| Purpose:Under the conditions of cell culture in vitro,observe the influence of Wenban Decoction serum containing drug on the expression of autophagy and apoptosis related proteins and genes of macrophages foamed,to explore the mechanisms of regulation of Wenban Decoction serum containing drug on autophagy and apoptosis pathway of macrophages foamed from the perspective of molecular biology,to further reveal the mechanism of Wenban Decoction in the prevention and treatment of atherosclerosis,so as to provide scientific theoretical basis for the clinical application of Wenban decoction,and enrich the scientific connotation of the theory of wind phlegm.Material and method:The 20 SD rats were randomly divided into 4 groups according to their weight,the blank group and the low,medium and high concentration groups of Wenban Decoction,5 in each group.The low,medium and high concentration groups of Wenban Decoction were filled with low,medium and high concentration 4.275g/kg?8.55g/kg?17.1g/kg of Wenban Decoction,and the blank group was given the same volume of distilled water to fill the stomach,and the stomach was continuously filled for 7 days,one time each day.In the eighth day of the gastric irrigation,after 2 hours,the abdominal aorta was taken out of blood,and the serum was separated after centrifugation.Mouse macrophages were subcultured,cryopreservation and resuscitation,divided into blank control group,model group,rapamycin group,Wenban Decoction in low,middle and high concentration groups,except blank control group,other groups of macrophage foamed intervention,after the intervention of the blank control group,model group,co culture 48 h with the blank serum and cells 10% rats;rapamycin group with rapamycin(final concentration 10nmol/l)and cells co cultured 48h;Wenban Decoction low,medium and high concentration group respectively with 10% Wenban Decoction low,high concentration of serum and cultured cells containing 48 h,detect related indexes after collecting cells.The ultrastructure of autophagy in each group was observed by transmission electron microscope;The proliferative activity of each group was detected by MTT method;the apoptosis rate of each group was detected by flow cytometry;The expression levels of autophagy and apoptosis related proteins LC3 B,BECLIN1,MTOR,BAX,BCL2 and CASPASE3 were detected by Western Blot and immunofluorescence;The expression levels of autophagy and apoptosis related genes Lc3 b,Beclin1,Mtor,Bax,Bcl2,Caspase3 were detected by RT-PCR.Results:1 Observation of ultrastructural autophago in each cell under transmission electron microscopeNo obvious autophagosomes appeared in the blank control group;model group showed a small amount of autophagy,autophagy is a double membrane vesicle like structure,wrapped with content;Wenban Decoction with different concentration groups are visible more autophagosomes,the number of autophagosomes were higher than that of model group;rapamycin group can also see more autophagosomes,the number of autophagosomes than Wenban Decoction with different concentration groups.2 Detection of proliferation activity of cells in each group by MTTCompared with the blank control group,model group,rapamycin group and OD Wenban Decoction with different concentration groups were significantly decreased(P<0.01);compared with the model group,rapamycin group and OD Wenban Decoction with different concentration groups were significantly increased(P<0.01);compared with the rapamycin group,OD Wenban Decoction low concentration group significantly decreased(P<0.01),no significant difference between the OD value of Wenban Decoction high concentration group(P>0.05).3 Detection of cell apoptosis rate in each group by flow cytometryCompared with the blank control group,model group and apoptosis of Wenban Decoction low concentration group were significantly increased(P<0.01),there were no significant difference between the rapamycin group and Wenban Decoction high concentration group(P>0.05);compared with the model group,the apoptosis of Wenban Decoction high concentration group had lower rate(P<0.05),rapamycin group decreased significantly(P<0.01),there were no obvious differences of Wenban Decoction low concentration group(P>0.05);compared with the rapamycin group,apoptosis rate in the Wenban Decoction concentration group increased(P<0.05),Wenban Decoction low concentration group increased significantly(P<0.01),no significant difference Wenban Decoction high concentration group(P>0.05).4 Detection of the expression level of autophagy and apoptosis related proteins in each cell by Western Blot4.1 Protein expression level of LC3 B in each groupCompared with the blank control group,the expression level of LC3 B protein increased in the model group(P<0.05),rapamycin group and Wenban Decoction groups were significantly increased(P<0.01);compared with the model group,the expression level of Wenban Decoction low concentration group of LC3 B protein increased(P<0.05),rapamycin group and the high Wenban Decoction concentration group were significantly increased(P<0.01);compared with the rapamycin group,the expression level of Wenban Decoction low concentration group of LC3 B protein were significantly decreased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).4.2 Protein expression level of BECLIN1 in each groupCompared with the blank control group,the expression level of BECLIN1 protein increased in the model group(P<0.05),rapamycin group and Wenban Decoction groups were significantly increased(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban Decoction groups with different concentration of BECLIN1 protein was significantly increased(P<0.01);compared