H2O2-preconditioned human adipose-derived stem cells (HC016) increase their resistance to oxidative stress by overexpressing Nrf2 and bioenergetic adaptation

Abstract

Background: Mesenchymal stem cells, including those derived from human adipose tissue (hASCs), are currently being widely investigated for cell therapy. However, when transplanted at the site of injury, the survival and engraftment rates of hASCs are low, mainly due to the harsh microenvironment they encounter, characterized by inflammation and oxidative stress. To overcome these therapeutic limitations, cell preconditioning with lowconcentration of hydrogen peroxide (H 2 O 2) has been proposed as a plausible strategy to increase their survival and adaptation to oxidative stress. Nonetheless, the underlying mechanisms of this approach are not yet fully understood. In this study, we analyzed molecular and bioenergetic changes that take place in H 2 O 2 preconditioned hASCs. Methods: Long-term exposure to a low concentration of H 2 O 2 was applied to obtain preconditioned hASCs (named HC016), and then, their response to oxidative stress was analyzed. The effect of preconditioning on the expression of Nrf2 and its downstream antioxidant enzymes (HO-1, SOD-1, GPx-1, and CAT), and of NF-κB and its related inflammatory proteins (COX-2 and IL-1β), were examined by Western blot. Finally, the Seahorse XF96 Flux analysis system was used to evaluate the mitochondrial respiration and glycolytic function, along with the total ATP production. Results: We found that under oxidative conditions, HC016 cells increased the survival by (i) decreasing intracellular ROS levels through the overexpression of the transcription factor Nrf2 and its related antioxidant enzymes HO-1, SOD-1, GPx-1, and CAT; (ii) reducing the secretion of pro-inflammatory molecules COX-2 and IL-1β through the attenuation of the expression of NF-κB; and (iii) increasing the total ATP production rate through the adaption of their metabolism to meet the energetic demand required to survive. Conclusions: H 2 O 2 preconditioning enhances hASC survival under oxidative stress conditions by stimulating their antioxidant response and bioenergetic adaptation. Therefore, this preconditioning strategy might be considered an excellent tool for strengthening the resistance of hASCs to harmful oxidative stress.

Hydrogen Peroxide-Preconditioned Human Adipose-Derived Stem Cells Enhance the Recovery of Oligodendrocyte-Like Cells after Oxidative Stress-Induced Damage

“…Finally, among the different co-cultures, the OX HOGd co-cultured with HC016 cells also exposed to oxidative stress showed a better recovery with enhanced viability (1.4-fold that in controls) and lower ROS levels (1.3-fold those in controls). As previously demonstrated, the 0.25 mM H 2 O 2 insult stimulated HC016 cell antioxidant response [ 18 ], and this seems to be beneficial for OX HOGd recovery.…”

“…The preconditioning process–sub-lethal exposure to cellular stressors–promotes the expression and secretion of certain molecules that are required to reduce damage and increase survival, giving cells the capacity to respond efficiently to a higher level of the same stressor [ 15 , 16 , 17 ]. In a previous study, we showed that hASCs preconditioned with low doses of H 2 O 2 (HC016 cells) increase their resistance to oxidative stress by enhancing their intrinsic antioxidant capacity and bioenergetic regulation [ 18 ].…”

Loganin Attenuates Septic Acute Renal Injury with the Participation of AKT and Nrf2/HO-1 Signaling Pathways

“…12 Under various pathologic conditions, the strong correlation between oxidative stress injury and nuclear factor E2-related factor 2 (Nrf2) has been previously proved. [13][14][15] The preceding study has pointed out that Nrf2/HO-1 pathway is one of the most recognized signaling closely associated with oxidative and anti-oxidative balance. 16,17 Under normal circumstances, the cap "n" collar subfamily of basic region-leucine zipper transcription factor Nrf2 is restricted in the cytoplasm by binding to its ligand Kelch-like ECH associating protein 1 (Keap1).…”

Antioxidant activity of mesenchymal stem cell-derived extracellular vesicles restores hippocampal neurons following seizure damage

“…MiRNA sequencing was used to identify the cargos that might be responsible for the antioxidant potential of MSC-EVs. We compared the differentially expressed miRNAs between MSC-EVs and conditioned EVs (derived from H 2 O 2 -stimulated MSCs) 22 and found enrichment of specific miRNAs in MSC-EVs (Figure 2 A and B; Table S1 ); differential expression analysis revealed upregulation of many miRNAs (log2-fold change > 5: miR-215-5p, miR-424-5p, miR-31-3p, miR-193b-3p and miR-200b-3p) in H 2 O 2 -conditioned EVs (Figure 2 B). Gene Ontology (GO) classification (Figure 2 C; Table S2 ) revealed that the exosomal miRNA target genes (n = 58) were associated with the antioxidant activity molecular function.…”

“…To detect the antioxidant cargoes in MSC-EVs, MSCs were preconditioned with H 2 O 2 22 , 23 in FBS-depleted media, and EVs were isolated as described above. Total RNA from MSC-EVs (Control group, n = 3) and H 2 O 2 -preconditioned MSC-EVs (H 2 O 2 group, n = 3) was extracted using the miRNeasy Serum/Plasma Kit (217184, Qiagen, Germany), and the RNA integrity and concentration were examined by the Agilent 2100 Bioanalyzer (Agilent Technology, USA) and NanoDrop 2000 (Thermo Fisher, USA).…”