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

Date Published: August 19, 2014

Publisher: Public Library of Science

Author(s): Beibei Luo, Bo Li, Wenke Wang, Xiangjuan Liu, Yanfei Xia, Cheng Zhang, Mingxiang Zhang, Yun Zhang, Fengshuang An, Philip Michael Bauer.

http://doi.org/10.1371/journal.pone.0104771

Abstract

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.

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.

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 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.

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.

Partial Text

Diabetic cardiomyopathy (DCM), characterized by consistent diastolic dysfunction and increased ventricular mass, is the leading cause of mortality among patients with diabetes [1], [2]. Hyperglycemia-induced reactive oxygen species (ROS) generation is considered to be responsible for progression and development of DCM [3], [4]. 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 [5], [6], [7]. Although inflammasome was shown to be involved in the pathogenic mechanisms of type 2 diabetes and its complications [8], [9], the potential role and regulatory mechanism of inflammasome in DCM has remained largely unexplored.

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-1β-related metabolic disorder and caspase-1-mediated pyroptosis in myocardium. In addition, we found that the NF-kB and TXNIP were involved in the ROS-induced NLRP3 inflammasome activation. Thus, NLRP3 inflammasome has a pivotal role in DCM and could be an attractive drug target for treating type 2 diabetes.

Our results suggest that the NLRP3 inflammasome is implicated in DCM. The activation of NLRP3 inflammasome and its effectors was regulated by NF-kB and TXNIP. Caspase-1 dependent pyroptosis had a distinct role in the process of DCM. NLRP3 gene silencing ameliorated the development of DCM in type 2 diabetic rat, by reducing cardiac inflammation, cardiomyocyte pyroptosis and left ventricular fibrosis. The cardioprotective effects with NLRP3 silencing suggest a potential role for NLRP3 antagonists in treating DCM in type 2 diabetes.

 

Source:

http://doi.org/10.1371/journal.pone.0104771