Explore The Mechanism Of Shenfu Injection In Promoting Cholesterol Efflux In RAW264.7 Derived Foam Cells Based On Autophagy

Objective RAW264.7 macrophage derived foam cells model was replicated in vitro,in order to analyze whether the effect of Shenfu Injection(SFI)in promoting cholesterol efflux was related to autophagy.The present study provide experimental evidence for basic research and clinical prevention on Atherosclerosis(AS).Method1.Copy the model and evaluate the efficacy RAW264.7 macrophage derived foam cell model was reproduced by ox-LDL intervention,and SFI was used as the research drug.Oil Red O staining was used to qualitatively analyze the effect of different concentrations of SFI on the accumulation of ox-LDL in foam cells and the content of oil red O dye was extracted with isopropanol to quantitatively analyze the effect of different drug concentrations;RT-q PCR was used to detect the cell cholesterol transport related factor ATP binding cassette transporter A1(ABCA1),ATP binding cassette transporter G1(ABCG1),Peroxisome proliferator-activated receptor gamma(PPARγ),Liver X receptor alpha(LXRα),scavenger receptor A1(SR-A1),and CD36 m RNA expression;2.Explore the mechanism of SFI based on autophagy SFI and autophagy inhibitor LY294002 interfered with the cells respectively,MDC fluorescence staining was used to observe the rate of autophagy;Oil Red O staining was used to evaluate the effect of each group on the accumulation of ox-LDL in RAW264.7 cells after intervention;RT-q PCR was used to detect cell cholesterol transport and autophagy-related factor m RNA expression levels.Western Blot was used to detect the expression of autophagy-related protein LC3 II / LC3 I ratio and P62 / SQSTM1 protein levels.The aim is to explore the possible mechanism of SFI in promoting cholesterol efflux,and to verify whether SFI promotes lipid transport by up-regulating autophagy.Result1.Evaluation of medicinal effect of SFI on cholesterol efflux from RAW264.7 derived foam cells(1)Oil red O staining was used to observe lipid accumulation in foam cells: There were almost no lipid droplets in control group;orange-red lipid droplets of different degrees were observed in model group and the different concentrations of the SFI group,except for the 8μl/ ml concentration of SFI group,the red stained area of the other SFI groups was smaller than that of the model group.(2)Quantitative results of isopropanol extraction: After the intervention with ox-LDL,the relative oil red O dye content in the cells increased significantly.Except for the 8μl / ml concentration of SFI group,the oil red O dye content in the other SFI concentration groups was lower than Model group(P <0.05).Combining oil red O staining and isopropanol extraction quantitative results showed that SFI can reduce ox-LDL accumulation in RAW 264.7 cells.(3)Effect of SFI on cholesterol transport-related factors m RNA expression: After ox-LDL intervention with a concentration of 50 μg / ml,SFI can promote the expression of four cholesterol efflux related factors ABCA1,ABCG1,PPARγ,LXRα m RNA in RAW 264.7 cells,and the effect of the SFI group with a concentration of 2μl/ ml was the best(P<0.01);the expression levels of scavenger receptors SR-A1 and CD36 had no significant effect(P > 0.05).2.The effect and mechanism of SFI on ox-LDL induced RAW 264.7 cells based on autophagy(1)MDC fluorescence staining was observed under a fluorescence microscope: Compared with the control group,the MDC fluorescence intensity in the model group was weaker,and the green particles in the cells were significantly reduced.Compared with the model group,the MDC fluorescence intensity was stronger in the SFI group,and the number of green particles in the cells was significantly increased.The results suggested that SFI can obviously induce autophagy in foam cells.(2)Effect of SFI on the expression of autophagy related factors m RNA and protein: Compared with the model group,the expression levels of the autophagy marker LC3 B m RNA expression and LC3 II / LC3 I protein ratio in the SFI group were significantly increased(P <0.05),and the expression level of autophagy factor P62 m RNA and protein was significantly reduced(P <0.05).The results were between these of the control group and the model group.(3)The reverse verification that autophagy affects cholesterol transport protein: Under a light microscope,there were almost no lipid droplets in the control group cells;oil red O staining of different degrees were seen in the cells of the other groups,and the staining area in the SFI group was significantly smaller than that of the model group.The results suggest that autophagy induced by SFI can promote the intracellular transport of cholesterol from foam cells to extracellular.The effect of autophagy on the m RNA and protein expression of cholesterol efflux-related factors in RAW 264.7 cells showed that the effect of SFI combined with LY294002 in inhibiting autophagy was not significantly different than that of LY294002 alone.Therefore,SFI can obviously induce autophagy levels in RAW 264.7 foam cells,and then promoted the expression of cholesterol transport related factors,thus facilitating the function of RCT.Conclusion1.SFI can reduce the accumulation of lipids in macrophages and inhibit the generation of foam cells.The mechanism is related to the up-regulation of the cholesterol transport factors ABCA1,ABCG1,PPARγ,LXRα m RNA expression;2.Inducing autophagy by increasing the LC3 II / LC3 I ratio and down-regulating P62 expression is a potential mechanism for SFI to up-regulate cholesterol transport factors and thereby promote the revers cholesterol transport.