The angiotensin‐(1‐7)/Mas receptor axis protects from endothelial cell senescence via klotho and Nrf2 activation

Endothelial cell senescence is a hallmark of vascular aging that predisposes to vascular disease. We aimed to explore the capacity of the renin–angiotensin system (RAS) heptapeptide angiotensin (Ang)‐(1‐7) to counteract human endothelial cell senescence and to identify intracellular pathways mediating its potential protective action. In human umbilical vein endothelial cell (HUVEC) cultures, Ang II promoted cell senescence, as revealed by the enhancement in senescence‐associated galactosidase (SA‐β‐gal+) positive staining, total and telomeric DNA damage, adhesion molecule expression, and human mononuclear adhesion to HUVEC monolayers. By activating the G protein‐coupled receptor Mas, Ang‐(1‐7) inhibited the pro‐senescence action of Ang II, but also of a non‐RAS stressor such as the cytokine IL‐1β. Moreover, Ang‐(1‐7) enhanced endothelial klotho levels, while klotho silencing resulted in the loss of the anti‐senescence action of the heptapeptide. Indeed, both Ang‐(1‐7) and recombinant klotho activated the cytoprotective Nrf2/heme oxygenase‐1 (HO‐1) pathway. The HO‐1 inhibitor tin protoporphyrin IX prevented the anti‐senescence action evoked by Ang‐(1‐7) or recombinant klotho. Overall, the present study identifies Ang‐(1‐7) as an anti‐senescence peptide displaying its protective action beyond the RAS by consecutively activating klotho and Nrf2/HO‐1. Ang‐(1‐7) mimetic drugs may thus prove useful to prevent endothelial cell senescence and its related vascular complications.     

Neuroprotective effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on ischaemia/reperfusion‐induced neuronal injury by activating the Nrf2/HO‐1 pathway

1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a lipophilic phenolic agent, has an antioxidant activity and reactive oxygen species (ROS) scavenging property. However, the role of HTHQ on cerebral ischaemic/reperfusion (I/R) injury and the underlying mechanisms remain poorly understood. In the present study, we demonstrated that HTHQ treatment ameliorated cerebral I/R injury in vivo, as demonstrated by the decreased infarct volume ration, neurological deficits, oxidative stress and neuronal apoptosis. HTHQ treatment increased the levels of nuclear factor erythroid 2–related factor 2 (Nrf2) and its downstream antioxidant protein, haeme oxygenase‐1 (HO‐1). In addition, HTHQ treatment decreases oxidative stress and neuronal apoptosis of PC12 cells following hypoxia and reperfusion (H/R) in vitro. Moreover, we provided evidence that PC12 cells were more vulnerable to H/R‐induced oxidative stress after si‐Nrf2 transfection, and the HTHQ‐mediated protection was lost in PC12 cells transfected with siNrf2. In conclusion, these results suggested that HTHQ possesses neuroprotective effects against oxidative stress and apoptosis after cerebral I/R injury via activation of the Nrf2/HO‐1 pathway.     

Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H₂O₂‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16^INK4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H₂O₂‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H₂O₂‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H₂O₂ treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.     

Rg1 protects H9C2 cells from high glucose‐/palmitate‐induced injury via activation of AKT/GSK‐3β/Nrf2 pathway

Our previous studies have assessed ginsenoside Rg1 (Rg1)‐mediated protection in a type 1 diabetes rat model. To uncover the mechanism through which Rg1 protects against cardiac injury induced by diabetes, we mimicked diabetic conditions by culturing H9C2 cells in high glucose/palmitate. Rg1 had no toxic effect, and it alleviated the high glucose/palmitate damage in a dose‐dependent manner, as indicated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay and lactate dehydrogenase release to the culture medium. Rg1 prevented high glucose/palmitate‐induced cell apoptosis, assessed using cleaved caspase‐3 and terminal deoxynucleotidyl transferase dUTP nick end labelling staining. Rg1 also reduced high glucose‐/palmitate‐induced reactive oxygen species formation and increased intracellular antioxidant enzyme activity. We found that Rg1 activates protein kinase B (AKT)/glycogen synthase kinase‐3 (GSK‐3β) pathway and antioxidant nuclear factor erythroid 2‐related factor 2 (Nrf2) pathway, indicated by increased phosphorylation of AKT and GSK‐3β, and nuclear translocation of Nrf2. We used phosphatidylinositol‐3‐kinase inhibitor Ly294002 to block the activation of the AKT/GSK‐3β pathway and found that it partially reversed the protection by Rg1 and decreased Nrf2 pathway activation. The results suggest that Rg1 exerts a protective effect against high glucose and palmitate damage that is partially AKT/GSK‐3β/Nrf2‐mediated. Further studies are required to validate these findings using primary cardiomyocytes and animal models of diabetes.     

