The Effect Of Yishen Jiedu Tongluo Method On PKC/ERK Signaling Pathway Of Astrocytes In EAE Mice

Objective:To study the effect of Yishen Jiedu Tongluo method on PKC/ERK signaling pathway of astrocytes in EAE mice.Methods:1.Cultivate primary astrocytes:Take healthy C57BL/6 suckling mice born for 1-2 days as the experimental object,aseptically isolate the cerebral cortex,and obtain primary astrocytes through culture,purification and passage.2.Preparation of serum containing Yishen Daluoyin:healthy C57BL/6 female mice were used as experimental objects.Grouping(5 groups):blank group(normal saline),Yishen Daluoyin high,medium and low concentration groups,PAT group(prednisone acetate suspension).For 7 consecutive days,blood was collected from the eyeball,and the supernatant was collected and stored at-20°C.3.Morphological observation and CCK-8 method to explore the best conditions for the experiment:①cells were seeded in 96-well plates at a concentration of 7,8,9,10,11,12×10~3 cells/well for 24h,48h,and 72h.Determine the optimal cell concentration for the experiment;②Inoculate the 96-well plate at the determined concentration for 48 hours,add LPS solution with concentrations of 0.25,0.5,1,2,4,and 8ug/ml respectively,and culture for 3,6,12,24,48,and 72 hours to determine the LPS-induced star shape.Optimal modeling conditions for EAE model of glial central nervous system inflammation.4.Immunofluorescence chemical staining method:①Detect the expression changes of GFAP in astrocytes after modeling;②To detect the expression levels of GFAP and P2Y1 in cells of each group after the in.5.Western-blot method:The expressions of PKC,ERK and P-ERK proteins in astrocytes of each group were detected under the intervention of Yishen Daluoyin.Results:1.①The primary astrocytes were successfully isolated and cultured,and after cell identification,their purity was above 95%,which could ensure the subsequent experiments;②The working concentration of astrocytes was 11×10~4 cells/ml,When the culture time is48h,the cell activity is the best under this condition;①The effect of lipopolysaccharide is:the concentration of 1ug/ml,the time of 24h,the best cell modeling effect;②The working conditions of the serum containing Yishen Daluoyin are::Concentration 10%,time 12h.2.Immunofluorescence results showed that after the intervention of Yishen Daluoyin-containing serum,compared with the model group,the protein expressions of GFAP and P2Y1 in the cells were inhibited.3.Western-blot results showed that compared with the model group,the protein expressions of PKC and P-ERK in astrocytes of the Chinese medicine compound Yishen Daluoyin-containing serum intervention group were significantly decreased.Conclusion:Yishen Jiedu Tongluo method can reduce the damage of the central nervous system by inhibiting the excessive proliferation and activation of astrocytes,reducing the expression of membrane receptor P2Y1,and then inhibiting the protein expression of the signaling pathway PKC/ERK.