| Objective:In this experiment,rat red blood cells were used as the research object,and the oxidative damage model of rat red blood cells in vitro and in vivo was established by using hydrogen peroxide（H2O2）and oxidative stress induced by exhaustive swimming.The effect of ginseng total saponin on resisting oxidative stress and enhancing energy metabolism of red blood cells was investigated.Based on the theory of"Qi and Blood Theory",the efficacy of ginseng"Dabu Yuanqi"was explained from the perspective of ginseng improving the antagonistic ability of red blood cells oxidative stress and promoting red blood cells energy metabolism,and the molecular mechanism was explored and the target was excavated.Methods:1.The ginseng total protein was extracted by alkali extraction and acid precipitation,and the protein content in the ginseng total protein was detected by BCA method.The ginseng total polysaccharides were extracted by water extraction and alcohol precipitation,and the content of polysaccharides in ginseng total polysaccharides was detected by phenol-sulfuric acid method.The total extract of ginseng was prepared by ultrasonic method,and the ginseng total saponin were isolated and purified by macroporous adsorption resin.The content of saponins in ginseng total saponin was detected by colorimetry,and the content of each monomer saponins in ginseng total saponin was determined by High Performance Liquid Chromatography（HPLC）.2.The H2O2was used to establish an in vitro oxidation damage model of red blood cells,and the optimal concentration of ginseng superior component was determined by red blood cells hemolysis rate.3.The in vivo oxidation model of red blood cells was established by exhaustive swimming in rats,and the superior components of ginseng were screened by H2O2induction.The levels of reactive oxygen species（ROS）and adenosine triphosphate（ATP）in red blood cells were detected at 0.5 h,1 h,2 h,4 h and 8 h after exhaustive administration,and the optimal administration time in vivo was screened out.Based on the optimal administration time,the levels of ROS and ATP in red blood cells of rats were detected when the concentration of superior components of ginseng was 10 mg/kg,20 mg/kg,30 mg/kg and 40mg/kg and the optimal concentration of superior components of ginseng in vivo was screened out.4.Scanning electron microscope observation of red blood cells morphology;Determination of reticulocyte number by staining.5.The level of red blood cells apoptosis was detected by flow cytometry,and the content of Ca2+in red blood cells was determined by colorimetry.6.The contents of met-hemoglobin（Met-Hb）,free-hemoglobin（F-Hb）,malonaldehyde（MDA）,glutataione（GSH）and erythropoietin（EPO）,the activities of superoxide dismutase（SOD）,catalase（CAT）,hexokinase（HK）,pyruvate kinase（PK）and Phosphofructokinase（PFK）,the levels of ROS and ATP,and the content of SH on the red blood cells membrane were detected by enzyme-linked immunosorbent assay（ELISA）.7.The activities of Na+K+-ATPase and Ca2+Mg2+-ATPase in red blood cells were detected by phosphorus determination method.8.SDS-PAGE method was used to detect the molecular weight distribution of red blood cells membrane proteins,and dithiothreitol（DTT）method was used to reduction the oxidative cross-linking of proteins induced by oxidative stress.9.Band 3 protein and its tyrosine phosphorylation distribution and fluorescence intensity were detected by Immunofluorescence（IF）method.10.The expressions of Band 3,Band 4.1,Spectrin,p Tyr protein,tyrosine phosphorylation kinase PTK,Syk and tyrosine phosphorylase SHP-2,PTP1B were detected by Western blotting（WB）.11.The interaction of Band 3 protein with key glycolytic enzymes HK,PFK,PK and HBA1 was examined by Co-Immunoprecipitation（Co-IP）method.Results:1.The ginseng total protein was extracted by alkali extraction and acid precipitation,and the yield was 2.8%.The protein content in the ginseng total protein was 86.8%by BCA method.The ginseng total polysaccharide was extracted by water extraction and alcohol precipitation,and the yield