Calcitonin Alleviates Sepsis-Induced Acute Lung Injury by Inhibiting the HMGB1/MyD88/NF-κB Pathway by Targeting CD3D.

BACKGROUND

Acute lung injury (ALI) triggered by sepsis continues to pose a significant difficulty in clinical practice. Due to its anti-inflammatory and antioxidant activities, calcitonin is considered a potential therapeutic option in sepsis.

METHODS

Bioinformatics analysis was performed using the GSE89376 and GSE67652 datasets. Serum levels of CD3D and NLR family pyrin domain containing 3 (NLRP3) inflammasome, as well as high-mobility group box 1 (HMGB1)/myeloid differentiation primary response gene 88 (MyD88)/nuclear factor-κB (NF-κB) pathway components, were evaluated in sepsis/ALI patients. The effects of calcitonin and CD3D knockdown on human pulmonary microvascular endothelial cells (hPMECs) activated by lipopolysaccharide (LPS) were investigated in vitro. Experimental assays, including quantitative real-time polymerase chain reaction (qRT-PCR), Cell Counting Kit-8 (CCK-8) assay, western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and flow cytometry, were used to assess cell viability, apoptosis, cell cycle, and oxidative stress markers.

RESULTS

CD3D was identified as a key sepsis/ALI-associated hub gene and correlated with NF-κB pathway activation in patients. CD3D silencing in hPMECs effectively suppressed LPS-induced inflammation, oxidative stress, apoptosis, and G1 phase arrest by downregulating the expression of NLRP3, phosphorylation (p)-NF-κB, MyD88, and HMGB1. Calcitonin alone mitigated LPS-induced injury in a dose-dependent way and further enhanced the protective impacts of CD3D knockdown. Co-treatment resulted in synergistic inhibition of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, a reduction in oxidative markers, restoration of antioxidant capacity (superoxide dismutase (SOD) and glutathione (GSH)), improved endothelial cell viability, and attenuation of apoptosis. Notably, combined treatment more robustly suppressed the HMGB1/MyD88/NF-κB pathway than either intervention alone.

CONCLUSION

CD3D exacerbates sepsis-induced ALI by potentiating the HMGB1/MyD88/NF-κB pathway and NLRP3 inflammasome, driving inflammation and oxidative stress. Combined CD3D knockdown and calcitonin treatment offers a novel synergistic therapeutic strategy for mitigating pulmonary endothelial injury in sepsis.