TUNEL staining showed that the apoptotic rate of the splenocytes in septic mice 20 h after CLP was significantly higher than that in sham and CLP+yohimbine groups (P＜0.05). Compared with CLP group, the proportion of apoptotic cells was decreased in septic mice in CLP+berberine+yohimbine and CLP+yohimbine groups (P＜0.05). Flow cytometry analysis demonstrated the similar results in the apoptosis of splenocytes and T lymphocytes. However, only yohimbine treatment reduced the apoptosis of B lymphocytes in the spleen of sepsis-challenged mice. Compared with CLP group, caspase-9 activity was significantly reduced in CLP+berberine group (P＜0.05), the activities of caspase-3, caspase-8 and caspase-9 were all statistically reduced (P＜0.05) in CLP+yohimbine group and CLP+yohimbine+berberine group. CLP significantly increased the expression of cytosolic Fas, Bim and mitochondrial Bax in the splenocytes, and decreased Bcl-2 expression compared with sham group. Compared with CLP group, the expression of cytosolic Bim and mitochondrial Bax in CLP+berberine group were reduced (P＜0.05). Fas expression decreased only in CLP+yohimbine group (P＜0.05). Berberine combined with yohimbine reduced the expression of cytosolic Fas, Bim and mitochondrial Bax in the septic mouse splenocytes (P＜0.05).CONCLUSION: Yohimbine reduces sepsis-induced splenic lymphocyte apoptosis in mice by inhibiting Fas expression and in turn blocking both extrinsic and intrinsic apoptosis pathways. Berberine reduces Bim expression and inhibits caspase-9 activation, but not caspase-3 activation and apoptosis in the septic mouse splenocytes. Berberine combined with yohimbine reduces splenocyte apoptosis in the septic mice by inhibiting both extrinsic and intrinsic apoptotic pathways.