Role of G protein‐coupled receptor kinase 2 in oxidative and nitrosative stress‐related neurohistopathological changes in a mouse model of sepsis‐associated encephalopathy

Abstract

Sepsis-associated encephalopathy (SAE), characterized as diffuse brain dysfunction and neurological manifestations secondary to sepsis, is a common complication in critically ill patients and can give rise to poor outcome, but understanding the molecular basis of this disorder remains a major challenge. Given the emerging role of G protein-coupled receptor 2 (GRK2), first identified as a G protein-coupled receptor (GPCR) regulator, in the regulation of non-G protein-coupled receptor-related molecules contributing to diverse cellular functions and pathology, including inflammation, we tested the hypothesis that GRK2 may be linked to the neuropathogenesis of SAE. When mouse MG6 microglial cells were challenged with lipopolysaccharide (LPS), GRK2 cytosolic expression was highly up-regulated. The ablation of GRK2 by small interfering RNAs (siRNAs) prevented an increase in intracellular reactive oxygen species generation in LPS-stimulated MG6 cells. Furthermore, the LPS-induced up-regulation of inducible nitric-oxide synthase expression and increase in nitric oxide production were negated by GRK2 inhibitor or siRNAs. However, GRK2 inhibition was without effect on overproduction of tumor necrosis factor-α, interleukin (IL)-6, and IL-1β in LPS-stimulated MG cells. In mice with cecal ligation and puncture-induced sepsis, treatment with GRK2 inhibitor reduced high levels of oxidative and nitrosative stress in the mice brains, where GRK2 expression was up-regulated, alleviated neurohistological damage observed in cerebral cortex sections, and conferred a significant survival advantage to CLP mice. Altogether, these results uncover the novel role for GRK2 in regulating cellular oxidative and nitrosative stress during inflammation and suggest that GRK2 may have a potential as an intriguing therapeutic target to prevent or treat SAE.

Editorial: G Protein-Coupled Receptor Kinases (GRKs) and β-Arrestins: New Insights Into Disease Regulators

“…They revealed that GRK2 strongly regulated the expression/activation of IRF1as well as the activation of the STAT pathway, leading to increased transcription of iNOS, when TLR-3, TLR-4, or TLR-9 was stimulated in microglial cells. This extends their previous report showing that GRK2 plays a critical role in iNOS gene t r a n s c r ip t i o n i n m i c r o g l i a l c e l l s s t im u l a t e d wi t h lipopolysaccharide (Kawakami et al, 2018). Their findings highlight a novel pathological role of GRK2 in regulating inflammatory signaling in microglia with a potential therapeutic window for some neuroinflammatory disorders.…”

Vascular endothelial growth factor contributes to lung vascular hyperpermeability in sepsis-associated acute lung injury

“…CLP-induced sepsis is regarded as a highly clinically relevant model of polymicrobial sepsis, because it reproduces many hallmarks of sepsis occurring in human patients (Hubbard et al 2005 ). The surgical procedure to generate CLP-induced sepsis was conducted according to our previous reports (Tomita et al 2015 ; Kawakami et al 2018 ; Yamashita et al 2018 ). In brief, male BALB/c mice (Sankyo Lab Service, Tokyo Japan), 8–12 weeks old, were anesthetized with 3–4% sevoflurane by inhalation, and a middle abdominal incision was made.…”

“…Values were normalized to the housekeeping gene GAPDH according to the manufacturer’s protocol (MX3000P real-time PCR system; Agilent Technologies Inc., Santa Clara, CA, USA). Additional details are described by our laboratory (Kawakami et al 2018 ; Yamashita et al 2018 ; Suzuki et al 2018 ). The PCR primers were designed as follows: forward 5’-TGCAGATTATGCGGATCAAACC-3′ and reverse 5′-TGCATTCACATTTGTTGTGCTGTAG-3′ for VEGF, forward 5′-CAGGCCCAGTTTCTGCCATT-3′ and reverse 5′-TTCCAGCTCAGCGTGGTCGTA-3′ for VEGFR1, forward 5′-CCAGCAAAAGCAGGGAGTCTGT-3′ and reverse 5′-TGTCTGTGTCATCGGAGTGATATCC-3′ for VEGFR2, and forward 5′-TGTGTCCGTCGTGGATCTGA-3′ and reverse 5′-TTGCTGTTGAAGTCGCAGGAG-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…”

Effect of incubation with lipopolysaccharide and interferon-γ on reactive astrogliosis

“…TNF-α and IL-6 are early indicators of sepsis (Kruttgen and Rose-John, 2012), and their concentrations are associated with outcomes in patients with sepsis. NO, and ROS play key roles in the pathogenesis of sepsis and also indicate oxidative stress and nitric stress in SAE, respectively (Kawakami et al, 2018;Neri et al, 2016). The results of this study showed that the concentrations of these indicators increased in the test groups compared with the control group.…”