| BackgroundDiabetic cardiomyopathy (DCM), an important complication of diabetes, is characterized by consistent diastolic dysfunction and ventricular mass. In addition, DCM is a common cause of heart failure. In the state of diabetes, hyperglycemia-induced reactive oxygen species (ROS) generation is considered to be responsible for progression and development of DCM. The increased ROS could induce a number of cytokine and inflammatory factors, such as nuclear factor-kB (NF-kB), thioredoxin interacting/inhibiting protein (TXNIP), and inflammasome. Although inflammasome was shown to be involved in the pathogenic mechanisms of type2diabetes and its complications, the potential role and regulatory mechanism of inflammasome in DCM has remained largely unexplored.Inflammasomes are multi-protein platforms that interact with various immune and cell death pathways. Different inflammasomes have been identified, including nucleotide-binding oligomerization domain-like receptors (NLRs) and absent in melanoma2(AIM2). NLRP3, the most extensively studied NLRs, forms a complexes comprised of the apoptosis associated speck like protein (ASC), and the serine protease caspase-1. Upon activation, NLRP3forms a complex with its adaptor ASC, which facilitates the autocatalytic activation of pro-caspase-1and the formation of an active caspase-1p10/20tetramer. The activated caspase-1can process pro-IL-1β into its mature form, which is important in cardiomyocyte apoptosis.The activation of NLRP3inflammasome consist of two characteristics, including the the up-regulation of NLRP3and the production of caspase-1and IL-1β. Previous research showed that NF-κB could induce NLRP3expression. Thioredoxin interacting/inhibiting protein (TXNIP) can bind NLRP3directly and lead to NLRP3inflammasome assembly in HEK293T cells. However, little is known about whether NF-κB and TXNIP participate in the regulation of NLRP3in hyperglycemia-treated cardiomyocyte.In addition to resulting in the maturation of IL-1β, activated caspase-1can induce a distinct form of programmed cell death called "pyroptosis". Pyroptosis, a highly inflammatory form of cell death, is dependent on caspase-1activity. The morphology of pyroptosis shares the unique characteristics with both apoptosis and necrosis. As in apoptotic cell, pyroptotic cells incur DNA damage and become positive in the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. As necrosis, pyroptosis results in pore formation in the cell membrane, release of pro-inflammatory cytosolic content, and cell lysis. Therefore, membrane impermeant dyes such as EthD-III stain pyroptotic cells by entering through the pores, but do not stain apoptotic cells. Pyroptosis is initially described in macrophages and dendritic cells infected with different pathogens. Recent studies showed that pyroptosis could also occur in non-myeloid cells induced by non-infectious stimuli. However, it is not clear whether pyroptosis participates in hyperglycemia-induced cardiomyocyte death.Electron microscopy studies of myocardium in diabetic mice and rats showed that the majority of dying cells had swollen fibril and mitochondria, which are the characteristics of cell swelling and lysis in pyroptosis. Activated caspase-1, the executor caspase of pyroptosis, is found to be elevated in DCM in a rat model. Thus, we hypothesized that pyroptosis, regulated by NLRP3inflammsome, might participate in the pathogenesis of DCM. We also hypothesized that NF-κB and TXNIP might be links between ROS and NLRP3activation.Objective (1) We established a type2diabetes rat model and explored the expression level of NLRP3inflammasome in different stages of IDCM;(2) We induced the cardiac NLRP3silencing by lenti-NLRP3-miRNA, and explore the role of NLRP3in DCM.