Effect Of Ginseng Total Protein On In Vitro And In Vivo PD Models And Its Effect On Intestinal Microflora

Objective: To explore the improvement effect of ginseng total protein on PD model in vitro and in vivo and its effect on intestinal microbial flora,so as to provide scientific and experimental basis for ginseng treatment of PD and lay a foundation for the further development of traditional Chinese medicine.Methods:(1)The optimum extraction and purification process of ginseng total protein were determined by single factor test and orthogonal test;The main proteins in ginseng total proteins were identified and analyzed by SDS-PAGE/secondary mass spectrometry.(2)In vitro PD models of PC12 cells,SH-SY5 Y cells and BV2 cells were constructed by MPP +,and the concentration of MPP+ and the concentration of total ginseng protein were determined by CCK-8 method;CCK-8 method,flow cytometry and mitochondrial membrane potential detection(JC-1)were used to detect the improvement of ginseng total protein on three cell PD models induced by MPP+.(3)The toxic effects of long-term feeding of low,medium and high doses of ginseng total protein on normal C57BL/6 mice were studied by brain histopathological experiment and intestinal microbiota analysis.(4)C57BL/6 mice were intraperitoneally injected with MPTP at a concentration of 35 mg/kg for 4 weeks to construct a chronic PD model.The improvement effect of long-term feeding of low,medium and high doses of ginseng total protein on MPTP induced chronic PD model C57BL/6 mice was explored through behavioral experiment,brain and intestinal histopathology experiment and intestinal microbiota analysis.(5)C57BL/6 mice were intraperitoneally injected with MPTP at a concentration of 35 mg/kg for 2 weeks to construct a subacute PD model.The improvement of low,medium and high doses of ginseng total protein on MPTP induced subacute PD model C57BL/6 mice was explored through behavioral experiment,brain histopathology experiment and intestinal microbiota analysis.Results:(1)It was determined that the best extraction method of ginseng total protein was cold soak extraction at 4 ℃,the extraction p H was 7.0,the extraction times were 3times,the solid-liquid ratio was 1:10,and the extraction time was 8 hours each time;Through the optimization of purification process,it is determined that the best purification process of ginseng total protein is ultrafiltration interception of ultrafiltration tube after isoelectric point precipitation,and the purity of purified protein reaches 80.80%;According to SDS-PAGE/secondary mass spectrometry,the main proteins of ginseng total protein are ribonuclease like main storage protein,flavin dependent oxidoreductase fox5 and class β-Amylase,latex like protein,glyceraldehyde 3-phosphate dehydrogenase,methionine synthase,ferredoxin dependent glutamate synthase 1,pre sequence protease 2.(2)The optimal modeling concentrations of MPP+ on PC12,SH-SY5 Y and BV2 cells were 1 m M,0.5 m M and 0.2 m M respectively.25-200 μg/m L ginseng total protein had no obvious toxicity to the three cells.CCK-8 method,flow cytometry and mitochondrial membrane potential detection(JC-1)were used.The results showed that each dose group(25 μg/m L、50 μg/m L and 100 μg/m L)ginseng total protein can improve the three cell PD models induced by MPP+.Compared with the model control group,except that the improvement effect of ginseng total protein in the medium dose group on BV2 cells was not statistically significant,the other differences were statistically significant.(3)Compared with the blank control group,the normal C57BL/6 mice treated with ginseng total protein in each dose group had no significant changes in body weight and other vital indicators,and there were no significant changes in hippocampus and striatum,there was no significant difference in the expression of α-Syn and TH,and there was no significant change in the diversity of intestinal microbiota.(4)The results of hanging rope experiment,rotating shaft experiment,hanging tail experiment,water maze experiment and open field experiment showed that the low,medium and high dose groups of ginseng total protein could significantly improve the behavioral disorder of chronic PD model mice.The results of brain tissue and intestinal histopathology showed that all dose groups could significantly improved the distribution of hippocampal cells and striate constitution,reduce α-Syn aggregation and increase TH expression in mice brain.The results of intestinal microbiota analysis showed that each dose group could improve the intestinal microbiota disorder of chronic PD model mice to a certain extent.(5)The results of hanging rope experiment and rotating shaft experiment showed that ginseng total protein could improve the behavioral disorder of subacute PD model mice to a certain extent.The experimental results of brain histopathology showed that ginseng total protein could improve the uneven arrangement of hippocampal cells and the distribution of striate constitution in the brain and reduce the enrichment of α-Syn enrichment and increased TH expression of subacute PD model mice.The results of intestinal microbiota analysis showed that ginseng total protein could change the intestinal microbiota disorder of subacute PD model mice to a certain extent.(6)The improvement effect of ginseng total protein on subacute PD model mice is weaker than that on chronic PD model mice.Conclusion:(1)The extraction and purification process of ginseng total protein established in this study has high extraction rate and purity.SDS-PAGE/secondary mass spectrometry identification showed that ribonuclease like main storage protein,flavin dependent oxidoreductase fox5 and β-Amylase is a monomer protein with high content.(2)Ginseng total protein can significantly improve the of PC12,SH-SY5 Y and BV2 cell PD models.(3)Ginseng total protein can improve the behavioral disorder,pathological injury of brain and intestinal tissue and disturbance of intestinal microbiota in C57BL/6 mice with chronic PD model.(4)Ginseng total protein can improve the behavioral disorder,brain pathological injury and intestinal microbial flora disorder of subacute PD model C57BL/6 mice to a certain extent.