Calycosin Ameliorates Neuroinflammation via TLR4-Mediated Signal Following Cerebral Ischemia/Reperfusion Injury in vivo and in vitro.

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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. In vivo, a rat CIRI model was established using middle cerebral artery occlusion (MCAO), inducing ischemia for 1.5h followed by 24h of reperfusion. Calycosin was administered intraperitoneally 1h 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. 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. These findings suggest that calycosin mitigates cerebral ischemia-reperfusion injury and neuroinflammation by inhibiting the HMGB1/TLR4/NF-κB signaling pathway, thereby providing neuroprotection.