Role Of CIC-3Chloride Channel In The Hippocampal Neuronal Apoptosis Induced By SIN-1in Culture

Ojective:To investigate the expression of C1C-3chloride channel in the hippocampal neuronal apoptosis induced by SIN-1(one kind of NO donors).To explore the role of chloride channel in the ischemic cerebral injury.Methods:The cultured neurons of12days from SD rats were randomly divided into three groups:Control、SIN-1and SIN-1+DIDS groups, each group contains six repeat pores. In the experiments, SIN-1stayed in culture medium for18h; In SIN-1+DIDS group, DIDS and SIN-1were co-added into the culture medium and sustained for18h. The neuronal viability was tested by MTT. Fluorescent staining with Hoechst33342, analysis of caspase-3, were used to detect neuronal apoptosis. The methods of immunocytochemical fluorescent stain, Western blot and real-time PCR were used to detect the expression of C1C-3chloride channel.Results:(1) SIN-1could induce cultured hippocampal neuronal apoptosis in dose-dependent manner. At the SIN-1concentration of0、0.2、0.4、0.8、1.0、1.5、2.0mmol/L, the neuronal viabilities were101.10±1.66%、88.60±2.01%、72.59±3.31%、69.52±1.00%、57.89±1.98%、44.30±2.66%、11.84±0.66%respectively, there were significantly difference in SIN-1concentration between0.2-2.Ommol/L compared with Ommol/L (P<0.001,n=6).1.0mmol/L of SIN-1could induce neuronal apoptosis obviously, and we used this concentration SIN-1to establish the cellular apoptotic models.(2) The chloride channel blocker of DIDS could enhance the neuronal viabilities and inhibit the neuronal apoptosis induced by SIN-1in dose-dependent manner. The neuronal viability in control group was100.20±0.92%, at the DIDS concentration of0、10、50、100、200、300μmol/L, the neuronal viabilities were58.99±3.70%、76.36±1.22%、73.74±0.93%、86.26±1.26%、78.79±1.60%、68.69±1.27%respectively, at the DIDS concentration between10-200μmol/L, compared with0μmol/L DIDS, the difference were significantly (P<0.001, n=6). We used the concentration of100μmol/L DIDS in later experiments.(3) Stained with Hoechst33342dye, we can see that the nucleus in the cultured normal neurons show olivary shape and in weak blue fluorescence, the apoptotic percentage was5.56±0.81%; However, in1.0mmol/L of SIN-1group, the nucleus were condense and became smaller and in strong white-blue fluorescence, the apoptotic percentage was51.06±2.38%, compared with control, the difference was significantly(P<0.001,n=6); In SIN-1+DIDS group, the apoptotic percentage was21.61±1.36%, compared with SIN-1group, the difference was significantly(P<0.001,n=6).(4) In immunohistochemical fluorescent stain experiment, the results showed that C1C-3chloride channel was positive on12DIV hippocampal neurons; The expression of C1C-3chloride channel was enhanced after the SIN-1induce neuronal apoptosis; In SIN-1+DIDS group, the expression of C1C-3chloride channel was weakened.(5) From Western blot result we can find that in control group, the C1C-3protein expression level was1.73±0.24; In1.0mmol/L of SIN-1group, the C1C-3protein expression level was1.73±0.24, compared with control group, the difference was significantly(P<0.001,n=6); In SIN-1+DIDS group, the C1C-3protein expression level was0.99±0.18, compared with SIN-1group, the difference was significantly(P<0.001,n=6).(6) The results of real-time PCR showed that, in control group the C1C-3mRNA expression level was0.33±0.11; In SIN-1(1.0mmol/L) group, the C1C-3mRNA expression level was11.99±4.76, compared with control group, the difference was significantly(P<0.05,n=3); In SIN-1+DIDS group, the C1C-3mRNA expression level was6.23±1.17, compared with SIN-1group, the difference was significanty(P<0.05,n=3).Conclusion:C1C-3chloride channel present high expresstion in hippocampal neuronal exposure to SIN-1, the excessive of NO take part in ischemic cerebral injury. C1C-3chloride channel perhaps participated in the hippocampal neuronal apoptosis induced by SIN-1in culture.