Ozone pretreatment alleviates ischemiareperfusion injury-induced myocardial ferroptosis by activating the Nrf2/Slc7a11/Gpx4 axis.

Myocardial ischemiareperfusion injury (MIRI) is defined as the additional damage that occurs during the process of restoring blood flow to the heart tissue after ischemia-induced damage. Ozone is a powerful oxidizer, but low concentrations of ozone can protect various organs from oxidative stress. Some studies have demonstrated a link between ozone and myocardioprotection, but the mechanism remains unclear. To establish an in vivo animal model of ischemiareperfusion injury (I/R), this study utilized C57 mice, while an in vitro model of hypoxia-reoxygenation (H/R) injury was developed using H9c2 cardiomyocytes to simulate ischemiareperfusion injury. Ozone pretreatment was used in in vitro and in vivo experiments. Through this research, we found that ozone therapy can reduce myocardial injury, and further studies found that ozone regulates the expression levels of these ferroptosis-related proteins and transcription factors in the H/R model, which were screened by bioinformatics. In particular, nuclear translocation of Nrf2 was enhanced by pretreatment with ozone, inhibited ferroptosis and ameliorated oxidative stress by initiating the expression of Slc7a11 and Gpx4. Significantly, Nrf2 gene silencing reverses the protective effects of ozone in the H/R model. In summary, our results suggest that ozone protects the myocardium from I/R damage through the Nrf2/Slc7a11/Gpx4 signaling pathway, highlighting the potential of ozone as a new coronary artery disease therapy.

Ozone treatment alleviates brain injury in cerebral ischemic rats by inhibiting the NF-κB signaling pathway and autophagy.

Stroke is the most frequent cause of disability in developed countries. A common phenomenon of stroke, cerebral ischemia, is threatening many lives worldwide. In addition, ozone treatment was previously reported to exert functions in relieving brain injury. In the current study, the therapeutic effects of ozone on cerebral ischemia are investigated. A rat model of middle cerebral artery occlusion (MCAO) was established. The brain water content was calculated by weighing brain tissues, and the 2, 3, 5-triphenyltetrazolium chloride staining was performed to measure brain infarction volume in rats. A colorimetric assay was conducted to examine expression levels of malondialdehyde, superoxide dismutase, catalase, and glutathione in the rat hippocampus. Reverse transcription quantitative polymerase-chain reaction and Western blot analyses were employed to evaluate expression levels of Beclin1, LC3B, p62, and critical factors implicated in the NF-κB signaling pathway. We found that ozone significantly improved the survival rate of MCAO model rats, reduced the cerebral water content, and decreased the neurological scores of ischemic rats. Ozone markedly reduced cerebral ischemia-induced infarction in ischemic rats. Ozone decreased MDA levels and increased SOD, catalase, and GSH levels in the hippocampus of rats. Ozone significantly inhibited autophagy by decreasing Beclin1 and LC3B expression and increasing p62 expression. The ozone inactivated the NF-κB signaling pathway by decreasing the protein levels of TLR4, p-IKKß, p-IKBα, and p-p65. We conclude that ozone treatment alleviates the brain injury in ischemic rats by suppressing autophagy and inactivating the NF-κB signaling pathway.

Ozone induces tolerance against cardiomyocytes oxygen-glucose deprivation/reperfusion through inhibition of autophagy pathway.

Previous studies have reported that excess activation of autophagy in cardiomyocytes is associated with an increase in myocardial oxygen-glucose deprivation/reperfusion (OGD/R) injury. Ozone therapy affords significant cardio-protection against myocardial OGD/R injury. The present study was designed to determine whether ozone-induced tolerance to myocardial OGD/R injury was mediated by inhibiting autophagy. Subsequently, the rat cardio myoblast H9C2 cell line was used in the present study. A model of H9C2 cells under OGD/R was established. The cells were incubated with different concentrations of ozone (10-60 µg/ml) during reperfusion. Furthermore, to investigate the role of autophagy in OGD/R-induced injury, the autophagy inducer and inhibitor were applied. Cell viability was detected by Cell Counting kit-8 assay. Cell apoptosis was evaluated by flow cytometry. Oxidative stress was examined by superoxide dismutase, lactate dehydrogenase and malondialdehyde levels. The expressions of apoptosis regulator B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (BAX), cleaved caspase-3, markers of autophagy microtuble-associated protein 1 light chain 3 (LC3), autophagy-related protein 5 (Atg5) and Beclin-1 were measured by western blot analysis. As a result, OGD/R notably decreased cell viability and induced apoptosis in H9C2 cells, while ozone (10-40 µg/ml) reversed the noxious effects of OGD/R on H9C2 cells, and 20 µg/ml ozone was the most effective. Ozone inhibited the decrease in the ratio of Bcl-2/BAX and the expression of cleaved caspase-3, and inhibited the increase in the ratio of LC3-II/LC3-I and the expression of Atg5 and Beclin-1 elicited by OGD/R, as well as dose-dependently preventing OGD/R-induced oxidative stress. Furthermore, rapamycin markedly reversed the effects of ozone (20 µg/ml) on OGD/R-induced expression of autophagy marker proteins and 3-methyladenine further improved the effect of ozone. Taken together, the results of the present study provided a credible mechanism by which ozone treatment at low concentrations could protect the myocardium from OGD/R-induced injury by inhibiting autophagy.

Ozone Attenuated H9c2 Cell Injury Induced by Doxorubicin.

ABSTRACT: Doxorubicin (DOX) is a commonly used drug in the treatment of cancers, whereas its application in the clinical stage is restricted because of side effects such as cardiomyocyte injury. Increasing studies indicated that ozone may protect cardiomyocytes from injuries. This study aimed to explore the effects of ozone on cardiotoxicity induced by DOX treatment. Rat heart myoblasts (H9c2) were treated with increasing concentrations of DOX (0.5, 1, 1.5, and 2 µM) to induce cell injury. 3-(4,5)-dimethylthiahiazo(-2)-3,5-diphenytetrazoliumromide assay and flow cytometry analysis were used to measure the viability and apoptosis of H9c2 cells. The mRNA and protein levels of proinflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-(IL)1ß, and IL-6, matrix metalloproteinases (MMP-2 and MMP-9), and the key factors on the TLR4/NF-kB signaling (TLR4, p-p65, and p65) were measured by reverse transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and western blot. The result showed that DOX promoted apoptosis and increased the expression of TNF-α (by 3.65-fold changes), IL-1ß (by 4.98-fold changes), IL-6 (by 3.44-fold changes), MMP-2 (by 1.98-fold changes), and MMP-9 (by 1.98-fold changes) levels in H9c2 cells. Moreover, the introduction of ozone reversed these changes in gene expression and suppressed the activation of the TLR4/NF-kB signaling, which indicated that ozone may exert protective effects on H9c2 heart myoblasts by relieving the cardiotoxicity induced by DOX. Our study provides theoretical basis for the significance of ozone in managing doxorubicin-induced H9c2 heart myoblast injury.