(3) We further explored the mechanism of the regulation of NLRP3activation in high glucose-treated H9c2cells.MethodsInduction of type2diabetes in ratsWe randomized120Sprague-Dawley rats into4groups (n=30per group):control, diabetes mellitus (DM), DM+vehicle, DM+NLRP3-miRNA. All rats were housed at22℃with12h light-dark cycles. The control group was fed the basal diet, and the other groups a high fat diet (HF diet,16%fat and0.25%cholesterol).4weeks later, the intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed to determine insulin resistance. In the groups of DM, DM+vehicle and DM+NLRP3-miRNA rats with reduced insulin sensitivity will receive a single intraperitoneal injection of streptozotocin (STZ;35mg/kg). After one week of STZ injection, blood glucose and insulin levels were measured after rats fasted overnight. Insulin sensitivity index (ISI) was calculated by ISI=In[(INS×BG)-1] method. Only rats with blood glucose level>11.1mmol/L and reduced insulin sensitivity were used as type2diabetes rats in the study.Lenti-NLRP3-miRNAAccording to RNAi principle, we designed4pieces of miRNA against rat NLRP3and constructed pcDNA6.2-GW/EmGFP-NLRP3-miRNA plasmid. Then we selected the most effective NLRP3-miRNA plasmid and then synthesized the shuttle pDONR221-EmGFP-NLRP3-miRNA plasmid. Finally, the lentiviral vector of pLenti6.3—EmGFP-NLRP3-miRNA was constructed.Gene silencing therapyAccording to previous studies, diabetic rats showed onset of cardiac dysfunction after8weeks of STZ injection. Our gene silencing treatment occurred after8weeks of STZ injection. The groups of DM+vehicle and DM+NLRP3-miRNA rats in our study received a jugular-vein injection of a total lentivector dose of1×108TU/rat vehicle (empty virus) or NLRP3-miRNA. After8weeks of lentivector injection, rats of DM+vehicle and DM+NLRP3-miRNA were killed. The heart was excised and immediately frozen to determine the transfection efficiency under fluorescence microscope. The age matched rats of control and DM were killed at the same time point (n=10per group).Cardiac expression pattern of NLRP3inflammasomeFor the analysis of NLRP3inflammasome and IL-1β expression, cardiac samples of DM rats were collected at0,4,8,12,16and20weeks (n=3) after STZ injection, and control rats (n=3) were collected at20weeks after diabetic rats received STZ.Serum measurementsAt the end point of gene silencing therapy, serum blood glucose, TG and TC analysis were tested in rats of the4groups.Enzyme Linked Immunosorbent Assay (ELISA)At the end point of gene silencing therapy, serum insulin was tested in rats of the4groups.EchocardiographyTransthoracic echocardiography analysis was performed after the rats anesthetized. And we collected the parameters of left ventricular end diastolic diameter (LVEDd), left ventricular ejection fraction (LVEF), fractional shortening (FS), peak E to peak A ratio (E/A), and early to late diastolic velocity ratio (E’/A’).Histological examinationThe hearts were arrested with10%KC1, and heart weight was measured immediately. The images of whole heart were obtained by camera. Paraffin sections from the midpoint of heart were stained with hematoxylin and eosin, and the images were obtained by camera.Measurement of myocardial fibrosisMasson’s trichrome and Sirius red staining were used for detecting collagen. The fibrotic area fraction was analyzed by automated image analysis.Transmission electron microscopy (TEM) About1mm3tissue was obtained from the left ventricle of rats and fixed in buffer. The following preparation of TEM was performed according to the guidance.ImmunohistochemistryImmunohistochemistry was performed in the paraffin section of the left ventricular tissue of different groups of rats. We used antibodies against caspase-1and IL-1β. Data were analyzed by Image-Pro Plus6.0.Real-time RT-PCRReal-time RT-PCR was used to explore the relative expression of NLRP3, ASC, caspase-1and IL-1β. The primer sequences were shown in table1. The relative expression of genes was calculated by the2-ΔΔCT method.Western blotWestern blot was used to explore the protein levels of NF-κB, TXNIP, NLRP3, ASC, caspase-1, IL-1β, collagen I, collagen III, and β-actin. The protein bands were developed