Hsp90β1 Regulates Pyroptosis via the AMPK/mTORC1 Pathway to Alleviate Alveolar Epithelial Barrier Dysfunction in Acute Lung Injury.

Abstract

Objective: Acute lung injury (ALI) is a severe and potentially life-threatening inflammatory disorder of the lungs. Given the limited efficacy and significant side effects of current treatments for ALI, there is an urgency for novel therapeutic strategies.

Methods: Potential pathogenic factors related to the mTOR complex 1 (mTORC1) pathway in ALI were identified through public database mining. In vivo and in vitro models of ALI were constructed through lipopolysaccharide induction. A549 cells were transfected with the constructed si-Hsp90β1, and AMPK activity in cells was inhibited using dorsomorphin. Hematoxylin-eosin (HE) staining, ELISA, lung wet/dry weight (W/D) ratio, and protein content in bronchoalveolar lavage fluid were employed to verify the successful establishment of the ALI animal model. The percentage of Caspase-1 positive cells was determined by the flow cytometry. The expression of Caspase-1 p20 in cells was quantified by immunofluorescence. Expression levels of Hsp90β1, epithelial barrier-related proteins (E-cadherin, ZO-1, and Occludin), pyroptosis-related proteins (Caspase-1, Caspase-1 p20, NLRP3), and proteins related to the AMPK/mTORC1 pathway were detected by western blot.

Results: Through bioinformatics analysis, seven hub genes were screened out, among which Hsp90β1 was selected for further investigation. Hsp90β1 was observed to be up-regulated in the ALI rat model, accompanied by significant histopathological changes, elevated lung W/D ratio, and increased levels of inflammatory factors observed in ALI rats. Both in vitro and in vivo models exhibited enhanced pyroptosis and impaired barrier. In addition, AMPK activity was inhibited and mTORC1 was activated in the ALI rat model. Inhibiting Hsp90β1 reduced inflammation and pyroptosis while restoring barrier function. However, further inhibition of AMPK activity reversed the effect induced by Hsp90β1 inhibition.

Conclusion: Low expression of Hsp90β1 alleviates inflammation, suppresses pyroptosis, and restores epithelial barrier function in ALI through the AMPK/mTORC1 pathway.