was 5.1%.The polysaccharide content in the ginseng total polysaccharide was 74.25%by phenol-sulfuric acid method.The total extract of ginseng was prepared by ultrasonic method,and then the ginseng total saponin were separated and purified by macroporous adsorption resin.The yield was 3.1%.The content of saponins in ginseng total saponin was 90.2%by colorimetry.The content of each monomer saponin in ginseng total saponin was determined by HPLC:Rg1（4.50%）,Re（15.22%）,Rf（1.74%）,Rg2（2.69%）,Ro（0.94%）,Rb1（21.51%）,Rc（15.84%）,Rb2（7.17%）,Rb3（3.00%）,Rd（12.62%）.2.The best modeling concentration of red blood cells induced by H2O2is 70 m M,and the ginseng superior component is ginseng total saponin and ginseng total saponin best in vitro drug concentration is 100μg/m L.3.After screening,it was found that the levels of ROS and ATP in red blood cells changed most significantly 2 h after exhaustive swimming and when the concentration of ginseng total saponin reached 20 mg/kg（p<0.001）.Therefore,the ginseng total saponin of20 mg/kg were administered 2 h after exhaustive swimming.4.The results of scanning electron microscopy showed that after H2O2and exhaustive swimming induction,the surface of red blood cells shrunk and a large number of ridge-shaped protrusions appeared.After administration of ginseng total saponin,the morphology of red blood cells gradually recovered smooth and became the original disc shape;Moreover,the treatment of ginseng total saponin significantly increased the number of reticulocytes in red blood cells injured by oxidative stress.5.Through flow cytometry detection,it was found that ginseng total saponin could significantly reduce the level of red blood cells apoptosis（p<0.01,p<0.001）and reduce the content of Ca2+（p<0.05,p<0.01）in red blood cells.6.Ginseng total saponin can restore the increase of Met-Hb（p<0.05,p<0.01）,F-Hb（p<0.05,p<0.01）,MDA（p<0.05,p<0.01）,EPO（p<0.01,p<0.001）content,the increase of ROS（p<0.01,p<0.001）level,the decrease of GSH（p<0.001）,Membrane protein SH（p<0.05,p<0.01）content,the decrease of SOD（p<0.05,p<0.001）,CAT（p<0.01,p<0.001）,HK（p<0.01）,PK（p<0.01,p<0.001）,PFK（p<0.05,p<0.01）activity and reduce of ATP（p<0.01,p<0.001）level caused by oxidative stress.7.Ginseng total saponin can significantly increase the activity of Na+K+-ATPase and Ca2+Mg2+-ATPase（p<0.05,p<0.01,p<0.001）in red blood cells induced by oxidative stress.8.The molecular weight distribution of red blood cells membrane protein was verified by SDS-PAGE,and the oxidative cross-linking of red blood cells membrane protein was improved after DTT reduction.9.The results of IF showed that after the intervention of ginseng total saponin,compared with the model group,the fluorescence intensity of Band 3 protein in red blood cells was significantly increased,and the fluorescence intensity of Band 3 protein p Tyr was significantly reduced.10.WB results showed that ginseng total saponin could restore the expression of Band3,Band 4.1,Spectrin（p<0.05,p<0.01）,p Tyr（p<0.05）protein,tyrosine phosphorylation kinase PTK,Syk（p<0.05,p<0.01）,tyrosine phosphorylase SHP-2,PTP1B（p<0.05,p<0.01）.11.The results of Co-IP showed that the interaction between Band 3 protein,glycolytic enzyme and HBA1 weakened after red blood cells oxidative stress,which further reduced the glycolytic rate of red blood cells and affected energy metabolism.After administration of ginseng total saponin,it returned to normal state.Conclusion:From the changes of hemolysis rate,morphology,oxygen carrying and releasing ability,apoptosis and antioxidant capacity of red blood cells,the protective effect of ginseng total saponin on oxidative stress-induced red blood cells injury was comprehensively confirmed.Ginseng total saponin may play a protective role by protecting SH in the red blood cells membrane from oxidative cross-linking caused by free radical damage,thereby reducing the p Tyr level of Band 3 protein,increasing the expression of Band 3 protein and enhancing the interaction with glycolytic key enzymes and HBA1,and improving the glycolytic ability of red blood cells. |
