N-acetylcysteine Protects Against Myocardial Ischemia-Reperfusion Injury Through Anti-ferroptosis in Type 1 Diabetic Mice.

The hearts of subjects with diabetes are vulnerable to ischemia-reperfusion injury (IRI). In contrast, experimentally rodent hearts have been shown to be more resistant to IRI at the very early stages of diabetes induction than the heart of the non-diabetic control mice, and the mechanism is largely unclear. Ferroptosis has recently been shown to play an important role in myocardial IRI including that in diabetes, while the specific mechanisms are still unclear. Non-diabetic control (NC) and streptozotocin-induced diabetic (DM) mice were treated with the antioxidant N-acetylcysteine (NAC) in drinking water for 4 week starting at 1 week after diabetes induction. Mice were subjected to myocardial IRI induced by occluding the coronary artery for 30 min followed by 2 h of reperfusion, subsequently at 1, 2, and 5 week of diabetes induction. The post-ischemic myocardial infarct size in the DM mice was smaller than that in NC mice at 1 week of diabetes but greater than that in the NC mice at 2 and 5 week of diabetes, which were associated with a significant increase of ferroptosis at 2 and 5 week but a significant reduction of ferroptosis at 1 week of diabetes. NAC significantly attenuated post-ischemic ferroptosis as well as oxidative stress and reduced infarct size at 2 and 5 week of diabetes. Application of erastin, a ferroptosis inducer, reversed the cardioprotective effects of NAC. It is concluded that increased oxidative stress and ferroptosis are the major factors attributable to the increased vulnerability to myocardial IRI in diabetes and that attenuation of ferroptosis represents a major mechanism whereby NAC confers cardioprotection against myocardial IRI in diabetes.

Propofol and salvianolic acid A synergistically attenuated cardiac ischemia-reperfusion injury in diabetic mice via modulating the CD36/AMPK pathway.

Background: Prevention of diabetic heart myocardial ischemia-reperfusion (IR) injury (MIRI) is challenging. Propofol attenuates MIRI through its reactive oxygen species scavenging property at high doses, while its use at high doses causes hemodynamic instability. Salvianolic acid A (SAA) is a potent antioxidant that confers protection against MIRI. Both propofol and SAA affect metabolic profiles through regulating Adenosine 5'-monophosphate-activated protein kinase (AMPK). The aim of this study was to investigate the protective effects and underlying mechanisms of low doses of propofol combined with SAA against diabetic MIRI. Methods: Diabetes was induced in mice by a high-fat diet followed by streptozotocin injection, and MIRI was induced by coronary artery occlusion and reperfusion. Mice were treated with propofol at 46 mg/kg/h without or with SAA at 10 mg/kg/h during IR. Cardiac origin H9c2 cells were exposed to high glucose (HG) and palmitic acid (PAL) for 24 h in the absence or presence of cluster of differentiation 36 (CD36) overexpression or AMPK gene knockdown, followed by hypoxia/reoxygenation (HR) for 6 and 12 h. Results: Diabetes-exacerbated MIRI is evidenced as significant increases in post-ischemic infarction with reductions in phosphorylated (p)-AMPK and increases in CD36 and ferroptosis. Propofol moderately yet significantly attenuated all the abovementioned changes, while propofol plus SAA conferred superior protection against MIRI to that of propofol. In vitro, exposure of H9c2 cells under HG and PAL decreased cell viability and increased oxidative stress that was concomitant with increased levels of ferroptosis and a significant increase in CD36, while p-AMPK was significantly reduced. Co-administration of low concentrations of propofol and SAA at 12.5 µM in H9c2 cells significantly reduced oxidative stress, ferroptosis and CD36 expression, while increasing p-AMPK compared to the effects of propofol at 25 µM. Moreover, either CD36 overexpression or AMPK silence significantly exacerbated HR-induced cellular injuries and ferroptosis, and canceled propofol- and SAA-mediated protection. Notably, p-AMPK expression was downregulated after CD36 overexpression, while AMPK knockdown did not affect CD36 expression. Conclusions: Combinational usage of propofol and SAA confers superior cellular protective effects to the use of high-dose propofol alone, and it does so through inhibiting HR-induced CD36 overexpression to upregulate p-AMPK.

Salvianolic acids and its potential for cardio-protection against myocardial ischemic reperfusion injury in diabetes.

