Downregulation of adiponectin (APN) multimerization is significantly correlated with the aggravation of myocardial ischemia/reperfusion (MI/R) injury in type 2 diabetes mellitus (T2DM). Resveratrol (RSV) upregulates APN multimerization in adipocytes, but whether RSV improves endogenous APN multimerization and thus attenuates MI/R injury in T2DM mice has never been investigated. T2DM mice were treated with 10 mg/kg RSV daily for 3 weeks, followed by 30 minutes of myocardial ischemia and 3 hours or 24 hours of reperfusion. RSV administration alleviated MI/R injury in diabetic mice, as evidenced by reduced infarct size, cardiomyocyte apoptosis, and caspase-3 activity, and improved cardiac function. Moreover, RSV reversed the downregulated APN levels and multimerization both in plasma and adipose tissue, accompanied by increased disulfide bond A oxidoreductase-like protein (DsbA-L) expression in T2DM mice. Conversely, serving as a key downstream molecule of APN in ameliorating MI/R injury, inhibition of AMP-activated protein kinase (AMPK) significantly attenuated the cardioprotective effects of RSV. In conclusion, long-term administration of RSV upregulates adiponectin levels and multimerization in T2DM mice, consequently attenuating MI/R injury partially through APN-AMPK signaling.

Tumor necrosis factor-α (TNF-α) antagonism alleviates myocardial ischemia-reperfusion (MI/R) injury. However, the mechanisms by which the downstream mediators of TNF-α change after acute antagonism during MI/R remain unclear. Adiponectin (APN) exerts anti-ischemic effects, but it is downregulated during MI/R. This study was conducted to investigate whether TNF-α is responsible for the decrease of APN, and whether antagonizing TNF-α affects MI/R injury by increasing APN. Male adult wild-type (WT), APN knockout (APN KO) mice, and those with cardiac knockdowns of APN receptors via siRNA injection were subjected to 30 min of MI followed by reperfusion. The TNF-α antagonist etanercept or globular domain of APN (gAD) was injected 10 min before reperfusion. Etanercept ameliorated MI/R injury in WT mice as evidenced by improved cardiac function, and reduced infarct size and cardiomyocyte apoptosis. APN concentrations were augmented in response to etanercept, followed by an increase in AMP-activated protein kinase phosphorylation. Etanercept still increased cardiac function and reduced infarct size and apoptosis in both APN KO and APN receptors knockdown mice. However, its potential was significantly weakened in these mice compared with the WT mice. TNF-α is responsible for the decrease in APN during MI/R. The cardioprotective effects of TNF-α neutralization are partially due to the upregulation of APN. The results provide more insight into the TNF-α-mediated signaling effects during MI/R and support the need for clinical trials to validate the efficacy of acute TNF-α antagonism in the treatment of MI/R injury.

AIM: To observe whether resveratrol(RSV) attenuates myocardial ischemia/reperfusion injury(MI/RI) in type Ⅱ diabetes mellitus(T2DM) and,if so,to further investigate the underlying mechanisms.METHODS: T2DM was induced by a high-fat diet(HD) plus low-dose i.p.streptozotocin(STZ) injection.Mice were treated with 10 mg/kg RSV daily by intragastric administration for 3 weeks after acknowledgement of T2DM.Mice were divided into six groups: sham group,I/R group,T2DM sham group,T2DM+I/R group,RSV group,and CpC group.After 30 min ischemia by slip-knot ligature of the left anterior descending coronary artery,myocardium was reperfused for 3 h after knot release(for apoptosis by TUNEL,caspase-3 activity by ELISA) or 24 h(for infarct size determination by TTC staining).At 1 h before MI/RI,compound C(an AMPK inhibitor) was administered i.p.(20 mg/kg).RESULTS: HD feeding plus low-dose STZ injection successfully induced T2DM.RSV alleviated MI/RI in diabetic mice as evidenced by decreased infarct size,cardiomyocyte apoptosis(P0.01) and caspase-3 activity(P0.05).RSV treatment also improved APN level both in plasma and adipose tissue in diabetic mice(all P0.01).Conversely,administration of AMPK inhibitor compound C significantly attenuated the cardioprotective effects of RSV(all P0.05).CONCLUSION: RSV upregulates adiponectin levels in both plasma and adipose tissue in T2DM,thus attenuating MI/RI.

