Alterations in necroptosis during ALDH2‑mediated protection against high glucose‑induced H9c2 cardiac cell injury

Abstract

The aim of the present study was to investigate whether necroptosis occurs in high glucose (HG)-induced H9c2 cardiac cell injury and whether the activation of aldehyde dehydrogenase 2 (ALDH2) can inhibit necroptosis. H9c2 cardiac cells were treated with 35 mM glucose to establish a HG-induced cell injury model. Alda-1 (20 µM), a specific activator of ALDH2 and necrostatin-1 (Nec-1, 100 µM), an inhibitor of necroptosis were used to treat H9c2 cardiac cells under HG conditions. Cell viability was measured using a Cell Counting Kit-8 assay and reactive oxygen species (ROS) generation was measured by the dihydroethidium staining method. ALDH2 activity was measured at 450 nm. The mRNA and protein expression of ALDH2, necroptosis-associated genes, receptor-interacting protein (RIP)1, RIP3 and mixed lineage kinase domain like pseudokinase (MLKL), were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting. The expression of cleaved caspase-3 protein was also examined by western blotting. The results demonstrated that under HG conditions, cell viability, ALDH2 activity, mRNA and protein expression were decreased. Furthermore, ROS generation, mRNA and protein expression of RIP1, RIP3, MLKL and the protein expression of cleaved caspase-3 were increased. Treatment with Alda-1 or Nec-1 attenuated HG-induced downregulation of ALDH2 activity, mRNA and protein expression. In addition, RIP1, RIP3, MLKL mRNA, and protein expression were downregulated. Furthermore, Alda-1 but not Nec-1 decreased cleaved caspase-3 protein expression. Collectively these data indicated that activation of ALDH2 protected H9c2 cardiac cells against HG-induced injury, partly by inhibiting the occurrence of necroptosis.

Necrostatin-1 and necroptosis inhibition: Pathophysiology and therapeutic implications

“…Liang et al firstly reported the contribution of necroptosis in HG-induced injury in cardiac cells and role of Nec-1 in reducing cytotoxicity, oxidative stress and dissipation of mitochondrial membrane potential (MMP) [ 39 ]. Similar results were obtained by later researches in both cardiac cells and endothelial cells [ 38 , 40 , 41 ]. However, current studies only provide an elusive description on this Nec-1 protective effect, and the underlying molecular mechanism remains to be investigated.…”

“…Besides, Han et al found that Nec-1 can enhance leukemia cell apoptosis induced by Shikonin [ 96 ], showing an anti-cancer effect; Jie et al found that Nec-1 could specifically induce neutrophils apoptosis rather than blockage [ 97 ], which implies an anti-inflammatory effect. Despite researches mentioned above, some researchers claim that Nec-1 does not affect apoptosis [ 38 , 48 ], which is probably RIA. When apoptosis is mainly RIP1-dependent, Nec-1 inhibits RIP1 kinase activity and formation of complex Ⅱa, and thus apoptosis is blocked; however, when apoptosis is RIP1-independent, Nec-1 has no inhibitory effect.…”

Necrotic enteritis challenge regulates peroxisome proliferator-1 activated receptors signaling and β-oxidation pathways in broiler chickens

“…2009 , 2016 ). Down-regulation of the gene can induce inflammation, apoptosis and necrosis by the activation of ROS pathway ( Fang et al., 2018 ; Liang et al., 2017 ).…”

Inhibition of microRNA-141-3p Reduces Hypoxia-Induced Apoptosis in H9c2 Rat Cardiomyocytes by Activating the RP105-Dependent PI3K/AKT Signaling Pathway

“…The dihydroethidium (DHE) probe (Beyotime, Shanghai, China) was used to measure ROS generation in H9c2 cells, as previously described [13]. Briefly, cells were adjusted to 1.5×10 5 cells per well and cultured in a 24-well culture plate.…”