[Resveratrol reduces apoptosis of mouse RAW264.7 macrophage via down-regulating HIF-1α].

Objective To investigate the protective effect of resveratrol (Res) against mouse RAW264.7 macrophage injury induced by cobalt chloride (CoCl2) and its mechanism. Methods Macrophages were divided into control group, CoCl2 group and Res pretreatment group. CoCl2 group was treated with 500 µmol/L CoCl2 for 8 hours, and Res pretreatment group was pretreated with 40 µmol/L Res for 2 hours followed by 500 µmol/L CoCl2 treatment for 8 hours. The cell vitality and apoptotic rate in every group were detected by CCK-8 assay and flow cytometry. The distributions of caspase-3 and hypoxia-inducible factor 1 alpha (HIF-1α), as well as the influences of CoCl2 and Res on their expression were detected by immunofluorescence cytochemistry. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in every group were measured by the corresponding kits. The expression levels of Bcl2, BAX, c-caspase-3 and HIF-1α in every group were observed by Western blot analysis. Results Compared with the CoCl2 group, pretreatment with Res increased cell vitality, decreased apoptosis, enhanced the activity of SOD, and reduced the level of MDA. The caspase-3 was mainly distributed in the cytoplasm, and HIF-1α was mainly distributed on the nucleus. Compared with the CoCl2 group, Res up-regulated the expression of Bcl2 and down-regulated the expression of BAX, cleaved caspase-3 and HIF-1α. Conclusion Res can decrease apoptosis in macrophages, which may occur via reducing the accumulation of intracellular reactive oxygen species, enhancing cell antioxidant capacity, and down-regulating the expression of HIF-1α on cells.

Vitamin K2 inhibits rat vascular smooth muscle cell calcification by restoring the Gas6/Axl/Akt anti-apoptotic pathway.

Vascular calcification is associated with cardiovascular disease as a complication of hypertension, hyperlipidemia, diabetes mellitus, and chronic kidney disease. Vitamin K2 (VK2) delays vascular calcification by an unclear mechanism. Moreover, apoptosis modulates vascular smooth muscle cell (VSMC) calcification. This paper aimed to study VK2-modified VSMC calcification and survival cell signaling mediated by growth arrest-specific gene 6 (Gas6) and its tyrosine kinase receptor Axl. Primary-cultured VSMCs were dose-dependently treated with VK2 in the presence of calcification medium for 8 days, or pre-treated for 1 h with/without the Axl inhibitor R428 (2 µmol/L) or the caspase inhibitor Z-VAD-fmk (20 µmol/L) followed by treatment with VK2 (10 µmol/L) or rmGas6 (200 nmol/L) in calcification medium for 8 days. Calcium deposition was determined by the o-cresolphthalein complexone assay and Alizarin Red S staining. Apoptosis was determined by TUNEL and flow cytometry using Annexin V-FITC and propidium iodide staining. Western blotting detected the expressions of Axl, Gas6, p-Akt, Akt, and Bcl2. VK2 significantly inhibited CaCl2- and ß-sodium glycerophosphate (ß-GP)-induced VSMC calcification and apoptosis, which was dependent on restored Gas6 expression and activated downstream signaling by Axl, p-Akt, and Bcl2. Z-VAD-fmk significantly inhibited CaCl2- and ß-GP-induced VSMC calcification and apoptosis. Augmented recombinant mouse Gas6 protein (rmGas6) expression significantly reduced VSMC calcification and apoptosis. Furthermore, the Gas6/Axl interaction was inhibited by R428, which abolished the preventive effect of VK2 on CaCl2- and ß-GP-induced apoptosis and calcification. These results suggest that Gas6 is critical in VK2-mediated functions that attenuate CaCl2- and ß-GP-induced VSMC calcification by blocking apoptosis.

Sirt3 deficiency exacerbates diabetic cardiac dysfunction: Role of Foxo3A-Parkin-mediated mitophagy.

