Relative risk by quartiles of serum Mg level according to different clinical presentation.

<p>95% CI, 95% confidence interval.</p><p>*<i>P</i><0.05, **<i>P</i><0.01.</p><p># adjusted for age, positive family history, smoking status, hypertension, hypercholesterolemia, and diabetes at baseline.</p

Factors of major adverse cardiac events (MACEs) and relative risk by quartiles of serum Mg level.

<p>PCI, percutaneous coronary intervention; 95% CI, 95% confidence interval.</p><p>*<i>P</i><0.05.</p><p># adjusted for positive family history, smoking status, hypertension, hypercholesterolemia, and diabetes at baseline.</p

Clinical characteristics of patients with acute coronary syndrome by quartiles of serum Mg level.

<p>Data are no. (%) unless indicated.</p

Risk factors of coronary artery disease by quartile (Q) of serum magnesium (Mg) level.

<p>BMI, body mass index; CAD, coronary artery disease.</p><p>Data are no. (%) unless indicated.</p

Cumulative major adverse cardiac event-free survival in 414 patients by quartiles of serum magnesium level.

<p>Cumulative major adverse cardiac event-free survival in 414 patients by quartiles of serum magnesium level.</p

NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy in a Type 2 Diabetes Rat Model

<div><p>Background</p><p>Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is associated with metabolic disorder and cell death, which are important triggers in diabetic cardiomyopathy (DCM). We aimed to explore whether NLRP3 inflammasome activation contributes to DCM and the mechanism involved.</p><p>Methods</p><p>Type 2 diabetic rat model was induced by high fat diet and low dose streptozotocin. The characteristics of type 2 DCM were evaluated by metabolic tests, echocardiography and histopathology. Gene silencing therapy was used to investigate the role of NLRP3 in the pathogenesis of DCM. High glucose treated H9c2 cardiomyocytes were used to determine the mechanism by which NLRP3 modulated the DCM. The cell death in vitro was detected by TUNEL and EthD-III staining. TXNIP-siRNA and pharmacological inhibitors of ROS and NF-kB were used to explore the mechanism of NLRP3 inflammasome activation.</p><p>Results</p><p>Diabetic rats showed severe metabolic disorder, cardiac inflammation, cell death, disorganized ultrastructure, fibrosis and excessive activation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), pro-caspase-1, activated caspase-1 and mature interleukin-1β (IL-1β). Evidence for pyroptosis was found <i>in vivo</i>, and the caspase-1 dependent pyroptosis was found <i>in vitro</i>. Silencing of NLRP3 <i>in vivo</i> did not attenuate systemic metabolic disturbances. However, NLRP3 gene silencing therapy ameliorated cardiac inflammation, pyroptosis, fibrosis and cardiac function. Silencing of NLRP3 in H9c2 cardiomyocytes suppressed pyroptosis under high glucose. ROS inhibition markedly decreased nuclear factor-kB (NF-kB) phosphorylation, thioredoxin interacting/inhibiting protein (TXNIP), NLRP3 inflammasome, and mature IL-1β in high glucose treated H9c2 cells. Inhibition of NF-kB reduced the activation of NLRP3 inflammasome. TXNIP-siRNA decreased the activation of caspase-1 and IL-1β.</p><p>Conclusion</p><p>NLRP3 inflammasome contributed to the development of DCM. NF-κB and TXNIP mediated the ROS-induced caspase-1 and IL-1β activation, which are the effectors of NLRP3 inflammasome. NLRP3 gene silencing may exert a protective effect on DCM.</p></div

Primer sequences for real-time RT-PCR.

<p>TXNIP, thioredoxin interacting/inhibiting protein; NLRP3, Nucleotide-binding oligomerization domain-like receptor protein 3; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; IL-1β, interleukin-1β.</p

ROS induced NLRP3 inflammsome activation.

<p>(A) Quantitative analysis of the ROS production in H9c2 cells treated with medium and high glucose. (B–D) Western blot analysis of pNF-kB and TXNIP. (E) The production of cellular ROS after NAC treatment. The protein levels of pNF-kB (F and G), TXNIP (G and H), NLRP3 (I and J), ASC (I and K), pro-caspase-1 (I and L), activated caspase-1(I and M), pro-IL-1β (I and N) and mature IL-1β (I and O). Data were presented as means±SEM, from 3 independent experiments. **p<0.01 vs. Ctrl; ^##p<0.01 vs. MG; ^§§p<0.01 vs. HG+vehicle.</p

NLRP3 gene silencing improved cardiac dysfunction in diabetes rats.

<p>(A) The cardiac function of rats in different groups shown by echocardiography. Representative images of 2D echocardiograms (A1), M-mode echocardiograms (A2), pulse-wave Doppler echocardiograms of mitral inflow (A3), tissue Doppler echocardiograms (A4). (B–F) Evaluation of LVEDd, LVEF, FS, E/A and E′/A′. Data were presented as means±SEM, n = 6. **p<0.01 vs. Ctrl; ^§p<0.05, ^§§p<0.01 vs. DM+vehicle. LV: left ventricle; IVS: interventricular septum; LVEDd: left ventricular end-diastolic dimension; LVEF: left ventricular ejection fraction; FS: fractional shortening; E/A: peak E to peak A ratio; E′/A′: early (E′) to late (A′) diastolic velocity ratio.</p

NLRP3 gene silencing suppressed activated caspase-1, inflammatory cytokines, and pyroptosis in high glucose-treated H9c2 cells.

<p>Relative mRNA expression of NLRP3 (A) and protein levels of NLRP3, pro-caspase-1, activated caspase-1, pro-IL-1β and mature IL-1β (B–G) in H9c2 cardiomyocytes with treatment. (H–K) The protein levels of IL-1β, IL-18, TNF-α and IL-6 in the cultured supernatants of H9c2 cardiomyocytes. (L) TUNEL staining of H9c2 cells, positive cell indicated by black arrow; scale bar: 20 µm. (M) Quantitative analysis of TUNEL positive cell in each group. (O) EthD-III (red) staining; scale bar: 50 µm. (N) Quantitative analysis of EthD-III positive cell. Data were presented as means±SEM, from 3 independent experiments. *p<0.05, **p<0.01 vs. Ctrl; ^§p<0.05, ^§§p<0.01 vs. HG+vehicle.</p