Abstract
BackgroundSecondary brain damage caused by the innate immune response and subsequent proinflammatory factor production is a major factor contributing to the high mortality of intracerebral haemorrhage (ICH). Nucleotide-binding oligomerization domain 1 (NOD1)/receptor-interacting protein 2 (RIP2) signalling has been reported to participate in the innate immune response and inflammatory response. Therefore, we investigated the role of NOD1/RIP2 signalling in mice with collagenase-induced ICH and in cultured primary microglia challenged with hemin.MethodsAdult male C57BL/6 mice were subjected to collagenase for induction of ICH model in vivo. Cultured primary microglia and BV2 microglial cells (microglial cell line) challenged with hemin aimed to simulate the ICH model in vitro. We first defined the expression of NOD1 and RIP2 in vivo and in vitro using an ICH model by western blotting. The effect of NOD1/RIP2 signalling on ICH-induced brain injury volume, neurological deficits, brain oedema, and microglial activation were assessed following intraventricular injection of either ML130 (a NOD1 inhibitor) or GSK583 (a RIP2 inhibitor). In addition, levels of JNK/P38 MAPK, IκBα, and inflammatory factors, including tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, and inducible nitric oxide synthase (iNOS) expression, were analysed in ICH-challenged brain and hemin-exposed cultured primary microglia by western blotting. Finally, we investigated whether the inflammatory factors could undergo crosstalk with NOD1 and RIP2.ResultsThe levels of NOD1 and its adaptor RIP2 were significantly elevated in the brains of mice in response to ICH and in cultured primary microglia, BV2 cells challenged with hemin. Administration of either a NOD1 or RIP2 inhibitor in mice with ICH prevented microglial activation and neuroinflammation, followed by alleviation of ICH-induced brain damage. Interestingly, the inflammatory factors interleukin (IL)-1β and tumour necrosis factor-α (TNF-α), which were enhanced by NOD1/RIP2 signalling, were found to contribute to the NOD1 and RIP2 upregulation in our study.ConclusionNOD1/RIP2 signalling played an important role in the regulation of the inflammatory response during ICH. In addition, a vicious feedback cycle was observed between NOD1/RIP2 and IL-1β/TNF-α, which could to some extent result in sustained brain damage during ICH. Hence, our study highlights NOD1/RIP2 signalling as a potential therapeutic target to protect the brain against secondary brain damage during ICH.
Highlights
Given the high morbidity and mortality of intracerebral haemorrhage (ICH) [1], concerns about identifying an effective treatment for ICH have been increasing
Nucleotide-binding oligomerization domain 1 (NOD1) and receptor-interacting protein 2 (RIP2) expression displayed dramatic increases following ICH To verify whether NOD1/RIP2 signalling contributed to haemorrhage-associated brain injury, we first investigated the expression of NOD1 and RIP2 in response to ICH
Western blot analysis showed that NOD1 and RIP2 expression was barely detectable within the sham brain, whereas significant overproduction was observed at 1 day following ICH surgery, with a maximum at 3 days and a gradual decrease thereafter (Fig. 2a–b)
Summary
Given the high morbidity and mortality of intracerebral haemorrhage (ICH) [1], concerns about identifying an effective treatment for ICH have been increasing. Accumulating evidence has demonstrated that the second wave of innate immune-induced inflammatory injury, rather than direct damage caused by hematoma-mediated compression, is the main contributor to a series of damaging events following ICH [2,3,4], including brain oedema, BBB damage, cell death, severe neurological dysfunctions, and even death. Secondary neuroinflammation is characterized by an innate immune response and subsequent inflammatory cytokine production. Microglia act as a major innate immune cell in the central nervous system (CNS) and can be induced to transition from the resting form to the activated form after sensing brain tissue injury [7, 8]. Secondary brain damage caused by the innate immune response and subsequent proinflammatory factor production is a major factor contributing to the high mortality of intracerebral haemorrhage (ICH). Nucleotide-binding oligomerization domain 1 (NOD1)/receptor-interacting protein 2 (RIP2) signalling has been reported to participate in the innate immune response and inflammatory response. We investigated the role of NOD1/RIP2 signalling in mice with collagenase-induced ICH and in cultured primary microglia challenged with hemin
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