by the use of chemiluminescence, and quantified by densitometric analysis.TUNEL AssayDetection of fragmentation of DNA was performed using an ApopTag in situ apoptosis detection kit. Deparaffinized sections were treated with3%hydrogen peroxide in methanol for10min. The sections were washed in PBS twice for5min each. After adding the equilibration buffer, sections were treated with TdT-enzyme at37℃for1h. The sections were then rinsed with PBS and incubated with digoxigenin-conjugated antibodies at37℃for30min. Then sections were colorized with DAB. Finally, the stained sections were examined under microscope.Cell culture and treatmentH9c2cardiomyocytes were cultured as described previously. During the treatment period, H9c2cells were cultured in normal glucose medium with minimal essential medium for12h, followed by the exposure of control glucose (Ctrl,5.5mM), medium glucose (MG,25mM), high glucose (HG,33.3mM), and osomotic control (OC,27.5mM mannose) for24,36, or48h. In ROS inhibition experiment, cells were treated with HG for36h in the presence of10mmol/L N-acetylcysteine or the relevant vehicle control (PBS).Lentivirus transfectionH9c2cardiomyocytes plated at a density of1×105/cm2were infected with vehicle (empty lentivirus) or lentivrius-NLRP3-miRNA at10multiplicity of infection (MOI) in serum-free media for2h, followed by incubation with DMEM containing10%FBS for an additional48h before processing. The transfection efficiency was observed by fluorescence microscope. In addition, NLRP3knockdown was assessed in transfected cells, and cells were used only if NLRP3mRNA was decreased by70%compared with vehicle.ImmunofluorescenceImmunofluorescence was used to detect the localization of caspase-1in H9c2cells. We used antibodies against caspase-1. The fluorescence was visualized by confocal microscopy and analyzed by Image-Pro Plus6.0.Caspase-1activity assayCaspase-1activity was measured by using colorimetric assay. This assay was designed in consideration of the ability of caspase-1to change acetyl-Tyr-Val-Ala-Asp p-nitroaniline (Ac-YVAD-pNA) into the yellow formazan product p-nitroaniline (pNA).ROS levelsIntracellular generation of ROS was tested by peroxide-sensitive fluorescent probe2’,7’-dichlorofluorescein diacetate (DCFH-DA).Cell death assayCell death was assessed by TUNEL assay and EthD-Ⅲ/calcein AM staining. The virtually nonfluorescent cell-permeant calcein AM is enzymically converted to the brightly fluorescent calcein, producing a bright green fluorescence in live cell. EthD-III enters dead cells, thereby producing an intense red fluorescence in dead cells. Statistical analysisData are expressed as mean±SEM. Differences among experimental groups were analyzed by ANOVA, followed by the Tukey-Kramer post hoc test and independent samples t test. Analysis involved use of SPSS v18.0. Significance was defined as p<0.05.ResultHF induced insulin resistanceAfter4weeks of HF diet, IPGTT and IPITT were performed in all groups. In IPGTT, the blood glucose in HF group was significantly higher than that in control group at all of the time points tested except at15min (p<0.05-p<0.01). The AUC for glucose level was higher in HF group than that in control (p<0.01). Similarly, the result observed by IPITT revealed impaired insulin sensitivity in rats with HF diet (p<0.05-p<0.01).NLRP3inflammasome and IL-1β were activated in DCMWhen compared with the control rats, diabetic rats showed elevated mRNA levels of NLRP3, ASC, caspase-1and IL-1β after4or8weeks of diabetes (all p<0.01). Moreover, NLRP3inflammasome components and IL-1β mRNA levels reached highest value after8weeks of diabetes, and stayed at relatively high value until20weeks of diabetes (all p<0.01). The protein levels of NLRP3, ASC, pro-caspase-1, activated caspasep-1(caspase-1p20) and mature IL-1β (IL-1β p17) were higher in DM than control group (all p<0.01). The protein expression of NLRP3, ASC and caspase-1achieved the highest levels after8weeks of diabetes and kept at