The Effect And Mechanism Of Dapagliflozin On Atherosclerosis In Diabetic Apoe^-/- Mice

Research backgroundWith the improvement of people’s living standard,the prevalence rate of type 2 diabetes mellitus(T2DM)assumes year by year all over the world,especially in developing countries.Compared with non-diabetes mellitus,the cardiovascular prognosis(such as atherosclerosis disease,cardiomyopathy,etc)of patients with T2 DM increases significantly,clinical progress develops rapidly[1].Atherosclerosis is one of the major vascular complications of diabetes,but specific mechanism of atherosclerosis is not yet clear,most experts consider it as the results of chronic inflammatory responses[2-4].The risk of myocardial infarction,and acute cerebral apoplexy is increased signific antly caused by atherosclerosis[5].The initial stages of atherosclerosis is due to the recruitment of inflammatory cells such as mononuclear macrophages caused by arterial intima,then these cells produce and release inflammatory mediator,such as interleukin 1β(IL-1β),interleukin 18(IL-18),tumor necrosis factor α(TNF-α),et al.,which is positively associated with cardiovascular risk.And the mononuclear macrophage in the lesion is the most critical inflammatory cells,for it is the protocell of the innate immune system,existing in every period of atherosclerotic lesions,and releasing the key proinflammatory factor,which contribute to the development of atherosclerosis[6].Macrophages are the major inflammatory cells producing IL-1β and IL-18,and which gathering in the vascular lesions is the main causes of local inflammatory response and plaque formation.A few studies indicated that nucleotide-binding and oligomerization domain-like receptors(NLR)in the cytoplasm of all eukaryotic cells can perceive the metabolites.The ligand-binding NLR can promote the formation of inflammasome and activate caspase-1,eventually facilitating the secretion of inflammation factors[7,8].NLRP3,which is most correlated with the chronic inflammation,can perceive the stimulation of aseptic metabolites in the body and then become activated[9].NLRP3 inflammasome is an intracellular pattern recognition receptor in innate immunity that has been more studied in the recent years,and it plays a key role in inherent immunity[10].The ligand-binding NLRP3 can promote the formation of inflammasomes and activate caspase-1,eventually facilitating the maturation and secretion of pro-IL-1β and pro-IL-18[11].Numerous studies have confirmed that IL-1β and IL-18 are both involved in inflammatory reaction of atherosclerosis and act an important role in the occurrence and development of atherosclerosis[12,13].Sodium glucose co-transporter 2(SGLT-2)inhibitors(e.g.,dapagliflozin and canagliflozin),as new reducing glucose drugs,achieve lowing blood glucose effects by inhibiting renal glucose reabsorption.There are trials showing that SGLT-2 inhibitors can inhibit inflammatory markers(e.g.,interleukin6,TNF-α and monocyte chemoattractant protein-1)in the serum and organs(e.g.,liver and kidneys)and thus reduce the degree of arteriosclerosis in the diabetic animal models[14,15].In addition,previous experiments demonstrated that dapagliflozin significantly reduced the macrophage infiltration,oxidative stress and cell apoptosis of kidneys and the expression of inflammation-associated genes(osteopontin,monocyte chemoattractant protein-1 and transforming growth factor-β),and delayed the occurrence and development of diabetic nephropathy in db/db mice[16].These findings prove that dapagliflozin can reverse diabetic renal injury and reduce renal inflammatory reaction,which supports its anti-inflammatory benefit.So we assume an improvement of dapagliflozin on atherosclerosis in diabetes,which may be related to the reduction of NLRP3 inflammasome,thus suppressing the production and release of IL-1β and IL-18.To confirm our hypothesis,the streptozotocin(STZ)induced diabetic Apo E-/-mice were fed with high-fat diet to establish the experimental diabetic atherosclerosis mice models.And