Electroacupuncture Alleviates Lung Injury in CpG1826-Challenged Mice via Modulating CD39-NLRP3 Pathway

Abstract

Purpose

Cytokine storm secondary lung injury (CSSLI) is the leading death cause in COVID-19 virus infection, and CD39-dominated purinergic brake drives NLRP3 inflammasome activation and pyroptosis, which plays a crucial role in the pathogenesis of CSSLI. Though electroacupuncture (EA) can alleviate lung injury caused by a variety of inducers, its effect on CSSLI and the underlying mechanism needs further investigation.

Methods

We established a widely recognized CSSLI mice model with CpG1826 (CpG), a TLR-9 agonist agent. Luminex liquid chip was employed to detect serum levels of 12 cytokines/chemokines to evaluate cytokine storm formation. H+E staining and transmission electron microscope were applied to examine pulmonary pathological injury and alveolar macrophage structure, respectively. IL-1β, IL-18, IL-1α, and HMGB-1 in BAL fluid were determined by ELISA kits. mRNA and protein levels of lung CD39 and NLRP3 were assessed by qRT-PCR and Western blotting. An in vitro model was also established by incubating PMA-differentiated THP-1 cells with serum samples obtained from relevant group of mice.

Results

Repeated CpG induced CSSLI together with the elevation of 11 cytokines/chemokines including GM-CSF, IL-16, IL-1α, MCP-1, IL-2, IL-10, CCL3, IL-1β, TNF-α, IL-6, and IL-17A, though not IFN-γ, which was reduced by EA pretreatment to a different extent. EA also alleviated lung injury and recovered lung macrophage structure. Moreover, CpG enhanced IL-1β and IL-18 level in BAL fluid, promoted NLRP3, while suppressing CD39 expression in lung, all of which were reversed by EA pretreatment. Of note, EA failed to further decrease BAL fluid IL-1β, IL-18, IL-1α, and HMGB-1 levels when A438079, a selective inhibitor of P2X7, was administered. However, both CD39 and NLRP3 are dispensable for EA decreasing multi-cytokine secretion in serum-incubated and CpG-stimulated THP-1 cells. Taken together, EA alleviated CSSLI in CpG-challenged mice by regulating the CD39-NLRP3 pathway in a P2X7-dependent way.

Conclusion

EA demonstrated potential to be applied in COVID-19 treatment.

Keywords:

• electroacupuncture
• cytokine storm
• CD39
• NLRP3 inflammasome
• lung injury
• CpG1826

Abbreviations

CSSLI, cytokine storm secondary lung injury; NLRP3, pyrin domain-containing 3; EA, electropuncture; IL, interleukin; HMGB-1, high mobility group box-1 protein; BAL, bronchoalveolar lavage; PMA, phorbol 12-myristate 13-acetate; GM-CSF, granulocyte-macrophage colony-stimulating factor; MCP-1, monocyte chemoattractant protein-1; CCL-3, macrophage inflammatory protein 1-α; TLR, Toll-like receptor.

Acknowledgments

This work is supported by the National Natural Science Foundation of China [82104491], the Natural Science Foundation of Sichuan [2023NSFSC0674], and the Postdoctoral Science Foundation of China [2021M693789].

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no competing interest in this work.