AT1R/GSK-3β/mTOR Signaling Pathway Involved in Angiotensin II-Induced Neuronal Apoptosis after HIE Both In Vitro and In Vivo

Abstract

Objective. The focus of the present study is to evaluate the effects of Angiotensin II (Ang II) on neuronal apoptosis after HIE and the potential underlying mechanisms. Methods. Primary neonatal rat cortical neurons were used to study the oxygen-glucose deprivation (OGD) cell model. The expressions of Ang II, AT1R, GSK-3β, p-GSK-3β, mTOR, p-mTOR, Bax, Bcl-2, and cleaved caspase-3 were detected via western blot. IF and flow cytometry were used to evaluate neuronal apoptosis. Hypoxic-ischemic encephalopathy (HIE) was established to evaluate the therapeutic effects of Ang II in vivo. Cerebral infarction areas were detected by 2,3,5-Triphenyltetrazolium chloride staining. The righting and geotaxis reflexes were also recorded. In addition, Fluoro-Jade C staining and TUNEL staining were performed to evaluate neuronal degeneration and apoptosis. Results. Ang II significantly increased the rate of neuronal apoptosis, upregulated the expression of cleaved caspase-3, and downregulated Bcl-2/Bax ratio after OGD insult. For vivo assay, the expressions of endogenous Ang II and AT1R gradually increased and peaked at 24 h after HIE. Ang II increased NeuN-positive AT1R cell expression. In addition, Ang II increased the area of cerebral infarction, promoted neuronal degeneration and apoptosis, aggravated neurological deficits on righting and geotaxis reflexes, and was accompanied by increased expressions of phosphorylated GSK-3β and mTOR. The application of valsartan (Ang II inhibitor) or SB216763 (GSK-3β inhibitor) reversed these phenomena triggered by Ang II following HIE. Conclusion. Ang II increased neuronal apoptosis through the AT1R/GSK-3β/mTOR signaling pathway after experimental HIE both in vitro and in vivo, and Ang II may serve as a novel therapeutic target to ameliorate brain injury after HIE.

Pre-Exposure to Environmental Enrichment Protects against Learning and Memory Deficits Caused by Infrasound Exposure

“…We used FJC (Chemicon, Temecula, CA, USA), which can specifically stain degenerating neurons in the CNS subject to various neurotoxin insults and neurological diseases [ 35 , 36 ].…”

“…Brain sections containing the hippocampus were prepared as in Section 2.9 . FJC staining was carried out using the following standard procedures [ 35 , 36 ]: (1) pretreatment with an alcohol-sodium hydroxide mixture. The sections were immersed in a solution containing 1% sodium hydroxide in 80% alcohol for 5 min, followed by 70% alcohol and distilled water each for 2 min.…”

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“…Western blot was performed as described previously (Li et al, 2020;Si et al, 2020). The whole-cell lysate was prepared using M-PER Protein Extraction Reagent (Pierce, Rockford, IL, United States) supplemented with protease inhibitor cocktail.…”