Angiotensin-converting enzyme 2 modulation of pyroptosis pathway in traumatic brain injury: A potential therapeutic target

Abstract

Dear Editor,

Traumatic brain injury (TBI) is a significant public health concern, with its severity largely influenced by secondary molecular damage like oxidative stress, cell death and neuroinflammation. ACE2 mediates the enzymatic conversion of AngII to Ang-(1−7) and interacts with the G protein-coupled receptor MasR, resulting in antagonistic biological effects to those of AngII. Research has shown that the ACE2/Ang-(1–7)/MasR pathway mitigates neuroimmune overactivation, thereby decreasing neural damage and brain inflammation associated with cerebral haemorrhage and ischemia-reperfusion.¹ Understanding the regulation of ACE2 could provide novel insights into its neuroprotective mechanisms of ACE2 and offer fundamental knowledge regarding its underlying molecular signalling pathways. Following TBI, ACE2 levels in the injured cortical area significantly decreased (Figure 1A), reaching their lowest point 24 h post-injury (Figure S1A,D). The ACE2 protein was found in MAP2, IBA1 and GFAP-positive brain cells (Figure 1B,C), with co-expression analysis of ACE2 and IBA1 further supporting this conclusion (Figure 1H–J). Additionally, TBI disrupts the normal function of the renin-angiotensin system (Figure 1D–G). Behavioural experiments were verified to confirm the neuroprotective effect of ACE2 in vivo (Figure 1K). AVE0991, a synthetic Mas receptor agonist, replicates Angiotensin-(1−7) effects by activating MasR and providing anti-inflammatory, anti-oxidative and anti-apoptotic benefits. AVE0991 treatments significantly mitigated cognitive decline caused by TBI, demonstrated by decreased escape latency time and distance in the learning curve (Figure 1L–N). Additionally, AVE0991 improved motor coordination and balance in TBI mice, evidenced by shorter completion times in the horizontal ladder and balance beam tests and better scores (Figure 1O–R). CRISPR/Cas9 successfully constructed a mouse ACE2-knockout (KO) model (Figure S1B–F). ACE2-KO exacerbated behavioural impairment in mice following TBI (Figure S1H–N).

Pyroptosis, a recently identified form of inflammatory cell death, has been linked to various central nervous system disorders, including TBI.^{2, 3} To investigate the impact of ACE2 depletion on TBI progression, transcriptome sequencing was performed, revealing 385 upregulated and 74 downregulated genes (Figure 2A). Enrichment analysis of these differentially expressed genes using Reactome highlighted the pyroptosis pathway (Figure 2B), with significant changes observed in pyroptosis-related factors such as Gsdmd, Casp1, Il18rap and illr2. The quantitative reverse transcriptase PCR (qRT-PCR) validation confirmed the upregulation of messenger ribonucleic acid levels for this pyroptosis (Figure S2C). Protein imprinting assessments revealed that ACE2 deletion exacerbated the activation of key pyroptosis factors GSDMD and CASP1 (Figure 2C–E), findings further supported by immunofluorescence double staining (Figure 2I). The absence of ACE2 significantly increased the activation levels of mature pro-inflammatory cytokines IL-1β and IL-18, both implicated in pyroptosis (Figure S2D–I). Sholl analysis, used to assess morphological changes in pericontusional microglial cells post-TBI, demonstrated a transition from a resting to an activated state (Figure 2F–H).

To further elucidate the role of ACE2 in TBI-induced pyroptosis,^4-6 we administered the ACE2 agonist AVE0991 to mice. AVE0991 treatment upregulated the expression levels of ACE2 and MasR, leading to an amelioration of cell pyroptosis (Figure S2J–N). Notably, AVE0991 demonstrated favourable outcomes in both animal models (Figure 2J–L). Lipopolysaccharide (LPS)-induced pyroptosis models provide a controlled and reproducible system to study inflammasome activation and inflammatory responses central to TBI pathogenesis. Moreover, we utilized LPS to induce a cellular pyroptosis model and supplemented it with Ang-(1–7) as an ACE2 adjunct. Transmission electron microscopy revealed cellular disintegration in the LPS group, characterized by extensive membrane rupture, cytoplasmic leakage, nuclear disintegration and chromatin condensation (Figure 2M). Ang-(1−7) primarily exerts protective effects by binding to the MasR and activating downstream anti-inflammatory signalling pathways. Conversely, Ang-(1–7) significantly alleviated LPS-induced cellular pyroptosis (Figure 2N). Assessment with nucleic acid dyes further corroborated the mitigating effect of Ang-(1–7) on cell pyroptosis severity (Figure S2P).

To explore the connection between ACE2 downregulation or deficiency and cell pyroptosis, we employed omics approaches and bioinformatics to uncover potential molecular mechanisms. Non-targeted metabolomics analysis identified 134 distinct metabolites, as illustrated in the volcano plot (Figure S3A–C). Notably, the metabolite thromboxane B2 (TXB2) was significantly elevated in the ACE2-TBI group, implicating the involvement of the acid metabolic pathway in pyroptosis regulation (Figure S3D). This pathway was further substantiated through lipid metabolism metabolomics analysis (Figure S4A,B), indicating a recurrence of differential lipid metabolites within the arachidonic acid metabolism pathway (Figure S3F). Notably, prostacyclin synthase (PTGIS) is a key metabolic enzyme in arachidonic acid metabolism,^{7, 8} displayed a significant downregulation in ACE2 deficiency (Figure 3A), corroborated by protein imprinting and immunohistochemistry (Figure S4E–G). Intriguingly, the Kyoto encyclopaedia of genes and genomes (KEGG) pathway analysis highlighted PTGIS as significantly enriched in the arachidonic acid metabolism pathway (Figure 3B). Collectively, these findings suggest that ACE2-KO disrupts the expression of arachidonic acid metabolism products and enzymes, shedding light on the intricate interplay between ACE2 and cell pyroptosis regulation.

