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.

Plasma levels of adiponectin (APN) are significantly increased in patients with renal dysfunction and are inversely related to the risk of cardiovascular mortality. The present study was designed to determine the role of APN in myocardial ischemia-reperfusion (MI/R) injury in mice with renal failure and delineate the underlying mechanisms. Renal failure was induced by subtotal nephrectomy (SN). Human recombinant globular domain of adiponectin (gAd) or full-length adiponectin (fAd) was administered via intraperitoneal injection once daily for 7 consecutive days after SN, and in vivo MI/R was introduced 3 wk later. Both plasma and urinary levels of APN increased significantly in SN mice. Compared with sham-operated mice, cardiac function was significantly depressed, and myocardial infarct size and apoptosis increased in SN mice following MI/R. The aggravated MI/R injury was further intensified in APN-knockout mice and markedly ameliorated by treatment with gAd but not fAd. Moreover, SN increased myocardial NO metabolites, superoxide, and their cytotoxic reaction product peroxynitrite, upregulated inducible NO synthase expression, and decreased endothelial NOS phosphorylation. In addition, SN mice also exhibited reduced APN receptor-1 (AdipoR1) expression and AMPK activation. All these changes were further amplified in the absence of APN but reversed by gAd treatment. The present study demonstrates that renal dysfunction increases cardiac susceptibility to ischemic-reperfusion injury, which is associated with downregulated APN/AdipoR1/AMPK signaling and increased oxidative/nitrative stress in local myocardium, and provides the first evidence for the protective role of exogenous supplement of gAd on MI/R outcomes in renal failure.