The incidence of diabetes and related mortality rate increase yearly in modern cities. Additionally, elevated glucose levels can result in an increase of reactive oxygen species (ROS), ferroptosis, and the disruption of protective pathways in the heart. These factors collectively heighten the vulnerability of diabetic individuals to myocardial ischemia. Reperfusion therapies have been effectively used in clinical practice. There are limitations to the current clinical methods used to treat myocardial ischemia-reperfusion injury. As a result, reducing post-treatment ischemia/reperfusion injury remains a challenge. Therefore, efforts are underway to provide more efficient therapy. Salvia miltiorrhiza Bunge (Danshen) has been used for centuries in ancient China to treat cardiovascular diseases (CVD) with rare side effects. Salvianolic acid is a water-soluble phenolic compound with potent antioxidant properties and has the greatest hydrophilic property in Danshen. It has recently been discovered that salvianolic acids A (SAA) and B (SAB) are capable of inhibiting apoptosis by targeting the JNK/Akt pathway and the NF-κB pathway, respectively. This review delves into the most recent discoveries regarding the therapeutic and cardioprotective benefits of salvianolic acid for individuals with diabetes. Salvianolic acid shows great potential in myocardial protection in diabetes mellitus. A thorough understanding of the protective mechanism of salvianolic acid could expand its potential uses in developing medicines for treating diabetes mellitus related myocardial ischemia-reperfusion.

Perspectives for Forkhead box transcription factors in diabetic cardiomyopathy: Their therapeutic potential and possible effects of salvianolic acids.

Diabetic cardiomyopathy (DCM) is the primary cause of morbidity and mortality in diabetic cardiovascular complications, which initially manifests as cardiac hypertrophy, myocardial fibrosis, dysfunctional remodeling, and diastolic dysfunction, followed by systolic dysfunction, and eventually end with acute heart failure. Molecular mechanisms underlying these pathological changes in diabetic hearts are complicated and multifactorial, including but not limited to insulin resistance, oxidative stress, lipotoxicity, cardiomyocytes apoptosis or autophagy, inflammatory response, and myocardial metabolic dysfunction. With the development of molecular biology technology, accumulating evidence illustrates that members of the class O of Forkhead box (FoxO) transcription factors are vital for maintaining cardiomyocyte metabolism and cell survival, and the functions of the FoxO family proteins can be modulated by a wide variety of post-translational modifications including phosphorylation, acetylation, ubiquitination, arginine methylation, and O-glycosylation. In this review, we highlight and summarize the most recent advances in two members of the FoxO family (predominately FoxO1 and FoxO3a) that are abundantly expressed in cardiac tissue and whose levels of gene and protein expressions change as DCM progresses, with the goal of providing valuable insights into the pathogenesis of diabetic cardiovascular complications and discussing their therapeutic potential and possible effects of salvianolic acids, a natural product.

Bioinspired Polypeptide Photonic Films with Tunable Structural Color.

We report a new synthetic strategy of combining N-carboxyanhydride (NCA) chemistry and photonic crystals for the fabrication of polypeptide structural color films. Driven by surface-initiated ring-opening polymerization, the di-NCA derivative of l-cystine (Cys) is introduced to replicate the functionalized colloidal crystal templates and construct freestanding P(Cys) films with tunable structural color. Furthermore, the feasibility of preparing patterned polypeptide photonic films is demonstrated via template microfabrication. Because of the incorporation of l-glutamate (Glu) components, the P(Cys-co-Glu) co-polypeptide films are endowed with a visual color responsiveness toward pH changes. Additionally, the polypeptide photonic films show on-demand degradability. Given the large family of amino acid building blocks, this powerful and versatile approach paves the way for chemical derivatization of multifunctional peptide-based optical platforms.

Propionate alleviates myocardial ischemia-reperfusion injury aggravated by Angiotensin II dependent on caveolin-1/ACE2 axis through GPR41.

Myocardial ischemia/reperfusion (I/R) injury is still a lack of effective therapeutic drugs, and its molecular mechanism is urgently needed. Studies have shown that the intestinal flora plays an important regulatory role in cardiovascular injury, but the specific mechanism has not been fully elucidated. In this study, we found that an increase in Ang II in plasma was accompanied by an increase in the levels of myocardial injury during myocardial reperfusion in patients with cardiopulmonary bypass. Furthermore, Ang II treatment enhanced mice myocardial I/R injury, which was reversed by caveolin-1 (CAV-1)-shRNA or strengthened by angiotensin-converting enzyme 2 (ACE2)-shRNA. The results showed that CAV-1 and ACE2 have protein interactions and inhibit each other's expression. In addition, propionate, a bacterial metabolite, inhibited the elevation of Ang II and myocardial injury, while GPR41-shRNA abolished the protective effects of propionate on myocardial I/R injury. Clinically, the propionate content in the patient's preoperative stool was related to Ang II levels and myocardial I/R injury levels during myocardial reperfusion. Taken together, propionate alleviates myocardial I/R injury aggravated by Ang II dependent on CAV-1/ACE2 axis through GPR41, which provides a new direction that diet to regulate the intestinal flora for treatment of myocardial I/R injury.

