Inhibition of the cGAS-STING Pathway Attenuates Lung Ischemia/Reperfusion Injury via Regulating Endoplasmic Reticulum Stress in Alveolar Epithelial Type II Cells of Rats

Abstract

Purpose

Endoplasmic reticulum stress (ERS) plays an important role in the pathogenesis of lung ischemia/reperfusion (I/R) injury. Cyclic GMP-AMP synthase (cGAS) is a cytosol dsDNA sensor, coupling with downstream stimulator of interferon genes (STING) located in the ER, which involves innate immune responses. The aim of our present study was to investigate the effects of cGAS on lung I/R injury via regulating ERS.

Methods

We used Sprague-Dawley rats to make the lung I/R model by performing left hilum occlusion-reperfusion surgery. cGAS-specific inhibitor RU.521, STING agonist SR-717, and 4-phenylbutyric acid (4-PBA), the ERS inhibitor, were intraperitoneally administered in rats. Double immunofluorescent staining was applied to detect the colocalization of cGAS or BiP, an ERS protein, with alveolar epithelial type II cells (AECIIs) marker. We used transmission electron microscopy to examine the ultrastructure of ER and mitochondria. Apoptosis and oxidative stress in the lungs were assessed, respectively. The profiles of pulmonary edema and lung tissue injury were evaluated. And the pulmonary ventilation function was measured using a spirometer system.

Results

In lung I/R rats, the cGAS-STING pathway was upregulated, which implied they were activated. After cGAS-STING pathway was inhibited or activated in lung I/R rats, the ERS was alleviated after cGAS was inhibited, while when STING was activated after lung I/R, ERS was aggravated in the AECIIs, these results suggested that cGAS-STING pathway might trigger ERS responses. Furthermore, activation of cGAS-STING pathway induced increased apoptosis, inflammation, and oxidative stress via regulating ERS and therefore resulted in pulmonary edema and pathological injury in the lungs of I/R rats. Inhibition of cGAS-STING pathway attenuated ERS, therefore attenuated lung injury and promoted pulmonary ventilation function in I/R rats.

Conclusion

Inhibition of the cGAS-STING pathway attenuates lung ischemia/reperfusion injury via alleviating endoplasmic reticulum stress in alveolar epithelial type II cells of rats.

Graphical Abstract

Keywords:

• cGAS-STING
• endoplasmic reticulum stress
• alveolar epithelial type II cells
• lung ischemia/reperfusion injury

Abbreviations

cGAS, cyclic GMP-AMP synthase; STING, stimulation of interferon genes; ERS, endoplasmic reticulum stress; SP-C, surfactant protein-C; 4-PBA, 4-phenylbutyric acid; DMSO, dimethyl sulfoxide; mtDNA, mitochondrial DNA; I/R, ischemia/reperfusion; W/D, lung wet/dry ratio; LW/BW, lung weight/body weight ratio; HE, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end-labeling; DHE, dihydroethidium; TEM, transmission electron microscopy; ROS, reactive oxygen species; DAMPs, damage-associated molecular patterns; PAMPs, pathogen-associated molecular patterns.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (no. 30800500, 81900007), the Shanghai Science and Technology Committee (18140903400), the Shanghai Municipal Health Commission (20204Y0082), the Project of the Xuhui District Science Committee of Shanghai (SHXH201838) and Fudan Undergraduate Research Opportunities Program (FDUROP, 21077). We sincerely thank Dr. Hongyang Gao’s technical support in electron micrographs taken by transmission electron microscopy (School of Basic Medical Sciences, Fudan University, Shanghai). We thank Miss. Yun Lin (Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai) for her excellent technical support in rat’s pulmonary ventilation function measurement and Western blot analysis.

Author Contributions

All the authors have made an important contribution to the present work, whether that is in the study design, conception, execution, acquisition of data, analysis and interpretation, or in all these areas: taking part in drafting, critically reviewing or revising the article. All the authors have given final approval of the version to be published and agreed on the journal to which the article has been submitted as well as agreed to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.