HomeHypertensionVol. 62, No. 2Hypoadiponectinemia and Endogenous Nitric Oxide Synthase Inhibitor in Hypertension Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBHypoadiponectinemia and Endogenous Nitric Oxide Synthase Inhibitor in Hypertension Kazushi Tsuda Kazushi TsudaKazushi Tsuda Cardiovascular and Metabolic Research Center, Kansai University of Health Sciences, Osaka, Japan Originally published10 Jun 2013https://doi.org/10.1161/HYPERTENSIONAHA.113.01641Hypertension. 2013;62:e4Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2013: Previous Version 1 To the Editor:We read with great interest the article by Xing et al1 dealing with the relationship between adiponectin and vascular insulin resistance in hypertension. The results of their study demonstrated that hypoadiponectinemia induced vascular insulin resistance in young spontaneously hypertensive rats. In addition, the authors indicated that downregulation of APPL1 (an adaptor protein that interacts directly with adiponectin receptors by the phosphotyrosine-binding domain) might partially contribute to vascular insulin resistance by differentially modulating the activation of Akt-dependent nitric oxide (NO) and ERK1/2-dependent endothelin-1 pathways in vascular endothelium in spontaneously hypertensive rats. Furthermore, the authors proposed that supplementation with exogenous adiponectin may have potential therapeutic value in the prevention and alleviation of endothelial dysfunction and vascular insulin resistance in hypertension.Current evidence indicates that adiponectin may improve NO bioavailability and restore endothelial dysfunction. In a study presented previously, we investigated the relationship between plasma adiponectin levels and plasma NO metabolites in hypertensive subjects.2 It was demonstrated that plasma adiponectin levels were significantly correlated with plasma NO metabolites.2 In the separate series of the study, we reported that reduced membrane fluidity of red blood cells was associated with hypoadiponectinemia in hypertensive subjects.3 Reduced membrane fluidity of red blood cells might cause a disturbance in the blood rheological behavior and microcirculation, which could contribute, at least in part, to the pathophysiology of circulatory disorders. It might be possible that adiponectin would be a defense against vascular complications in circulatory disorders through increased NO production.However, it has been shown that, in an in vitro study, adiponectin significantly inhibited the tumor necrosis factor-α–induced asymmetrical dimethylarginine (an endogenous NO synthase inhibitor) accumulation in both human umbilical vein endothelial cells and human coronary artery endothelial cells, which was accompanied by an increase in dimethylarginine dimethylaminohydrolase activity.4 Heilman et al5 also have reported the elevated plasma adiponectin and decreased plasma asymmetrical dimethylarginine levels in children with type 1 diabetes mellitus. In this context, it is strongly suggested that adiponectin would protect against endothelial dysfunction, at least in part, by influencing asymmetrical dimethylarginine metabolism. Therefore, we would like to know whether asymmetrical dimethylarginine might actively participate in hypoadiponectinemia-induced alterations in NO signaling and vascular insulin resistance in the study of Xing et al. It would be important to assess more precisely the relationships between adiponectin and endogenous NO synthase inhibitors and their role in the progression of endothelial dysfunction and insulin resistance in hypertension.Kazushi TsudaCardiovascular and Metabolic Research CenterKansai University of Health SciencesOsaka, JapanDisclosuresNone.FootnotesLetters to the Editor will be published, if suitable, as space permits. They should not exceed 500 words (typed double-spaced) plus 5 references in length and may be subject to editing or abridgment. References 1. Xing W, Yan W, Liu P, Ji L, Li Y, Sun L, Tao L, Zhang H, Gao F. A novel mechanism for vascular insulin resistance in normotensive young SHRs: hypoadiponectinemia and resultant APPL1 downregulation.Hypertension. 2013; 61:1028–1035.LinkGoogle Scholar2. Tsuda K. Adiponectin and nitric oxide production in normotensive and hypertensive men.Clin Exp Pharmacol Physiol. 2007; 34(suppl 1):S64–S66.CrossrefGoogle Scholar3. Tsuda K. Adiponectin and membrane fluidity of erythrocytes in normotensive and hypertensive men.Obesity (Silver Spring). 2006; 14:1505–1510.CrossrefMedlineGoogle Scholar4. Eid HM, Lyberg T, Arnesen H, Seljeflot I. Insulin and adiponectin inhibit the TNFα-induced ADMA accumulation in human endothelial cells: the role of DDAH.Atherosclerosis. 2007; 194:e1–e8.CrossrefMedlineGoogle Scholar5. Heilman K, Zilmer M, Zilmer K, Kool P, Tillmann V. Elevated plasma adiponectin and decreased plasma homocysteine and asymmetric dimethylarginine in children with type 1 diabetes.Scand J Clin Lab Invest. 2009; 69:85–91.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Abdel-Fadeil M, Abedelhaffez A, Makhlouf H and Al Qirshi G (2017) Obstructive sleep apnea: Influence of hypertension on adiponectin, inflammatory markers and dyslipidemia, Pathophysiology, 10.1016/j.pathophys.2017.08.003, 24:4, (305-315), Online publication date: 1-Dec-2017. Xing W, Ji L, Li Y, Gao F, Yan W, Liu P, Sun L, Tao L and Zhang H (2013) Response to Hypoadiponectinemia and Endogenous Nitric Oxide Synthase Inhibitor in Hypertension, Hypertension, 62:2, (e5-e5), Online publication date: 1-Aug-2013. August 2013Vol 62, Issue 2 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/HYPERTENSIONAHA.113.01641PMID: 23753414 Originally publishedJune 10, 2013 PDF download Advertisement SubjectsEndothelium/Vascular Type/Nitric Oxide

Sphingosine-1-phosphate (S1P) signaling directs a diverse array of biological processes, such as anti-apoptosis, promoting in[[Unable to Display Character: fl]]ammatory, and inducing fibrosis. Therefore, S1P levels need to be tightly controlled through the delicate interplay of its generating enzymes and its degrading enzymes. S1P lyase (SPL1) is a stress-activated enzyme responsible for irreversible S1P catabolism. This study was designed to determine the role of SPL1 in ischemia/reperfusion (I/R) induced cardiac dysfunction and the underlying mechanisms. Mice were subjected to 40 minutes left coronary artery occlusion followed by reperfusion for 24 hours, 1 week, or 4 weeks. First, we observed that SPL1 expression in cardiac tissue was upregulated by about 4-fold in response to I/R (n=6, P <0.01 versus sham). Further, oral administration of THI (an SPL1 inhibitor, 25 mg/L, daily) increased plasma S1P level and deteriorated I/R-induced apoptosis and fibrosis determined 1 week after I/R (all P <0.05 versus I/R + saline group), and cardiac dysfunction (ejection fraction: 39.1 ± 3.1% in I/R + THI group versus 50.7 ± 2.8% in I/R + saline group determined 4 weeks after I/R, P <0.05). After 1 week of I/R, hearts from THI-fed mice exhibited increased hypoxia-inducible factor-1α (HIF-1α) expression, along with significant oxidative/nitrative damage as evidenced by gp91 phox , inducible nitric oxide synthase (iNOS), and 3-nitrotyrosine abundance (all P <0.05 versus vehicle). To define the underlying mechanisms of SPL1 in I/R-induced heart injury, neonatal rat ventricular myocytes were cultured and submitted to hypoxia/reoxygenation (H/R, 9 hours/3 hours) with or without THI treatment. Importantly, THI significantly increased gp91 phox , iNOS, and 3-nitrotyrosine level, as well as apoptosis (n=8, all P <0.05 versus H/R), all of which were inhibited by pretreatment with HIF-1α siRNA (all P <0.05 versus scramble RNA). The present study demonstrated for the first time that SPL1 is remarkably upregulated during I/R and protects heart from I/R-induced S1P/HIF-1α-mediated oxidative/nitrative stress, suggesting SPL1 as a potential targets of therapy for ischemic heart disease.