ApoE-/-mice were administrated with dapagliflozin or vehicle for 12 w treatment.Then observe the activation of inflammsome,formation of atherosclerosis and activation of NLRP3 signaling pathway in vivo and in vitro.ObjectiveTo observe the effects of dapagliflozin(a SGLT-2 inhibitor)on atherosclerosis of aorta in streptozotocin(STZ)induced diabetic ApoE-/-mice and the relevant mechanisms.MethodsThe STZ induced diabetic ApoE-/-mice were fed with a high-fat diet to establish the experimental diabetic atherosclerosis mouse models.C57BL/6J mice were served as control group and they were fed with a general diet.ApoE-/-mice were intragastrically administrated with dapagliflozin 1mg/kg.d or vehicle for 12 w treatment,the blood was collected via eyepit to test the levels of cholesterol(TCH),triglyceride(TG),free fatty acid(FFA),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C);and thereafter their aortas were collected for oil red O staining to analyze the area of lesion and for hematoxylin eosin(HE)staining to observe the pathologically morphological changes.The NLRP3,IL-1β and IL-18 in the serum were tested by ELISA,the macrophage infiltration in the lesion and the stability of lesion were detected by immunofluorescence,and the expression levels of NLRP3 inflammasome,IL-1β and IL-18 in aorta were analyzed by Western blot.At the same time,the reactive oxygen species(ROS)were measured by Dihydroethidium(DHE).Besides,The effects of dapagliflozin on the IL-1β production in culturing primary macrophages of wild type and NLRP3-/-knockout mice were investigated for mechanism analyses.ResultsDapagliflozin decreased the blood glucose in diabetic Apo E-/-mice(p<0.01)and in non-diabetic Apo E-/-mice(p<0.05),respectively;and it lowered FFA and TG in diabetic ApoE-/-mice(P<0.05).HE and oil red O staining results of aorta showed that dapagliflozin inhibited the formation of aortic atherosclerosis in diabetic ApoE-/-mice(P<0.01).The serum ELISA results demonstrated that dapagliflozin decreased the serum levels of IL-1β,IL-18 and NLRP3 in diabetic ApoE-/-mice(P<0.05,P<0.05 and P<0.01),while it only reduced the serum IL-1β level in non-diabetic Apo E-/-mice(P<0.05).The immunofluorescence results of aortic root indicated that dapagliflozin reduced macrophage infiltration in the lesion,prevented the reduction of smooth muscle cells and increased the stability of lesion in diabetic mice.To investigate aortic atherosclerotic lesions ROS levels,isolated arteries were loaded with DHE,the result showed that dapagliflozin suppressed the production of ROS by DM mice.The Western blot results of abdominal aorta indicated that dapagliflozin lowered the expression of NLRP3 inflammasome in abdominal aorta of diabetic Apo E-/-mice and decreased the production and release of mature IL-1β and IL-18.The mechanism may be associated with the inhibition of ROS.In cellular experiments,we showed that NLRP3 inflammasome can be activated by both palmitic acid(PA)and high glucose(HG)to generate pro-inflammatory cytokines IL-1β in macrophage,however,the activation of NLRP3 could not be suppressed by dapagliflozin in macrophage,which indicated that dapagliflozin can’t directly activate the NLRP3.Moreover,we used the PA and HG to stimulate bone marrow-derived macrophages from NLRP3-/-and wild type(WT)mice,and the result showed that compared with macrophage from WT,the generation of IL-1β was significantly reduced,which indicated that the inhibitory effect on the secretion of IL-1β stimulated by PA and HG via the NLRP3-ASC-caspase-1 pathway.ConclusionDapagliflozin can improve the formation of atherosclerosis by lowing blood glucose and regulating lipids,and reduce the macrophage infiltration in aorta,block the generation of inflammasome by ROS-NLRP3-Caspase-1,thus reduce the producing and releasing of IL-1β and IL-18,and then alleviate the inflammation response of atherosclerosis.Dapagliflozin maybe have a role on inhibiting the activation of NLRP3 inflammsome via the inhibition of ROS,besides lowing blood glucose and regulating lipids.