Objective:To construct prokaryotic expression system for mass production of recombinant human thioredoxin and establish the purification process of thioredoxin.Methods: Total RNA was extracted from HEK293(human embryonic kidney cells).The thioredoxin coding sequence was subcloned into the pET-22b(+) vector after amplified by PCR.The recombinant plasmids were transformed into E.coli BL21(DE3),and the thioredoxin was expressed with IPTG induction.The expressed thioredoxin was purified by two-step ion exchange chromatography and tested by SDS-PAGE,Western blotting,MALDI-TOF-M,HPLC,and insulin disulfide reduction assay for identification,purity assay and activity determination,respectively.Results: Gene sequencing demonstrated that thioredoxin coding sequence was cloned into pET-22b(+) vector successfully.The prokaryotic expression system achieved high yield of thioredoxin(180 mg/5L of fermentation broth),which was identified by Western blotting and MALDI-TOF-MS,with an estimated the molecular weight of 12 000.The purity of thioredoxin is more than 95%.The activity of purified thioredoxin had the same activity as the standard control.Conclusion: The prokaryotic expression system could achieve mass production of recombinant human thioredoxin,which can be highly purified by two-steps ion exchange chromatography.This preliminary study provides the foundation for the large-scale industrial production of thioredoxin.

目的:阐明心肌缺血/再灌注（MI/R）时,脂联素（APN）与肿瘤坏死因子-α（TNF-α）的关系,以及使用中和抗体阻断TNF-α可否提高血浆APN,进而发挥心肌保护作用。方法:96只成年雄性C57小鼠和36只ob/ob小鼠均采用30 min缺血/再灌注（I/R）建立MI/R模型。96只C57小鼠分为假手术组、手术+盐水对照组及手术+抗TNF-α中和抗体治疗组（n=32）;36只ob/ob小鼠分为ob/ob假手术组、ob/ob手术+盐水对照组及ob/ob手术+抗TNF-α中和抗体治疗组（n=12）。假手术或缺血20 min后,腹腔注射给予单次抗TNF-α中和抗体或盐水干预。分别采用ELISA检测TNF-α与APN血浆水平;小鼠心脏超声评估心脏LVEF;伊文氏蓝/TTC染色检测心脏梗死面积;以及TUNEL/Caspase-3活性检测观察心肌细胞凋亡。结果:血浆ELISA测定发现,MI/R后,小鼠血浆TNF-α水平在再灌后1 h即显著升高,后缓慢下降。注射抗TNF-α中和抗体可在再灌后1 h即中和TNF-α（P＜0.01）,同时在再灌注3 h、8 h、1 d及3 d后4个时间点,较给予盐水对照显著升高血浆APN（P＜0.01）。通过小鼠心脏超声、伊文氏蓝/TTC染色和TUNEL/Caspase-3活性检测发现,与给予盐水的对照相比,腹腔注射抗TNF-α中和抗体可提高小鼠的心肌功能（P＜0.05）、减少梗死面积（P＜0.01）及心肌细胞的凋亡（P＜0.01）。而在ob/ob小鼠中,通过以同样的实验方法证实,单次注射抗TNF-α中和抗体已不能提高血浆APN的含量,其减轻心肌损伤的作用同样被显著削弱,但给予APN球状片段仍可发挥心肌保护作用。结论:以抗TNF-α的中和抗体阻断TNF-α可逆转MI/R后血浆APN的降低并发挥心肌保护作用,提示抗TNF-α中和抗体发挥的心肌保护作用可能部分通过提高APN实现。

目的:观察心肌中插头转录因子O1（FoxO1）在糖尿病（DM）小鼠心肌中表达量变化及对小鼠心肌缺血/再灌注（I/R）损伤的影响。方法:将90只健康雄性Swiss小鼠随机分为5组:假手术（Sham）组、I/R组、DM+Sham组、DM+I/R组及DM+FoxO1SiRNA+I/R组,每组18只。采用高糖高脂饮食加链脲菌素（Streptozocin,STZ）腹腔注射诱导建立DM小鼠模型。采用FoxO1SiRNA心肌点注射下调心肌FoxO1表达。心肌I/R损伤模型的建立,采用结扎心脏冠状动脉左前降支30 min后再灌注方案实施。心肌再灌注3 h后,用原位缺口末端标法（TUNEL）检测心肌细胞凋亡。用ELISA法检测心肌中Caspase-3的活性。用Western blot法检测心肌中FoxO1的表达量。心肌再灌注24 h后,用2,3,5-三苯基氯化四氮唑（TTC）染色法检测心肌梗死（MI）的面积。结果:与Sham组比较,DM+Sham组心肌中FoxO1的表达量明显增高（P＜0.01）。与I/R组比,DM+I/R组MI的面积增大（P＜0.05）,心肌细胞凋亡数量及Caspase-3活性明显增加（P＜0.01）。与DM+I/R组相比,DM+FoxO1SiRNA+I/R组心肌FoxO1的表达量下调（P＜0.05）,MI面积及Caspase-3的活性减小（P＜0.05）,心肌细胞凋亡数量减少（P＜0.01）。结论:DM小鼠心肌中FoxO1表达量的增加可加重心肌I/R损伤;而下调心肌中FoxO1的表达量后,心肌I/R损伤减轻。