Objectives The circulating, adipocyte-secreted hormone adiponectin (APN) exerts protective effects on the heart under stress conditions. Recent study demonstrated that APN induces a marked Ca2+ influx in skeletal muscle. However, whether APN modulates [Ca2+]i activity, especially modulates [Ca2+]i transients in cardiomyocytes is still unknown. This study was designed to determine whether and how APN modulates [Ca2+]i transients in cardiomyocytes. Methods Adult male wild-type (WT) and APN knock-out (APN KO) mice were subjected to myocardial ischaemia/reperfusion (I/R, 30 min/30 min) injury.We observed CaMKII-PLB phosphorylation and SR Ca2+-ATPase (SERCA2) activity were downregulated in I/R heart of WT mice (all P <0.01) and further decreased in I/R heart of APN KO mice (P <0.05). Administration of globular adiponectin domain and full-length adiponectin 20 min before reperfusion significantly reversed the decrease in CaMKII-PLB phosphorylation and SERCA2 activity in WT and APN KO mice (all P <0.05). Compared with WT littermates, single myocytes isolated from APN KO mice displayed equal cell lengths, caffeine-induced Ca2+ transient peaks, and number of Ca2+ sparks (all P >0.05), but decreased [Ca2+]i transients (P <0.001), decreased cell shortening (P <0.01), and a prolonged Ca2+ decay rate (P <0.05), all of which were reversed by APN exposure (all P <0.05). Preincubation with KN-93 (a CaMKII inhibitor), but not Rp-cAMP (a PKA inhibitor), completely abrogated the APN-mediated improvement in [Ca2+]i transients (P <0.05). Interestingly, pretreatment with the sphingosine 1-phosphate (S1P) receptor (S1PR1/3) antagonist VPC23019, but not AMPK inhibitor compound C, virtually abolished APN-stimulated CaMKII-PLB-SERCA2 activation during I/R (P <0.05). A short incubation with APN also reversed the decreases in S1P content and CaMKII-PLB signalling in cultured cardiomyocytes from APN KO mice (all P <0.05). Concomitantly, S1P activated CaMKII-PLB signalling in neonatal rat ventricular myocytes dose-dependently and improved [Ca2+]i transients in APN KO myocytes via S1PR1/3 (all P <0.05). Results Treatment of myocytes after hypoxia/reoxygenation with S1P also increased cell viability and reduced caspase-3 activity and apoptosis (all P <0.05); this effect was partially abolished by siRNAs against CaMKIIδ/PLB/SERCA2 (all P <0.05). More importantly, pharmacological inhibition of S1P/S1PR and siRNA-mediated silencing of SERCA2 could suppress APN-mediated cardioprotection during I/R (all P <0.05). Conclusions These data demonstrate that S1P is a novel regulator of SERCA2 by activating CaMKII-PLB signalling and mediates APN-induced cardioprotection.

