Genistein Attenuates Isoflurane-Induced Neuroinflammation by Inhibiting TLR4-Mediated Microglial-Polarization in vivo and in vitro

BACKGROUND: Isoflurane, a widely used anesthetic in surgery, has been found to induce neurotoxicity. In parallel, genistein is thought to attenuate isoflurane-induced neurotoxicity, although underlying molecular mechanisms are still unclear. In this study, we studied the protective effects of genistein on isoflurane-induced neuroinflammation in rats and BV2 cells. METHODS: Sprague-Dawley rat pups were exposed to 0.75% isoflurane for 6 hours at postnatal day 7 (P7), and genistein (20, 40, or 80 mg/kg/day) or saline administered from P3 to P15. Hippocampal single-cell suspensions were prepared and apoptosis analyzed by flow cytometry. mRNA expression was determined by RT-qPCR, while protein expression was assessed using Western blot, immunochemistry and immunofluorescence. TLR4 was knocked-out in BV2 cells through CRISPR-Cas9. RESULTS: Genistein treatment reduced isoflurane-induced apoptosis and inflammation in rat hippocampus. Importantly, genistein promoted M2 and suppressed M1 microglia polarization in rat hippocampus after stimulation with isoflurane. In addition, genistein reduced isoflurane-induced protein expression levels of TLR4, MyD88, TRAF6, p-TAK1, p-p38, p-ERK, p-IκBα and p-NF-κB in rat hippocampus. In BV2 cells exposed to isoflurane, genistein treatment decreased IL-1β, TNF-α, IL-6 and IL-8 mRNA expressions, promoted M2 and suppressed M1 microglia polarization. Similarly, genistein also decreased TLR4 protein levels in isoflurane-induced BV2 cells. However, genistein did not affect CD16, iNOS, CD206 and Arg1 protein levels in TLR4-KO BV2 cells exposed to isoflurane. CONCLUSION: Genistein attenuates isoflurane-induced neurotoxicity by inhibiting TLR4-mediated microglial inflammation in vivo and in vitro.