Objectives type 2 diabetes (T2DM) exacerbated myocardial ischemia/reperfusion (MI/R) injury, accompanied by significantly lower adiponectin (APN) level and diminished APN multimerisation. Resveratrol, a natural polyphenol, promotes APN up-regulation and multimerisation both in adipocytes and mice by up-regulation of DsbA-L (a recently identified protein that facilitates APN multimerisation and stability). Therefore, the present study aimed to observe whether RSV attenuates MI/R injury in T2DM, and if so, to further investigate the underlying mechanisms. Methods T2DM was induced by high-fat diet (HD) feeding plus low-dose streptozotocin (STZ) injection. Mice received an HD since three weeks old for eight weeks. After three weeks of HD feeding, mice were intraperitoneally injected with 100 mg/kg STZ (Sigma). T2DM was confirmed by markedly elevated fasting-blood glucose level (>11.1 mmol/l) five weeks after injection. Mice were treated with 10 mg/kg RSV daily by intragastric administration for three weeks since five weeks of HD feeding. Compound C (an AMPK inhibitor) was administrated by intraperitoneal injection with 20 ug/g one h before MI/R. Mice were subjected to 30 min of ischemia and three h or 24 h of reperfusion. Results HD feeding plus low-dose STZ injection successfully induced T2DM. Compared to normal control, diabetic mice manifested higher fasting-blood glucose level, lower glucose tolerance in OGTT examination (n=12, p<0.05), but there was no difference in plasma insulin levels. RSV alleviated MI/R injury in both normal and diabetic mice, as evidenced by decreased infarct size, cardiomyocytes apoptosis, caspase-3 activity, improved cardiac function (n=10, all p<0.05). Moreover, RSV treatment improved APN level, upregulated APN multimerisation both in plasma and adipose tissue, and increased DsbA-L expression in adipose tissue in diabetic mice (all p<0.01). Conversely, administration of AMPK inhibitor Compound C significantly attenuated the cardioprotective effects of RSV (all p<0.05). Conclusions RSV upregulates adiponectin level and multimerisation in both plasma and adipose tissue in T2DM, and therefore attenuates MI/R injury.

Objective Adiponectin is a protein secreted predominantly by differentiated adipocytes, which plays a protective role in the development of insulin resistance and exerts beneficial actions on the hearts subjected to ischemia/reperfusion (I/R) insult. Insulin has been proved to reduce myocardial I/R injury in both normal and diabetic ones. These suggest the relationship between the insulin and adiponectin effects in diabetes. The present study aimed to investigate the effect of insulin treatment on the systemic and local cardiac adiponectin levels and on the myocardial I/R injury in type 1 diabetic mice. Methods type 1 diabetic mice were rendered through 5 days of daily intraperitoneal injection with 40 mg/kg STZ, and confirmed by markedly elevated fasting-blood glucose levels (>11.1 μmol/l). Insulin (5 IU/kg·d) was administrated for a 14, 7 or 1 day duration, respectively, since diabetic models were established. MI/R was performed by Langendorff perfusion ex vivo in hearts from the 14 day duration mice, in which the hearts were subjected to 40 min of ischemia and 60 min of reperfusion. Results The plasma adiponectin level in type 1 diabetic mice increased on the day 7 but decreased on the day 14 (p<0.01 vs control). At the same time, cardiac adiponectin mRNA level in type 1 diabetic mice gradually decreased (p<0.01 vs control). The diabetic hearts showed an increased infarct size and cardiomyocytes apoptosis (p<0.01). Insulin treatment increased both the plasma and cardiac adiponectin level in diabetic mice (p<0.05), and decreased the infarct size and cardiomyocytes apoptosis in type 1 diabetic mice (p<0.01 vs diabetic). Compound C pretreatment (an inhibitor of AMPK) partly inhibited the effect of insulin treatment (p<0.01). Conclusions Insulin treatment increases both plasma and cardiac adiponectin levels, which is at least partly responsible for the cardioprotective effects of insulin treatment in diabetes.