relatively high levels until20weeks of diabetes (all p＜0.01). The activated caspase-1and IL-1β exhibited the similar protein expression pattern (both p＜0.01).NLRP3gene silencing improved metabolism abnormalitiesThe metabolic characteristic of the experimental animals analyzed in this study are shown in Table2. In the DM group, blood glucose, TC, TG and INS were remarkably higher than the control (all p＜0.01). ISI in DM group was lower than control group (p＜0.01). NLRP3gene silencing was insufficient to improve the systemic metabolic disturbance. Cardiac NLRP3expression was suppressed by gene silencingAfter10weeks of NLRP3silencing treatment, transfection efficiency was checked in all groups. As compared with vehicle treatment, NLRP3-miRNA treatment decreased the mRNA and protein levels of cardiac NLRP3(both p<0.01). In addition, the protein levels of activated caspaspe-1and mature IL-1β were lower in NLRP3-miRNA treated diabetic rats than the vehicle treated rats (both p<0.01). Immunohistochemistry revealed that increased caspase-1predominantly localized in perinuclear area, while elevated IL-1β showed diffused distribution pattern in DCM (both p<0.01).NLRP3gene silencing alleviated left ventricular disfunction in DCMThe results of echocardiography showed that LVEDd of DM rat was larger than control (p<0.01). LVEF, FS, E/A and E’/A’were lower in DM than control p<0.01). When compared with vehicle, increased LVEF, FS, E/A, E’/A’and decreased LVEDd were observed in NLRP3-miRNA group (p<0.05-p<0.01).NLRP3gene silencing reversed myocardial remodelingThe DM group showed the phenotype of eccentric ventricular hypertrophy, which was characterized with larger chamber size and thicker ventricular wall. The ratio of heart weight to body weight was larger for DM than the control group (p<0.01). TUNEL result showed that the percentage of dead cell was obviously higher in DM group than control (p<0.01). The ultrastructure of cardiomyocyte in control rats showed typical symmetric myofibrils, well-organized Z lines with sarcomeres, and packed mictochontria beside the fibers. In contrast, DM rats showed severe damage of the left ventricular ultrastructure, including destruction of myofibrils, swollen mitochondria with disorganized cristae, excess glycogen lysis, and accumulated lipids. Moreover, the diabetic group showed increased interstitial cardiac fibrosis as compared with the control (p＜0.01). Coincident with cardiac fibrosis, the protein levels of collagen Ⅰ and collagen Ⅲ, and the collagen I-to-III ratio were significantly higher in DM group than the control (all p＜0.01).With NLRP3gene silencing, heart weight to body weight ratio and cell death were significantly decreased in the NLRP3-miRNA group versus the vehicle group (both p<0.01). NLRP3-miRNA treatment normalized alterations in myofilaments and mitochondria, along with reduced glycogen lysis and lipid accumulation in diabetic rats. Moreover, cardiac fibrosis area, collagen I, collagen III, and the collagen I to III ratio were lower in the DM+NLRP3-miRNA group than vehicle group (all p<0.01).The increased expression of NLRP3inflammasome and IL-1β were induced by high glucoseDifferent levels of glucose caused a concentration-dependent increase of NLRP3, ASC, caspase-1and IL-1β in H9c2cells in24to48h (all p<0.01). Except for caspase-1, the mRNA levels of NLRP3inflammasome and IL-1β were increased in a time-dependent pattern with high glucose (both p<0.05-p<0.01). Likewise, the protein levels of all NLRP3inflammasome components and mature IL-1β were increased significantly at high glucose as compared with control and medium glucose in36and48h (all p<0.01). Moreover, the increase of NLRP3, ASC and mature IL-1β protein showed the highest level at36h, and persisted for48h with high glucose incubation (all p<0.05). Thus, we chose high glucose as the stimulation, and chose36h as the stimulated time in subsequent experiments. The expression of NLRP3inflammasome and mature IL-1β in H9c2cells