| Part one: Toxic effects of scorpion polypeptide from BmK venom, on PC12 cellsThe technique of MTT was applied to explore toxic effects of the scorpion polypeptide on PC 12 cells. PC 12 cells were treated by the scorpion polypeptide with different doses for 24h, and their changes were estimated by MTT assay. The cell growth inhibition rates (%) were 3.39, 11.82, 26.65, 69.38, according to the concentration of the scorpion polypeptide 2, 8, 32, 128μg/mL. The result suggested that the inhibition rates of cells increased with the concentration of the scorpion polypeptide increased(P < 0.05 ) .Part two: Effect of scorpion polypeptide from BmK venom, on membrane potential of PC12 cells and its correlation with sodium channelPC 12 cells were labeled with DiBAC4 (3). Laser scanning confocal microscope was used to monitor the changes of membrane potential in real-time on these cells that were treated with different concentrations of the scorpion polypeptide. The effect of sodium channel blocker TTX (tetrodotoxin) on the changes was also observed. The results indicated that membrane potential depolarization induced by the scorpion polypeptide peaked at 3 min and became stabilized after 5 min of the treatment. After comparing to fluorescence intensity without treatment, the normalized fluorescence intensity was determined to be 1.375±0.048, 1.504±0.034 and 1.839±0.022 in cells that were treated with 10, 20, 40μg·mL-1 scorpion polypeptide respectively. These numbers are significantly lower than those from untreated control cells( P < 0.01 ).When PC12 cells were co-incubated with 1μmol/L TTX for 20min, then treated with 40μg/ml scorpion polypeptide, the intensity changed to 1.035±0.028, which was similar to that of the control (P>0.05 ) . This indicates that the effect of scorpion polypeptide can be completely inhibited by TTX. The scorpion polypeptide induces membrane depolarization of PC12 cells in a dependent manner concentration, suggesting that the sodium channel is likely involved. |
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