Our study explored the role of PTGIS in brain injury and its co-expression with IBA1. The results indicated that PTGIS expression decreases as microglial activation increases following TBI (Figure 3D). Moreover, the ACE2-TBI group exhibited a heightened level of pyroptosis alongside reduced PTGIS expression levels (Figure 3C). Enzyme-linked immunosorbent assay (ELISA) results confirmed a significant increase in the arachidonic acid metabolite TXB2 following TBI in comparison to normal mice, with ACE2-TBI mice exhibiting strong expression (Figure 3E). Conversely, low PTGIS expression led to a notable decrease in the levels of the PGI2 metabolite 6-keto-PGF1a (Figure 3F). The findings from arachidonic acid-targeted metabolomics further supported our initial hypothesis, as illustrated by metabolic clustering diagrams and analysis (Figure 3G). As indicated in the diagram, red arrows denote elevations in metabolic levels, while green arrows represent reductions. The absence of ACE2 significantly disrupts the balance equilibrium between TXA2 and PGI2, exacerbating the pyroptosis mechanism (Figure 3H).

Based on the findings presented, it is hypothesized that supplementing with PTGIS may mitigate the dysregulated metabolism of arachidonic acid and reduce pyroptosis progression. LPS was used to induce cell pyroptosis, and a microglial PTGIS overexpression vector was utilized to assess the impact of PTGIS on pyroptosis (Figure 4A). PTGIS effectively suppressed the release of inflammatory mediators associated with pyroptosis, as well as the maturation of GSDMD and Caspase-1 (Figure 4B). Scanning electron microscopy further confirmed that PTGIS inhibited pyroptosis progression and delayed cell disintegration (Figure 4C). This conclusion was further supported by immunofluorescence and transcript-level analyses (Figure 4D,E). Although the inhibitory effect of LPS on 6-keto-PGF1a levels was not pronounced, PTGIS intervention restored these levels to normal (Figure 4F). PTGIS intervention promoted arachidonic acid metabolism, reduced cellular expression and normalized PGE2 levels. Nucleic acid dyes were utilized to assess cellular morphology. The findings indicated that LPS increased the permeability of the cellular membrane, resulting in the entry of YO-PRO-1 into the cell nucleus and an increase in fluorescence (Figure 4G). EthD-2 was observed to penetrate the damaged membrane and bind to a greater amount of nucleic acids (Figure 4G). Besides, PTGIS was found to inhibit the process of pyroptosis.

TXA2 serves as a counterbalance to PGI2. U46619, a TXA2 agonist, directly exacerbates cell pyroptosis,⁹ leading to increased cell membrane permeability and upregulating the expression of a pyroptosis-related protein (Figure S5A,B,E,F). This progression is linked to dysregulation in the levels of TXB2 and 6-keto-PGF1a (Figure S5C−E). Ozagrel, a TXA2 inhibitor, was used to explore the role of TXA2 in TBI. In vivo, ozagrel administration to TBI mice significantly mitigated cognitive and motor deficits associated with TBI (Figure S5H–O). These results indicated that TXA2 signalling contributes to inflammation and pyroptosis following TBI, and inhibiting this pathway can help reduce these effects.

This study identified ACE2 deficiency as the initiator of disrupted TXA2/PTGIS balance, contributing to pyroptosis and inflammation (Figure 4H). Our research elucidates the dual function of ACE2 in attenuating inflammation and modulating pyroptosis through the metabolism of arachidonic acid, thereby expanding its recognized anti-inflammatory properties. In contrast to the AngII/AT1R axis, which intensifies injury, the ACE2 axis alleviates inflammation and metabolic disturbances, directly impacting secondary injury mechanisms in TBI. Our findings distinctly associate the ACE2-mediated restoration of PTGIS and the regulation of TXA2 levels with a reduction in pyroptosis, offering a novel perspective on its neuroprotective effects in TBI.

Jinxiu Guo, Shiyuan Zhao, Jing Chen and Pei Jiang designed this experiment. Xue Chu, Shanshan Wei and Junjun Meng carried out animal models and performed the behaviour tests. Ruili Dang, Changshui Wang, Jinxiu Guo and Mengqi Yang conducted the biochemical experiments. Jinxiu Guo wrote this manuscript. Jianhua Wang, Yujin Guo, Weihua Kong, Mengqi Yang, Tao Zhang and Wenxue Sun carried out analysis and interpretation of the data. All authors have read and approved the final version of the manuscript.

The authors declare no conflict of interest.

The study was supported by the National Natural Science Foundation of China (No. 82272253), the Natural Science Foundation of Shandong Province (No. ZR2022MH007) and the Key R&D Program of Jining (No. 2023YXNS016, No. 2022YXNS148 and No. 2023YXNS037).

All animal studies were conducted following the Care and Use of Laboratory Animals, with the approval of the Ethics Committee of Jining First People's Hospital (JNRM-2022-DW-011).