Inflammasome Activation Regulates Apoptosis and Pyroptosis in Astrocytes under Ischemic Conditions

Introduction

Ischemic stroke is one of the main mechanisms leading to brain damage, characterized by hypoxia and energy deprivation in local brain regions due to interrupted blood flow. In recent years, research has found that inflammatory responses play a crucial role in ischemic stroke. Inflammasomes are intracellular multiprotein complexes that drive inflammatory responses mainly by activating pro-inflammatory caspases and promoting the maturation and secretion of pro-inflammatory cytokines such as IL-1β and IL-18. Currently, numerous studies have identified the role of inflammasomes in neuronal death and microglial activation and cell death caused by ischemic stroke, but the regulatory mechanisms in astrocytes are still poorly understood.

Research Background and Motivation

In the central nervous system (CNS), astrocytes are the most abundant type of glial cells, mainly divided into fibrous astrocytes and protoplasmic astrocytes. Fibrous astrocytes are mainly located in the white matter of the brain and spinal cord, responsible for regulating the extracellular environment around axons; while protoplasmic astrocytes are mainly located in the gray matter, providing metabolic support for neurons. Under pathological conditions such as hypoxia and ischemia, the activation, functional abnormalities, and death of astrocytes accelerate conduction damage and the expansion of the infarct core. Reactive astrocyte proliferation (astrogliosis) is a typical response of astrocytes to ischemic damage, manifested as changes in morphology and function, as well as increased expression of glial fibrillary acidic protein (GFAP) and inflammatory markers. However, there is a lack of research on the expression and activation of inflammasomes in astrocytes under ischemic conditions.

This article was written by a multi-unit research team led by Lap Jack Wong, including scientists from the National University of Singapore, La Trobe University, and Sungkyunkwan University. The paper was published online in the journal “Neuromolecular Medicine” on August 30, 2023.

Research Methods

Research Subjects and Experimental Design

The research team selected protoplasmic astrocyte cell line (C8-D30) and fibrous astrocyte cell line (C8-D1a) from mice. These cells were cultured in specific culture media, and when the cells reached 80-90% confluence, they were exposed to oxygen-glucose deprivation (OGD) conditions to simulate an ischemic environment.

By replenishing glucose-free Locke’s buffer and placing the cells in an atmosphere of 95% nitrogen and 5% carbon dioxide, the cells were then treated at different time points from 1 hour to 24 hours. In addition, cells were intervened with different concentrations (1 µM, 3 µM, 10 µM, 30 µM) of caspase-1 inhibitor to observe its effects on inflammasome components and apoptosis and pyroptosis markers.

Biochemical Analysis

After the experiment, cells were washed with PBS and scraped, protein extraction was performed with RIPA lysis buffer, and cell lysates were fully homogenized by ultrasonic treatment. Total protein concentration was determined using the BCA protein quantification kit. The same method was also used for protein precipitation and collection in cell culture media. The expression levels of inflammasome components, pro-cytokines, and cell death markers were analyzed by SDS-PAGE and immunoblotting techniques, and data quantification and analysis were performed using ImageJ and GraphPad Prism software.

Statistical Analysis

Significant differences between groups were determined by one-way analysis of variance (ANOVA) and Bonferroni post-hoc test. Experimental data were expressed as mean ± standard error (SEM), with the statistical significance threshold set at p < 0.05.

Research Results

Expression of Inflammasome Components and Pro-cytokines

Under oxygen-glucose deprivation conditions, the expression levels of pattern recognition receptors (PRRs) such as NLRP1, NLRC4, and AIM2 in protoplasmic astrocytes increased significantly. Furthermore, time-series analysis revealed that oxygen-glucose deprivation led to significantly increased expression of NLRP1, NLRC4, and AIM2 in protoplasmic astrocytes, with NAIP showing an upward trend at the 9-hour point.

In contrast, fibrous astrocytes showed no significant changes in the expression levels of NLRP1 and NLRC4 under oxygen-glucose deprivation conditions, while the expression of NAIP relatively decreased. The adaptor protein ASC and total caspase-1, -8 showed significant increases over time points, while the expression of pro-IL-1β decreased.

Inflammasome Activation and Its Promotion of Astrocyte Death

Under high-brightness electrochemiluminescence (ECL) immunoblot analysis, cleaved caspase-1, -8, and mature IL-1β and IL-18, which mark inflammasome activation, all showed increased expression under oxygen-glucose deprivation conditions, indicating inflammasome activation. Further analysis revealed that under oxygen-glucose deprivation conditions, the expression of cell death markers including apoptosis (cleaved caspase-3) and pyroptosis (N-terminal gasdermin D) as well as secondary pyroptosis (N-terminal gasdermin E) also increased significantly.

In addition, this experiment also found that astrocytes can release inflammasome components into the culture medium under oxygen-glucose deprivation conditions, thereby further achieving extracellular inflammasome activation to drive inflammatory responses.

Effects of Caspase-1 Inhibition on Inflammasome Activation and Astrocyte Death

Through progressive concentration treatment with caspase-1 inhibitor, it was found that it could effectively inhibit inflammasome activation and cell death under oxygen-glucose deprivation conditions. Specifically, the expression of cleaved caspase-1 and mature IL-1β decreased, while the expression of apoptosis and pyroptosis markers also significantly decreased.

Conclusion

In summary, this study establishes inflammasomes as important regulatory factors for inflammatory responses and cell death in astrocytes under oxygen-glucose deprivation conditions. By inhibiting caspase-1, apoptosis and pyroptosis cell death can be effectively reduced, suggesting that inflammasomes may become a new target for treating ischemic stroke-induced astrocyte death. However, future in vivo studies are necessary to further validate these findings to deepen the understanding of the dynamic role of inflammasomes in astrocytes under cerebral ischemic conditions. This study lays a solid foundation for future related research while pointing out important directions for future research.

Research Highlights

1. Different effects of inflammasome activation on protoplasmic and fibrous astrocytes under ischemic conditions: The study for the first time thoroughly explored the expression and activation effects of inflammasomes in different subtypes of astrocytes under ischemic conditions.
2. Revealing the extracellular role of inflammasomes: It was demonstrated that astrocytes can further drive inflammatory responses by releasing inflammasome components under oxygen-glucose deprivation conditions.
3. Effective application of caspase-1 inhibitors: The regulation of inflammasomes through pharmacological means provides new research directions and potential therapeutic approaches for treating astrocyte death after ischemic stroke.

Research Value

This study not only reveals the role of inflammasomes under ischemic conditions but also provides important clues for future development of anti-inflammatory treatment strategies. Especially in terms of astrocyte protection and neuroinflammation reduction, the research results show the potential application value of caspase-1 inhibitors, providing a new perspective for improving the prognosis of ischemic stroke patients.