Vascular insulin resistance contributes to elevated peripheral vascular resistance and subsequent hypertension. Clinical observation showed that lower plasma adiponectin concentration is significantly associated with hypertension. This study was aimed to determine whether hypoadiponectinemia induces vascular insulin resistance before systemic hypertension and the underlying mechanisms. Four-week-old young spontaneously hypertensive rats (ySHRs, normotensive) and adiponectin knockout (KO; APN -/- ) mice were used to evaluate the role of hypoadiponectinemia in insulin-induced vasodilation of resistance vessels. ySHRs showed significant vascular insulin resistance as evidenced by the blunted vasorelaxation response to insulin in mesenteric arterioles compared with that of age-matched Wistar-Kyoto controls. Serum adiponectin and mesenteric arteriolar APPL1 (an adaptor protein that mediates adiponectin signaling) expression of ySHRs were significantly reduced. In addition, Akt and endothelial NO synthase phosphorylation and NO production in arterioles were markedly reduced, whereas extracellular signal-regulated protein kinases 1/2 (ERK1/2) phosphorylation and endothelin-1 secretion were augmented in ySHRs. APN -/- mice showed significantly decreased APPL1 expression and vasodilation evoked by insulin. More importantly, treatment of ySHRs in vivo with the globular domain of adiponectin for 1 week increased APPL1 expression and insulin-induced vasodilation, and restored the balance between insulin-stimulated endothelial vasodilator NO and vasoconstrictor endothelin-1. In cultured human umbilical vein endothelial cells, globular domain of adiponectin upregulated APPL1 expression. Suppression of APPL1 expression with small interfering RNA markedly blunted the globular domain of adiponectin-induced insulin sensitization as evidenced by reduced Akt/endothelial NO synthase and potentiated ERK1/2 phosphorylations. In conclusion, hypoadiponectinemia induces APPL1 downregulation in the resistance vessels, contributing to the development of vascular insulin resistance by differentially modulating the Akt/endothelial NO synthase/NO and ERK1/2/endothelin-1 pathways in vascular endothelium in normotensive ySHRs.

Impaired mitochondrial biogenesis causes skeletal muscle damage in diabetes. However, whether and how mitochondrial biogenesis is impaired in the diabetic heart remains largely unknown. Whether adiponectin (APN), a potent cardioprotective molecule, regulates cardiac mitochondrial function has also not been previously investigated. In this study, electron microscopy revealed significant mitochondrial disorders in ob/ob cardiomyocytes, including mitochondrial swelling and cristae disorientation and breakage. Moreover, mitochondrial biogenesis of ob/ob cardiomyocytes is significantly impaired, as evidenced by reduced Ppargc-1a/Nrf-1/Tfam mRNA levels, mitochondrial DNA content, ATP content, citrate synthase activity, complexes I/III/V activity, AMPK phosphorylation, and increased PGC-1α acetylation. Since APN is an upstream activator of AMPK and APN plasma levels are significantly reduced in ob/ob mice, we further tested the hypothesis that reduced APN in ob/ob mice is causatively related to mitochondrial biogenesis impairment. One week of APN treatment of ob/ob mice activated AMPK, reduced PGC-1α acetylation, increased mitochondrial biogenesis, and attenuated mitochondrial disorders. In contrast, knocking out APN inhibited AMPK-PGC-1α signaling and impaired both mitochondrial biogenesis and function. The ob/ob mice exhibited lower survival rates and exacerbated myocardial injury after MI, when compared to controls. APN supplementation improved mitochondrial biogenesis and attenuated MI injury, an effect that was almost completely abrogated by the AMPK inhibitor compound C. In high glucose/high fat treated neonatal rat ventricular myocytes, siRNA-mediated knockdown of PGC-1α blocked gAd-enhanced mitochondrial biogenesis and function and attenuated protection against hypoxia/reoxygenation injury. In conclusion, hypoadiponectinemia impaired AMPK-PGC-1α signaling, resulting in dysfunctional mitochondrial biogenesis that constitutes a novel mechanism for rendering diabetic hearts more vulnerable to enhanced MI injury.

Contrast-induced nephropathy (CIN) is one of major complications in patients undergoing PCI. We prospectively examined the association of urinary adiponectin (UAPN), a sensitive marker for early renal function impairment, with CIN. We enrolled 208 patients without severe hepatic and renal function

