Neuroprotective Effect Of Lycium Barbarum Polysaccharide Against In Vitro Ischemic Neuronal Damage: Evidence For Inhibition Of PARP-1Activation And AIF Translocation

Objectives In this study we investigated the protective effects of Lyciumbarbarum polysaccharide (LBP) on hippocampal neurons injured by oxygen-glucosedeprivation/reperfusion (OGD/RP) in vitro and elucidated the related mechanisms.Methods1. Cultured hippocampal neurons were exposed to oxygen-glucosedeprivation (OGD) for2h followed by a24h reperfusion (RP) were used as an invitro model of ischemia and reperfusion. Hippocampal neurons fluorescense stainingwith rabbit anti-rat neuron specific enolase (NSE) were performed to identify theneurons.2. The survival rate of nerve cells was measured by MTT assay. The nerve celllactate dehydrogenase (LDH) leakage rate and NAD lever was measured by chemicalcolorimetry.3. The neuron apoptosis was evaluated by Hoechst33342staining andAnnexinV-FITC/PI.4. The concentration of intracellular free calcium [Ca2+]iand mitochondrialmembrane potential (MMP) were determined by laser scanning confocal microscopy.5. The DNA Ladder were separated using gel electrophoresis.6. Western blotting assay and Quantitative real-time PCR assay were used toevaluate protein and mRNA expression of PARP-1，AIF and Endo G.Results1. Compared with control group, OGD/RP group can significantlyincrease the lactate dehydrogenase leakage rate (P<0.01), reduce the survival rate of nerve cells (P<0.01) and NAD level (P<0.001). Compared with the OGD/RP group,treatment with LBP (10、20、40mg/L) significantly inhibited LDH release (P<0.05,0.01), increased cell viability (P<0.01) and NAD level (P<0.01,0.001).2. In order to determine apoptosis of neonatal rat primary cultured hippocampalneurons injured by OGD/RP were investigated with Hoechst33342and AnnexinV-FICT/PI double staining. Compared with the OGD/RP group, treatment with LBP(10、20、40mg/L) significantly attenuated neuronal damage, with evidence ofdecreased cell apoptosis (P<0.01，0.001).3. Compared with the OGD/RP group, treatment with LBP (10、20、40mg/L)significantly attenuated these increased of [Ca2+]i(P<0.05,0.01) and decreased MMP(P<0.01).4. DNA laddering was found in hippocampal neurons subjected to OGD/RP, butnot in control group. What’s more, it was significantly decreased after LBP treatments.5. Compared with the OGD/RP group, treatment with LBP (40mg/L) couldsignificantly down regulate the expression of PARP-1(P<0.001) and AIF (P<0.01) inprotein level, induce an increase of Endo G (P<0.05) in protein level.6. Compared with the OGD/RP group, treatment with LBP (40mg/L) couldsignificantly down regulate the expression of PARP-1(P<0.01), AIF (P<0.05) and ofEndo G (P<0.01) in mRNA level.Conclusion1. LBP have significantly protective effects on OGD/RP-inducedneuronal damages in rat primary neuron cultures.2. Neuroprotective effects of LBP on brain ischemic injury-induced apoptosismight be associated with inhibition of PARP/AIF.