Abstract
We have previously shown that biochanin A exhibits neuroprotective properties in the context of cerebral ischemia/reperfusion (I/R) injury. The mechanistic basis for such properties, however, remains poorly understood. This study was therefore designed to explore the manner whereby biochanin A controls endoplasmic reticulum (ER) stress, apoptosis, and inflammation within fetal rat primary cortical neurons in response to oxygen-glucose deprivation/reoxygenation (OGD/R) injury, and in a rat model of middle cerebral artery occlusion and reperfusion (MCAO/R) injury. For the OGD/R in vitro model system, cells were evaluated after a 2 h OGD following a 24 h reoxygenation period, whereas in vivo neurological deficits were evaluated following 2 h of ischemia and 24 h of reperfusion. The expression of proteins associated with apoptosis, ER stress (ERS), and p38 MAPK phosphorylation was evaluated in these samples. Rats treated with biochanin A exhibited reduced neurological deficits relative to control rats following MCAO/R injury. Additionally, GRP78 and CHOP levels rose following I/R modeling both in vitro and in vivo, whereas biochanin A treatment was associated with reductions in CHOP levels but further increases in GRP78 levels. In addition, OGD/R or MCAO/R were associated with markedly enhanced p38 MAPK phosphorylation that was alleviated by biochanin A treatment. Similarly, OGD/R or MCAO/R injury resulted in increases in caspase-3, caspase-12, and Bax levels as well as decreases in Bcl-2 levels, whereas biochanin A treatment was sufficient to reverse these phenotypes. Together, these findings thus demonstrate that biochanin A can alleviate cerebral I/R-induced damage at least in part via suppressing apoptosis, ER stress, and p38 MAPK signaling, thereby serving as a potent neuroprotective agent.
Highlights
Ischemic and hemorrhagic strokes remain a leading cause of death and disability globally
We evaluated the impact of biochanin A treatment on endoplasmic reticulum stress by assessing C/EBP-homologous protein; also known as GADD153 (CHOP) and GRP78 mRNA and protein levels in Middle Cerebral Artery Occlusion (MCAO)/R model rats
The results of this analysis indicated that biochanin A, which is an isoflavone phytoestrogen derived from chickpeas and other leguminous plants, is neuroprotective in the context of ischemia/ reperfusion (I/R) injury both in vitro and in vivo through mechanisms associated with endoplasmic reticulum (ER) stress alleviation, suppression of ER stress-related apoptosis, and inhibition of p38 MAPK signaling
Summary
Ischemic and hemorrhagic strokes remain a leading cause of death and disability globally. The ER is an organelle that serves as a site of protein folding and as a regulator of calcium homeostasis Stressful conditions such as ATP depletion, calcium overload, or hypoxia can disrupt normal ER homeostasis and result in misfolded or unfolded protein accumulation. Such ER stress can, when mild, activate the unfolded protein response (UPR) in an effort to restore homeostasis and promote cell survival. The UPR is governed by three regulatory proteins expressed within the ER membrane: activating transcription factor 6 (ATF6), protein kinase RNAlike endoplasmic reticulum kinase (PERK), and inositolrequiring enzyme 1 (IRE1) [5] At baseline, these three proteins bind to the ER chaperone glucose regulated protein 78 (GRP78, known as binding immunoglobulin protein, BIP) and thereafter remain in an inactive state. Caspase-12 serves as an important regulator of ER stressinduced apoptosis, as activated caspase-12 release into the cytoplasm induces the pro-apoptotic activation of caspase-3/9, in turn resulting in DNA fragmentation and apoptotic cell death [10]
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