Inhibition of Macrophage Migration Inhibition Factor Combined with PCSK9 Silencing Attenuates Atherosclerosis in ApoE-/-Mice
Objective To investigate the comprehensive therapeutic effect of macrophage migration inhibition factor(MIF)inhibitor on ApoE-/-mouse atherosclerosis(AS)based on the silencing of Pro-protein convertase subtilisin/kexin 9(PCSK9).Methods ApoE-/-mice were fed general diet for 6 months to establish AS model.Mice in PCSK9si group were injected with 2 × 1011 vg/AAV8.2-Lbcpf-CrRNA2 via tail vein.Mice in PCSK9si+ISO-1 group were injected with 2 × 1011 vg/AAV8.2-Lbcpf-CrRNA2 via caudal vein,while mice in control group were intraperitoneally injected with 5mg/kg/d MIF inhibitor ISO-1(without therapeutic intervention).Blood samples were collected from the internal canthal vein at 0,2,4,and 6 months during the AS model to detect the levels of PCSK9 and MIF.After 6 months of modeling,blood samples were collected to detect serum lipids and interleukin 1β(IL-1β)levels.Flow cytometry was used to detect circulating Ly-6Chigh proinflammatory mononuclear cells.The plaque area,fibrosis and lipid infiltration,and the expression of CD68(macrophage marker)and nuclear factor κB(NF-κB)p65 protein in the lipid plaques were determined histologically.The protein interaction between PCSK9 and MIF was retrieved in STRING database.Results Compared with the control group,the total cholesterol level in PCSK9si group and PCSK9si+ISO-1 group was reduced by 49％and 44％,respectively;low density lipoprotein levels decreased by 54％and 52％(both P＜0.000 1);triglyceride levels were reduced by 25％and 27％(both P＜0.05);high density lipoprotein cholesterol levels increased by 93％and 77％(both P＜0.01).Serum PCSK9 levels in PCSK9si group and PCSK9si+ISO-1 group were reduced by 59％and 46％,respectively.MIF levels fell by 28％and 44％.The percentage of Ly-6Chigh pro-inflammatory monocytes and plasma IL-1β in PCSK9si group were not significantly different,while the percentage of Ly-6Chigh proinflammatory monocytes and plasma IL-1β in PCSK9si+ISO-1 group were reduced by 50％,respectively(both P＜0.05)and 48.4％(both P＜0.05).Aortic AS plaque area decreased by 16％(P＞0.05)and 31.2％in PCSK9si and PCSK9si+ISO-1 groups,respectively(P＜0.05),the aortic sinus plaque area was reduced by 20％and 46.9％,respectively(both P＜0.05),the area of Sirius red staining positive area in aortic sinus plaques decreased by 23.6％(P＞0.05)and 57.3％,respectively(P＜0.05).Protein interaction analysis showed that MIF interacts with PCSK9 through chemokine receptor 4(CXCR4)and epidermal cell growth factor(EGF).Conclusion On the basis of silencing PCSK9,the application of MIF inhibitor ISO-1 can significantly reduce the degree of AS lesions in ApoE-/-mice.
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