FNDC5 is a hormone secreted by myocytes that could reduce obesity and insulin resistance, However, the exact effect of FNDC5 on glucose and lipid metabolism remain poorly identified; More importantly, the signaling pathways that mediate the metabolic effects of FNDC5 is completely unknown. Here we showed that FNDC5 stimulates β-oxidation and glucose uptake in C2C12 cells in a dose- and time-dependent fashion in vitro (n=8, all P<0.01). In vivo study revealed that FNDC5 also enhanced glucose tolerance in diabetic mice and increased the glucose uptake evidenced by increased [18F] FDG accumulation in hearts by PET scan (n=6, all P<0.05). FNDC5 decreased the expression of gluconeogenesis related molecules (PEPCK and G6Pase) and increased the phosphorylation of ACC, a key modulator of fatty-acid oxidation, both in hepatocytes and C2C12 cells (n=3, all P<0.05). In parallel with its stimulation of β-oxidation and glucose uptake, FNDC5 increased the phosphorylation of AMPK both in hepatocytes and C2C12 cells in a dose- and time-dependent fashion in vitro and in vivo. More importantly, the β-oxidation and glucose uptake, the expression of PEPCK and G6Pase and the phosphorylation of ACC induced by FNDC5 were attenuated by AMPK inhibitor in hepatocytes and C2C12 cells (P<0.05). Most importantly, the FNDC5 induced glucose uptake and phosphorylation of ACC were attenuated in AMPK-DN mice (n=6, all P<0.05). The glucose-lowering effect of FNDC5 in diabetic mice was also attenuated by AMPK inhibitor. Our data presents the direct evidence that FNDC5 stimulates glucose utilization and fatty-acid oxidation by AMPK signaling pathway, suggesting that FNDC5 be a novel pharmacological approach for type 2 diabetes.

Objective: To construct prokaryotic expression vector pET-22b(+)-gAd of human globular domain of adiponectin(gAd) gene, express and purify no-tagged recombinant human gAd protein. Methods: Total RNA was extracted from human adipose tissue. The gAd coding sequence was subcloned into the pET-22b(+) after ampli?fied by PCR. The recombinant plasmids were transformed into E.coli BL21(DE3), and the gAd was soluble ex?pressed in suitable temperature, by IPTG induction. The expressed gAd was purified by three-step procedure: am?monium sulfate precipitation, gel filtration chromatography and anion exchange chromatography. SDS-PAGE, West?ern blot, HPLC and the ability to induce the phosphorylation of AMPK in HUVEC were used for identification, pu?rity and biological activity assay. Results: The human gAd coding sequence was cloned into pET-22b(+) vector. After expression and purification, the purity of gAd was more than 95%. The recombinant human gAd significantly induced the phosphorylation of AMPK in HUVEC. Conclusion: The no-tagged recombinant human gAd was suc?cessfully expressed and purified by prokaryotic expression system and three-step purification procedure with high purity and biological activity..

Oxidative/nitrative stress plays an important role in myocardial ischemia/reperfusion (MI/R) injury. Notch1 participates in the regulation of cardiogenesis and cardiac response to hypertrophic stress, but the function of Notch1 signaling in MI/R has not been explored. This study aims to determine the role of Notch1 in MI/R, and investigate whether Notch1 confers cardioprotection. Notch1 specific small interfering RNA (siRNA, 20 μg) or Jagged1 (a Notch ligand, 12 μg) was delivered through intramyocardial injection. 48 h after injection, mice were subjected to 30 min of myocardial ischemia followed by 3 h (for cell apoptosis and oxidative/nitrative stress), 24 h (for infarct size and cardiac function), or 2 weeks (for cardiac fibrosis and function) of reperfusion. Cardiac-specific Notch1 knockdown resulted in significantly aggravated I/R injury, as evidenced by enlarged infarct size, depressed cardiac function, increased myocardial apoptosis and cardiac fibrosis. Downregulation of Notch1 increased expression of inducible NO synthase (iNOS) and gp 91phox , enhanced the production of NO metabolites and superoxide, as well as their cytotoxic reaction product peroxynitrite. Moreover, Notch1 blockade also reduced phosphorylation of endothelial NO synthase (eNOS) and Akt, and increased expression of PTEN, a key phosphatase involved in the regulation of Akt phosphorylation. In addition, activation of Notch1 by Jagged1 or administration of peroxynitrite scavenger reduced production of peroxynitrite and attenuated MI/R injury. These data indicate that Notch1 signaling protects against MI/R injury partly though PTEN/Akt mediated anti-oxidative and anti-nitrative effects.

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.

