LBP Diminished the Effects of NR2A Antagonist and NR2B Co-Agonist during OGD
To further confirm that LBP-induced neuroprotection was mediated by inhibiting NR2B signaling or activating the NR2A signaling pathway, we employed a NR2B co-agonist (D-serine) and a NR2A antagonist (NVP-AAM077) in this study. Our results showed that 4 h OGD caused twice the neuronal mortality (205.1% ± 12.6, p < 0.01 vs. control; Figure 7A). This mortality was further enhanced by administration of D-serine (259.1% ± 14.6) or NVP-AAM077 (264.5% ± 10.6; all p < 0.05 vs. OGD alone). LBP treatment markedly blocked the increased cell mortality caused by D-serine or NVP-AAM077 and reduced neural death to 140.8% ± 2.3 in the LBP+D-serine group or 156.4% ± 4.5 in the LBP+NVP-AAM077 group (p < 0.01 vs. D-serine or NVP-AAM077 group, respectively). Consistent with the observed neuron death results, cell viability decreased to 45.9% ± 9.7 in the OGD group (Figure 7B), which was further reduced by application of D-serine (38.8% ± 1.3) or NVP-AAM077 (38.6% ± 2.4). However, LBP treatment considerably prohibited reduced neuronal viability induced by D-serine or NVP-AAM077 and increased neural viability to 67.4% ± 1.5 in the LBP+D-serine group and 65.7% ± 2.6 in the LBP+NVP-AAM077 group (p < 0.01 vs. D-serine or NVP-AAM077 group, respectively). These results indicated that LBP’s neuroprotection was mediated by its dual roles in blocking NR2B signaling and activating NR2A signaling. To further confirm this conclusion, we determined the effects of D-serine or NVP-AAM077 on expression of nNOS (a major protein of NR2B signaling pathway) and pAkt (a major protein of NR2A signaling pathway). As shown in Figures 7C,D, the nNOS expression increased to 174.0% ± 15.2 after 4 h OGD exposure, and this expression was further enhanced to 280.3 ± 11.9% in the presence of D-serine (p < 0.01 between OGD alone group and D-serine group). However, LBP treatment dramatically blocked the expression of nNOS to 122.5% ± 9.0 (p < 0.01 vs. D-serine group). LBP+NVP-AAM077 group invariably showed a slight reduction of nNOS expression compared with NVP-AAM007 alone group, although not statistically significant. These results suggest that LBP reduced expression of nNOS through inhibiting NR2B signaling pathway. Furthermore, pAkt expression significantly decreased in response to OGD alone (75.3% ± 1.4, p < 0.01 vs. control), which was further downregulated with administration of NVP-AAM077 to 46.4% ± 3.3 (p < 0.01 vs. OGD alone group). However, LBP reduced the inhibition of NVP-AAM077 and increased pAkt expression to 58.8% ± 3 (p < 0.05, compared between NVP-AAM077 group and LBP- NVP-AAM077 group). There was no significant difference of pAkt expression between D-serine group (64.2% ± 2.3) and LBP+D-serine group (67.1% ± 4.5, p > 0.05). Taken together, these results indicated that LBP upregulates expression of pAkt by stimulating NR2A signaling pathway as well as downregulates nNOS expression by inhibiting NR2B signaling pathway after OGD.
Figure 7  Neuroprotective effect of LBP was confirmed by using NR2B agonist D-serine and NR2A antagonist NVP-AAM077. LBP treatment dramatically antagonized the increase in cellular mortality caused by D-serine and NVP-AAM077 (A) and the decrease in cellular viability in response to 4 h OGD (B). Furthermore, LBP primarily blocked the increase in expression of nNOS caused by D-serine and inhibited the decrease in expression of pAkt caused by NVP-AAM077 when cultured neurons were exposed to OGD for 4 h (C,D; *P < 0.05; **P < 0.01).