with rapamycin group,the expression level of Wenban Decoction low concentration group of BECLIN1 protein were significantly decreased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).4.3 Protein expression level of MTOR in each groupCompared with the blank control group,the expression level of MTOR protein decreased in the model group(P<0.05),rapamycin group and Wenban Decoction groups were significantly decreased(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban Decoction of different concentrations of MTOR protein were significantly lower(P<0.01);compared with rapamycin group,the expression level of Wenban Decoction low concentration group of MTOR protein were significantly increased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).4.4 Protein expression level of BAX in each groupCompared with the blank control group,the expression level of model group,rapamycin group and Wenban Decoction of different concentrations of BAX protein were significantly increased(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban Decoction of different concentrations of BAX protein were significantly decreased(P<0.01);compared with the rapamycin group,expression the level of Wenban Decoction low concentration group of BAX protein were significantly increased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).4.5 Protein expression level of BCL2 in each groupCompared with the blank control group,the expression level of model group,rapamycin group and Wenban Decoction of different concentrations of BCL2 protein were significantly decreased(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban Decoction of different concentrations of BCL2 protein were significantly increased(P<0.01);compared with the rapamycin group,The expression level of Wenban Decoction high concentration group of BCL2 protein decreased(P<0.05),Wenban Decoction low concentration group were significantly decreased(P<0.01).4.6 Protein expression level of CASPASE3 in each groupCompared with the blank control group,the expression level of model group,rapamycin group and Wenban Decoction of different concentrations of CASPASE3 protein were significantly increased(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban Decoction of different concentrations of CASPASE3 protein were significantly decreased(P<0.01);compared with the rapamycin group,the expression level of Wenban Decoction high concentration group CASPASE3 protein increased(P<0.05),Wenban Decoction low concentration group were significantly increased(P<0.01).5 Expression of autophagy and apoptosis related proteins in each group by immunofluorescence5.1 Protein expression level of LC3 B in each groupCompared with the blank control group,the expression level of model group,rapamycin group and Wenban Decoction of different concentrations of LC3 B protein were significantly increased(P<0.01);compared with the model group,the steady concentration of group Wenban Decoction the protein LC3 B expression level increased(P<0.05),rapamycin group and Wenban Decoction high dosage group were significantly increased(P<0.01),no significant difference of Wenban Decoction low concentration group(P>0.05);compared with the rapamycin group,Wenban Decoction group concentration the protein LC3 B expression level decreased(P<0.05),Wenban Decoction low concentration group decreased significantly(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).5.2 Protein expression level of BECLIN1 in each groupCompared with the blank control group,the expression level of BECLIN1 protein increased in the model group(P<0.05),rapamycin group and Wenban Decoction groups were significantly increased(P<0.01);compared with the model group,the steady concentration of Wenban Decoction group the protein BECLIN1 expression level increased(P<0.05),rapamycin group and Wenban Decoction the concentration of group were significantly increased(P<0.01),no significant difference of Wenban Decoction low concentration group(P>0.05);compared with the rapamycin group,the expression level of Wenban Decoction low concentration group of BECLIN1 protein were significantly decreased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).5.3 Protein expression level of MTOR in each groupCompared with the blank control group,the expression level of MTOR protein decreased in the model group(P<0.05),rapamycin group and Wenban Decoction groups were significantly decreased(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban Decoction of different concentrations of MTOR protein were significantly lower(P<0.01);compared with rapamycin group,the expression level of Wenban Decoction low concentration group of MTOR protein were significantly increased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).5.4 Protein expression level of BAX in each groupCompared with the blank control group,the expression level of Wenban Decoction high concentration group of BAX protein increased(P<0.05),model group and Wenban Decoction in low concentration group were significantly increased(P<0.01),no significant difference in rapamycin group(P>0.05);compared with the model group,the expression level of rapamycin group and Wenban Decoction the high concentration group of BAX protein were significantly decreased(P<0.01),no significant difference of Wenban Decoction low concentration group(P>0.05);compared with the rapamycin group,the expression level of Wenban Decoction low concentration group of BAX protein were significantly increased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).5.5 Protein expression level of BCL2 in each groupCompared with the blank control group,model group,and the expression level of Wenban Decoction low