Elsevier

Brain, Behavior, and Immunity

Volume 91, January 2021, Pages 587-600
Brain, Behavior, and Immunity

INT-777 attenuates NLRP3-ASC inflammasome-mediated neuroinflammation via TGR5/cAMP/PKA signaling pathway after subarachnoid hemorrhage in rats

https://doi.org/10.1016/j.bbi.2020.09.016Get rights and content

Highlights

Abstract

Background

Inflammasome-mediated neuroinflammation plays an important role in the pathogenesis of early brain injury (EBI) following subarachnoid hemorrhage (SAH). The activation of the TGR5 receptor has been shown to be neuroprotective in a variety of neurological diseases. This study aimed to investigate the effects of the specific synthetic TGR5 agonist, INT-777, in attenuating NLRP3-ASC inflammasome activation and reducing neuroinflammation after SAH.

Methods

One hundred and eighty-four male Sprague Dawley rats were used. SAH was induced by the endovascular perforation. INT-777 was administered intranasally at 1 h after SAH induction. To elucidate the signaling pathway involved in the effect of INT-777 on inflammasome activation during EBI, TGR5 knockout CRISPR and PKA inhibitor H89 were administered intracerebroventricularly and intraperitoneally at 48 h and 1 h before SAH. The SAH grade, short- and long-term neurobehavioral assessments, brain water content, western blot, immunofluorescence staining, and Nissl staining were performed.

Results

The expressions of endogenous TGR5, p-PKA, and NLRP3-ASC inflammasome were increased after SAH. INT-777 administration significantly decreased NLRP3-ASC inflammasome activation in microglia, reduced brain edema and neuroinflammation, leading to improved short-term neurobehavioral functions at 24 h after SAH. The administration of TGR5 CRISPR or PKA inhibitor (H89) abolished the anti-inflammation effects of INT-777, on NLRP3-ASC inflammasome, pro-inflammatory cytokines (IL-6, IL-1β, and TNF-a), and neutrophil infiltration at 24 h after SAH. Moreover, early administration of INT-777 attenuated neuronal degeneration in hippocampus on 28 d after SAH.

Conclusions

INT-777 attenuated NLRP3-ASC inflammasome-dependent neuroinflammation in the EBI after SAH, partially via TGR5/cAMP/PKA signaling pathway. Early administration of INT-777 may serve as a potential therapeutic strategy for EBI management in the setting of SAH.

Introduction

Aneurysmal subarachnoid hemorrhage (SAH) is a life-threatening hemorrhagic stroke, accounting for 5–10% of all stroke types, with a mortality and disability rate of 25–50% (Lawton and Vates, 2017, Kamp et al., 2020). Early brain injury (EBI) begins immediately after an aneurysm ruptures, and lasts up to 72 h; EBI has been proven to be the primary cause of poor outcomes after SAH (Cahill et al., 2006, Peng et al., 2019a).

Over the past few decades, researchers have been elucidated the pathological mechanisms underlying EBI including sharply elevated intracranial pressure, decreased cerebral perfusion pressure and cerebral blood flow as well as cerebral vasospasm, all of which consecutively initiate various of cascades of pathophysiological events such as oxidative stress, neuroinflammation, blood–brain barrier (BBB) dysfunction, and apoptosis (Conzen et al., 2019, Koseki et al., 2020). SAH-induced neuroinflammation has been considered one of the key devastating pathophysiological processes in EBI induced by SAH (Pang et al., 2018, Suzuki, 2019). Thus, therapeutically targeting neuroinflammation would attenuate EBI and benefit neurological outcomes in the setting of SAH.

Previous studies have demonstrated that the brain’s residual microglial/macrophage activation and neutrophil infiltration releases massive pro-inflammatory cytokines which would magnify the inflammatory responses and further aggravate the neurobehavioral deficits and delayed cognitive dysfunctions (Zuo et al., 2019b, Xu et al., 2020). While microgliosis following neuronal damage is a normal physiologic response to injury, however, persistent and self-propagating microgliosis can result in sustained neurological dysfunctions (Alawieh et al., 2018, Alawieh et al., 2020, Figueiredo et al., 2019). The selective polarization of microglia toward anti-inflammatory phenotype attenuated neuroinflammatory response and white matter injury in EBI after SAH (Peng et al., 2019a, Zheng et al., 2020).

