Regulation Of Macrophage Polarization And Cholesterol Accumulation By Atorvastatin Induced HO-1 Expression

Objective: To explore the effect of atorvastatin induced heme oxygenase-1 expression on the ox-LDL stimulated RAW264.7 macrophage polarization,inflammatory symptom state and cholesterol level through in vitro experiments,and provide a new direction for the follow-up research on the prevention and treatment of atherosclerosis.Methods: Experiment 1: Firstly,RAW264.7 cells cultured in vitro were randomly divided into 6 groups,and were given different concentrations of atorvastatin(0,1,5,10,20,40 μmol/L)incubation for 24 h,Western blot was used to detect the expression of HO-1 protein,CCK8 method was used to detect cell activity,and the optimal dose of atorvastatin treatment was explored by combining cell activity and HO-1 protein expression.Experiment 2:RAW264.7 cells were randomly divided into control group,atorvastatin group,and HO-1 inhibition group,and were treated with the same amount of pure culture medium,atorvastatin 20 μmol/L and Zn PPIX 10 μmol/L+atorvastatin20 μmol/L Cells were pre-incubated with for 24 hours,and then incubated with50 mg/L ox-LDL for 48 hours.Collect the RAW264.7 macrophage supernatant after grouping intervention,and detect the TGF-β,IL-10,IL-1β,TNF-α by ELISA.The cells were lysed to extract protein.β-actin was used as the internal reference.LC3Ⅱ/Ⅰ,P62,PPARγ,and ABCA1 were detected by Western-blot.Determination of cellular FC and TC by oxidase method,then calculate the difference between TC and FC.To explore the polarization phenotype of RAW264.7,CD86 antibody was used to label M1,and CD206 antibody was used to label M2.Flow cytometry was used to sort RAW264.7 cells.The quantity of M1 and M2 in macrophages of mice in each group was detected and the ratio of M1 to M2 was calculated(M1/M2).Results:1.Atorvastatin induced HO-1 protein expression in macrophages in a dose-dependent manner.2.OX-LDL can induce macrophages to polarize to M1 and secrete pro-inflammatory factors,increase intracellular cholesterol accumulation and foam cell formation.3.Compared with the control group,the expression of HO-1 protein in macrophages increased after atorvastatin treatment,and the cell polarization turned to M2 type and TGF was mainly secreted-β,IL-10 and other anti-inflammatory factors,meanwhile,PPARγ,The expression of signal molecules reflecting cholesterol efflux and autophagy such as ABCA1 and LC3II/I increased,and the content of intracellular cholesterol and lipid droplets decreased significantly(P<0.05).4.The HO-1 inhibition group pretreated with Zn PPIX simultaneously effectively reversed the above changes in atorvastatin group.Conclusion:1.Atorvastatin can induce the expression of HO-1 protein in RAW264.7cells in a dose-dependent manner.2.Atorvastatin may promote the polarization of macrophages stimulated by oxidized low density lipoprotein to M2 type and inhibit inflammation by up-regulating the expression of HO-1.3.Atorvastatin may up-regulate HO-1 expression,which in turn up-regulates PPARγ/ABCA1 signaling pathway and enhanced autophagy increase intracellular cholesterol outflow,reduce intracellular lipid accumulation and foam cell formation.