| Atherosclerosis(AS)is a chronic inflammatory disease caused by the imbalance of lipid metabolism in the arterial wall and immune dysfunction.Among them,macrophage foaming leads to the accumulation of lipids in the vascular intima,which is a key link in the development of AS.Cellular autophagy can resist external stimuli,degrade the damaged components in cells into small molecules for the body to reuse,and maintain the balance of energy metabolism.Related research shows that moderate autophagy of macrophages promotes the outflow of cholesterol in cells and delays the occurrence and development of AS.Obesity is a high risk factor for AS,and treating obesity can return arterial stiffness to normal.Qi deficiency constitution and phlegm and dampness constitution are the two main factors affecting obesity.Wang Qi Master of Traditional Chinese Medicine believes that obesity treatment should follow the two main treatments:"Yiqi Jianyun" and "Hua tan Qu shi".Therefore,in order to systematically study the therapeutic effect and mechanism of Yiqi Jianyun Recipe and Huatan Qushi Recipe on atherosclerosis,this paper builds a foam cell model through mouse macrophage RAW264.7 to simulate the formation process of atherosclerosis,Add Yiqi Jianyun Fang and Huatan Qushi Fang to intervene to explore the molecular mechanism of treating atherosclerosis.Purpose:To simulate the formation of atherosclerosis by treating mouse macrophage RAW264.7 with ox-LDL.To detect the changes of autophagy function of macrophages in the process of atherosclerosis after drug intervention,and to explore the regulatory effect and mechanism of AS by fu Yuan granule and Yifat granule.Methods:The mouse macrophage RAW264.7 was cultured to construct a foam cell model.The cells were randomly divided into 9 groups,namely normal control group,oxidized low density lipoprotein(ox-LDL)model group,Yiqi Jianyunfang 25ug/ml group,50mg/ml group,100ug/ml group;Huatanqu Wet prescription 25ug/ml group,50mg/ml group,100ug/ml group and Yiqi Jianyun prescription and Huatan Qushi prescription combined prescription group(both drug concentrations are 100ug/ml),detected by oil red O staining method Cell lipid content in each group.CD3 6 was used as a marker protein for lipid metabolism of macrophages,and the protein was collected.Westren-blot was used to detect the change of CD36.In order to study the effects of Yiqi Jianyun Fang and Huatan Qushi Fang on macrophage autophagy,proteins were collected to detect the expression of Beclin-1,P62 and LC3.In order to further study the effect of Yiqi Jianyun Fang and Huatan Qushi Fang on the autophagy function of foam cells,an autophagy protein chip was used for the study.Results:1.The result of oil red O staining showed that at the concentration of ox-LDL 40 ng/μl,the lipid content of the cells increased significantly,and the foam cell modeling was successful.After adding Yiqi Jianyun Fang and Huatan Qushi Fang medicine intervention,compared with the foam cell successfully modeled,the cellular lipid content of the medicine intervention was significantly reduced.2.Western blot results showed that after drug intervention,the expression of CD36 in both Yiqi Jianyun Fang group and Huatan Qushi Fang group was increased.The drug stimulated the expression of CD36 to enhance the ability of macrophages to process lipids.The ratio of LC3Ⅱ/LC3Ⅰ in Huatan Qushifang 50ng/ul group and Yiqi Jianyunfang 50,100,150ng/ul three concentration groups increased.Compared with the model group,the expression of P62 in the 150 ng/ul concentration group of Huatan Qushi Decoction decreased,but there was no significant difference between the 50,100 ng/ul concentration group of Huatan Qushi Decoction and each concentration group of Yiqi Jianyun Decoction.Compared with the model group,there was no significant difference in the expression of Beclin1 in each concentration group of Huatan Qushi Decoction and Yiqi Jianyun Decoction.3.The results of autophagy chip showed:① Compared with the blank control DMEM group,the expression of lysosomal marker LAMP1 and autophagy substrate P62 increased after induction with ox-LDL,indicating that after adding ox-LDL,macrophages Cellular autophagy does indeed suffer from impairment.②Compared with the ox-LDL group,the expression of P62 decreased and the expression of ATG10 increased in the group of Huatan Qushi Recipe.It shows that the autophagy of the medicine is enhanced after adding Huatan Qushi Fang.③Compared with the ox-LDL group,the expressions of the three differential genes ATG10,ATG12,and ATG4A in the Yiqi Jianyun prescription drug group were all increased.This indicates that the autophagy is enhanced after the intervention of Yiqi Jianyun Recipe.④ Compared with the ox-LDL group,the expression of ATG10,ATG4A,ATG12,and GAB ARAP genes in the Hefang group was enhanced,indicating that autophagy was enhanced after the drug intervention of Hefang was added.It showed that Yiqi Jianyun Fang and Huatan Qushi Fang enhanced macrophage autophagy by up-regulating ATG series genes in the process of autophagy.Conclusion:① After adding the medicines of Yiqi Jianyun and Huatan Qushi for 24h and 48h,25,50,100,150ug/ml concentration drugs had no significant effect on cell viability,and 200ug/ml drug concentration would reduce cell viability.②After adding 50,100 and 150ug/ml drugs,it can be clearly seen that the lipid content of the cells is reduced by oil red O staining,indicating that both Yiqi Jianyun Fang and Huatan Qushi Fang can increase fat Qualitative metabolism,reduce the lipid content of foam cells.③The results of autophagy protein chip showed that after adding ox-LDL,the autophagy of the cells was blocked,and the autophagy could be restored after the intervention of drugs.④Western blot results showed that the expression of LC3 Ⅱ/Ⅰ was enhanced and the expression of CD36 was enhanced after drug intervention,indicating that Yiqi Jianyun Fang and Huatan Qushi Fang can enhance the autophagy of macrophages.Lipid metabolism of phagocytes reduces the formation of foam cells. |
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