Inflammasomes are a part of the innate immune system, and plays a central role in microglia activation, which involves multimeric protein complexes sensing various environmental and cellular stress signals (Freeman et al., 2017, Luo et al., 2019). The NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome activation leads to oligomerization, which can trigger the helical fibrillar assembly of a downstream adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). ASC results in caspase1 activation and promotes pro-interleukin-1 beta (pro-IL-1β) processing and release of the mature cytokine IL-1β (Latz et al., 2013, Broz and Dixit, 2016). NLRP3–ASC inflammasome activation plays a central role in microglia activation that induces an increase in cytokine and chemokine concentrations (Heneka et al., 2018, Stancu et al., 2019). Microglial NLRP3-ASC inflammasome activation exacerbates Tau‐associated neuroinflammatory changes in Alzheimer’s disease (AD) (Stancu et al., 2019). The inhibition of NLRP3–ASC inflammasome reduces the innate immune response ameliorates neuroinflammation, attenuates neuronal pyroptosis, and delays neuronal degeneration in animal models of diabetic cardiomyopathy (Ye et al., 2017), AD (Venegas et al., 2017, Stancu et al., 2019), cerebellar ataxias (Kojic et al., 2018), neurodegenerative disorders (Gordon et al., 2018), intracerebral hemorrhage (Yang et al., 2018), and ischemic stroke (She et al., 2019). Furthermore, the activation of NLRP3 inflammasome has been demonstrated to take part in neuronal apoptosis following SAH (Dong et al., 2016). The signaling pathway that suppressed NLRP3 inflammasome activation could promote neurogenesis after SAH (Zuo et al., 2018). However, there is currently no research exerting the role of TGR5 targeting the NLRP3-ASC inflammasome‐mediated neuroinflammation in EBI after SAH.

Trans-membrane G protein-coupled receptor-5 (TGR5), is a novel membrane-bound bile acid receptor found in the gastrointestinal tract and in immune cells; has pleiotropic actions in metabolic diseases and immunomodulation (Guo et al., 2016, Shapiro et al., 2018). The expression of TGR5 was also reported in neurons, astrocytes, and microglia cells (Keitel et al., 2010). Recent studies have demonstrated that the TGR5 activation reduces BBB breakdown after ischemic stroke (Liang et al., 2020a), alleviates microglial activation in hepatic encephalopathy (McMillin et al., 2015) and acute brain injury (Yanguas-Casas et al., 2017), as well as provides a beneficial effect against neuronal apoptosis and neuroinflammation in an animal model of AD (Wu et al., 2018). On the contrary, the reduction of endogenous TRG5 expressions exacerbated neuroinflammation associated with microglial activation and reduced postsynaptic plasticity (Jena et al., 2018). INT-777, as a novel specific semisynthetic TGR5 agonist, has been shown to alleviate cardiomyocyte injury by inhibiting inflammatory responses and oxidative stress (Deng et al., 2019), attenuated NLRP3 inflammasome‐mediated inflammatory bowel diseases (Chen et al., 2019b), mitigated impaired urinary concentration in lithium-induced nephrogenic diabetes insipidus (Wang et al., 2016), and improved cognitive impairment and synaptic dysfunction in mice model of AD (Wu et al., 2018). In our previous study, we have shown that activation of the TGR5 receptor with INT-777 attenuated oxidative stress and neuronal apoptosis in a rat model of endovascular perforation model of SAH (Zuo et al., 2019a). Specifically, INT-777 administration significantly increased intracellular cAMP levels and the phosphorylation of PKCε (Zuo et al., 2019a). An elevation of intracellular cAMP levels can result in the phosphorylation of PKA and further induced the phosphorylation and the ubiquitination of NLRP3, which blocks NLRP3 inflammasome-dependent inflammation and NLRP3 inflammasome-related metabolic disorders (Guo et al., 2016). The inhibition of cAMP or PKA increases bone marrow-derived macrophages-mediated inflammation (Song et al., 2018).

