NLRP3 gene silencing ameliorates diabetic cardiomyopathy in a type 2 diabetes rat model.

Beibei Luo,Yanfei Xia,Yun Zhang,Fengshuang An,Bo Li,Mingxiang Zhang,Wenke Wang,Cheng Zhang,Xiangjuan Liu,Philip Michael Bauer

doi:10.1371/journal.pone.0104771

Abstract

BackgroundNucleotide-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.MethodsType 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.ResultsDiabetic 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 in vivo, and the caspase-1 dependent pyroptosis was found in vitro. Silencing of NLRP3 in vivo 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.ConclusionNLRP3 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.

Highlights

Diabetic cardiomyopathy (DCM), characterized by consistent diastolic dysfunction and increased ventricular mass, is the leading cause of mortality among patients with diabetes [1,2]
Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome and IL-1b were activated in diabetic cardiomyopathy (DCM) When compared with the control rats, diabetic rats showed elevated mRNA and protein levels of NLRP3, associated speck like protein (ASC), caspase-1 and IL-1b beginning at 4 or 8 weeks of diabetes (Fig. 2A–C, all p, 0.01)
The present study provided new evidence to show the direct contribution of NLRP3 inflammasome in the pathogenesis of DCM, which was associated with the IL-1b-related metabolic disorder and caspase-1-mediated pyroptosis in myocardium

Summary

Introduction

Diabetic cardiomyopathy (DCM), characterized by consistent diastolic dysfunction and increased ventricular mass, is the leading cause of mortality among patients with diabetes [1,2]. The increased ROS could induce a number of cytokine and inflammatory factors, such as nuclear factor-kB (NFkB), thioredoxin interacting/inhibiting protein (TXNIP), and inflammasome [5,6,7]. Different inflammasomes have been identified, including nucleotide-binding oligomerization domain-like receptors (NLRs) and absent in melanoma 2 (AIM2) [12]. NLRP3, the most extensively studied NLRs, forms a complexes comprised of the apoptosis associated speck like protein (ASC), and the serine protease caspase-1 [13]. The activated caspase-1 can process pro-IL-1b into its mature form, which is important in cardiomyocyte apoptosis [11,14]. 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

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Discussion

Conclusion