Propofol postconditioning ameliorates hypoxia/reoxygenation induced H9c2 cell apoptosis and autophagy via upregulating forkhead transcription factors under hyperglycemia.

BACKGROUND: Administration of propofol, an intravenous anesthetic with antioxidant property, immediately at the onset of post-ischemic reperfusion (propofol postconditioning, P-PostC) has been shown to confer cardioprotection against ischemia-reperfusion injury, while the underlying mechanism remains incompletely understood. The FoxO transcription factors are reported to play critical roles in activating cardiomyocyte survival signaling throughout the process of cellular injuries induced by oxidative stress and are also involved in hypoxic postconditioning mediated neuroprotection, however, the role of FoxO in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown. METHODS: Rat heart-derived H9c2 cells were exposed to high glucose (HG) for 48 h (h), then subjected to hypoxia/reoxygenation (H/R, composed of 8 h of hypoxia followed by 12 h of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol (P-PostC) at the onset of reoxygenation. After having identified the optical concentration of propofol, H9c2 cells were subjected to H/R and P-PostC in the absence or presence of FoxO1 or FoxO3a gene silencing to explore their roles in P-PostC mediated protection against apoptotic and autophagic cell deaths under hyperglycemia. RESULTS: The results showed that HG with or without H/R decreased cell viability, increased lactate dehydrogenase (LDH) leakage and the production of reactive oxygen species (ROS) in H9c2 cells, all of which were significantly reversed by propofol (P-PostC), especially at the concentration of 25 µmol/L (P25) (all P < 0.05, NC vs. HG; HG vs. HG + HR; HG + HR + P12.5 or HG + HR + P25 or HG + HR + P50 vs. HG + HR). Moreover, we found that propofol (P25) decreased H9c2 cells apoptosis and autophagy that were concomitant with increased FoxO1 and FoxO3a expression (all P < 0.05, HG + HR + P25 vs. HG + HR). The protective effects of propofol (P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a (all P < 0.05, HG + HR + P25 vs. HG + HR + P25 + siRNA-1 or HG + HR + P25 + siRNA-5). CONCLUSION: It is concluded that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.

Allopurinol ameliorates liver injury in type 1 diabetic rats through activating Nrf2.

Hyperglycemia-induced oxidative stress plays important roles in the development of non-alcoholic fatty liver disease (NAFLD), which is a common complication in diabetic patients. The Nrf2-Keap1 pathway is important for cell antioxidant protection, while its role in exogenous antioxidant mediated protection against NAFLD is unclear. We thus, postulated that antioxidant treatment with allopurinol (ALP) may attenuate diabetic liver injury and explored the underlying mechanisms. Control (C) and streptozotocin (STZ)-induced diabetes rats (D) were untreated or treated with ALP for 4 weeks starting at 1 week after diabetes induction. Serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), production of lipid peroxidation product malondialdehyde (MDA), and serum superoxide dismutase (SOD) were detected. Liver protein expressions of cleaved-caspase 3, IL-1ß, nuclear factor-erythroid-2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1), P62, Kelch-like ECH-associated protein 1 (Keap1), and LC3 were analyzed. In vitro, cultured rat normal hepatocytes BRL-3A were grouped to normal glucose (5.5 mM, NG) or high glucose (25 mM, HG) and treated with or without allopurinol (100 µM) for 48 h. Rats in the D group demonstrated liver injury evidenced as increased serum levels of ALT and AST. Diabetes increased apoptotic cell death, enhanced liver protein expressions of cleaved-caspase 3 and IL-1ß with concomitantly increased production of MDA while serum SOD content was significantly reduced (all P < 0.05 vs C). In the meantime, protein levels of Nrf2, HO-1, and P62 were reduced while Keap1 and LC3 were increased in the untreated D group as compared to control (P < 0.05 vs C). And all the above alterations were significantly attenuated by ALP. Similar to our findings obtained from in vivo study, we got the same results in in vitro experiments. It is concluded that ALP activates the Nrf2/p62 pathway to ameliorate oxidative stress and liver injury in diabetic rats.

Astragalus polysaccharide ameliorates H2O2-induced human umbilical vein endothelial cell injury.

