Excessive nNOS/NO/AMPK signaling activation mediated by the blockage of the CBS/H2S system contributes to oxygen‑glucose deprivation‑induced endoplasmic reticulum stress in PC12 cells

Abstract

Hypoxic‑ischemia stress causes severe brain injury, leading to death and disability worldwide. Although it has been reported that endoplasmic reticulum (ER) stress is an essential step in the progression of hypoxia or ischemia‑induced brain injury, the underlying molecular mechanisms are and have not yet been fully elucidated. Accumulating evidence has indicated that both nitric oxide (NO) and hydrogen sulfide (H2S) play an important role in the development of cerebral ischemic injury. In the present study, we aimed to investigate the effect of the association between NO signaling and the cystathionine β‑synthase (CBS)/H2S system on ER stress in a cell model of cerebral hypoxia‑ischemia injury. We found that oxygen‑glucose deprivation (OGD) markedly increased the NO level and neuronal NO synthase (nNOS) activity. 3‑Bromo‑7‑nitroindazole (3‑Br‑7‑NI), a relatively selective nNOS inhibitor, abolished the OGD‑induced inhibition of cell viability and the increased expression of ER stress‑related proteins, including glucose‑regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and cleaved caspase‑12 in PC12 cells, indicating the contribution of excessive nNOS/NO signaling to OGD‑induced ER stress. Furthermore, we found that OGD increased the phosphorylated AMP‑activated protein kinase (p‑AMPK)/AMPK ratio, and the AMPK activator, 5‑aminoimidazole‑4‑carboxamide‑1‑β‑D‑ribofuranoside (AICAR), attenuated the effects on OGD‑induced ER stress, suggesting that OGD‑induced NO overproduction results in AMPK activation in PC12 cells. We also found that OGD induced the downregulation of the CBS/H2S system, as indicated by the decreased H2S level in the culture supernatant and CBS activity in PC12 cells. In addition, we found that treatment with NaHS (a H2S donor) or S‑adenosyl‑L‑methionine (SAM, a CBS agonist) mitigated OGD‑induced ER stress, as well as the NO level, nNOS activity and AMPK phosphorylation in PC12 cells. On the whole, these results suggest that the inhibition of the CBS/H2S system, which facilitated excessive nNOS/NO/AMPK activation, contributes to OGD‑induced ER stress.