with control glucose or isotonic mannose had no significant change at the same time point tested.High glucose induced caspase-1activation and cell death in H9c2cellsHigh glucose significantly increased the level of activated caspase-1as compared with control and medium glucose (p<0.05-p<0.01). Immunofluorescence result showed the accumulation of caspase-1in the cytoplasm of H9c2cells incubated with medium and high glucose. Coincident with caspase-1activation in high glucose, TUNEL result revealed increased cell death with nucleus DNA damage (p<0.01), and EthD-Ⅲ/calceim AM staining showed elevated cell death with damaged cell membrane as compared with control and medium glucose (p<0.01).NLRP3was involved in cell death induced by high glucoseTo explore the role of NLRP3in high glucose-induced cell death of H9c2, we inhibited the expression of NLRP3by lentivirial NLRP3-miRNA. The transfection efficacy of lentivirial NLRP3-miRNA in H9c2cardiomyocytes reached80%. Both the mRNA and protein levels of NLRP3in cells transfected with NLRP3-miRNA were significantly lower than the vehicle (both p<0.01). After inhibiting the expression of NLRP3, the protein levels of activated caspase-1and mature IL-1β induced by high glucose decreased significantly as compared with vehicle (p<0.05-p<0.01). In keeping with these observations, the dead cell rate detected by TUNEL was obviously lower in HG+NLRP3-miRNA than HG+vehicle (p<0.01). To avoid the interference of lentivector fluorescence, we did not use calcein AM in the detection of live cells. The high glucose induced-cell-death rate detected by EthD-Ⅲ were significantly decreased in NLRP3-miRNA treated cells as compared with vehicle (p<0.01).NF-κB and TXNIP were associated with the ROS-induced NLRP3inflammasome activationGlucose treatment promoted ROS generation in H9c2. The increase of ROS was dose dependent (all p<0.01). Coincidence with ROS production, the phosphorylation of NF-kB p65was increased in medium and high glucose treated cells. And the expression of TXNIP also exhibited a dose-dependent manner (all p<0.01). Pretreatment of cells with NAC inhibited high glucose-induced increase in intracellular ROS activity (p<0.01). Intriguingly, the levels of NF-kB phosphorylation and TXNIP were also lower in the NAC treated group than the PBS treated group (all p<0.01). In keeping with the decreased expression of NF-kB and TXNIP, the expression of all component of NLRP3, ASC, pro-caspaspe-1, activated caspase-1and mature IL-1β were down-regulated with pretreatment of NAC as compared with PBS (all p<0.01).Conclusion(1) High glucose-induced ROS accelerated the expression of NLRP3inflammasome. NF-kB and TXNIP might be involved in the inhibition of RSV on NLRP3inflammasome.(2) We found the increased expression of NLRP3inflammasome in cardiac tissue at the early stage of diabetes. NLRP3inflammasome accelerated the process of DCM by inducing cardiac inflammation, cardiomyocyte dysfunction and interstitial fibrosis. (3) The cardiac NLRP3gene silencing was effective by using the lenti-NLRP3-miRNA in vivo.(4) The NLRP3gene silencing therapy ameliorated the structural and functional disorder in DCM. BackgroundDiabetic cardiomyopathy (DCM), a significant contributor to morbidity and mortality in diabetes, is characterized by early-onset diastolic and late-onset systolic dysfunction without coronary artery disease, hypertension, or valvular heart disease. Accumulating evidence indicates that inflammation is an important pathogenic factor in DCM. Therefore, investigation of the inflammatory mechanism is the keystone in the management of DCM.Previous studies showed that interleukin-1β (IL-1β) is an important proinflammatory cytokine in the development of DCM. IL-1β activation is mainly mediated the protein platform "the inflammasome". Inflammasome is a multiprotein complex which exists in the cytoplasm. Different inflammasomes have been identified, including nucleotide-binding oligomerization domain-like