Endothelial dysfunction caused by reactive oxygen species (ROS) has been implicated in numerous cardiovascular diseases. Astragalus polysaccharide (APS), an important bioactive component extracted from the Chinese herb Astragalus membranaceus, has been widely used for the treatment of cardiovascular disease. The present study aimed to investigate the effects of APS on hydrogen peroxide (H2O2)­induced human umbilical vein endothelial cell (HUVEC) injury. Following treatment with 400 µM H2O2 for 24 h, cell viability was decreased and apoptosis was increased. However, pretreatment with APS for 1 h significantly attenuated H2O2­induced injury in HUVECs. In addition, APS decreased intracellular ROS levels, increased the protein expression of endothelial nitric oxide synthase and copper­zinc superoxide dismutase, elevated intracellular cyclic guanosine monophosphate (an activity marker for nitric oxide) levels and restored the mitochondrial membrane potential, compared with cells treated with H2O2 only. In conclusion, the results of the present study suggested that APS may protect HUVECs from injury induced by H2O2 via increasing the cell antioxidant capacity and nitric oxide (NO) bioavailability, which may contribute to the improvement of the imbalance between ROS and NO levels.

Pretreatment with Astragaloside IV protects human umbilical vein endothelial cells from hydrogen peroxide induced oxidative stress and cell dysfunction via inhibiting eNOS uncoupling and NADPH oxidase - ROS - NF-κB pathway.

Endothelial cell injury caused by reactive oxygen species (ROS) plays a critical role in the pathogenesis of cardiovascular disorders. Astragaloside IV (AsIV) possesses potent antioxidant properties against oxidative stress through undefined mechanism(s). We sought to investigate whether AsIV protects human umbilical vein endothelial cells (HUVECs) from hydrogen peroxide (H2O2) induced oxidative stress focusing on eNOS uncoupling and the NADPH oxidase - ROS - NF-κB pathway. Compared with HUVECs incubated with H2O2 alone, pretreatment with AsIV significantly increased the viability of HUVECs, which was accompanied with apparent increase in nitric oxide (NO) production and decrease in intracellular superoxide anion production. Furthermore, pretreatment with AsIV increased endothelial nitric oxide synthase (eNOS) dimer/monomer ratio and its critical cofactor tetrahydrobiopterin (BH4) content, decreased Nox4 protein expression (the most abundant Nox isoform in HUVECs), inhibited translocation of NF-κB p65 subunit into nuclear fraction while enhanced the protein expression of IκB-α (the inhibitor of NF-κB p65), reduced the levels of IL-1ß, IL-6, and TNF-α in HUVECs medium, and decreased iNOS protein expression. These results suggest that AsIV may protect HUVECs from H2O2-induced oxidative stress via inhibiting NADPH oxidase - ROS - NF-κB pathway and eNOS uncoupling.

Protective effects of Astragalus polysaccharides against endothelial dysfunction in hypertrophic rats induced by isoproterenol.

Astragalus polysaccharide (APS) is an important bioactive component extracted from Chinese herb Astragalus membranaceus. It has been widely used in treatment of cardiovascular diseases. We have previously reported that APS could inhibit isoproterenol-induced cardiac hypertrophy. The present study was designed to evaluate the protective effect of APS on vascular endothelia in cardiac hypertrophy rats induced by isoproterenol (ISO). ISO (10mg×kg(-1)) was intraperitoneally injected once daily for 2weeks to induce cardiac hypertrophy. APS (400 and 800mg×kg(-1)) was intragastrically injected once daily along with ISO. The results showed that combination with APS significantly ameliorates the endothelial dysfunction while attenuates cardiac hypertrophy induced by ISO. We found that administration with APS could attenuate the increase in number of circulating endothelial cell (CEC). APS also decreases the superoxide anion generation and the protein expression of p65 and the levels of TNF-α and IL-6; while increases the cGMP levels, an activity marker for nitric oxide (NO) in aortas. In addition, APS improves the relaxation dysfunction in isolated aortic rings and increases the protein expression of IκBα and Cu/Zn-SOD in aortas. In conclusion, our results suggested that APS had a protective effect against endothelial dysfunction in hypertrophic rats induced by ISO. The underlining mechanisms may be contributed to the anti-inflammatory effects and the improvement of the imbalance between reactive oxygen species (ROS) and NO.

Inhibition of TNF-α-mediated NF-κB Activation by Ginsenoside Rg1 Contributes the Attenuation of Cardiac Hypertrophy Induced by Abdominal Aorta Coarctation.

