Oxidative Stress-induced Autophagy Compromises Stem Cell Viability.

Stem cell therapies have emerged as a promising treatment strategy for various diseases characterized by ischemic injury such as ischemic stroke. Cell survival after transplantation remains a critical issue. We investigated the impact of oxidative stress, being typically present in ischemically challenged tissue, on human dental pulp stem cells (hDPSC) and human mesenchymal stem cells (hMSC). We used oxygen-glucose deprivation (OGD) to induce oxidative stress in hDPSC and hMSC. OGD-induced generation of O2•- or H2O2 enhanced autophagy by inducing the expression of activating molecule in BECN1-regulated autophagy protein 1 (Ambra1) and Beclin1 in both cell types. However, hDPSC and hMSC pre-conditioning using reactive oxygen species (ROS) scavengers significantly repressed the expression of Ambra1 and Beclin1 and inactivated autophagy. O2•- or H2O2 acted upstream of autophagy, and the mechanism was unidirectional. Furthermore, our findings revealed ROS-p38-Erk1/2 involvement. Pre-treatment with selective inhibitors of p38 and Erk1/2 pathways (SB202190 and PD98059) reversed OGD effects on the expression of Ambra1 and Beclin1, suggesting that these pathways induced oxidative stress-mediated autophagy. SIRT3 depletion was found to be associated with increased oxidative stress and activation of p38 and Erk1/2 MAPKs pathways. Global ROS inhibition by NAC or a combination of polyethylene glycol-superoxide dismutase (PEG-SOD) and polyethylene glycol-catalase (PEG-catalase) further confirmed that O2•- or H2O2 or a combination of both impacts stems cell viability by inducing autophagy. Furthermore, autophagy inhibition by 3-methyladenine (3-MA) significantly improved hDPSC viability. These findings contribute to a better understanding of post-transplantation hDPSC and hMSC death and may deduce strategies to minimize therapeutic cell loss under oxidative stress.

Oxidative Stress Enhances Autophagy-Mediated Death Of Stem Cells Through Erk1/2 Signaling Pathway - Implications For Neurotransplantations.

Stem cell therapies are becoming increasingly popular solutions for neurological disorders. However, there is a lower survival rate of these cells after transplantation. Oxidative stress is linked to brain damage, and it may also impact transplanted stem cells. To better understand how transplanted cells respond to oxidative stress, the current study used H2O2. We briefly illustrated that exogenous H2O2 treatment exaggerated oxidative stress in the human dental pulp and mesenchymal stem cells. 2',7'-Dichlorofluorescin diacetate (DCFDA), MitoSOX confirms the reactive oxygen species (ROS) involvement, which was remarkably subsided by the ROS inhibitors. The findings showed that H2O2 activates autophagy by enhancing pro-autophagic proteins, Beclin1 and Atg7. Increased LC3II/I expression (which co-localized with lysosomal proteins, LAMP1 and Cathepsin B) showed that H2O2 treatment promoted autophagolysosome formation. In the results, both Beclin1 and Atg7 were observed co-localized in mitochondria, indicating their involvement in mitophagy. The evaluation of Erk1/2 in the presence and absence of Na-Pyruvate, PEG-Catalase, and PD98059 established ROS-Erk1/2 participation in autophagy regulation. Further, these findings showed a link between apoptosis and autophagy. The results conclude that H2O2 acts as a stressor, promoting autophagy and mitophagy in stem cells under oxidative stress. The current study may help understand better cell survival and death approaches for transplanted cells in various neurological diseases. The current study uses human Dental Pulp and Mesenchymal Stem cells to demonstrate the importance of H2O2-driven autophagy in deciding the fate of these cells in an oxidative microenvironment. To summarise, we discovered that exogenous H2O2 treatment causes oxidative stress. Exogenous H2O2  treatment also increased ROS production, especially intracellular H2O2. H2O2 stimulated the ErK1/2 signaling pathway and autophagy. Erk1/2 was found to cause autophagy. Further, the function of mitophagy appeared to be an important factor in the H2O2-induced regulation of these two human stem cell types. In a nutshell, by engaging in autophagy nucleation, maturation, and terminal phase proteins, we elucidated the participation of autophagy in cell dysfunction and death.

Gelatin-Coated Polycaprolactone Nanoparticle-Mediated Naringenin Delivery Rescue Human Mesenchymal Stem Cells from Oxygen Glucose Deprivation-Induced Inflammatory Stress.

Ischemic stroke involves pro-inflammatory species, which implicates inflammation in the disease mechanism. Recent studies indicate that the prevalence of therapeutic choice such as stem cell transplantation has seen an upsurge in ischemic stroke. However, after transplantation the fate of transplanted cells is largely unknown. Human mesenchymal stem cells (MSCs), due to their robust survival rate upon transplantation in brain tissue, are being widely employed to treat ischemic stroke. In the present study, we have evaluated naringenin-loaded gelatin-coated polycaprolactone nanoparticles (nar-gel-c-PCL NPs) to rescue MSCs against oxygen glucose deprived insult. Naringenin, due to its strong anti-inflammatory effects, remains a therapeutic choice in neurological disorders. Though, the low solubility and inefficient delivery remain challenges in using naringenin as a therapeutic drug. The present study showed that inflammation occurred in MSCs during their treatment with oxygen glucose deprivation (OGD) and was well overturned by treatment with nar-gel-c-PCL NPs. In brief, the results indicated that nar-gel-c-PCL NPs were able to protect the loss of cell membrane integrity and restored neuronal morphology. Then nar-gel-c-PCL NPs successfully protected the human MSCs against OGD-induced inflammation as evident by reduced level of pro-inflammatory cytokine (TNF-α, IFN-γ, and IL-1ß) and other inflammatory biomarkers (COX2, iNOS, and MPO activity). Therefore, the modulation of inflammation by treatment with nar-gel-c-PCL NPs in MSCs could provide a novel strategy to improve MSC-based therapy, and thus, our nanoformulation may find a wide therapeutic application in ischemic stroke and other neuro-inflammatory diseases.

Chick Embryo: A Preclinical Model for Understanding Ischemia-Reperfusion Mechanism.

Ischemia-reperfusion (I/R)-related disorders, such as stroke, myocardial infarction, and peripheral vascular disease, are among the most frequent causes of disease and death. Tissue injury or death may result from the initial ischemic insult, primarily determined by the magnitude and duration of the interruption in blood supply and then by the subsequent reperfusion-induced damage. Various in vitro and in vivo models are currently available to study I/R mechanism in the brain and other tissues. However, thus far, no in ovo I/R model has been reported for understanding the I/R mechanisms and for faster drug screening. Here, we developed an in ovo Hook model of I/R by occluding and releasing the right vitelline artery of a chick embryo at 72 h of development. To validate the model and elucidate various underlying survival and death mechanisms, we employed imaging (Doppler blood flow imaging), biochemical, and blotting techniques and evaluated the cell death mechanism: autophagy and inflammation caused by I/R. In conclusion, the present model is useful in parallel with established in vitro and in vivo I/R models to understand the mechanisms of I/R development and its treatment.