receptors (NLRs) and absent in melanoma2(AIM2). NOD like receptor3(NLRP3) is a member of NLRs. NLRP3inflammasome consists of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1. Recently, several studies suggested that NLRP3inflammasome plays a critical role in the inflammatory process of diabetes and diabetic complications, such as diabetic nephropathy and diabetic retinopathy. Recent observations indicate that thioredoxin interacting/inhibiting protein (TXNIP) can bind NLRP3directly and lead to NLRP3inflammasome assembly under oxidative stress. And our previous research indicated that NLRP3inflammasome could contribute to the process of DCM.In DCM, mitogen-activated protein kinases (MAPKs) are the most important pathways induced by high glucose levels and are involved in inflammation. Also, activation of MAPKs can contribute to the development of cardiac fibrosis.Rosuvastatin (RSV), a member of3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, has a number of pleiotropic properties, such as anti-inflammation, antioxidation and cardiac remodeling attenuation. The cardioprotective effect of RSV has been demonstrated in animal models of hypertrophy, myocardial infarction and experimental autoimmune myocarditis. However, the mechanisms by which RSV protects against DCM are incompletely understood.The main purpose of the present study was to determine whether NLRP3and MAPKs were important targets for RSV in exerting its protective effect on DCM. And we tried to explore the potential value of NLRP3inflammasome in the treatment of DCM.Objective(1) We established a type2diabetes rat model and evaluated the effect of RSV on the process of DCM.(2) We also evaluated whether NLRP3was important target for RSV in exerting its protective effect on DCM.(3) We tried to explore the mechanism by which RSV inhibites NLRP3inflammasome in the process of DCM.MethodsInduction of type2diabetes in ratsWe randomized105Sprague-Dawley rats (100-120g) into7groups (n=15per group):control, high fat (HF), diabetes mellitus (DM), HF+RSV10mg/kg (10mg/kg, body weight, daily), HF+RSV15mg/kg, DM+RSV10mg/kg and DM+RSV15mg/kg. The control group was fed the basal diet, and the other groups a high fat diet (HF diet,16%fat and0.25%cholesterol).4weeks later, the intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed to determine insulin resistance. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ;35mg/kg) to rats in the groups of DM, DM+RSV10mg/kg and DM+RSV15mg/kg. One week later, blood glucose levels were measured after rats fasted overnight. Only rats with blood glucose level≥11.1mmol/L were used as diabetes in the study. The oral administration of RSV was started in the ninth week after STZ injection, and was continued for8weeks. Rats were weighted before they were killed after8weeks of RSV treatment. The experiments complied with the Animal Management Rule of Chinese Ministry of Health (documentation no.55,2001), and experimental protocols were approved by the Shandong University Animal Care Committee.Lenti-NLRP3-miRNAAccording to RNAi principle, we design4pieces of miRNA against rat NLRP3and constructed pcDNA6.2-GW/EmGFP-NLRP3-miRNA plasmid. Then we selected the most effective NLRP3-miRNA plasmid and then synthesized the shuttle pDONR221-EmGFP-NLRP3-miRNA plasmid. Finally, the lentiviral vector of pLenti6.3—EmGFP-NLRP3-miRNA was constructed.Gene silencing therapyAnother90Sprague-Dawley rats (100-120g) were randomized into6groups (n=15per group):vehicle+control, vehicle+DM, vehicle+DM+RSV15mg/kg (the dose of15mg/kg was the most effective in our drug experiment), NLRP3microRNA (NLRP3-miRNA)+control, NLRP3-miRNA+DM, and NLRP3-miRNA+RSV15mg/kg. After8weeks of STZ injection, diabetic rats showed onset of cardiac dysfunction. Then all6groups of rats received a jugular-vein injection of a total lentivector dose of1×108TU/rat NLRP3-miRNA or vehicle. RSV supplementation for the2groups was performed at the same time for8weeks. After8weeks of lentivector