Mitochondrial Peptide SHLP2 Limits Poly(I: C)-Induced Alveolar Epithelial Injury by Modulating Redox, Apoptotic,...

Type II alveolar epithelial (ATII) cells play a central role in pulmonary homeostasis, epithelial repair, and innate antiviral defense. Polyinosinic: polycytidylic acid [Poly(I: C)], a synthetic analog of viral double-stranded RNA, induces a viral-like epithelial injury phenotype characterized by oxidative stress, mitochondrial dysfunction, inflammation, and inflammasome-associated responses. Small humanin-like peptide 2 (SHLP2), a mitochondrial-derived peptide, has been associated with cytoprotective and mitochondrial regulatory effects; however, its role in viral-like alveolar epithelial injury remains unclear. This study investigated the effects of SHLP2 on Poly(I: C)-induced cellular damage in human ATII cells. Human ATII cells were assigned to four groups: control, SHLP2 alone, Poly(I: C) alone, and subsequent SHLP2 treatment during continued Poly(I: C) exposure. Cell viability was assessed by MTT assay. Apoptosis was evaluated by Annexin V-FITC/PI flow cytometry and by measuring BCL2/BAX expression. Mitochondrial membrane potential and intracellular reactive oxygen species (ROS) production were analyzed. Oxidative stress-related markers, inflammatory mediators and antiviral response-associated markers, and inflammasome-associated molecules were evaluated by qPCR and ELISA. In silico protein-peptide docking was performed to predict possible interactions with SHLP2. Poly(I: C) reduced cell viability and promoted apoptosis, mitochondrial depolarization, and ROS accumulation in human ATII cells. These effects were accompanied by altered BCL2/BAX expression, increased SOD2 and GPX1 expression, changes in total NRF2 and KEAP1 protein levels, elevated inflammatory mediators and Poly(I: C)-induced antiviral response-associated markers, including TNF-α, IL-6, NF-κB, TICAM1, and IFN-β, and increases in NLRP3 inflammasome-associated markers, including NLRP3, cleaved caspase-1, IL-1β, and IL-18. SHLP2 alone did not exert cytotoxic effects. Notably, subsequent SHLP2 treatment during continued Poly(I: C) exposure attenuated apoptosis, mitochondrial depolarization, ROS accumulation, inflammatory mediator production, TICAM1 and IFN-β levels, and several inflammasome-associated markers and outputs. Docking analyses predicted possible interactions of SHLP2 with TICAM1 and KEAP1, providing hypothesis-generating in silico evidence for potential associations with inflammatory and redox-related proteins. SHLP2 treatment was associated with reduced mitochondrial depolarization, oxidative stress, and apoptosis, together with changes in inflammatory and antiviral response-associated markers and NLRP3 inflammasome-related outputs in Poly(I: C)-exposed human ATII cells. These findings identify SHLP2 as an early-stage candidate for further investigation in Poly(I: C)-induced viral-like alveolar epithelial injury.

The online version contains supplementary material available at https://doi.org/10.1007/s10616-026-01070-z.