Study On The Effect And Mechanism Of The Effective Components Of Astragalus And Leech On Lipid Accumulation In Macrophages

Objective: In recent years,coronary atherosclerotic heart disease has become one of the main chronic diseases that affect human health.The clinical application of a large number of Chinese medicine compounds has achieved significant effects in the treatment of coronary heart disease.On this basis,exploring the active ingredients of the main drugs in the compound is of great significance for the development of new drugs for the treatment of coronary heart disease.Atherosclerosis is the main pathological basis of coronary heart disease.The foaming and apoptosis of macrophages runs through the development of atherosclerosis.Therefore,this study used OX-LDL to stimulate the mouse Raw264.7 cells to establish an atherosclerotic foam cell model.Astragalus polysaccharide,the active ingredient of Astragalus membranaceus,the representative medicine for replenishing Qi,and hirudin,the active ingredient of Leech,the representative medicine for Zhuyu Tongluo,were used for joint intervention.And then reveal the mechanism of the combined intervention of astragalus polysaccharide and hirudin on OX-LDL-induced macrophage foaming lipid accumulation and apoptosis from the cellular level,providing scientific experimental basis for the development of new clinical drugs for the treatment of atherosclerosis.Methods: The mouse Raw264.7 cell line was selected for subculture,and 100ug/ml OX-LDL stimulated Raw264.7 cells for 24 hours to establish a foam cell model.The safe drug concentration of hirudin and astragalus polysaccharide was screened by CCK-8,and the low,medium and high dose groups of hirudin were set up as 10ug/ml,20ug/ml and40ug/ml respectively,and the low,medium and high dose groups of astragalus polysaccharide were 25ug/ml and 50ug/ml respectively.In the experiment,a blank group,a model group,and a drug low,medium and high dose group were set up in the experiment.Oil red O staining and oxidase method were used to detect the cholesterol content in OX-LDL-induced macrophages to determine the best effective concentration of the drug.The optimal effective concentration of hirudin was combined with astragalus polysaccharide to intervene,and the experiment was divided into blank group,model group and drug compatibility group.Flow cytometry was used to detect the early,late and total apoptosis rates of OX-LDL-induced macrophages;Confocal laser microscope and flow cytometry were used to detect the changes of mitochondrial membrane potential;Western blotting was used to detect and analyze the expression levels of anti-apoptotic protein Bcl-2 and pro-apoptotic proteins Caspase3 and Bax.Results: 1.CCK-8 detection found that with the increase of hirudin drug concentration,it has a significant inhibitory effect on the growth of macrophages.Compared with the normal group of cells,cell growth is inhibited when the hirudin drug concentration is greater than 80ug/ml,which is statistically significant.Difference(P<0.05),so hirudin solutions with concentrations of 10,20,and 40ug/ml were selected as low,medium,and high drug dosage groups;the increase in the concentration of astragalus polysaccharide drug has a proliferative effect on the growth of macrophages.In related literature,we choose astragalus polysaccharide solutions with concentrations of 25,50,and 100ug/ml as low,medium and high dose groups.2.After co-cultivating Raw264.7 cells with 100ug/ml OX-LDL for 24 hours,OX-LDL can stimulate Raw264.7 cells to transform into foam cells.Compared with the normal group,the cholesterol content in the cells of the model group increased significantly.There was statistical significance(P<0.05);compared with the model group in the low,middle and high dose groups of the drug,it was found that after intervention in the high-dose group of hirudin 40ug/ml and the high-dose group of astragalus polysaccharide100ug/ml,the cholesterol content was lower and moderate.The dose group decreased more significantly(P<0.05),the red lipid droplets in the cytoplasm decreased,and the oil red O staining effect was weaker.3.Compared with the hirudin 40ug/ml and astragalus polysaccharide 100ug/ml combined intervention group,the cholesterol content was lowered more significantly than the hirudin 40ug/ml dosage group and the astragalus polysaccharide 100ug/ml dosage group,which was statistically significant(P<0.05).Red O dyeing effect is weak.4.Compared with the normal group,the model group can significantly increase the early,late and total apoptosis rate of macrophages induced by OX-LDL,with statistical significance(P<0.01);compared with the model group,the combined intervention group of hirudin 40ug/ml and astragalus polysaccharide 100ug/ml can significantly reduce the early apoptosis rate and total apoptosis rate of macrophages induced by OX-LDL,with statistical significance(P<0.01).5.Compared with the normal group,the mitochondrial membrane potential of macrophages in the model group decreased after OX-LDL stimulation,which was statistically significant(P<0.01);compared with the model group,the combination of hirudin 40ug/ml and astragalus polysaccharide 100ug/ml increased the mitochondrial membrane potential of macrophages,which was statistically significant(P<0.01).6.Compared with the normal group,the expression of Caspase3 protein and Bax protein in the model group increased significantly,and the expression of Bcl-2 protein decreased significantly(P<0.01);compared with the model group,after the combined intervention of 40μg/ml hirudin and 100μg/ml astragalus polysaccharide,the expression of Caspase3 and Bax protein in the cells decreased,and the expression of Bcl-2 protein increased,which was statistically significant(P<0.05).Conclusion: 1.Hirudin 40ug/ml,astragalus polysaccharide 100ug/ml and the combined intervention of the two all reduce lipid accumulation in macrophages,but the combined intervention of hirudin 40ug/ml and astragalus polysaccharide 100ug/ml is better than the effect of single drugs.2.The combined intervention of hirudin 40ug/ml and astragalus polysaccharide100ug/ml can reduce the rate of OX-LDL-induced macrophage apoptosis,and its mechanism of treatment of AS may be related to the regulation of mitochondrial membrane potential and related pro-apoptotic proteins Caspase3,Bax and anti-apoptotic proteins.The expression of apoptosis protein Bcl-2 is related.