injection, rats were killed. The heart was excised and immediately frozen to determine the transfection efficiency under fluorescence microscope.Serum measurementsAfter rats fasted overnight, we collected jugular blood samples. Serum triglycerides (TG), total cholesterol (TC), insulin (INS) were determined. The insulin sensitivity index (ISI) was calculated by ISI=In[(FINS×FBG)-1] method.EchocardiographyTransthoracic echocardiography was performed in rats anesthetized with10%chloral hydrate. The derived echocardiography parameters included left ventricular ejection fraction (LVEF), fractional shortening (FS), peak E to peak A ratio (E/A), and early (E’) to late (A’) diastolic velocity ratio (E’/A’).Measurement of myocardial fibrosisMasson’s trichrome and Sirius red staining were used for detecting collagen. The fibrotic area fraction was analyzed by automated image analysis.Transmission electron microscopy (TEM)About1mm3tissue was obtained from the left ventricle of rats and fixed in buffer. The following preparation of TEM was performed according to the guidance.Real-time RT-PCRReal-time RT-PCR was used to explore the relative expression of NLRP3, ASC, caspase-1, IL-1β, collagen I and collagen III. The relative expression of genes was calculated by the2-ΔΔCT method.Western blotWestern blot was used to explore the relative protein expression of NLRP3, ASC, pro-caspase-1, caspase-1, IL-1β, and the phosphorylation of ERK1/2, P38, and JNK. The protein bands were developed by the use of chemiluminescence, and quantified by densitometric analysis.Statistical analysisAnalysis involved use of SPSS v18.0. Data are expressed as mean±SEM. Differences among experimental groups were analyzed by ANOVA, followed by the Tukey-Kramer post hoc test and independent samples t test. Significance was defined as p<0.05. ResultsHF induced insulin resistanceAfter4weeks of HF diet, insulin tolerance was determined by IPGTT and IPITT. In IPGTT, the blood glucose in HF group was significantly higher than that in control group at all of the time points tested except at15min (p<0.05-p<0.01). The AUC for glucose level was higher in HF group than that in control one (p<0.01). Similarly, the result observed by IPITT revealed blood glucose in HF group was significantly higher than that in control group at all of the time points tested except at15min (p<0.05-p<0.01). The AUC for glucose level was higher in HF group than that in control one (p<0.01).RSV had limited effect on systemic metabolic abnormitiesDM rats showed more severe hyperglycemia, hyperlipidemia and impaired insulin sensitivity than control and HF rats (both p<0.01). The metabolic state was worse for HF group than control group (p<0.05-p<0.01).RSV affected TC regulation. TC level was lower in DM rats receiving10and15mg/kg RSV than no treatment (p<0.05). TC level was lower with HF+RSV15mg/kg group than HF group (p<0.05). Neither10nor15mg/kg RSV improved glucose dysregulation, TG level disturbance, insulin imbalanee, or impaired insulin sensitivity in DM and HF rats.RSV alleviated DM-induced left-ventricular dysfunctionLVEF, FS, E/A and E’/A were lower with DM than control. E/A and E’/A’was lower with HF group than control (p<0.05-p<0.01).After treatment, LVEF was improved with DM+RSV15mg/kg as compared with DM alone, with no significant improvement with DM+RSV10mg/kg, or HF+RSV10or15mg/kg over DM and HF, respectively (p<0.05). RSV treatment did not significantly prevent FS in DM or HF rats. For diastolic function, DM+RSV15mg/kg increased both E/A and E’/A’as compared with DM alone (both p<0.05). DM+RSV10mg/kg also improved E’/A’(p<0.05).RSV alleviated myocardial remodelingThe ratio of heart weight to body weight was larger for DM and HF than the control group (p<0.05-p<0.01). Diabetes induced interstitial fibrosis, increased collagen mRNA expression, and increased the collagen I to III ratio (p<0.05-p<0.01). Similarly, HF rats showed increased fibrosis and collagen content as compared with control