Objective: This study was to investigate the role of Chloride Channel-3 (ClC-3) in apoptosis of primary rat hippocampal neurons.
Methods: SIN-1 (3-morpholinosyndomine), a nitric oxide donor, was used to induce apoptosis in the hippocampal neurons. The cell viability was assessed by the MTT (3-(4, 5-Dimethythiazol-2 yl)- diphenytetra zolium bromide) assay. Hoechst 33342 staining was performed to detect apoptosis. Immunofluorescence staining, Western blot and quantitative real-time PCR were used to evaluate protein and mRNA expression levels.
Results: MTT assay showed that SIN-1 could decrease the cell viability of rat hippocampal neurons, which could be restored by the chloride channel inhibitor, DIDS (4, 4’-diisothiocyanostilbene-2, 2’- disulfonic acid). Results from the immunofluorescence and Hoechst 33342 staining showed that the expression level of ClC-3 was dramatically elevated, and the apoptosis rate was significantly increased, in the SIN-1 group. However, the DIDS treatment could decrease the expression levels of ClC-3, and inhibited SIN-1-induced apoptosis of neurons. Moreover, Western blot analysis showed that the levels of cleaved caspase-3 were significantly elevated following SIN-1 induction, which could be inhibited by the DIDS treatment.
Conclusion: ClC-3 is involved in SIN-1-induced apoptosis in rat hippocampal neurons. These findings might provide theoretical and experimental evidence for the development and application of antiapoptotic drugs in clinical practice.