Tetrahydroxystilbene Glucoside (TSG) Restores the Effect of Transient Hypoxia on Reperfusion Injury in Senescent H9c2 Cells by Regulating Mitochondrial Energy Metabolism

Tetrahydroxystilbene glucoside (TSG) is extracted from a famous Chinese herbal medicine which is widely used as an antiaging agent in history. Lots of studies gave evidence that TSG exhibited benefits to brain, like improvement of learning and memory and synaptic plasticity. Moreover, the polyphenolic structure of TSG enables its capability to prevent cerebral ischemia/reperfusion injury (IRI) by reducing apoptosis and ROS/RNS generation. Due to its antioxidant profile, TSG had been demonstrated to alleviate cardiac toxicity by regulating biochemical indexes and ROS. However, whether TSG exhibited cardioprotective effects via mitochondrial energy metabolic functions, which played crucial role in IRI, remained unclear. Here, we used an in vitro aging model of cardiomyocytes to evaluate the effects of TSG on transient hypoxia-pretreated hypoxia/reoxygenation (H/R) injury and mitochondrial energy metaolism. Our results showed that TSG enhanced cardioprotective effect of transient hypoxia on H/R by reducing excessive ROS production and calcium overloading. Significant improvements of mitochondrial respiratory functions and ketone body metabolism elucidated that TSG restored the effect of transient hypoxia on H/R injury in aging cardiomyocytes via upregulating mitochondrial energy metabolism.

Abstract 254: 14, 15-EET Protects Heart Pericytes by Delaying I/R Injury-induced Calcium Overloading

Background - Pericytes are an important cellular component of the blood vessel wall of the arteries, arterioles, and microvessels of the heart; they provide structural integrity and regulate vessel diameter by contracting and relaxing dynamically in response to vasoactive stimuli. It has been suggested that pericytes contribute to coronary no-flow due to pericyte constriction following myocardial infarction, thus worsening outcome. It has also been demonstrated that intracellular calcium is involved in perciyte constriction. Our previous findings indicate that cardiomyocytes are protected following ischemia/ reperfusion injury (I/R) by the eicosanoid 14,15-EET. Since 14,15-EET is protective following I/R and a vasodilator, we tested the hypotheses that I/R injury induces calcium overloading, which injures peciytes, and that 14, 15-EET is able to block this process. Methods and Results - We isolated and cultured pericytes from the mouse heart ventricle by 3G5 antibody Dynabead sorting. Pericytes were characterized by multiple immunocytochemical markers for contractile proteins, cytoskeletal protein, and cell surface receptors (alpha-smooth muscle actin, calponin-1, NG2, vimentin, CD31,smoothlin, and fibroblast protein-1). Cultured pericytes were subjected to 5 hours of oxygen and glucose deprivation, with or without 14,15-EET, followed by 15 hours of re-oxygenation in the absence of 14,15-EET. Calcium imaging and cell death during re-oxygenation were assessed by Fluo-4 and propidium iodide respectively. Digital images were taken with confocal microscope (Nikon Eclipse Tie-A1RSi). The brightness of the green fluorescent signals represents the relative level of intracellular calcium and the red fluorescent signals represent the cell death. We found that calcium signal peak (overloading) occurred during re-oxygenation, immediately followed by cell death. This process was delayed by 14,15-EET treatment during oxygen and glucose deprivation. The cell death at 5h, 10h, and 15h of re-oxygenation was 57.2%, 71.1%, and 85.3% in control group, and 19.9%, 35.3%, and 58.3% in 14,15-EET treated group. Conclusions - Our data suggests that 14,15-EET-induced protection in pericytes is mediated through the calcium signaling pathway.

Bioassay-Guided Isolation of Neuroprotective Compounds fromUncaria rhynchophyllaagainst Beta-Amyloid-Induced Neurotoxicity

Uncaria rhynchophyllais a component herb of many Chinese herbal formulae for the treatment of neurodegenerative diseases. Previous study in our laboratory has demonstrated that an ethanol extract ofUncaria rhynchophyllaameliorated cognitive deficits in a mouse model of Alzheimer’s disease induced by D-galactose. However, the active ingredients ofUncaria rhynchophyllaresponsible for the anti-Alzheimer’s disease activity have not been identified. This study aims to identify the active ingredients ofUncaria rhynchophyllaby a bioassay-guided fractionation approach and explore the acting mechanism of these active ingredients by using a well-established cellular model of Alzheimer’s disease, beta-amyloid- (Aβ-) induced neurotoxicity in PC12 cells. The results showed that six alkaloids, namely, corynoxine, corynoxine B, corynoxeine, isorhynchophylline, isocorynoxeine, and rhynchophylline were isolated from the extract ofUncaria rhynchophylla. Among them, rhynchophylline and isorhynchophylline significantly decreased Aβ-induced cell death, intracellular calcium overloading, and tau protein hyperphosphorylation in PC12 cells. These results suggest that rhynchophylline and isorhynchophylline are the major active ingredients responsible for the protective action ofUncaria rhynchophyllaagainst Aβ-induced neuronal toxicity, and their neuroprotective effect may be mediated, at least in part, by inhibiting intracellular calcium overloading and tau protein hyperphosphorylation.

Abstract P25: Hypothermia Treatment Inhibits Cardiomyocytes Calcium Overload and Mitochondria Transition Permeability after Hypoxia- Reoxygenation Injuries

Introduction: Hypothermia treatment can provide cardiac protection against ischemia reperfusion injuries, but underlying mechanisms remain unclear. Hypothesis: Hypothermia-related cardiomyocyte protection is through the mitochondrial dependent pathways. Methods : H9c2 rat cardiomyocytes were cultured in 37°C, 5% CO 2 incubators. After initiation of hypoxia-reoxygenation (H-R) treatment, the H9c2 cells were moved to hypothermia (31°C) or kept in normothermia (37°C) environments until cells harvested. Cell damage, intracellular and mitochondria calcium loads were studies. Mitochondria permeability transition and transmembrane potentials were studies by flowcytometric studies. Results: Hypothermia treatment ameliorates H9c2 cardiomyocytes survival after H-R injuries (68.1±11.8% vs. 85.0±12.7 %, P=0.025). Intracellular and mitochondria calcium overloading after H-R injuries was improved under 31°C environment (153.5±16.4 % vs. 957.1±311.7 %, P<0.01 for intracellular calcium; 101.8±28.5% vs. 159.4±32.5%, P=0.014 for mitochondria calcium). Mitochondria reductase activity were more preserved under hypothermia treatment after H-R injuries (55.7±10.9% vs. 8.5±1.2%, P<0.01). Hypothermia treatment decreased the continuous opening of mitochondria permeability transition pore after H-R damage by less reduction of mitochondria calcein fluorescence (15.6±13.7 % vs. 52.8±28.1 %, P=0.003). Release of cytochrome c into the cytoplasm from mitochondria after H-R injuries was more evident in normothermia condition by confocal microscopy study. Activation of caspase-9, which is down-streaming to cytochrome c, was down-regulated under hypothermia (62.1±21.9% vs. 87.5±7.3%, P=0.019). Loss of mitochondria integrity with decreasing of mitochondria transmembrane potential was less evident in 31°C than 37°C environments (55.9±23.3% vs. 102±20.2%, P=0.016). Conclusion: Hypothermia treatment at 31°C provides cardiomyocyte protection against hypoxia-reoxygenation injuries. The mechanisms are related to the decreasing intracellular and mitochondria calcium overloading, preserving the integrity of mitochondria by reduction of mitochondria permeability transition pore opening and cytochrome c release.