Aldehyde dehydrogenase 2 activation ameliorates CCl4‐induced chronic liver fibrosis in mice by up‐regulating Nrf2/HO‐1 antioxidant pathway

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is critical in the pathogenesis of alcoholic liver cirrhosis. However, the effect of ALHD2 on liver fibrosis remains to be further elucidated. This study aimed to demonstrate whether ALDH2 regulates carbon tetrachloride (CCl₄)‐induced liver fibrosis and to investigate the efficacy of Alda‐1, a specific activator of ALDH2, on attenuating liver fibrosis. ALDH2 expression was increased after chronic CCl₄ exposure. ALDH2 deficiency accentuated CCl₄‐induced liver fibrosis in mice, accompanied by increased expression of collagen 1α1, α‐SMA and TIMP‐1. Moreover, ALDH2 knockout triggered more ROS generation, hepatocyte apoptosis and impaired mitophagy after CCl₄ treatment. In cultured HSC‐T6 cells, ALDH2 knockdown by transfecting with lentivirus vector increased ROS generation and α‐SMA expression in an in vitro hepatocyte fibrosis model using TGF‐β1. ALDH2 overexpression by lentivirus or activation by Alda‐1 administration partly reversed the effect of TGF‐β1, whereas ALDH2 knockdown totally blocked the protective effect of Alda‐1. Furthermore, Alda‐1 administration protected against liver fibrosis in vivo, which might be mediated through up‐regulation of Nrf2/HO‐1 cascade and activation of Parkin‐related mitophagy. These findings indicate that ALDH2 deficiency aggravated CCl₄‐induced hepatic fibrosis through ROS overproduction, increased apoptosis and mitochondrial damage, whereas ALDH2 activation through Alda‐1 administration alleviated hepatic fibrosis partly through activation of the Nrf2/HO‐1 antioxidant pathway and Parkin‐related mitophagy, which indicate ALDH2 as a promising anti‐fibrotic target and Alda‐1 as a potential therapeutic agent in treating CCl₄‐induced liver fibrosis.     

Hypothesis: cell signalling influences age‐related risk of colorectal cancer

We propose that ageing is linked to colonic carcinogenesis through crosstalk between Wnt activity and signalling pathways related to ageing and senescence: progerin, klotho and mTOR. Mutations in the Wnt signalling pathway are responsible for the majority of colorectal cancers (CRCs); however, hyperactivation of Wnt signalling by butyrate, a breakdown product of dietary fibre, induces CRC cell apoptosis. This effect of butyrate may in part explain the protective action of fibre against CRC. Hutchinson–Gilford progeria syndrome is a premature ageing disorder caused by accumulation of the progerin protein; however, healthy individuals also produce progerin in the course of their normal ageing. Progerin activates expression of the Wnt inhibitors HES1 and TLE1. Thus, we hypothesize that with age, the increasing expression of progerin suppresses butyrate‐mediated Wnt hyperactivation and apoptosis, leading to increased CRC risk. Wild‐type klotho contributes to a significantly increased lifespan; however, Klotho gene variants differ significantly between newborns and elderly. Klotho inhibits basal Wnt signalling activity; thus, the protein may function as a tumour suppressor for CRC. However, similar to progerin, klotho variants associated with lifespan differences may repress butyrate‐mediated Wnt hyperactivation, and thus increase the risk of CRC. Finally, mTOR signalling has also been linked to human ageing, and crosstalk between Wnt and mTOR signalling may influence colonic tumourigenesis. Understanding how progerin, klotho and mTOR link ageing with colonic neoplastic development may lead to novel preventive and therapeutic strategies against CRC associated with age.     

