The SIRT1-Rab7 Axis Regulates NLRP3 and STING Activation in Sepsis-Induced Acute Lung Injury via Terminal Endosome-Dependent Mitophagy

Academic Background

Acute lung injury (ALI) is one of the leading causes of death in septic patients, primarily due to the pro-inflammatory changes and permeability defects in endothelial cells (ECs). Mitochondrial dysfunction plays a crucial regulatory role in the pathogenesis of sepsis-induced ALI. While mitophagy is widely recognized for regulating mitochondrial quality, its specific role in sepsis-induced ALI remains unclear. SIRT1 (Sirtuin 1) is a histone deacetylase involved in regulating inflammation, mitophagy, and cellular senescence. This study demonstrates a terminal endosome-dependent mitophagy mechanism through which the SIRT1 signaling pathway suppresses NLRP3 and STING activation in sepsis-induced ALI.

Research Source

This work was jointly conducted by Jiang Tao, Liu Enran, Li Zhiyuan, and others at Harbin Medical University and Northeast Forestry University and published in the International Journal of Surgery, Volume 110, 2024.

Research Details

Experimental Procedures

Animal Studies: Healthy adult male C57BL/6J mice weighing 20-25 grams were used, and all animal experiments strictly adhered to animal ethics regulations. To establish a sepsis-induced ALI model, the cecal ligation and puncture (CLP) method was employed. The study used the SIRT1 inhibitor EX527 (10mg/kg), administered intraperitoneally 30 minutes before CLP surgery to investigate SIRT1’s role in sepsis-induced ALI. In different experimental groups, various pharmacological interventions were introduced, including SIRT1 activator SRT1720 (20mg/kg), NLRP3 inhibitor (MCC950), and STING inhibitor (C176) before the CLP model. Euthanasia was performed 24 hours after CLP surgery for all experimental groups. Lung tissue was fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. Three pathologists blindly evaluated the degree of tissue injury. Methods for assessing vascular permeability, Evans blue staining, enzyme-linked immunosorbent assay (ELISA) for measuring serum cytokines and myeloperoxidase (MPO) activity are described in detail within the paper.

Cell Experiments: Mouse lung endothelial cells (ECs) were cultured under different experimental conditions, including LPS (1μg/mL) treatment to mimic sepsis-induced ALI. To inhibit autophagy, cells were pre-treated with 20μM chloroquine (CQ). The roles of SIRT1 and Rab7 in mitophagy were also investigated. Additionally, techniques such as immunofluorescence staining, flow cytometry, and Western blotting were employed to explore the specific mechanisms of SIRT1 in cellular autophagy and inflammatory responses.

Key Research Findings

SIRT1 Activity Deficiency Exacerbates Sepsis-Induced ALI and Sepsis Severity: Histological analysis revealed that SIRT1 activity and expression significantly decreased in the CLP model, and the addition of the SIRT1 inhibitor EX527 further exacerbated lung injury and tissue damage in other organs. Increased lung MPO activity, weight loss, decreased core temperature, and reduced survival rates further supported these findings. Further electron microscopy observations confirmed that SIRT1 activity deficiency in mice increased vascular fluid leakage and pro-inflammatory cytokine levels (IL-1β, IL-6, TNF-α) in the CLP model.

SIRT1 Deficiency Disrupts Mitochondrial Homeostasis and Activates Inflammatory Responses: In vitro studies showed that lung ECs treated with SIRT1 siRNA exhibited significantly decreased mitochondrial membrane potential, increased damaged mitochondria, and accumulated mtDNA in the cytosol, further activating the NLRP3 inflammasome and STING signaling pathways. The use of specific mitochondrial ROS scavengers (Mitotempo) and mtDNA removal methods demonstrated the crucial role of mtDNA in mitochondrial damage and inflammatory responses.

SIRT1 Mediates Mitophagy via Rab7: Further research revealed that the interaction between SIRT1 and Rab7 is essential for mitophagy activation. SIRT1 deficiency prevented the transport of mitophagy vesicles to lysosomes, leading to the accumulation of damaged mitochondria and activation of inflammatory pathways. In vitro experiments confirmed that Rab7 overexpression could restore mitophagy flux, reduce damaged mitochondria, and decrease inflammatory cytokine secretion.

Pharmacological Intervention Unveils SIRT1’s Crucial Role in Sepsis-Induced ALI: Pharmacological restoration of SIRT1 activity significantly improved symptoms of sepsis-induced ALI and sepsis severity. SRT1720 treatment significantly reduced NLRP3 and STING pathway activation, improved lung vascular barrier function, alleviated lung injury, and mitigated sepsis severity.

Conclusion

This study revealed that SIRT1 regulates excessive mitochondrial ROS generation and mtDNA leakage through terminal endosome-dependent mitophagy, which is critical for limiting NLRP3 and STING pathway activation under LPS stimulation. SIRT1 deficiency exacerbated endothelial injury and sepsis severity in the CLP model. These findings provide potential therapeutic targets for treating sepsis-induced ALI and highlight the crucial roles of SIRT1 and mitochondria in endothelial cell dysfunction caused by sepsis at the molecular level.

Surprising Findings and Innovations

1. First demonstration that SIRT1 deficiency leads to mtDNA and mtROS accumulation, activating the NLRP3 inflammasome and STING pathways, by affecting terminal endosome-dependent mitophagy.
2. Discovery of the interaction between Rab7 and SIRT1 and its importance in mitophagy flux.
3. Pharmacological activation of SIRT1 provides an effective therapeutic strategy, significantly improving sepsis-induced ALI and sepsis severity.

This research provides new insights into the molecular mechanisms underlying sepsis-induced ALI and may pave the way for developing novel therapeutic strategies to reduce mortality in septic patients.