The Study Of Effect In Main Effective Components In AST And PNS Compatibility On Oxidative Damage Of PC12Cell

Objective:To investigate the Effective compatibility dose of Astragaloside IV,GinsenosideRb1, GinsenosideRgl and NotoginsenosideRl to anti-PC12cells oxidative damage, then study the Mechanism mainly on oxidative stress and mitochondrial pathway of apoptosis.Methods:1. Transdifferentiation of PC12cells by NGF were used as nerve cells, The model of oxidative damage of PC12cells were induced by CoC12. Cell lactate dehydrogenase (LDH) leakage rate was used as index to study the effective inhibitory concentration of the four kinds of active ingredients of PC12cells to oxidative damage. The effective compatibility dose of the four kinds of active ingredients were identified according to the U8*(85) uniform experimental design by the basic of this.2. Experimental cell were divided into black group (Just add serum-free media600μl), alcohol-PBS group(add ethanol-PBS with50%concentration60μL+serum-free media540μT), CoCl2damage group (add the optimal concentration of CoCl260μL+serum-free media540 μL), alcohol-PBS-CoCl2group (add ethanol-PBS with50%concentration60μL+serum-free media480μL+the optimal concentration of CoCl260μL), GinsenosideRgl group(add serum-free media480μL+GinsenosideRgl60μL3h,then add CoCl260μL), GinsenosideRb1group(add serum-free media480μL+GinsenosideRb160μL3h,then add CoCl260μL), NotoginsenosideRl group(add serum-free media480μL+NotoginsenosideRl60μL3h, then add CoCl260μL), Astragaloside IVgroup(add serum-free media480μL+Astragaloside IV60μL3h, then add CoCl260μL), the positive control medicine edaravone group (add serum-free media480μL+edaravone with a final concentration of20μmoL/L60μL3h, then add CoCl260μL), active ingredients compatibility group (add serum-free media480μL+active ingredient parties60μL3h, then add CoCl260μL). Fluorescence staining has been done with Hoechst33258with a final concentration of5μg/mL.Five non-repeating fields of vision were randomly photographed for each picture with fluorescence microscopy. The number of apoptotic cells and total cell were calculated in five fields of vision. Then the apoptosis rate was calculated on the basic of this.3. Cells were packet processing as2. DCFH-DA were added in with a final concentration of10umol/l in37℃for incubation for60minutes. Five non-repeating fields of vision were randomly photographed for each picture with fluorescence microscopy. The average of the green fluorescence intensity of the five fields of vision was calculated with Color Histogram template in ImageJ1.410software. Changes of intracellular reactive oxygen species were statistical analysis with the average of the green fluorescence intensity.4. Cells were packet processing as2, then incubated in37℃for45minutes with Rh123as a final concentration of100μg/L. Five non-repeating fields of vision were randomly photographed for each picture with fluorescence microscopy. The average of the green fluorescence intensity of the five fields of vision was calculated with Color Histogram template in ImageJ1.410software. Changes of intracellular mitochondrial membrane potential were statistical analysis with the average of the green fluorescence intensity.Results:1. Six different concentrations of the four active ingredients can effectively inhibit the leakage of LDH(p<0.05). Furthermore, the inhibition rate of the LDH leakage increases with the increase of doses. The inhibition rate of LDH leakage reach to80%when the concentration of Astragalside Ⅳ and GinsenosideRb1in50μmol/L, GinsenosideRg1and NotoginsenosideR1in100μmol/L. The result of multiple regression analysis for the uniform design of U8*(85) showed that eight different concentrations of compatibility of four active ingredients can effectively inhibit the leakage of LDH (p<0.05).The best effective components dose to against the Oxidative damage of PC12cells induced by CoCl2is GinsenosideRgl in50μmol/L, Astragaloside IV in0.78125μmol/L GinsenosideRbl and NotoginsenosideRl each in1.5625μmol/L2. The apoptosis rate was significantly increased in CoCl2damage group and alcohol-PBS-CoCl2group compared with black group (p<0.01). The apoptosis rate was significantly decreased after using the four active ingredients and their compatibility (p<0.01).The effect of anti-apoptotic of active ingredients compatibility group was better than Astragaloside IV,GinsenosideRbl and NotoginsenosideRl group, but it had on significance compared with GinsenosideRgl group.3. The average of the green fluorescence intensity in DCF was significantly increased in CoCl2damage group and alcohol-PBS-CoCl2group compared with black group (p<0.01). The average of the green fluorescence intensity in DCF was decreased in varying degrees after using the four active ingredients and their compatibility (p<0.01). It had on significance in active ingredients compatibility group compared with the four active ingredients group.4. The average of the green fluorescence intensity in Rhl23was significantly increased in CoCl2damage group and alcohol-PBS-CoCl2group compared with black group (p<0.01). The average of the green fluorescence intensity in DCF was decreased in varying degrees after singly using the four active ingredients, but did not have a significant (p>0.05). The average of the green fluorescence intensity in DCF was significantly decreased in active ingredients compatibility group (p<0.01).Conclusion:Compatibility with GinsenosideRgl in50μmol/L, Astragaloside IV in0.78125μmol/L, GinsenosideRb1and each in1.5625}μmol/L has the effect of anti-PC12cells oxidative damage. The mechanism applies to resistance to apoptosis, increase mitochondrial membrane potential and the release of the less reactive oxygen species.