Diabetic cardiomyopathy (DCM) is often associated with suppressed cardiac autophagy, mitochondrial structural and functional impairment. Sirtuin-3 (Sirt3) has been reported to play a crucial role in mitochondrial homeostasis and confers a protective role against the onset and development of DCM although the precise mechanism(s) remains elusive. Here we hypothesized that Sirt3 exerts cardioprotection against DCM by activating Parkin-mediated mitophagy, en route to preserved mitochondrial homeostasis and suppressed cardiomyocyte apoptosis. Adult male wild-type (WT) and Sirt3 knockout (Sirt3KO) mice were treated with streptozotocin (STZ) or vehicle for 3months prior to assessment of echocardiographic property, interstitial fibrosis, cardiomyocyte apoptosis, mitochondrial morphology, cardiac autophagy and cell signaling molecules. Our findings revealed that STZ-induced diabetes mellitus prompted cardiac dysfunction, interstitial fibrosis, cardiomyocyte apoptosis and mitochondrial injury, accompanied with suppressed autophagy and mitophagy, the effects of which were aggravated by Sirt3KO. To the contrary, Sirt3 overexpression in vitro activated autophagy and mitophagy, inhibited mitochondrial injury and cardiomyocyte apoptosis, the effects of which were attenuated by autophagy inhibition using 3-MA. Moreover, deacetylation of Foxo3A and expression of Parkin were decreased by Sirt3KO, while these effects were facilitated by Sirt3OE in diabetic and high glucose settings. Taken together, our data suggested that suppressed Sirt3-Foxo3A-Parkin signaling mediated downregulation of mitophagy may play a vital role in the development of diabetic cardiomyopathy. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure edited by Dr. Jun Ren & Yingmei Zhang.

Vitamin K2 regression aortic calcification induced by warfarin via Gas6/Axl survival pathway in rats.

The aim of this study was to investigate the effect of vitamin K2 on aortic calcification induced by warfarin via Gas6/Axl survival pathway in rats. A calcification model was established by administering 3mg/g warfarin to rats. Rats were divided into 9 groups: control group (0W, 4W, 6W and 12W groups), 4W calcification group, 6W calcification group, 12W calcification group, 6W calcification+6W normal group and 6W calcification+6W vitamin K2 group. Alizarin red S staining measured aortic calcium depositions; alkaline phosphatase activity in serum was measured by a kit; apoptosis was evaluated by TUNEL assay; protein expression levels of Gas6, Axl, phosphorylated Akt (p-Akt), and Bcl-2 were determined by western blotting. The calcium content, calcium depositions, ALP activity and apoptosis were significantly higher in the calcification groups than control group. Gas6, Axl, p-Akt and Bcl-2 expression was lower in the calcification group than control group. 100µg/g vitamin K2 treatment decreased calcium depositions, ALP activity and apoptosis significantly, but increased Gas6, Axl, p-Akt and Bcl-2 expression. 100µg/g vitamin K2 reversed 44% calcification. Pearson correlation analysis showed a positive correlation between formation calcification and apoptosis (R(2)=0.8853, P<0.0001). In conclusion, we established a warfarin-induced calcification model and showed vitamin K2 can inhibit warfarin-induced aortic calcification and apoptosis. The regression of aortic calcification by vitamin K2 involved the Gas6/Axl axis. This data may provide a theoretical basis for future clinical treatments for aortic calcification.

Notch3/Akt signaling contributes to OSM-induced protection against cardiac ischemia/reperfusion injury.

Oncostatin M (OSM) exhibits many unique biological activities by activating the Oß receptor. However, its role in myocardial ischemia/reperfusion injury (I/R injury) in mice remains unknown. We investigated whether Notch3/Akt signaling is involved in the regulation of OSM-induced protection against cardiac I/R injury. The effects of OSM were assessed in mice that underwent myocardial I/R injury by OSM treatment or by genetic deficiency of the OSM receptor Oß. We investigated its effects on cardiomyocyte apoptosis and mitochondrial biogenesis and whether Notch3/Akt signaling was involved in the regulation of OSM-induced protection against cardiac I/R injury. The mice underwent 30 min of ischemia followed by 3 h of reperfusion and were randomized to be treated with Notch3 siRNA (siNotch3) or lentivirus carrying Notch3 cDNA (Notch3) 72 h before coronary artery ligation. Myocardial infarct size, cardiac function, cardiomyocyte apoptosis and mitochondria morphology in mice that underwent cardiac I/R injury were compared between groups. OSM alleviated cardiac I/R injury by inhibiting cardiomyocyte apoptosis through promotion of Notch3 production, thus activating the PI3K/Akt pathway. OSM enhanced mitochondrial biogenesis and mitochondrial function in mice subjected to cardiac I/R injury. In contrast, OSM receptor Oß knock out exacerbated cardiac I/R injury, decreased Notch3 production, enhanced cardiomyocyte apoptosis, and impaired mitochondrial biogenesis in cardiac I/R injured mice. The mechanism of OSM on cardiac I/R injury is partly mediated by the Notch3/Akt pathway. These results suggest a novel role of Notch3/Akt signaling that contributes to OSM-induced protection against cardiac I/R injury.