rats (p<0.05-p<0.01). TEM revealed typical symmetric myofibrils, well-organized Z-lines with sarcomeres, and packed mitochondria beside fibers in control rats. In contrast, DM rats showed severe damage of the left ventricular ultrastructure, including destruction of myofibrils, degenerated Z-lines, swollen mitochondria with vacuoles and disorganized cristae, coalescence of irregular mitochondria, and accumulated lipids. The HF rats showed moderate destruction of the left ventricular ultrastructure.RSV15mg/kg treatment decreased heart weight to body weight ratio, fibrosis area and collagen disorders in DM rats (all p<0.01), but had little effect on HF rats. RSV10mg/kg restored only the disordered fibrosis and collagen I content in DM rats (p<0.05-p<0.01). After treatment with RSV, the diabetic and HF rats showed improved cardiac ultrastructure.RSV inhibited NLRP3inflammasome activationWe investigated the mRNA and protein levels of TXNIP, NLRP3, ASC, pro-caspase-1, activated caspase-1and activated IL-1β in left ventricular tissue of rats. The mRNA expression of TXNIP, NLRP3, ASC, caspase-1and IL-1β was significantly higher in DM than control rats (all p<0.01). The protein levels of TXNIP, NLRP3, ASC, pro-caspase-1, activated caspase-1and maturation of IL-1β were higher in DM than control rats (p<0.05-p<0.01). Similarly, the mRNA and protein levels of TXNIP, NLRP3inflammasome and IL-1β were higher in HF than control rats (p<0.05-p<0.01).RSV15mg/kg suppressed the increased mRNA levels of TXNIP, NLRP3inflammasome and IL-1β induced by diabetes (p<0.05-p<0.01). The mRNA levels of TXNIP and IL-1β were lower with DM+RSV10mg/kg than DM alone (both p<0.05). Except for caspase-1mRNA, similar reduced levels were observed in HF with RSV treatment than HF alone and control group (p<0.05-p<0.01). Protein levels of TXNIP, NLPR3inflammasome and mature IL-1β were lower with DM+RSV15mg/kg than DM alone (p<0.05-p<0.01). DM+RSV10mg/kg only decreased pro-caspase-1protein level as compared with DM alone (p<0.05). In addition, DM+RSV15mg/kg decreased protein levels of TXNIP and ASC, total and activated caspase-1, and activated IL-1β as compared with DM+RSV10mg/kg (all p<0.01). Similar results were observed in RSV-treated HF groups (p<0.05-p<0.01).RSV inhibited the phosphorylation of MAPK signaling pathwaysWe further investigated the activation of MAPK signaling pathways. DM rats showed the greatest activation of ERK1/2, p38and JNK (p<0.05-p<0.01). The HF group showed increased phosphorylation of MAPKs as compared with control group (p<0.05-p<0.01).The phosphorylation of MAPKs was suppressed in RSV-treated DM rats as compared with DM alone (p<0.05-p<0.01), and RSV15mg/kg was better than10mg/kg in inhibiting ERK1/2phosphorylation (p<0.01). Similar results were found in RSV-treated HF rats (p<0.05-p<0.01).Detection of cardiac NLRP3expression with gene silencingAfter8weeks of NLRP3silencing treatment, transfection efficiency was checked in all groups. As compared with vehicle treatment, NLRP3-miRNA treatment decreased the mRNA and protein levels of cardiac NLRP3and mature IL-1β (p<0.05-p<0.01).RSV alleviated diabetes-induced cardiac dysfunction by inhibiting NLRP3expressionAfter8weeks of NLRP3-miRNA treatment, LVEF, FS, E/A and E’/A’were improved in the DM rats as compared with vehicle treatment (all p<0.05). NLRP3miRNA treatment increased E/A and E’/A’in DM+RSV15mg/kg rats as compared with vehicle treatment (both p<0.05).In vehicle-treated rats, diabetes-induced severe systolic and diastolic dysfunction was ameliorated with RSV supplementation (all p<0.05). The cardioprotective effect of RSV in diabetes was abrogated more in NLRP3-miRNA-treated rats than vehicle-treated rats.RSV reversed diabetes-induced myocardial disorder with inhibited NLRP3 In DM rats, NLRP3-miRNA treatment reduced the heart weight to body weight ratio, interstitial fibrosis, collagen mRNA expression, and collagen I to collage... |
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