Effect And Mechanism Of Probucol On Atherosclerosis By Regulating M1 Macrophages Polarization In ApoE^-/- Mice

Objective:To study the role of probucol on atherosclerotic plaques in ApoE^-/- mice;To explore the molecular mechanisms of probucol treatment on the progression of atherosclerosis from the perspective of macrophage polarization.Methods:（1）Twenty 8-weeks-old male ApoE^-/-mice were fed with a high-fat diet（HFD）to build the atherosclerosis model.Mice were randomly divided into control group（10 mice）and probucol group（10 mice）.Mice in the observation group were given probucol（30 mg/Kg）by gavage,once every other day;the mice in the control group were given the same dose of normal saline by gavage.After the gavage of probucol or normal saline for 12 weeks,the mice were sacrificed and the serum,heart and aortas were collected.The aortas were stained with Oil Red O.The aortic Frozen section of aortic root.The atherosclerotic plaque area was detected by hematoxylin-eosin（HE）staining.The content of collagen fiber in the plaque was detected by Masson staining,and immunohistofluorescence was used to detect the staining area of smooth muscle cells,macrophages and the marker of M1 polarization（i NOS）in the plaque.The content of lipid in plaques was measured by Oil red O staining.The total protein of mouse blood vessels was extracted,and the markers of macrophage M1 polarization（i NOS,CD86,IL-1β）were detected by Western Blot.ELISA was used to detect blood lipids,liver and kidney function and inflammatory factors（IL-1β,IL-6,TNF-α）.（2）The primary peritoneal macrophages were extracted from C57BL/6J mice.Macrophage M1 polarization was induced by lipopolysaccharide（LPS）combined with interferon-γ（IFN-γ）,following stimulated with PBS or Probucol.Western Blot,ELISA and Q-PCR were used to measure the protein and m RNA levels of M1 polarization markers.Results:Compared with the control group,serum lipids was modified,and the plaque area of the whole aorta and the aortic root was significantly reduced in the probuco group.Probucol treatment reduced the content of macrophages and lipid deposition in the plaque,while the content of smooth muscle and collagen fiber was increased,indicating that the plaque was more stable in the probucol group.Compared the control group,the serum levels of inflammatory factors including IL-1β,IL-6 and TNF-α,the proportion of M1 macrophages（mom A-2⁺i NOS⁺）in aortic root plaque,and the levels of M1 polarization markers（i NOS,CD86 and IL-1β）in total protein extracted from aorta all significantly decreased.Furthermore,the in vitrol experiments showed that probucol inhibited M1 polarization of macrophages induced by LPS combined with IFN-γ,demonstrated by decreased protein and m RNA levels of M1 polarization markers.Besides,probucol inhibited STAT1phosphorylation,which may underlies the mechanisms of the inhibitory effenct of probucol on M1 macrophages polarization.Conclusion:Probucol mitigated the progression of atherosclerosis and promoted plaque stability in ApoE^-/-mice,possibly by inhibiting M1 polarization of macrophages and alleviating plaque inflammation.