In this present study, we hypothesized that INT-777 would attenuate the neurological deficits and suppress NLRP3‐ASC inflammasome dependent neuroinflammation via TGR5/cAMP/PKA signaling pathway in EBI after SAH. Moreover, these beneficial effects of INT-777-attenuated EBI improved delayed neurological impairments after experimental SAH (shown in Fig. s1).

Section snippets

Animals

Adult male Sprague-Dawley rats (n = 184; weighting = 280–320 g; Harlan, Indianapolis, USA) were used in this study. All animals were housed in a room with controlled humidity (60 ± 5%) and temperature (25 ± 1 °C), under a 12 h light and dark cycle with libitum access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Loma Linda University, which comply with the National Institutes of Health Guidelines for the Care and Use of

Expression levels of endogenous TGR5, p-PKA, NLRP3, ASC and cellular locations of TGR5 after SAH

The protein levels of endogenous TGR5, p-PKA, NLRP3, and ASC in ipsilateral (left) hemisphere among groups of sham, 3 h, 6 h, 12 h, 24 h, and 72 h after SAH were measured by western blot. The results showed that the expression of endogenous TGR5, p-PKA, NLRP3, and ASC increased in a time-dependent manner, and peaked at 24 h after SAH (P < 0.05 compared with sham group, Fig. 1A-E).

Double immunofluorescence staining of TGR5 receptors with microglia (Iba-1), neurons (NeuN), and astrocytes (GFAP)

Discussion

EBI describes the immediate injury to the brain after SAH before onset of delayed vasospasm. During the EBI period, a ruptured aneurysm brings on many physiological derangements such as elevated intracranial pressure (ICP), decreased cerebral blood flow (CBF), and global cerebral ischemia. These events initiate secondary injuries such as blood–brain barrier disruption, neuroinflammation, and oxidative cascades that all ultimately lead to cell death (Fujii et al., 2013). High intracranial

Conclusions

Our results showed that INT-777 administration suppressed NLRP3‐ASC inflammasome-mediated neuroinflammation and improved neurological deficits partially through activating the TGR5/cAMP/PKA signaling pathway in a rat model of SAH. Early administration of INT-777 may be a therapeutic and preventive strategy against delayed hippocampal injury after SAH due to its attenuation of EBI.

Acknowledgements

Not applicable.

Authors’ contributions

XH, JY, LH, GZ, and JHZ participated in the experimental design, data interpretation and analysis. GZ, XH, and JY performed the SAH surgeries, western blotting, immunofluorescence staining, Nissl staining, and neurobehavioral test. XH, YJ, and JP collected and analyzed the data and GZ drafted the manuscript. LH and CA revised the paper and proofread the language. All authors read and approved the final manuscript.

Funding

This study was supported by grants from the National Institutes of Health NS081740 and NS 082184 to John H. Zhang, grants QNRC2016263, H201654, and GSWS2019080 from Jiangsu provincial health and family planning commission of China to Dr. G. Zuo, and grants [2017]5631, [2018]5764, [2017]5724 from Guizhou Provincial Science and Technology Platform and Talent Team Project of China to Dr. X. Hu.

Ethics approval and consent to participate

All animal experiments performed in this study were accrodance with the National Institutes of Health Guide for the Care and Use of Laboratory Aniamls which was approved by Loma Linda Univesity Institutional Animal Care and Use Committee.

Consent for publication

Not applicable.

Conflict of Interest

The authors declare no conflict of interest.

Availability of data and materials

The data support the findings of this study and are available from the corresponding author upon reasonable request.

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    Xiao Hu and Jun Yan contributed equally to this work.

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