| BackgroundsIt is well recognized that hyperglycaemia or hypertriglyceridemia as marksof diabetes, and that will lead to the increased vulnerability of hearts tomyocardial ischemia/reperfusion (MI/R) injury. The mechanism is that thenumber of cell death, heart dysfunction and no-reflow phenomenon afterrefusion are increased in diabetic patients compared with non-diabetes inischemic heart deseases. However, the mechanism of worsened MI/R injury indiabetes and further leading to increased mortality has not been fully elucidated.So, it is most important to clarify the molecular mechanisms of MI/R injury indiabetes. Moreover, that will provide new ideas and new targets for theprevention and treatment of diabetic MI/R injury.Adiponectin is an adipocytokine secreted from adipose tissues, which playsimportant roles in insulin-sensitizing, anti-inflammation, anti-atherogenic andheart protection. It is markedly downregulated in obestity-linked diseases suchas coronary artery disease and type2diabetes. However, administrated with exogenous adiponectin or stimulating adiponectin production can reduce glucoselevels via increasing insulin sensitivity. Moreover, several studies publishedrecently demonstrated that human and murine cardiomyocytes also synthesizeand secrete adiponectin. It is suggested that cardiomyocyte-derived adiponectinplays an important role in protection of MI/R injury.FoxO family is a research focus in recent years which are crucial for amultitude of biological processes, including the cell cycle, cell death,differentiation, andmetabolism, and have prominent roles in insulin signalingpathways. FoxO1is one of the most powerful isoform of FoxO and studied mostprevalence. Many studies have proved that hypertriglyceridemia in diabetes isclosely related with the diaturbance of glucose and fat metabolism caused byFoxO1. Although studies have shown that FoxO1can increase adiponectinexpression in adipose tissues, however, others showed that FoxO1inhibit theexpression of PPARγ gene in diabetes. Thus, how FoxO1to regulate adiponectinexpression may be related to cell type and upstream signals. Therefore, weassume that decreased adiponectin expression caused by FoxO1overproductionin diabetes may due to the aggravated myocardial ischemic injury. In this study,we will identify the hypotheses and thus clarify the molecular mechanisms. Thatwill provide a new experimental basis to further elucidate the mechanism ofaggravated myocardial ischemic injury in diabetes.Aims1. Establish MI/R model of diabetic mice, to observe whether FoxO1activationexisted in diabetic mice. Whether there is a relationship between FoxO1activation and aggravated MI/R injury in diabetic mice.2. In vivo FoxO1siRNA transfection to clearify the change of adiponectin andits receptors expression as well as changes in the MI/R injury. To clearify the action of FoxO1to MI/R injury in type2diabetes;3. Cardiomyocytes cells were cultured with high glucose in vitro experiment.The changes of FoxO1expression was observed in high glucose cluturedcardiomyocytes and after FoxO1siRNA transfection. The changes ofadiponectin were indentified after transfected with FoxO1siRNA. Furthermore,the changes of AdipoR1expression and cell apoptosis were indentified aftersubsequence SI/R. Then to clarify the molecular mechanism that FoxO1how toregulate adiponectin and AdipoR1signal in vitro as in diabetic animals afterMI/R;4. To obsearve the changes of MI/R injury after FoxO1siRNA transfection invivo in APN-/-mice. Further validation to verify that whether the reversal ofadiponectin and their receptors downregulation and thus play a role in protectionof heart by supressing FoxO1in the diabetic animals with MI/R injury.Methods1. Male C57B16/J mice,20-25g, dieted with high glucose and high cholesterolfor2weeks. Diabetes was induced with a single intraperitoneal injection ofstreptozotocin (50mg/kg, Sigma).Then dieted with high glucose and highcholesterol for6weeks. Random serum glucose greater than10mmol/L wereconsidered success.2. In vivo transfection of siRNA: Mice were anesthetized with2%isoflurane,skin disinfection and extrusion heart. To inject the FoxO1siRNA/RNAi-Mate inmyocardial that below the ligation site with30G needle (20μg/50μL,2-3points).MI/R