This study was to investigate the protective effect of hyperbaric oxygen (HBO) on HT22 and PC12 cell damage caused by oxygen-glucose deprivation/reperfusion-induced ferroptosis. A 2-h oxygen-glucose deprivation and 24-h reperfusion model on HT22 and PC12 cells was used to simulate cerebral ischemia-reperfusion injury. Cell viabilities were detected by Cell Counting Kit-8 (CCK-8) method. The levels of reactive oxygen species (ROS) and lipid reactive oxygen species (Lipid ROS) were detected by fluorescent probes Dihydroethidium (DHE) and C11 BODIPY 581/591. Iron Colorimetric Assay Kit, malondialdehyde (MDA) and glutathione (GSH) activity assay kits were used to detect intracellular iron ion, MDA and GSHcontent. Cell ferroptosis-related ultrastructures were visualized using transmission electron microscopy (TEM). Furthermore, PCR and Western blot analyses were used to detect the expressions of ferroptosis-related genes and proteins. After receiving oxygen-glucose deprivation/reperfusion, the viabilities of HT22 and PC12 cells were significantly decreased; ROS, Lipid ROS, iron ions and MDA accumulation occurred in the cells; GSH contents decreased; TEM showed that cells were ruptured and blebbed, mitochondria atrophied and became smaller, mitochondrial ridges were reduced or even disappeared, and apoptotic bodies appeared. And the expressions of Nrf2, SLC7A11 and GPX4 genes were reduced; the expressions of p-Nrf2/Nrf2, xCT and GPX4 proteins were reduced. Notably, these parameters were significantly reversed by HBO, indicating that HBO can protect HT22 cells and PC12 cells from damage caused by oxygen-glucosedeprivation/reperfusion via the inhibition of Nrf2/System Xc-/GPX4 axis-mediated ferroptosis.

Our previous study demonstrated that hyperbaric oxygen (HBO) improves heart function predominantly through reducing oxygen stress, modulating energy metabolism and inhibiting cell apoptosis. The present study aimed to investigate the protective effects of HBO on mitochondrial function and autophagy using rats with a ligated left anterior descending artery. The cardioprotective effects of HBO were mainly evaluated using ELISA, fluorescent probes, transmission electron microscopy and reverse transcription-quantitative PCR (RT-qPCR). HBO pretreatment for 14 days (once a day) using a 0.25 MPa chamber improved mitochondrial morphology and decreased the number of autophagic vesicles, as observed using a transmission electron microscope. HBO pretreatment significantly increased the levels of ATP, ADP, energy charge and the opening of the mitochondrial permeability transition pore, but decreased the levels of AMP, cytochromecand reactive oxygen species. Moreover, HBO pretreatment significantly increased the gene or protein expression levels of eIF4E-binding protein 1, mammalian target of rapamycin (mTOR), mitochondrial DNA, NADH dehydrogenase subunit 1, mitofusin 1 and mitofusin 2, whereas it decreased the gene or protein expression levels of autophagy-related 5 (Atg5), cytochromec, dynamin-related protein 1 and p53, as determined using RT-qPCR or immunohistochemistry. In conclusion, HBO treatment was observed to protect cardiomyocytes during myocardial ischemia-reperfusion injury (MIRI) by preventing mitochondrial dysfunction and inhibiting autophagy. Thus, these results provide novel evidence to support the use of HBO as a potential agent for the mitigation of MIRI.

Background: 2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) has been reported to inhibit a variety of cancer cell lines. The purpose of this study was to investigate the effects of DMDD on 4T1 breast cancer cells and the effects of DMDD on 4T1 breast cancer in mice and its molecular mechanisms. Methods: 4T1 breast cancer cells were treated with different concentrations of DMDD, and their proliferation, apoptosis, cell-cycle distribution, migration, and invasion were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, Acridine orange and ethidium bromide dual staining analysis (AO/EB) dual staining, flow cytometry, scratch test, and the Transwell assay. Relative quantitative real-time qPCR analysis and Western blot were applied to examine the expression levels of related genes and proteins. In animal experiments, we established a xenograft model to assess the anti-breast cancer effects of DMDD by evaluating the inhibition rate. The apoptotic activity of DMDD was evaluated by hematoxylin-eosin (HE) staining, transmission electron microscope (TEM) analysis and TdT-mediated dUTP nick end labeling (TUNEL) assays. The mRNA expression levels of MAPK pathway components were detected by relative quantitative real-time qPCR. In addition, the protein expression levels of MAPK pathway components were assessed through immunohistochemical assays and Western blotting. Results: Experiments showed that DMDD could inhibit the proliferation, migration, invasion of 4T1 cells and induce cellular apoptosis and G1 cell cycle arrest. Moreover, DMDD down-regulated the mRNA expressions of raf1, mek1, mek2, erk1, erk2, bcl2, and up-regulated the mRNA expression of bax. DMDD reduced the protein expressions of p-raf1, p-mek, p-erk, p-p38, Bcl2, MMP2, MMP9 and increased the protein expressions of Bax and p-JNK. The results showed that DMDD can effectively reduce the tumor volume and weight of breast cancer in vivo, up-regulate the expression of IL-2, down-regulate the expression of IL-4 and IL-10, induce the apoptosis of breast cancer cells in mice, and regulate the expression of genes and proteins of the MAPK pathway. Conclusion: Our study indicates that DMDD can inhibit proliferation, migration, and invasion and induces apoptosis and cell-cycle arrest of 4T1 breast cancer cells. Also, our findings indicate that DMDD induces the apoptosis of breast cancer cells and inhibits the growth in mice. Its mechanism may be related to the MAPK pathway.

In this study, we investigated the protective effect of hyperbaric oxygen (HBO) on PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion and its possible mechanism. PC12 and H9C2 cell oxygen-glucose deprivation/reperfusion model were established. Cells were divided into a control group, model group, hyperbaric air (HBA) group and HBO group. The cell viability was detected by the CCK8 assay. Hoechst 33342 and PI staining assays and mitochondrial membrane potential (MMP) assays were used to detect cell apoptosis. The ultrastructure of cells, including autophagosomes, lysosomes, and apoptosis, were examined using a transmission electron microscope. The expression of autophagy-related proteins was detected by cellular immunofluorescence and immunocytochemistry. Our results showed that HBO can significantly improve the vitality of damaged PC12 and H9C2 cells caused by oxygen–glucose deprivation/reperfusion. HBO can significantly inhibit apoptosis of PC12 and H9C2 cells caused by oxygen-glucose deprivation/reperfusion. Importantly, we found that the protective mechanism of PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion may be related to the inhibition of the autophagy pathway. In this study, the results of cellular immunofluorescence and immunocytochemistry experiments showed that the 4E-BP1, p-AKt and mTOR levels of PC12 and H9C2 cells in the model group decreased, while the levels of LC3B, Atg5 and p53 increased. However, after HBO treatment, these autophagy-related indexes were reversed. In addition, observation of the cell ultrastructure with transmission electron microscopy found that in the model group, a significant increase in the number of autophagic vesicles was observed. In the HBO group, a decrease in autophagic vesicles was observed. The study demonstrated that hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy.