Effects Of Eugenol On Hypoxic Injure In PC12 Cells Via Affacting The Expression Of NMDA Receptor Subunits

ObjectiveTo investigate the protective effects of eugenol in PC12 cells, and the gene, the protein expression of NMDA recoptor subunits.MethodsThe research was divided into three groups:normal group, model group and eugenol group. The normal group was cultured in low sugar DMEM medium without serum, the model group was disposed by lOmmol/L Na2S204 in low sugar DMEM medium without serum, and the eugenol group was dealed with 250nmol/L eugenol in low sugar Na2S2O4 DMEM medium without serum. Then the morphological changes of PC12 cells were observed by inverted microscope; cell activity was evaluated by CCK-8 assay; the changes of cell apoptosis, calcium ion concentration and mitochondrial membrane potential were detected by flow cytometry;the expression of synaptophysin was detected by immunohistochemistry;the gene expression of NMDA receptor subunits-Grinl, Grin2a, Grin2b and Grin2c and Grin2d, Grin3a, Grin3b-were tested by RT-PCR; the protein expression of NR1, NR2A, NR2B were operated by Western Blot.ResultsCCK-8 assay showed that, the cell viability of model group decreased compared with normal, the difference was extremely significant (P<0.01). Compared with model group, the cell activity of eugenol group was enhanced markedly (P<0.01); while compared with normal group, cell viability in eugenol group decreased significantly (P< 0.01). Compared with normal group, the apoptosis rate which detected by flow cytometry increased significantly in model group (P<0.05). Compared with model group, the apoptosis rate dereased significantly in eugenol group(P<0.05); while compared with normal group, the apoptosis rate of eugenol group had no significant difference(P> 0.05). When compared model group with normal group, the [Ca2+] increased remarkably(P<0.01). Compared with model group, the [Ca2+] in eugenol group dereased remarkably (P<0.01), while it increased remarkably(P<0.01) when compared with normal group(P<0.01). In model group, the MMP had a remarkable decrease compared with normal group (P<0.01). In eugenol group, the MMP had a extremely significant increase compared with model group(P<0.01), while it had no significant difference when compared with normal group. The results of the expression of synaptophysin tested by immunohistochemistry were that: the expression of synaptophysin was lower remarkably in model group than normal group(P<0.01); and it was higher remarkably in eugenol group than model group(P<0.01), while it has no significant difference compared eugenol group with normal group. The RT-PCR assay revealed that, the gene Grin2a had a significant higher expression in model group compared with normal group (P <0.05);compared with model group, the eugenol group had a lower expression of Grin2a significantly(P<0.05), while it had a higher expression compared with noemal group. These genes Grinl, Grin2b, Grin2c, Grin2d, Grin3a had no significant difference in model group compared with normal group; the expressions of them in eugenol group had no significant difference in eugenol group compared with model group, and the same result to comparing with normal group. Under current experimental conditions, the expression of Grin3b hadn’t been detected.The protein expressions of NMDA receptor were tested by Western-Blot assay, the results showed that, the protein expressions of NR1, NR2A, NR2B increased remarkably in model group compared with normal group (P <0.01); the protein expression of NR2A decreased remarkably in eugenol group compared with model group (P<0.05), but the expressions of protein NR1,NR2B has no significant difference; the protein expression of NR2A significant increased in eugenol group compared with normal group(P<0.05), and the NR2B remarkable increased (P<0.01), the NR1 had no significant difference.ConcilsionThe protective effects of eugenol on PC12 cells injured by hypoxic may play a highly relevant part in affecting the expression of the NMDAr subunit especially NR2A.