N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. In this study, we hypothesized the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by inhibiting autophagy in H9c2 cells. The degree of autophagy by treatment with F2 exposed to H/R in H9c2 cell was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Our results indicated that treatment with F2 inhibited autophagy in H9c2 cells exposed to H/R. 3-methyladenine, an inhibitor of autophagy, suppressed H/R-induced autophagy, and decreased apoptosis, whereas rapamycin, a classical autophagy sensitizer, increased autophagy and apoptosis. Mechanistically, macrophage migration inhibitory factor (MIF) was inhibited by F2 treatment after H/R. Accordingly, small interfering RNA (siRNA)-mediated MIF knockdown decreased H/R-induced autophagy. In summary, F2 protects cardiomyocytes during H/R injury through suppressing autophagy activation. Our results provide a new mechanistic insight into a functional role of F2 against H/R-induced cardiomyocyte injury and death.

BackgroundReal-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a critical tool for evaluating the levels of mRNA transcribed from genes. Reliable RT-qPCR results largely depend on normalization to suitable reference genes. Middle cerebral artery occlusion (MCAO) and oxygen–glucose deprivation/reoxygenation (OGD/R) are models that are commonly used to study ischemic stroke. However, the proper reference genes for RNA analysis in these two models have not yet been determined.ResultsIn this study, we evaluated the expression levels of six candidate housekeeping genes and selected the most suitable reference genes for RT-qPCR analyses of the cortices of MCAO mice and OGD/R-injured N2a cells. Four software programs, geNorm, NormFinder, BestKeeper and RefFinder, were used to validate the stabilities of the candidate reference genes. The results revealed that HPRT and 18S were the most stable reference genes in the cortices of MCAO mice and that β-actin and cyclophilin were the most stable reference genes in the OGD/R-injured N2a cells; in contrast, GAPDH and Sdha were the least stable genes in the cortices of MCAO mice and the OGD/R-injured N2a cells, respectively. Moreover, a combination of HPRT, 18S and cyclophilin was most suitable for normalization in analyses of the cortices of MCAO mice, and a combination of β-actin, cyclophilin, GAPDH, and 18S was most suitable for analyses of the OGD/R-injured N2a cells.ConclusionsThis study provides appropriate reference genes for further RT-qPCR analyses of in vivo and in vitro ischemic stroke and demonstrates the necessity of validating reference genes for RNA analyses under variable conditions.Electronic supplementary materialThe online version of this article (10.1186/s12868-018-0403-6) contains supplementary material, which is available to authorized users.

Endothelial cells are highly sensitive to hypoxia and contribute to myocardial ischemia/reperfusion injury. We have reported that N-n-butyl haloperidol iodide (F2) can attenuate hypoxia/reoxygenation (H/R) injury in cardiac microvascular endothelial cells (CMECs). However, the molecular mechanisms remain unclear. Neonatal rat CMECs were isolated and subjected to H/R. Pretreatment of F2 leads to a reduction in H/R injury, as evidenced by increased cell viability, decreased lactate dehydrogenase (LDH) leakage and apoptosis, together with enhanced AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1) phosphorylation in H/R ECs. Blockade of AMPK with compound C reversed F2-induced inhibition of H/R injury, as evidenced by decreased cell viability, increased LDH release and apoptosis. Moreover, compound C also blocked the ability of F2 to reduce H/R-induced reactive oxygen species (ROS) generation. Supplementation with the ROS scavenger N-acetyl-L-cysteine (NAC) reduced ROS levels, increased cell survival rate, and decreased both LDH release and apoptosis after H/R. In conclusion, our data indicate that F2 may mitigate H/R injury by stimulating LKB1/AMPK signaling pathway and subsequent suppression of ROS production in CMECs.

Cardiac microvascular endothelial cell (CMEC) dysfunction is considered as a major contributor to the cardiovascular complications in diabetes mellitus, with oxidative stress caused by hyperglycemia playing a critical role in the progression of CMEC dysfunction. Melatonin is a kind of hormone well known for its antioxidant properties, which has potential protective effects against diabetes mellitus and its complications. However, the role of melatonin on CMEC dysfunction caused by hyperglycemia and its molecular mechanisms underlying these effects has not been clarified. Herein, we investigate the protective effects of melatonin on high glucose- (HG-) evoked oxidative stress and apoptosis in CMECs and underlying mechanisms. Our results revealed that melatonin ameliorated the injury caused by HG in primary cultured rat CMECs. Injury can be accompanied by reduced reactive oxygen species (ROS) and malondialdehyde (MDA) production, and enhanced superoxide dismutase (SOD) activity. Meanwhile, melatonin treatment significantly inhibited HG-induced CMEC apoptosis. Moreover, melatonin increased the activity of the AMPK/SIRT1 signaling axis in CMECs under HG condition, whereas administration of the AMPK inhibitor compound C or SIRT1 silencing partially abrogated the beneficial effects of melatonin. In streptozotocin- (STZ-) evoked diabetic mice, melatonin notably ameliorated cardiac dysfunction and activated the AMPK/SIRT1 signaling. In conclusion, our findings revealed that melatonin attenuates HG-induced CMEC oxidant stress, apoptosis injury, and STZ-induced cardiac dysfunction through regulating the AMPK/SIRT1 signaling pathway.

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