Mechanism Of Lycium Barbarum Polysaccharides On Primarycultured Rat Hippocampal Neurons : Possible Involvement Of Toll-like Receptor 4 And Nuclear Factor Kappa B Pathway

Objectives In this study we investigated the protective effects of Lycium barbarum polysaccharide(LBP) on hippocampal neurons injured by oxygen-glucose deprivation/reperfusion(OGD/RP) in vitro and elucidated the related mechanisms.Methods 1. Cultured hippocampal neurons were exposed to oxygen-glucose privation(OGD) for 2h followed by a 24 h reperfusion(RP) were used as an in vitro model of ischemia and reperfusion.2. The survival rate of nerve cells was measured by MTT assay. The nerve cell lactate dehydrogenase(LDH) leakage rate.3. The neuron apoptosis was evaluated by Hoechst 33342 staining TUNEL staining.4. The concentration of intracellular free calcium [Ca2+]i and mitochondrial membrane potential(MMP) were determined by laser scanning confocal microscopy.5. Western blotting assay and Quantitative real-time PCR assay were used to evaluate protein and m RNA expression of TLR4,NF-κB,IL-6 and IκB-α.Results 1. Compared with control group, OGD/RP group can significantly reduce the survival rate of nerve cells(P<0.01), increase the lactate dehydrogenase leakage rate(P<0.01). Compared with the OGD/RP group, treatment with LBP(10、20、40 mg/L) significantly increased cell viability(P<0.01) and inhibited LDH release(P<0.01).2. In order to determine apoptosis of neonatal rat primary cultured hippocampal neurons injured by OGD/RP were investigated with Hoechst 33342 and TUNEL staining. Compared with the OGD/RP group, treatment with LBP(10、20、40 mg/L) significantly attenuated neuronal damage, with evidence of decreased cell apoptosis(P<0.05,0.01).3. Compared with the OGD/RP group, treatment with LBP(10 、 20 、 40 mg/L) significantly attenuated these increased of [Ca2+]i(P<0.05, 0.01)and decreased MMP(P<0.01).4. Compared with the OGD/RP group, treatment with LBP(40 mg/L) could significantly down regulate the expression of TLR4(P<0.05), NF-κB(P<0.05)and IL-6(P<0.01) in protein level, induce an increase of IκB-α(P<0.01) in protein level.5. Compared with the OGD/RP group, treatment with LBP(40 mg/L) could significantly down regulate the expression of NF-κB(P<0.01),IL-6(P<0.05) and TLR4(P<0.01) and induce an increase of IκB-α(P<0.01) in m RNA level.Conclusion 1. LBP have significantly protective effects on OGD/RP-induced neuronal damages in rat primary neuron cultures.2. Neuroprotective effects of LBP on brain ischemic injury-induced apoptosis might be associated with TLR4/NF-κB signaling way.