The Effect And Mechanism Research Of Disulfiram On High Fat Diet Induced Atherosclerosis In ApoE Knockout Mice

BackgroundAtherosclerotic cardiovascular disease(ASCVD)remains one of the leading causes of death worldwide which has become a huge burden on the global economy.At present,despite the definite reduction in atherosclerotic risk with statins and additional triglyceridelowering therapies,patients continue to be at potential residual risk for recurrent events.Admittedly,inflammation plays an important role in all stages of the atherosclerotic process proved by a mass of experiments,including the novel research spot NLRP3 inflammasome pathway.Previous studies have revealed that lipid deposition induces the NLRP3 inflammasome activating the Caspase-1,which can simultaneously promote the activation of inflammatory factors(IL-1β and IL-8)and promote the formation of membrane pores by cutting Gasdermin D(GSDMD),triggering the subsequent release of inflammatory factors and cell pyrosis.Therefore GSDMD is a key target to inhibit the inflammatory response triggered by the NLRP3 inflammasome pathway and cell pyroptosis.Disulfiram is a drug for chronic alcoholism in the clinic and is shown to be effective in inhibiting GSDMD protein expression in human or mouse cells.ObjectiveIn this experiment,we constructed high-fat diet-induced ApoE deficient female mouse atherosclerotic models,used disulfiram to suppress GSDMD proteins in mice,and then observed the effects of the disulfiram on atherosclerosis in tested mice and explored the mechanisms behind it.MethodsThe C57BL/6 mice were divided into a control group which was fed normal chow.The ApoE knockout mice were randomly divided into three groups: a high-fat diet group(HFD),a high-fat diet with a low dose of DSF group(HFDL),and a high-fat diet with a high dose of DSF group(HFDH).The ApoE knockout mice were fed the high-fat diet to model atherosclerosis in mice(except the control group).The drug groups used disulfiram irrigation and the other groups used carboxymethyl cellulose-na(CMC-Na)irrigation.Monitored the weight and the four kinds of lipid levels of all groups of the tested mice to compare the severity of atherosclerosis.ELISA method was used to detect the concentration of IL-18 and IL-1β in the serum and to evaluate local pathological changes in the aortic valve by HE staining,oil-red-o staining,collagen fiber staining,immunohistochemistry,and western blot.ResultsSignificantly,the HFD group mice’s body weight,four kinds of plasma lipid levels,the expression of GSDMD and GSDMD-N,the serum levels of IL-1β and IL-18,and macrophages infiltration level were higher than those of the control group(P<0.01).Compared with the HFD group,a high dose of DSF inhibit the increase in body weight(P<0.01).There was no significant difference in plasma lipid levels between the high-fat diet groups of mice(P>0.05).Pathological staining show that the mild pathological changes and elastic fibers which were arranged neatly and closely together without complete rupture were observed in the mice in HFDL and HFDH groups.DSF increased the collagen fiber and muscle fiber contents of atherosclerotic plaques(P<0.01),suggesting a more stable plaque structure.Of note,the fibrotic thickness was 2.14-fold increased in HFDH group mice plaques(P<0.01).Indeed,the ratio of collagen to the necrotic core in HFDH group mice,an important indicator of plaque stability,was 1.52-fold increased(P<0.01)compared to the HFD group.Western blot showed that disulfiram restrained the expression of mouse GSDMD and GSDMD-N(P<0.01).The serum IL-1β and IL-18 concentrations of mice in the HFDH group were markedly lower than that of the HFD group(P<0.01).Immunohistochemistry showed that the inflammatory-related indicators,CD68 infiltration score of the disulfiram group,were dramatically reduced(P<0.01).ConclusionDisulfiram inhibited weight gain but not lipid levels in ApoE knockout mice.Disulfiram improved the stability of atherosclerotic plaques induced by a high-fat diet in ApoE mice by increasing the fiber cap thickness and collagen fiber and muscle fiber content of atherosclerotic plaques and also reduced local aortic valve macrophage infiltration.The mechanism may be bound up with the inhibition of GSDMD and GSDMD-N expression in tested mice and the downtrend of IL-1β and IL-18 concentrations in the serum,which is the pyroptosis pathway.