Kallistatin reduces vascular senescence and aging by regulating microRNA‐34a‐SIRT1 pathway

Kallistatin, an endogenous protein, protects against vascular injury by inhibiting oxidative stress and inflammation in hypertensive rats and enhancing the mobility and function of endothelial progenitor cells (EPCs). We aimed to determine the role and mechanism of kallistatin in vascular senescence and aging using cultured EPCs, streptozotocin (STZ)‐induced diabetic mice, and Caenorhabditis elegans (C. elegans). Human kallistatin significantly decreased TNF‐α‐induced cellular senescence in EPCs, as indicated by reduced senescence‐associated β‐galactosidase activity and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked TNF‐α‐induced superoxide levels, NADPH oxidase activity, and microRNA‐21 (miR‐21) and p16^INK4a synthesis. Kallistatin prevented TNF‐α‐mediated inhibition of SIRT1, eNOS, and catalase, and directly stimulated the expression of these antioxidant enzymes. Moreover, kallistatin inhibited miR‐34a synthesis, whereas miR‐34a overexpression abolished kallistatin‐induced antioxidant gene expression and antisenescence activity. Kallistatin via its active site inhibited miR‐34a, and stimulated SIRT1 and eNOS synthesis in EPCs, which was abolished by genistein, indicating an event mediated by tyrosine kinase. Moreover, kallistatin administration attenuated STZ‐induced aortic senescence, oxidative stress, and miR‐34a and miR‐21 synthesis, and increased SIRT1, eNOS, and catalase levels in diabetic mice. Furthermore, kallistatin treatment reduced superoxide formation and prolonged wild‐type C. elegans lifespan under oxidative or heat stress, although kallistatin's protective effect was abolished in miR‐34 or sir‐2.1 (SIRT1 homolog) mutant C. elegans. Kallistatin inhibited miR‐34, but stimulated sir‐2.1 and sod‐3 synthesis in C. elegans. These in vitro and in vivo studies provide significant insights into the role and mechanism of kallistatin in vascular senescence and aging by regulating miR‐34a‐SIRT1 pathway.     

GRP78 effectively protect hypoxia/reperfusion‐induced myocardial apoptosis via promotion of the Nrf2/HO‐1 signaling pathway

Myocardial infarction is a major cause of death worldwide. Despite our understanding of the pathophysiology of myocardial infarction and the therapeutic options for treatment have improved substantially, acute myocardial infarction remains a leading cause of morbidity and mortality. Recent findings revealed that GRP78 could protect myocardial cells against ischemia reperfusion injury‐induced apoptosis, but the exact function and molecular mechanism remains unclear. In this study, we aimed to explore the effects of GRP78 on hypoxia/reperfusion (H/R)‐induced cardiomyocyte injury. Intriguingly, we first observed that GRP78 overexpression significantly protected myocytes from H/R‐induced apoptosis. On mechanism, our work revealed that GRP78 protected myocardial cells from hypoxia/reperfusion‐induced apoptosis via the activation of the Nrf2/HO‐1 signaling pathway. We observed the enhanced expression of Nrf2/HO‐1 in GRP78 overexpressed H9c2 cell, while GRP78 deficiency dramatically antagonized the expression of Nrf2/HO‐1. Furthermore, we found that blocked the Nrf2/HO‐1 signaling by the HO‐1 inhibitor zinc protoporphyrin IX (Znpp) significantly retrieved H9c2 cells apoptosis that inhibited by GRP78 overexpression. Taken together, our findings revealed a new mechanism by which GRP78 alleviated H/R‐induced cardiomyocyte apoptosis in H9c2 cells via the promotion of the Nrf2/HO‐1 signaling pathway.     

The apolipoprotein A‐I mimetic peptide,D‐4F,restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.     

Suppression of microRNA‐144‐3p attenuates oxygen–glucose deprivation/reoxygenation‐induced neuronal injury by promoting Brg1/Nrf2/ARE signaling

Accumulating evidence has reported that microRNA‐144‐3p (miR‐144‐3p) is highly related to oxidative stress and apoptosis. However, little is known regarding its role in cerebral ischemia/reperfusion‐induced neuronal injury. Herein, our results showed that miR‐144‐3p expression was significantly downregulated in neurons following oxygen–glucose deprivation and reoxygenation (OGD/R) treatment. Overexpression of miR‐144‐3p markedly reduced cell viability, promoted cell apoptosis, and increased oxidative stress in neurons with OGD/R treatment, whereas downregulation of miR‐144‐3p protected neurons against OGD/R‐induced injury. Brahma‐related gene 1 (Brg1) was identified as a potential target gene of miR‐144‐3p. Moreover, downregulation of miR‐144‐3p promoted the nuclear translocation of nuclear factor erythroid 2‐related factor 2 (Nrf2) and increased antioxidant response element (ARE) activity. However, knockdown of Brg1 significantly abrogated the neuroprotective effects of miR‐144‐3p downregulation. Overall, our results suggest that miR‐144‐3p contributes to OGD/R‐induced neuronal injury in vitro through negatively regulating Brg1/Nrf2/ARE signaling.     