Ginsenoside Rg1 (Rg1), a protopanaxadiol saponin extracted from Chinese medicine Panax ginseng C.A. Meyer, has been demonstrated to inhibit the cardiac hypertrophy. However, the molecular mechanisms underlying the inhibition remain poorly understood. Activation of nuclear factor-kappa B (NF-κB) mediated by tumor necrosis factor α (TNF-α) gets involved in the cardiac hypertrophy. This study is designed to investigate the effects and the potential mechanism of Rg1 on the abdominal aorta coarctation (AAC)-induced cardiac hypertrophy with focus on TNF-α/NF-κB signaling pathway. The results showed that oral administration of Rg1 dose-dependently improved the pathological changes, decreased the ratios of left ventricular weight/body weight (LVW/BW) and heart weight/BW (HW/BW), corrected the dysfunction of the cardiac hemodynamics by decreasing the left ventricular systolic pressure and left ventricular end-diastolic pressure and increasing the maximal rate of left ventricular systolic and diastolic pressure (±dp/dtmax) compared with the AAC alone. Rg1 also downregulated the atrial natriuretic peptide mRNA expression and decreased the mRNA and protein expression of TNF-α in the heart tissue of rats compared with the AAC alone. In addition, Rg1 and BAY, the specific inhibitor of NF-κB, decreased the protein content and downregulated the mRNA expression of atrial natriuretic peptide in neonatal rat ventricular myocytes treated with TNF-α. Furthermore, Rg1 increased the protein expression of p65, the subunit of NF-κB, in cytoplasm and decreased the expression p65 in nucleus of the heart tissue of rats undergoing the AAC and of neonatal rat ventricular myocytes treated with TNF-α. The results suggested that Rg1 attenuates the AAC-induced cardiac hypertrophy through inhibition of TNF-α/NF-κB signaling pathway.

Astragaloside IV improves the isoproterenol-induced vascular dysfunction via attenuating eNOS uncoupling-mediated oxidative stress and inhibiting ROS-NF-κB pathways.

OBJECTIVE: Oxidative stress and inflammation are regarded as two important triggers of endothelial dysfunction and play pivotal role in progression of vascular damage associated with cardiac hypertrophy. Our previous studies demonstrated that astragaloside IV (AsIV) could protect against cardiac hypertrophy in rats induced by isoproterenol (Iso), but its effects on the aorta are not known. In present study, we aimed to assess the effects of AsIV on Isoinduced vascular dysfunction. METHODS: Sprague-Dawley (SD) rats were treated with Iso (10mg/kg/d) alone or in combination with AsIV (50mg/kg/d). RESULTS: Compared with Isotreated alone, AsIV significantly reduced the ratios of heart weight/body weight and left ventricular weight/body weight. AsIV ameliorated the increased vasoconstriction response to phenylephrine induced by Iso and suppressed superoxide anion generation in rat aorta, increased endothelial nitric oxide synthase (eNOS) dimer/monomer ratio and its critical cofactor tetrahydrobiopterin (BH4) content in aorta as well as the NO production in the serum, reduced the plasmatic peroxynitrite (ONOO-). Moreover, in contrast with Isotreatment alone, AsIV decreased the ratio of nuclear-to-cytosolic protein expression of the NF-κB p65 subunit while enhanced its inhibited protein expression of IκB-α, down-regulated mRNA expression of IL-1ß, IL-6 and TNF-α of the aorta. CONCLUSIONS: The present study suggested that AsIV protects against Isoinduced vascular dysfunction probably via attenuating eNOS uncoupling-mediated oxidative stress and inhibiting ROS-NF-κB pathways.

Astragaloside IV attenuates apoptosis of hypertrophic cardiomyocyte through inhibiting oxidative stress and calpain-1 activation.

Calpain-1 activation and oxidative stress are two critical factors contributing to apoptosis of hypertrophic cardiomyocyte. Astragaloside IV (ASIV) exhibits protective effect against various heart diseases. The present study was designed to investigate whether the inhibitory effect of ASIV on isoproterenol (ISO)-induced apoptosis of hypertrophic cardiomyocyte was associated with the anti-oxidation and calpain-1 inhibition. Hypertrophy, apoptosis, mitochondrial oxidative stress and calpain-1 expression were measured in the heart tissue of Sprague-Dawley (SD) rats and H9C2 cells treated with ISO alone or combination with ASIV. The results showed that ASIV attenuated apoptotic rate, increased Bcl-2 expression, decreased Bax expression, ameliorated the integrity of mitochondrial structure and improved mitochondrial membrane potential (MMP). Moreover, ASIV combination reduced both calpain-1 protein expression and calpain activity, down-regulated mitochondrial NOX4 (mito-NOX4) expression, increased activity of mitochondrial superoxide dismutase (mito-SOD) and mitochondrial catalase (mito-CAT) compared to ISO treated alone. The results suggested that ASIV exerted anti-apoptosis effect on ISO-induced hypertrophic cardiomyocyte by attenuating oxidative stress and calpain-1 activation.