Protective Effect Of Traditional Chinese Medicine Raspberry On Cultured Retinal Ganglion Cells In Vitro

Purpose:To investigate the protective effect of raspberry on retinal ganglion cells?RGCs?,and screen their effective components,to study the effects of raspberry on the expression of Bcl-2 and Bax proteins during the process of apoptosis induced by hydrogen peroxide?H202?.To further develop the use of Chinese medicine raspberry to provide theoretical basis for the study of optic nerve protection.Material and method:Major instruments and reagents:SW-CJ-1F clean bench?Jiangsu Sujing Group,Suzhou Antai Air Technology Co.,Ltd.?,Forma 3111 CO2 incubator ?MK3 enzyme standard quantitative tester?Thermo Fisher Scientific China Co.,Ltd.?,Inverted phase difference fluorescence microscopy?Nikon,Japan?,High-speed refrigerated centrifuge?American sigma?,Petri dishes?96 well culture plate?American Life Technologies?,-80 ? ultra-low temperature refrigerator?the United States Thermo company?,Retinal ganglion cell RGC-5 cell line?ATCC?,Raspberry?raspberry-20,raspberry-40,raspberry-70?Shenyang Pharmaceutical University?,Bax antibody,Bcl-2antibody?Santa Cruz Biotechnology,USA?,Horseradish enzyme labeled goat anti-mouse Ig G?Beijing Zhongyu Jinqiao Biotechnology Co.,Ltd.?,High glucose DMEM liquid medium,PBS,trypsin?Thermo Fisher Scientific Biotechnology Co.,Ltd.Beijing?,Fetal bovine serum?MTS kit?Double resistance?American Life Technologies?,Volume fraction of 30%hydrogen peroxide solution?Sinopharm Group Chemical Reagent Co.,Ltd.?.Method:The rat retinal ganglion cell line RGC-5 was cultured in vitro,different concentrations of H₂O₂?100?50?25?15?5?mol/L?and RGC-5 were used for 24 h,MTS colorimetric method in the microplate reader on the test,select the wavelength of 492 nm to absorbance of each hole is detected,cell viability was calculated.The optimum concentration of H₂O₂ model was determined according to the corresponding mass-effect,curve RGC-5 was divided into normal control group,raspberry drug concentration group?100?50?25?10?1?0.1ug/ml?,After incubation for 24 h,the absorbance value was measured on the microplate reader by MTS colorimetric method,the effects of different concentrations of raspberry on RGC-5 were analyzed.RGC-5 was divided into normal control group,H₂O₂ group model group,H₂O₂ + raspberry drug concentration group?100?50?25?10?1?0.1ug/ml?,H₂O₂ model 24 hours later,administration for 24 hours,the absorbance value was measured on the microplate reader by MTS colorimetric method,effects of different concentrations of raspberry on H₂O₂-induced oxidative stress injury in RGC-5cells.At the same time in the inverted phase contrast microscope under the general morphological observation.Western blot was used to detect the effects of different concentrations of raspberry?25?10? 1? 0.1 ?g / ml?on H₂O₂-induced oxidative stress,RGC-5 regulates protein expression of apoptosis-related genes Bcl-2 and Bax.The components of the raspberry:raspberry-20?1.5?1.25?1?0.5?0.25?0.1ug/ml?,raspberry-40?50?5?1?0.5?0.25?0.1ug/ml?,raspberry-70?50?25?10?1?0.1?0.01ug/ml?repeat the above MTS experimental method,screening the effective components to determine the optimal effective concentration of proliferation and anti-apoptosis in the active component.Results:1.H₂O₂ injury model can reduce the survival rate of RGC-5 cells,and the inhibitory effect of H₂O₂ on RGC-5 was enhanced with the increase of the concentration.H₂O₂ at a concentration of 25 ?mol / L was applied to RGC-5 cells for 24 hours,its survival rate reduced to 50.7%?P<0.05?.In this experiment,we selected the concentration of RGC-5 oxidative stress injury model.2.MTS results show:The concentration of 1ug / ml,10 ug / ml raspberry drug group and RGC-5 cultured 24 hours,a significant proliferation,The survival rates were 109.37%,105.56%?P<0.05?.There was no significant difference in the rest concentration group.3.MTS results show:the survival rate of the normal control group was 100.30%,The survival rate of the model group was 52.08%?P<0.01?.The survival rates of the raspberry drug group at concentrations of 25?10? 1 ug / ml were 61.18%,62.79%,76.62%?P<0.05?,They were significantly higher than the survival rates of the model group,the protective effect of RGC-5 on H₂O₂ injury was the best when the concentration of raspberry was 1ug / ml.4.Western blot results showed:compared with the normal control group,the expression of Bax protein in H₂O₂ model group was significantly up-regulated,while the expression of Bcl-2 protein decreased?P<0.01?.Compared with the model group,the concentration of 1?g / ml and 10 ug / ml raspberry drug concentration group significantly inhibited the upregulation of Bax protein and increased the expression of Bcl-2 protein.5.Screening of effective components of raspberry:the raspberry-20 group at a concentration of 0.25 ug / ml and 0.5 ug / ml had a proliferative effect on RGC-5 cells,Their survival rates were: 111.93 %,116.00%?P<0.05?.Concentration of 0.25 ug / ml,0.5ug / ml,1ug / ml,5ug / ml drug concentration was significant anti-apoptotic effect,The survival rates were70.93%,72.76%,66.98% and 66.57%,respectively?P <0.05?.There was no significant difference in the proliferation of RGC-5 cells between the two groups,and there was no significant difference in the anti-apoptotic effect of anti-H₂O₂-induced cells.The effect of raspberry-70 on RGC-5 cells was not obvious The survival rate of the raspberry-70 group was61.02%,78.05%,67.45%?P <0.05?at the concentration of 1 ug / ml,10 ug / ml and 10 ug/ml.Conclusion:1.Raspberry has protective effect on oxidative stress injury of cultured rat retinal ganglion cells in vitro.2.Raspberry can resist H₂O₂-induced apoptosis,,which may be related to its up-regulation of Bcl-2 protein expression and down-regulation of Bax protein expression.3.The effective components of raspberry-ruthenin were: raspberry-20 had the effect of proliferation and anti-H₂O₂ apoptosis on RGC-5 cells.raspberry-70 had no obvious effect on the proliferation of RGC-5 cells H₂O₂ apoptosis was significant.raspberry-40 has no protective effect on RGC-5 cells.