The Protective Effect Of Nec-1 On Al-poisoned Nerve Cells

[Objective] To observe and investigate the effect of Nec-1 on nerve cells poisoned by alumnium.[Methods] 1. The study of aluminum in vitro:cultured primary neurocytes of 1-3 days Kunming mouse and dissociated cerebral cortex, cell density was adjusted at 1×105 cells/ml.2. Manufacturing Al injury model of nerve cells:After 5 days, neurocytes were poisoned with Al3+ of dose 2mmol/L.3. Adding different doses of Nec-1(Oμmol/L,30μmol/L,60μmol/L,90μmol/L). After 48 hours, (1) Observating the morphological changes of nerve cells and the cell viability of neurocytes was quantified by method of Cell Counting Kit-8; (2) the necrotic and apoptosis rate of neurocytes was measured by method of fluorescent analysis; (3) the genes expression of necrosis, apoptosis and autophagy were detected by qRT-PCR; (4) the expressions of necrosis, apoptosis and autophagy in protein levels were detected by Western blot.[Results] The results presented here, indicate that 1. (1) In light microscopy, normal nerve cells growed vigorously, they had long and relatively homogeneous axons and there were more dendrites which became tapered from cell body to peripheral,and there were rich connection between cell bodies. While in aluminum (2mmol/L) group, nucleus splitted and synaptic became fewer and shorter or even disappeared. When the doses of Nec-1 increased, cell volume gradually increased, axons became longer, and the dendritic markedly increased, and the connection between cell body to cell body and cell body to synaptic became significantly increased. (2) Chromatin of normal cells showed green and a normal structure, while adding Al3+(2mmol/L), nerve cells appeared apoptosis and necrosis. With the dose of Nec-1 increasing, apoptosis and necrosis of cells decrease. (3) Compared with aluminum group, Nec-1 (60μmol/L.90μmol/L) increased the viability of cultured neurocytes significantly (P<0.01).2. (1) FCM indicated that after Al3+poisoning nerve cells and with Nec-1 dose increasing, the necrotic rate of cells declined(P<0.01). (2) The result of qRT-PCR showed that compared with Al3+(2mmol/L), the expressions of mRNA of RIP1,NFκB genes decreased significantly in the doses of Nec-1 (60μmol/L,90μmol/L)(P<0.01). (3) The Western-blot demonstrated that compared with Al3+(2mmol/L) group, Nec-1 (60μmol/L,90μmol/L) declined the expression of NFκB(P<0.01); the expression of RIP 1 protein decrease significantly in Nec-1 (90μmol/L) group(P＜0.01).3. (1) JC-1 staining showed that normal nerve cells had a relative high mitochondrial membrane potential, resulting in red fluorescence, and in 2mM aluminum group of nerve cells, which had low mitochondrial membrane potential, resulting in an increase in green fluorescence, while membrane potential of neve cells of treated with Nec-1 (60μmol/L) had increased, resulting in red fluorescent cells were significantly increased compared with anodized aluminum. (2) FCM indicated that after Al3+ poisoning nerve cells and with Nec-1 dose increasing, the apoptosis rate of cells declined (P＜0.01). (3) The result of qRT-PCR showed that compared with Al3+(2mmol/L), the expressions of mRNA of caspase-3,caspase-8,caspase-9 genes decreased significantly in the doses of Nec-1 (60μmol/L,90μmol/L) (P<0.01). (4) The Western-blot demonstrated that compared with Al3+(2mmol/L) group, Nec-1 (60μmol/L,90μmol/L) declined the expression of caspase-3,caspase-8,caspase-9 (P<0.01).4. Compared with Al3+(2mmol/L), Nec-1 (60μmol/L) declined the expression of LC3-Ⅱgene (P＜0.05), and Nec-1 (90μmol/L) decreased the protein expression of LC3-Ⅱ(P＜0.05).[Conclusion] Nec-1 can reduce aluminum-induced death of nerve cells, especially in the pathway of apoptosis and necrosis, accordingly protect nerve cells.