4-Octyl itaconate inhibits hypoxia-induced injuries on β-cell via reducing LDHA-mediated ROS generation
Jianmin Wu1, Xingshi Gu1, Wei Wang1, Xiaoqian Ma1.
1The institute for cell transplantation and gene therapy, The third hospital of Central South University, Changsha, People's Republic of China
Introduction: Islet transplantation has been shown as a potentiate way to cure Type 1 Diabetes. However, isolation and purification inevitably devascularize the islets. Hypoxia stress has been observed up to 60% of newly transplanted tissue during the first couple of days which increase the hypoxia induced β-cell death. Reactive oxygen species (ROS) are key factors mediating hypoxic damage in β-cell and are mainly generated in NADPH or NADH dependent pathways. Itaconate is a by-product of the tricarboxylic acid cycle that protects cell from inflammation and oxidative stress induced damage. However, the effect of itaconate for β-cell in hypoxia condition still unknown. In this study, we aimed to explore the effect of 4-OI, a cell-permeable derivative of itaconate, on protection beta cells from hypoxia-induced damage and its mechanism.
Method: MIN6 cells were cultured under hypoxic conditions (1% O2) with or without 125μM 4-OI in vitro for 72h. The viability of cells was examined using MTS assays and flow cytometric analysis. The levels of inflammatory cytokines were detected by qRT-PCR and ELISA assays. Intracellular ROS content was measured by DCFH-DA probe. LDH activity assay kit was used to test the activity of LDHA. Finally, cells were treated with ROS scavenger N-acetylcysteine (NAC) and LDHA specific inhibitor FX-11 in hypoxia condition for 72h to verify the role of 4-OI in regulating ROS.
Results: Hypoxia-induced injuries on MIN6 cells were confirmed by decreased cell viability, increased oxidative stress and apoptosis, and the production of inflammatory cytokines when compared with normoxia. After the treatment of 4-OI, those changes caused by hypoxia injury were effectively reversed. The viability of MIN6 cells under hypoxia was improved, the intracellular ROS level, inflammatory cytokines and apoptosis were decreased. Similarly, when we used ROS scavenger NAC to treat the cells with hypoxia, cell viability was increased while the quantity of intracellular ROS, and inflammatory cytokines were reduced. Then, we found that the level of NADPH was not significantly altered under hypoxia, whereas NADH was dramatically increased. When the cells treated with 4-OI, the level of NADH was down-regulated. In addition, we found that 4-OI decreased LDHA activity, which suggested the production of ROS triggered the cell death was from the interaction of LDHA and NADH. Finally, to confirm our findings, we treated hypoxic cells with FX-11, a specific inhibitor of LDHA. The treatment of FX-11 enhanced cell survival, inhibited the production of ROS, and reduced the level of inflammatory cytokines.
Conclusion: In this study, we provide evidence that 4-OI can decrease the interaction of LDHA-NADH then reduce the generation of ROS which finally protect the β cell under hypoxia condition.
Natural Science Foundation of Hunan Province, China (Grant No.:2021JJ31018).
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