As an inhibitor of the antioxidant thioredoxin, thioredoxin-interacting protein (Txnip) is linked to insulin resistance. NLRP3 inflammasome, a major regulator of innate immunity, has been reported to be activated by Txnip, thus contributing to the pathogenesis of type 2 diabetes mellitus. However, the role of Txnip and its NLRP3 inflammasome activation in the myocardial ischemia/reperfusion (MI/R) injury has not been previously investigated. C57BL/6J mice were subjected to 30 min of ischemia and 3 or 24 hrs of reperfusion. The ischemic heart exhibited increased Txnip and NLRP3 expressions, increased interaction between Txnip and NLRP3 (by immunoprecipitation, 1.8-fold increase over sham), and increased IL-1β, IL-18 and caspase-1 expressions (%increase: 80%, 77% and 110%, respectively) (n=8, all P <0.05). Compared with vehicle group, those mice either receiving intramyocardial small-interfering RNA (siRNA) injection to specifically knockdown the myocardial NLRP3 or intraperitoneal injection of the inflammasome inhibitor (BAY 11-7082) exhibited significantly improved cardiac function (by 28% and 25%), decreased the infarct size (by 40% and 38%), and decreased the cardiomyocytes apoptosis (all P <0.05). NLRP3 knockdown or inflammasome inhibitor also decreased the inflammatory cells infiltration (macrophages and neutrophils) and cytokines (TNF-α, INF-γ and IL-6) production (all P <0.05). To elucidate the role of Txnip in the NLRP3 activation in MI/R, intramyocardial injection of Txnip siRNA was performed to specifically knockdown the myocardial Txnip expression. Compared with vehicle, the Txnip knockdown significantly decreased Txnip/NLRP3 interaction and NLRP3activation as evidenced by lower expressions of IL-1β and caspase-1, decreased inflammatory cells infiltration and cytokines expressions, and consequently decreased the myocardial infarct size and increased the heart function (all P <0.05). Collectively, we demonstrated for the first time that Txnip mediatedNLRP3 inflammasome activation is a novel mechanism of MI/R injury. Interventions targeted to blocking the activation of NLRP3 by inhibiting Txnip may have therapeutic potential for preventing MI/R injury.

Edaravone has been recognized as a potential protective agent for cardiovascular diseases. It is well known that edaravone can attenuate oxidative stress and inhibit the production of proinflammatory cytokines such as TNF-α. However, the mechanisms behind the cardioprotection of edaravone are still incompletely understood yet. As a primary adipose-derived protein, adiponectin exhibits protective properties on the heart and blood vessels. Accumulative studies have shown that there exists a reciprocal relationship between adiponectin and TNF-α, i.e., adiponectin negatively regulates TNF-α expression, whereas adiponectin expression is inhibited by TNF-α. So we hypothesize that edaravone plays a role in cardioprotection partly through modulation of adiponectin level by suppression of TNF-α.

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。

The receptor for advanced glycation end products (RAGE) and thioredoxin (Trx) play opposing roles in diabetic myocardial ischemia-reperfusion (MI/R) injury. We recently demonstrated nitrative modification of Trx leads to its inactivation and loss of cardioprotection. The present study is to determine the relationship between augmented RAGE expression and diminished Trx activity pertaining to exacerbated MI/R injury in the diabetic heart. The diabetic state was induced in mice by multiple intraperitoneal low-dose streptozotocin injections. RAGE small-interfering RNA (siRNA) or soluble RAGE (sRAGE, a RAGE decoy) was via intramyocardial and intraperitoneal injection before MI/R, respectively. Mice were subjected to 30 min of myocardial infarction followed by 3 or 24 h of reperfusion. At 10 min before reperfusion, diabetic mice were randomized to receive EUK134 (peroxynitrite scavenger), recombinant hTrx-1, nitrated Trx-1, apocynin (a NADPH oxidase inhibitor), or 1400W [an inducible nitric oxide synthase (iNOS) inhibitor] administration. The diabetic heart manifested increased RAGE expression and N(ε)-(carboxymethyl)lysine (CML, major advanced glycation end product subtype) content, reduced Trx-1 activity, and increased Trx nitration after MI/R. RAGE siRNA or administration of sRAGE in diabetic mice decreased MI/R-induced iNOS and gp91(phox) expression, reduced Trx nitration, preserved Trx activity, and decreased infarct size. Apocynin or 1400W significantly decreased nitrotyrosine production and restored Trx activity. Conversely, administration of either EUK134 or reduced hTrx, but not nitrated hTrx, attenuated MI/R-induced superoxide production, RAGE expression, and CML content and decreased cardiomyocyte apoptosis in diabetic mice. Collectively, we demonstrate that RAGE modulates the MI/R injury in a Trx nitrative inactivation fashion. Conversely, nitrative modification of Trx blocked its inhibitory effect upon RAGE expression in the diabetic heart. This is the first direct evidence demonstrating the alternative cross talk between RAGE overexpression and nitrative Trx inactivation, suggesting that interventions interfering with their interaction may be novel means of mitigating diabetic MI/R injury.