Mechanism Of Astragaloside Ⅳ Improving Diabetic Cardiomyopathy By Inhibiting Pyroptosis

BackgroundDiabetic cardiomyopathy(DCM)is a pathophysiological condition caused by hyperglycemia and refers to the development of cardiac insufficiency in the absence of hypertension,coronary artery disease and other cardiovascular diseases.With the increasing prevalence of obesity and type 2 diabetes,the prevalence of DCM is also increasing year by year.It is urgent to fully understand the pathogenesis of DCM and find new therapeutic methods.Recent studies have shown that cell pyroptosis plays an important role in the pathological process of DCM.Pyroptosis is a type of programmed cell death associated with inflammatory activation.The NOD-like receptor protein 3(NLRP3)inflammasome is considered to be the most important triggers of pyroptosis,and their activation can induce a series of inflammatory cascade responses.Astragaloside Ⅳ(ASIV),the main pharmacological extract of Astragalus,has various pharmacological effects in the cardiovascular system,including anti-oxidative stress,anti-inflammatory,anti-apoptotic,immunomodulatory and cardioprotective effects.Previous studies have shown that ASIV can ameliorate myocardial damage in diabetic rats,but whether it can inhibit the development of DCM by regulating pyroptosis remains unclear.Therefore,in this experiment,the effects of ASIV on DCM and its related mechanisms were investigated by taking pyroptosis as the entry point,so as to provide a new theoretical basis and action target for the prevention and treatment of DCM.ObjectivesThis study is proposed to clarify the role of pyroptosis in high glucose-induced myocardial injury by establishing a H9c2 cell model with high glucose injury and a DCM mouse model.And to investigate whether ASIV can affect DCM myocardial injury by regulating NLRP3/cysteine asparticase-1(Caspase-1)/gasdermin D(GSDMD)pathway.Methods1.H9c2 cardiomyocytes were cultured in vitro to establish a high glucose injury model.The experiment was divided into 4 groups:control(CON)group,high glucose(HG)group,high glucose+low dose ASIV(HG+ASIV-L)group,and high glucose+high dose ASIV(HG+ASIV-H)group.The control and high glucose groups were incubated with culture medium containing 5.5 mmol/L and 33.3 mmol/L glucose,respectively.The concentrations of low and high dose ASIV were 50 μmol/L and 100 μmol/L,respectively.After the intervention in each group,the cell activity was detected by CCK-8 method and the pyroptosis was detected by TUNEL staining.2.The levels of reactive oxygen species(ROS)in H9c2 cells were detected by dihydroethidium(DHE).The activity of lactate dehydrogenase(LDH),superoxide dismutase(SOD),and malondialdehyde(MDA)concentration in cell supernatants were measured.3.The fluorescence intensity of NLRP3 and Caspase-1 in H9c2 cells was determined by immunofluorescence.The protein expression of NLRP3,apoptosis-associated speck-like protein(ASC),precursor Caspase-1(pro-Caspase-1),Caspase-1,GSDMD,GSDMD-N,pro-interleukin-1β(pro-IL-1β),and IL-1β was detected by Western Blot.4.Eighteen 8-week-old male db/db mice were randomly divided into three groups:model(db/db)group,low-dose ASIV treatment(db/db+ASIV-L)group,and high-dose ASIV treatment(db/db+ASIV-H)group,with six mice in each group.Another six db/m mice of the same age and sex were used as the control group.The low and high dose ASIV treatment groups were given 20 mg/kg/d and 40 mg/kg/d ASIV gavage treatment,respectively.The model and control groups were given an equal volume of 0.5%sodium carboxymethylcellulose solvent by gavage.The treatment lasted for 12 weeks.Body weight and fasting blood glucose were measured every two weeks during the treatment period.5.At the end of feeding,mice were anesthetized with isoflurane and echocardiography was performed to detect cardiac function in each group,including left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),left ventricular internal diameter at end-diastole(LVIDd),left ventricular internal diameter at end-systole(LVIDs),and E/A ratio.Blood was collected from mice and serum was isolated to measure total cholesterol(TC),triacylglycerol(TG),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),LDH,and creatine kinase MB isoenzyme(CK-MB)levels.6.HE,Masson staining and wheat germ agglutinin(WGA)immunofluorescence were used to observe myocardial histopathological changes.Transmission electron microscopy was used to observe the ultrastructure of myocardial tissue.7.Serum levels of IL-1β,IL-18,IL-6 and tumor necrosis factor-α(TNF-α)in mice were detected by ELISA.8.Immunohistochemical staining was performed to observe the expression of NLRP3 and Caspase-1 in mouse myocardial tissues.The protein expression of NLRP3,ASC,pro-Caspase-1,Caspase-1,GSDMD,GSDMD-N,pro-IL-1β and IL-1β in myocardial tissues were detected by Western Blot.Results1.ASIV significantly increased the viability of H9c2 cells damaged by high glucose in a concentration-dependent manner.TUNEL staining showed that the pyroptosis level of H9c2 cells was increased in the high glucose group,and ASIV reduced pyroptosis.2.Compared with the control group,H9c2 cells in the high glucose group had increased ROS levels,increased LDH activity,increased MDA content and decreased SOD activity.ASIV can reverse the above changes and reduce oxidative stress response.3.The body weight,fasting glucose and lipid levels of mice in the db/db model group were significantly higher than those in the db/m group.ASIV reduced body weight,fasting glucose and serum TC and TG levels in the db/db mice,but had no significant effect on serum LDL-C and HDL-C levels.4.The mice in the db/db group had decreased cardiac function,decreased LVEF,LVFS,E/A,increased LVIDd and LVIDs,and increased heart weight to tibial length ratio compared with the db/m group.ASIV improved the above cardiac systolic and diastolic function indices and decreased heart weight to tibial length ratio in the db/db mice.Compared with the db/m group,serum LDH and CK-MB were elevated in the db/db group,and they were decreased after ASIV treatment.5.Compared with the db/m group,mice in the db/db group had disturbed myocardial cell arrangement,increased myocardial interstitial fibrosis,and increased myocardial cell cross-sectional area.All these pathological changes were improved after ASIV treatment.Transmission electron microscopy showed that ASIV treatment reduced myofibrillar lysis breakage and mitochondrial swelling in db/db mice.6.The levels of serum inflammatory factors(IL-1β,IL-18,IL-6 and TNF-α)were increased in the db/db group of mice compared with the db/m group,and ASIV treatment reduced the levels of serum inflammatory factors in the db/db mice.7.Cellular and animal experiments showed that the expression of pyroptosis-related proteins NLRP3,ASC,Caspase-1,GSDMD,GSDMD-N,pro-IL-1β,and IL-1β was elevated in the DCM model group compared with the control group.ASIV reduced the expression of the above proteins and attenuated myocardial injury in DCM.ConclusionASIV can improve dysglycolipid metabolism,inhibit myocardial hypertrophy and fibrosis,and improve cardiac function in db/db mice.ASIV may exert these protective effects by inhibiting NLRP3/Caspase-1/GSDMD-mediated pyroptosis.