Objectives Evidence exists that the increase of TNFα is associated with reperfusion injury in patients with myocardial infarction after PCI. Adiponectin (APN) is anti-inflammatory and cardioprotective protein suppressed by TNFα, and is significantly reduced during MI/R. Whether neutralising TNFα protects against MI/R injury through upregulation of APN level has not been previously investigated. Methods and Results Adult male C57 mice were subjected to 30 min MI followed by 3h or 24h reperfusion or sham MI/R for this study. Etanercept, a TNFα neutralising drug for treating rheumatoid arthritis in clinic, was intraperitoneally injected 10 min before reperfusion. Etanercept administration ameliorated MI/R injury evidenced by increased cardiac function (1 day, 7 day, 14 day p<0.05 vs vehicle), reduced infarct size (p <0.05 vs vehicle) and apoptosis (p<0.01 vs vehicle). Etanercept significantly increased plasma APN concentration at 3 h, 8 h, 1 day and 3 days after reperfusion, respectively (all p<0.05 vs vehicle) and pAMPK/AMPK ratio at 3 h in myocardium (p<0.05 vs vehicle). To further investigate whether Etanercept attenuated MI/R injury is related to the APN signalling, additional experiments were performed. Firstly, the siRNA was used to knockdown (KD) the APN receptor 1 & receptor 2 (APN-R1&2) by intramyocardial injection 48 h before MI/R in vivo. APN receptor knockdown attenuated the cardioprotective effect induced by Etanercept supported by decreased infarct size (10% decrease in KD+Etanercept vs 25% decrease in WT+Etanercept), apoptosis (caspase-3 activity 21% vs 35% reduction) and lower pAMPK/AMPK ratio. second, the ob/ob mice, which APN signalling is impaired, were subjected to the MI/R following the same procedure. The cardioprotective effect of Etanercept was attenuated in the ob/ob mice compared with the wild type mice as well. Conclusions Overall, we have demonstrated for the first time that upregulating of adiponectin is involved in the cardioprotective effect of Etanercept, suggesting that using a single administration of Etanercept during PCI might improve the outcome of myocardial infarction patients.

BackgroundThioredoxin-1 (Trx) is an important antioxidant and antiapoptotic small molecule and its activity can be altered by modification on different amino acid sites. After myocardial ischemia/reperfusion (MI/R), the increase of oxidative stress stimulates nitrative inactivation of Trx. In turn, nitrated Trx intensifies MI/R injury by mediating postischemic myocardial apoptosis. However, the precise mechanism of how nitrated Trx fails to exert a cardioprotective effect is to be determined.Methods and resultsIn this study, we examined if Y49 is the nitrative modification site and whether the single mutation of Trx at site Y49 (Trx Y49F) can resist nitrative inactivation and strengthen the inhibition of ASK-1 mediated apoptosis. In contrast to Trx, in vitro exposure of Trx Y49F to SIN-1 (a protein nitration reagent) did not result in protein nitration. The interactions of Trx/Trx Y49F and ASK-1 were studied by cotransfection of Trx/Trx Y49F and ASK-1 plasmids into HEK293A cells. By treating cells with H2O2 of adequate concentration, Trx-ASK-1 complex formation was reduced and activity of ASK-1 and p38 mitogen-activated protein kinase (MAPK) increased. In contrast, mutation Y49 prevented Trx nitration, increased Trx activity, restored Trx-ASK-1 interaction, reduced ASK-1 and P38 MAPK activity, and attenuated caspase 3 activation (p<0.01) under the same treatment of H2O2. Further animal experiments confirmed that compared with Trx, Trx Y49F is more effective on protecting MI/R injury by enhancing Trx-ASK-1 interaction, inhibiting p38 MAPK signalling pathway, and subsequent decreasing postischemic cardiomyocyte apoptosis (p<0.05).ConclusionsOur results demonstrated that the increase of oxidative stress will result in nitration of Trx at site Y49. Nitrative inactivation of Trx can be prevented by Y49F mutation, which strengthens the inhibition of ASK-1 mediated pathological apoptosis. This finding may lead to a novel therapeutic method to attenuate MI/R injury in patients.