Lipopolysaccharide (LPS) Aggravates High Glucose- and Hypoxia/Reoxygenation-Induced Injury through Activating ROS-Dependent NLRP3 Inflammasome-Mediated Pyroptosis in H9C2 Cardiomyocytes

Abstract

Diabetes aggravates myocardial ischemia-reperfusion (I/R) injury because of the combination effects of changes in glucose and lipid energy metabolism, oxidative stress, and systemic inflammatory response. Studies have indicated that myocardial I/R may coincide and interact with sepsis and inflammation. However, the role of LPS in hypoxia/reoxygenation (H/R) injury in cardiomyocytes under high glucose conditions is still unclear. Our objective was to examine whether lipopolysaccharide (LPS) could aggravate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by upregulating ROS production to activate NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes. H9C2 cardiomyocytes were exposed to HG (30 mM) condition with or without LPS, along with caspase-1 inhibitor (Ac-YVAD-CMK), inflammasome inhibitor (BAY11-7082), ROS scavenger N-acetylcysteine (NAC), or not for 24 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). The cell viability, lactate dehydrogenase (LDH) release, caspase-1 activity, and intracellular ROS production were detected by using assay kits. The incidence of pyroptosis was detected by calcein-AM/propidium iodide (PI) double staining kit. The concentrations of IL-1β and IL-18 in the supernatants were assessed by ELISA. The mRNA levels of NLRP3, ASC, and caspase-1 were detected by qRT-PCR. The protein levels of NF-κB p65, NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18 were detected by western blot. The results indicated that pretreatment LPS with 1 μg/ml not 0.1 μg/ml could efficiently aggravate HG and H/R injury by activating NLRP3 inflammasome to mediate pyroptosis in H9C2 cells, as evidenced by increased LDH release and decreased cell viability in the cells, and increased expression of NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18. Meanwhile, Ac-YVAD-CMK, BAY11-7082, or NAC attenuated HG- and H/R-induced H9C2 cell injury with LPS stimulated by reversing the activation of NLRP3 inflammasome-mediated pyroptosis. In conclusion, LPS could increase the sensitivity of H9C2 cells to HG and H/R and aggravated HG- and H/R-induced H9C2 cell injury by promoting ROS production to induce NLRP3 inflammasome-mediated pyroptosis.

Nucleotide-Binding Oligomerization Domain-Like Receptor 3 Deficiency Attenuated Isoproterenol-Induced Cardiac Fibrosis via Reactive Oxygen Species/High Mobility Group Box 1 Protein Axis

“…In the present study, we found that NLRP3 overexpression increased the ROS production, and NLRP3 knockdown decreased the ROS production in NRVMs stimulated by ISO, which indicated that NLRP3 could regulate the generation of ROS. However, most of the previous studies have considered that ROS is a redox signaling molecule that could trigger NLRP3 inflammasome activation in the heart ( Minutoli et al, 2016 ; Zhang H. et al, 2018 ; Zhang X. et al, 2018 ; Qiu et al, 2019 ), which were contradictory with our study. In fact, ROS may act as not only a triggering factor to activate NLRP3 inflammasomes but also an effector molecule ( Abais et al, 2015 ); early ROS generation may be a signaling mechanism to activate the formation and activation of NLRP3 due to its action on thioredoxin-interacting protein (TXNIP) binding ( Abais et al, 2014 ) but may not yet have major damage to cell function or activity.…”

Clemastine Fumarate Protects Against Myocardial Ischemia Reperfusion Injury by Activating the TLR4/PI3K/Akt Signaling Pathway

“…When myocardial ischemia-reperfusion injury occurs, the expression of pro-inflammatory factors increased, mainly due to the increased release of TNF-a and IL-1b (Qiu et al, 2019;Xu et al, 2019). CLE can inhibit the release of inflammatory factors such as TNF-a and IL-1b, revers the inflammatory response of MIRI.…”

N‑acetyl cysteine inhibits the lipopolysaccharide‑induced inflammatory response in bone marrow mesenchymal stem cells by suppressing the TXNIP/NLRP3/IL‑1β signaling pathway

“…By contrast, less attention has been paid to the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in studies on LPS-induced inflammation. LPS is widely known to stimulate the NLRP3 inflammasome in different cell types (16)(17)(18). Additionally, the NLRP3 inflammasome serves a significant role in LPS-mediated inflammation, as its upregulation is conducive…”