Inhibition of bromodomain‐containing protein 4 ameliorates oxidative stress–mediated apoptosis and cartilage matrix degeneration through activation of NF‐E2–related factor 2‐heme oxygenase‐1 signaling in rat chondrocytes

During the progression of osteoarthritis, dysregulation of extracellular matrix (ECM) anabolism, abnormal generation of reactive oxygen species, and proteolytic enzymes have been shown to accelerate the degradation process of cartilage. The purpose of the current study was to investigate the functional role of bromodomain‐containing protein 4 (BRD4) in hydrogen peroxide (H₂O₂)–stimulated chondrocyte injury and delineate the underlying molecular mechanisms. We observed that the expression BRD4 was markedly elevated in rat chondrocytes after H₂O₂ stimulation. Additionally, inhibition of BRD4 using small interfering RNA or JQ1 (a selective potent chemical inhibitor) led to repression of H₂O₂‐induced oxidative stress, as revealed by a decrease in the reactive oxygen species production accompanied by a decreased malondialdehyde content, along with increased activities of antioxidant markers superoxide dismutase, catalase, and glutathione peroxidase on exposure of chondrocytes to H₂O₂. Meanwhile, depletion of BRD4 led to repress the oxidative stress–induced apoptosis of chondrocytes triggered by H₂O₂ accompanied by an increase in the expression of anti‐apoptotic Bcl‐2 and a decrease in the expression of pro‐apoptotic Bax and caspase 3 as well as attenuated caspase 3 activity. Moreover, knockdown of BRD4 or treatment with JQ1 markedly attenuated ECM deposition, reflected in a marked upregulation of proteoglycans collagen type II and aggrecan as well as downregulation of ECM–degrading enzymes matrix metalloproteinase 13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‐5). More importantly, inhibition of BRD4‐activated NF‐E2–related factor 2 (Nrf2)–heme oxygenase‐1 signaling. Mechanistically, the protective effect of BRD4 inhibition on H₂O₂‐stimulated apoptosis and cartilage matrix degeneration was markedly abrogated by Nrf2 depletion. Altogether, we concluded that the protective effect of BRD4 inhibition against oxidative stress–mediated apoptosis and cartilage matrix degeneration occurred through Nrf2–heme oxygenase‐1 signaling, implying that BRD4 inhibition may be a more effective therapeutic strategy against osteoarthritis.     

Dimethyl fumarate protects thioacetamide‐induced liver damage in rats: Studies on Nrf2, NLRP3, and NF‐κB

The present study was designed to investigate the hepatoprotective potential of dimethyl fumarate (DMF) against thioacetamide (TAA)‐induced liver damage. Wistar rats were treated with DMF (12.5, 25, and 50 mg/kg/day, orally) and TAA (200 mg/kg intraperitoneally, every third day) for 6 consecutive weeks. TAA exposure significantly reduced body weight, increased liver weight and index, and intervention with DMF did not ameliorate these parameters. DMF treatment significantly restored TAA‐induced increase in the levels of aspartate aminotransferase, alanine aminotransferase, γ‐glutamyl transferase, total bilirubin, uric acid, malondialdehyde, reduced glutathione, and histopathological findings such as inflammatory cell infiltration, deposition of collagen, necrosis, and bridging fibrosis. DMF treatment significantly ameliorated TAA‐induced hepatic stellate cell activation, increase in inflammatory cascade markers (NACHT, LRR, and PYD domains‐containing protein 3; NLRP3, apoptosis‐associated speck like protein containing a caspase recruitment domain; ASC, caspase‐1, nuclear factor‐kappa B; NF‐κB, interleukin‐6), fibrogenic makers (α‐smooth muscle actin; ɑ‐SMA, transforming growth factor; TGF‐β1, fibronectin, collagen 1) and antioxidant markers (nuclear factor (erythroid‐derived 2)‐like factor 2; Nrf2, superoxide dismutase‐1; SOD‐1, catalase). The present findings concluded that DMF protects against TAA‐induced hepatic damage mediated through the downregulation of inflammatory cascades and upregulation of antioxidant status.