High-glucose-induced apoptosis, ROS production and pro-inflammatory response in cardiomyocytes is attenuated by metformin treatment via PP2A activation

Metformin has been shown to ameliorate diabetic cardiomyopathy. In the present research we investigatedwhether metformin would reduce cardiomyocyte apoptosis that was induced by high-glucose stimulation in vitrovia activation of PP2A. Primary human and rat cardiomyocytes were subject to high-glucose stimulation. Okadaicacid was used to inhibit PP2A activity. Cell viability and apoptosis was assessed using CCK-8 and by flowcytometry, respectively. Release of HMGB1, TNFa or IL-6 was analyzed by ELISA. Oxidative stress wasevaluated by measuring cellular ROS and mitochondrial superoxide level. PP2A activity was evaluated by Serine/Threonine phosphatase assay system or analyzing Y307 phosphorylation level of PP2A catalytic domain (PP2Ac)by Western blot and the association between PP2Ac and a4 by co-immunoprecipitation. Activation of the NF-jBsignaling pathway was assessed by detecting Ser32 phosphorylation level of IjBa as well as nuclear entry of p65protein by Western blot. Activation of the GSK3b/MCL1 signaling pathway was assessed by detecting Ser9phosphorylation level of GSK3b and protein level of MCL1. We found Metformin pre-treatment attenuatedhuman and rat cardiomyocytes apoptosis, HMGB1, TNFa and IL-6 release and ROS production that were inducedby high-glucose stimulation, and these effects of metformin could be blocked by okadaic acid treatment. Metformin reduced the upregulation of PP2Ac pY307 and the PP2Ac-a4 association, which was not affected byokadaic acid treatment. Metformin pre-treatment reduced NF-jB activation in human and rat cardiomyocytesapoptosis that was elicited by high-glucose stimulation, and this effect of metformin could be blocked by okadaicacid treatment. GSK3b/MCL1 is not part of metformin activating PP2A induced myocardial cell death inhibition.In conclusion, metformin reduced apoptosis, ROS production and inflammatory response in primary human andrat cardiomyocytes in vitro in a PP2A dependent manner.

Effects of nicorandil on neurobehavioral function, BBB integrity, edema and stereological parameters of the brain in the sub-acute phase of stroke in a rat model

Blood–brain barrier (BBB) disruption, inflammation, and cell death are the pathogenic mechanisms of cerebralischemia/reperfusion (I/R) injury. Nicorandil protects ischemic injury via some of these mechanisms. The aimof this study was to investigate the therapeutic effects of this drug on the brain ischemia after transient middlecerebral artery occlusion (MCAO) and clarify the NF-jB and Nrf2-dependent mechanisms modulated by thisdrug. Sixty-six rats were randomized into sham, MCAO and MCAO ? nicorandil groups with oral gavage for3 days. Cerebral I/R injury were induced by a transient MCAO for 1 h and neurobehavioral scores wereperformed for 3 days. In addition to measurement of BBB disruption and brain water content, the total andinfarct volume, density, and total number of neurons, non-neurons and dead neurons in the right cortex wereestimated by unbiased stereological methods. RT-PCR was performed to analyze the expression levels of NFjB and Nrf2. Although nicorandil treatment in the sub-acute brain ischemia did not have a prominent effect onneurobehavioral function and number of neurons, non-neurons and dead neurons probably through up-regulation of NF-jB, it, however, improved ischemia-induced BBB disruption and brain edema and showed asignificant reduction in the infarction volume probably through up-regulation of Nrf2.

Resveratrol protects H9c2 cells against hypoxia-induced apoptosis through miR-30d-5p/SIRT1/NF-κB axis

Previous studies have demonstrated the cardioprotective role of resveratrol (Res). However, the underlyingmolecular mechanisms involved in the protective role of Res are still largely unknown. H9c2 cells weredistributed into five groups: normal condition (Control), DMSO, 20 mMRes (dissolved with DMSO), hypoxia(Hyp), and Res?Hyp. Cell apoptosis was evaluated using flow cytometry and protein analysis of cleavedcaspase 3 (cle-caspase 3). qRT-PCR assay was performed to measure the expression of microRNA-30d-5p(miR-30d-5p). MTT assay was performed to evaluate the cell proliferation. The relationship between miR-30d5p and silent information regulator 1 (SIRT1) was confirmed by luciferase reporter, RNA immunoprecipitation(RIP), and western blot assays. Western blot was performed to analyze NF-jB/p65 and I-jBa expressions. Ourdata showed that hypoxia enhanced apoptosis and NF-jB signaling pathway, which was alleviated by Restreatment. Hypoxia increased the expression of miR-30d-5p while decreased the SIRT1expression, which wasalso attenuated by Res treatment. Furthermore, miR-30d-5p depletion inhibited the proliferation, reducedapoptosis and decreased the expression of cle-caspase 3 in H9c2 cells with hypoxia treatment. Luciferasereporter, RIP, and western blot assays further confirmed that miR-30d-5p negatively regulated the expression ofSIRT1. Interestingly, the rescue-of-function experiments further indicated that knockdown of SIRT1 attenuatedthe effect of miR-30d-5p depletion on proliferation, apoptosis NF-jB signaling pathway inH9c2 cells withhypoxia treatment. In addition, the suppression of NF-jB signaling pathway increased cell viability whiledecreased cell apoptosis in hypoxia-mediatedH9c2 cells. Our data suggested Res mayprotectH9c2 cells againsthypoxia-induced apoptosis through miR-30d-5p/SIRT1/NF-jB axis