FTZ Ameliorates Atherosclerosis By Suppressing Macrophage Pyroptosis And M1 Polarization

Objective:Atherosclerosis is a long-term inflammatory disease,which is recognized as the main cause of incidence rate and mortality associated with cardiovascular diseases.Macrophages undergo M1polarization and pyroptosis during the development of atherosclerosis,which lead to the rupture of plaque and the formation of thrombosis.Therefore,inhibiting the pyroptosis of macrophages and reducing the number of M1 macrophages are effective ways to enhance plaque stability.Fufang Zhen Zhu Tiaozhi capsule（FTZ）is a traditional Chinese medicine for treating glycolipid metabolic disorders.Previous studies have reported that FTZ can ameliorate atherosclerosis,but its potential mechanism is unclear.Our study focused on whether FTZ could improve the stability of plaque by inhibiting NLR family pyrin domain containing 3（NLRP3）inflammasome to inhibit the pyroptosis and M1 polarization of macrophages.Methods:ApoE^-/-mice were randomly divided into four groups（6mice in each group）:Normal diet group（ND group）,High-fat diet group（HFD group）,FTZ treatment group（HFD+FTZ group）and simvastatin treatment group（HFD+SIM group）.ApoE^-/-mice were fed with a high-fat diet for 8 weeks to establish a mouse model of atherosclerosis.Mice in the FTZ treatment group and simvastatin treatment group were respectively administrated with FTZ（1.2 g/kg/d）and simvastatin（5mg/kg/d）.And they were subsequently maintained on high-fat diet for 14weeks.The content of cholesterol in serum was detected by blood lipid detection kit.The lipid deposition at the aortic and aortic arch of mice were detected by oil red O staining and hematoxylin eosin staining.The levels of interleukin-18 and interleukin-1βin the serum of ApoE^-/-mice were detected by ELISA kits.In vitro,the oxidative damage and pyroptosis of macrophages were detected by immunofluorescence staining and flow cytometry.Western blot and qRT-PCR were used to evaluate the expression of pyroptosis and macrophage polarization related genes（NLRP3,ASC,GSDMD-N,Caspase-1,Arginase-1（Arg1）,CD206 and i NOS）.Results:In vivo experiments,we found that intragastric administration of FTZ could improve the levels of total cholesterol（12.22±2.92 mmol/L vs 6.35±0.29 mmol/L,P<0.01）,triglyceride（6.30±0.41 mmol/L vs 1.83±0.19 mmol/L,P<0.01）and low-density lipoprotein cholesterol（13.29±1.90 mmol/L vs 5.53±0.39 mmol/L,P<0.01）in serum of ApoE^-/-mice fed with a high-fat diet（HFD）.Compared with HFD group,immunohistochemical staining showed that FTZ treatment reduced vascular lipid deposition and plaque inflammatory infiltration,and the plaque stability index significant increased（0.75±0.04 vs 3.25±0.22,P<0.01）.Immunofluorescence results showed that FTZ treatment inhibited the activation of NLRP3 inflammasome and M1polarization of macrophages,and reduced the secretion of IL-18 and IL-1βin serum of high-fat diet mice（HFD）.In vitro experiments,FTZ treatment of bone marrow-derived macrophages（BMDM）could inhibit the increase of reactive oxygen species and mitochondrial oxidative damage induced by oxidized low-density lipoprotein（ox-LDL）.In addition,ox-LDL activated NLRP3-ASC inflammasome,upregulated pyroptosis related proteins expression（NLRP3,Caspase-1,ASC and GSDMD-N）,increased the release of lactate dehydrogenase and propidium iodide positive cells,（4.43±0.41）%vs（9.85±1.09）%,P<0.01.The above phenomenon was reversed after FTZ treatment.Moreover,compared with ox-LDL group,FTZ treatment of BMDM promoted the conversion from M1 macrophages to M2 macrophages and weakened inflammatory related factor（IL-1β,IL-18,TNF-αand IL-6）m RNA expression levels.Conclusion:FTZ improved the lipid metabolism disorder,reduced inflammatory infiltration and increased the stability of plaque in ApoE^-/-mice;FTZ enhanced the plaque stability by regulating M1/M2macrophage polarization and inhibiting NLRP3 mediated macrophage pyroptosis to delay the process of atherosclerosis.