Background/Aims: Emerging evidence suggests that autophagy plays important roles in the pathophysiological processes of cerebral ischemia and reperfusion injury. Calycosin, an isoflavone phytoestrogen, possesses neuroprotective effects in cerebral ischemia and reperfusion in rats. Here, we investigated the neuroprotective effects of calycosin against ischemia and reperfusion injury, as well as related probable mechanisms behind autophagy pathways. Methods: A cerebral ischemic and reperfusion injury model was established by middle cerebral artery occlusion in male Sprague-Dawley rats. Neurological scores, infarct volumes, and brain water content were assessed after 24 h reperfusion following 2 h ischemia. Additionally, the expression of the autophagy-related protein p62 and NBR1 (neighbor of BRCA1 gene 1), as well as Bcl-2, and TNF-α in rat brain tissues was measured by RT-PCR, western blotting and immunohistochemical analyses. Results: The results showed that calycosin pretreatment for 14 days markedly decreased infarct volume and brain edema, and ameliorated neurological scores in rats with focal cerebral ischemia and reperfusion. It was observed that levels of p62, NBR1 and Bcl-2 were greatly decreased, and levels of TNF-α significantly increased after ischemia and reperfusion injury. However, calycosin administration dramatically upregulated the expression of p62, NBR1 and Bcl-2, and downregulated the level of TNF-α. Conclusions: All data reveal that calycosin exerts a neuroprotective effect on cerebral ischemia and reperfusion injury, and the mechanisms maybe associated with its anti-autophagic, anti-apoptotic and anti-inflammatory action.

BACKGROUND:Oxidative stress and neuroinflammation are 2 pivotal mechanisms in the progression of cerebral ischemia/reperfusion injury. Biochanin A, a natural phytoestrogen, has been reported to protect against ischemic brain injury in animal experime...

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.

Background: Cerebral ischemia-reperfusion injury (CIRI) is a key pathophysiological process that leads to stroke mortality, with TLR4-mediated inflammation playing a crucial role. Our previous research highlighted the neuroprotective effects of the phytoestrogen calycosin on CIRI, although the precise mechanism remains unclear. This study aimed to explore the effects of calycosin on the HMGB1/TLR4/NF-κB signaling pathway in rat models of CIRI, both in vivo and in vitro. Methods: In vivo, a rat CIRI model was established using middle cerebral artery occlusion (MCAO), inducing ischemia for 1.5 h followed by 24 h of reperfusion. Calycosin was administered intraperitoneally 1 h after ischemia. Neurological deficits and brain infarct volumes were evaluated. Histological changes and key protein expressions around the ischemic penumbra were assessed by H&E staining and immunofluorescence. In vitro, primary neurons and PC12 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic CIRI. Cell viability was measured using a CCK8 assay, and alterations in HMGB1/TLR4/NF-κB pathway components were analyzed using qRT-PCR, Western blotting, and ELISA. Results: In the MCAO rat model, calycosin significantly reduced neurological deficits and infarct sizes, and improved brain tissue damage following reperfusion. Similarly, in the OGD/R model, calycosin attenuated neuronal injury in PC12 cells and in primary neurons. Additionally, calycosin inhibited LPS-induced activation of the HMGB1/TLR4/NF-κB signaling pathway in PC12 cells. Both in vitro and in vivo studies have shown that calycosin effectively downregulates HMGB1 and TLR4 expression, decreases NF-κB and IκB phosphorylation, and reduces the secretion of inflammatory cytokines such as IL-6 and IL-18. Conclusion: These findings suggest that calycosin mitigates cerebral ischemia-reperfusion injury and neuroinflammation by inhibiting the HMGB1/TLR4/NF-κB signaling pathway, thereby providing neuroprotection.