concentration group of BCL2 protein were significantly decreased(P<0.01),no significant difference between the group and rapamycin Wenban Decoction high dose group(P>0.05);compared with the model group,the expression level of rapamycin group and Wenban Decoction groups with different concentration of BCL2 protein increased significantly(P<0.01);compared with the rapamycin group,the expression level of Wenban Decoction low concentration group of BCL2 protein were significantly decreased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).5.6 Protein expression level of CASPASE3 in each groupCompared with the blank control group,the expression level of CASPASE3 protein increased in rapamycin group(P<0.05),the model group and the concentration of Wenban Decoction group were significantly increased(P<0.01);compared with the model group,the expression of rapamycin group and Wenban Decoction group and high concentration group of CASPASE3 protein were significantly lower(P<0.01),no significant differences of Wenban Decoction in low concentration group(P>0.05);compared with the rapamycin group,stable plaque group CASPASE3 Decoction concentration the protein expression level increased(P<0.05),Wenban Decoction low concentration group increased significantly(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).6 Detection of the expression level of autophagy and apoptosis related genes in each cell by RT-PCR6.1 m RNA expression level of Lc3 b in each groupCompared with the blank control group,the expression level of model group,rapamycin group and Wenban Decoction groups with different concentration of Lc3 b m RNA increased significantly(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban decoction,high concentration group Lc3 b m RNA were significantly increased(P<0.01),no significant difference of Wenban decoction the low concentration group(P>0.05);compared with the rapamycin group,the expression level of Wenban Decoction low concentration group Lc3 b m RNA were significantly decreased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).6.2 mRNA expression level of Beclin1 in each groupCompared with the blank control group,the expression level of model group,rapamycin group and Wenban Decoction groups with different concentration of Beclin1 m RNA increased significantly(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban decoction,high concentration group Beclin1 m RNA were significantly increased(P<0.01),no significant difference of Wenban decoction the low concentration group(P>0.05);compared with the rapamycin group,the expression level of Wenban Decoction low concentration group Beclin1 m RNA were significantly decreased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).6.3 m RNA expression level of Mtor in each groupCompared with the blank control group,the expression level of model group,rapamycin group and Wenban Decoction groups with different concentration of Mtor m RNA decreased significantly(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban decoction,high concentration group Mtor m RNA were significantly decreased(P<0.01),no significant difference of Wenban decoction the low concentration group(P>0.05);compared with the rapamycin group,the expression level of Wenban Decoction low concentration group Mtor m RNA were significantly increased(P<0.01),no significant difference of Wenban Decoction high concentration group(P>0.05).6.4 m RNA expression level of Bax in each groupCompared with the blank control group,the expression level of model group,rapamycin group and Wenban Decoction groups with different concentration of Bax m RNA increased significantly(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban Decoction groups with different concentration of Bax m RNA decreased significantly(P<0.01);and rapamycin group,expression the level of Wenban Decoction high concentration group Bax m RNA increased(P<0.05),Wenban Decoction low concentration group increased significantly(P<0.01).6.5 m RNA expression level of Bcl2 in each groupCompared with the blank control group,the expression level of model group,rapamycin group and Wenban Decoction groups with different concentration of Bcl2 m RNA decreased significantly(P<0.01);compared with the model group,the expression level of Wenban Decoction low concentration group Bcl2 m RNA increased(P<0.05),rapamycin group and Wenban Decoction group and high concentration group are increased significantly(P<0.01);compared with the rapamycin group,the expression level of Wenban Decoction in different concentrations of Bcl2 m RNA were significantly lower(P<0.01).6.6 m RNA expression level of Caspase3 in each groupCompared with the blank control group,the expression level of model group,rapamycin group and Wenban Decoction groups with different concentration of Caspase3 m RNA increased significantly(P<0.01);compared with the model group,the expression level of rapamycin group and Wenban Decoction groups with different concentration of Caspase3 m RNA decreased significantly(P<0.01);compared with the rapamycin group,the expression level of Wenban Decoction concentration of group Caspase3 were significantly higher m RNA(P<0.01).Conclusion:1 Wenban Decoction serum containing drug has the effect of promoting autophagy and inhibiting apoptosis on the macrophage foamed.2 Wenban Decoction serum containing drug can play a role in promoting autophagy and inhibiting apoptosis by regulating the expression of autophagy and apoptosis related proteins and genes on macrophage foamed,which may be one of the mechanism of action of Wenban Decoction in the prevention and treatment of atherosclerosis. |
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