was administrated after48hours of transfection.3. Mice MI/R model: A slipknot was placed at the halfway point of the leftanterior descending artery (LAD) with6-0silk sutures and ischemia wasinduced by ligation of the slipknot. Reperfusion was performed by releasing the slipknot30min after the ligation. Cardiomyocyte apoptosis and related proteinlevels were determined at3h, and myocardial infarction and cardiac functiondetermined at24h, after the conclusion of the reperfusion. Sham operation wasperformed in a same manner except that the LAD will be left unligated.4. Neonatal mouse cardiomyocytes cultured in high glucose (25mmol/L) for24h before transfection of FoxO1/RNAi-Mate complex. Then it was subjected toSI/R24h later.5. Determination of myocardial infarction: Myocardial infarct size wasdetermined by Evans blue/2,3,5-triphenyl tetrazolium chloride (TTC) doublestaining.6. Determination of myocardial apoptosis: Myocardial apoptosis was determinedby terminal deoxynucleotidyltransferase-mediated dUTP nick end labelingstaining (TUNEL) and caspase-3activity assay (ELISA).7. Expression of FoxO1, adiponectin and AdipoR1was determined byWestern-blot.8. The expression levels of FoxO1mRNA were determined by Real Time-PCR.Results1. Compared to normal mice, myocardial FoxO1protein expression wasincreased (P<0.05) and cardiomyocyte-derived adiponectin (P<0.05), AdipoR1expression (P<0.05) reduced in type2diabetic mice. Meanwhile, the type2diabetic mice has increased myocardial infarct size after ischemia/reperfusion(P<0.05), cardiomyocyte apoptosis and caspase-3activity (P<0.05).2. Myocardial injection of FoxO1siRNA effectively reduced the excessiveproduction of FoxO1mRNA and protein expression (P<0.05) in diabetic mice.Supressing cardiac expression of FoxO1promoted the recovery of cardiac-derived adiponectin and AdipoR1expression (P<0.05). 3. Inhibition of the excessive production of FoxO1in diabetic mice cansignificantly reduce myocardial infarct size (P<0.05), improve cardiac function(P<0.05) and reduce cardiomyocyte apoptosis (P<0.05) and caspase-3activity(P<0.05) after MI/R. In addition, consistent with the changes in adiponectin, itsdownstream AMPK activity recovered too.(4) Compared to normal conditioned medium, the FoxO1mRNA and proteinlevels in cardiomyocytes increased (P<0.05) in high glucose cultured condition.However, transfection of FoxO1siRNA can effectively suppress FoxO1mRNAand protein expression (P<0.05) in high glucose cultured mouse cardiomyocytes.Moreover, inhibition of FoxO1expression can promote the recovery of reducedadiponectin levels (P<0.05) in high glucose condition. It will also can reducecardiomyocyte apoptosis and promote the recovery of AdipoR1after SI/Rcultured in high glucose cultured condition.5. FoxO1siRNA injection decreased mRNA expression (P<0.05) in APN-/-mice.However, MI/R injury of APN-/-mice didn’t have significant change.Conclusions1. Cardiomyocyte-derived FoxO1expression increased in diabetic micecompared with normal mice. However, cardio-derived adiponectin and AdipoR1which have cardioprotective effcts decreased in diabetic mice. It has beensuggested that FoxO1may aggravate MI/R injury by reducingcardiomyocyte-derived adiponectin signal in diabetes.2. Supressing FoxO1with in vivo FoxO1siRNA transfection can alleviate MI/Rinjury in diabetes. Moreover, the expression of cardiomyocyte-derivedadiponectin and AdipoR1which decreased before were recoverd. It has beenconfirmed that the effect of FoxO1overproduction reduced adiponectin is aleading cause to the exacerbation of MI/R injury in diabetic mice. 3. The experiment of high glucose cultured cardiomyocytes further elucidatedthe the relationship between high glucose caused FoxO1excessive productionand dreased adiponectin production.4. Supressing FoxO1with in vivo FoxO1siRNA transfection can effectivelyinhibit cardiomyocyte-derived FoxO1mRNA level. However, there is nosignificant impact on MI/R injury in APN-/-mice. That futher confirmed thatFoxO1played an important role in the regulation of adiponectin prodution.Our results suggested that supressing excessive production of FoxO1hassignificant effect in alleviating MI/R injury of diabetic mice. |
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