NOS1 knockdown attenuates traumatic brain injury‑induced acute lung injury through the NO-cGMP-PKG1 signaling axis to restore calcium homeostasis and enhance antioxidant defense

Abstract Traumatic brain injury (TBI) represents a devastating public health problem frequently complicated by secondary acute lung injury (ALI), which severely worsens clinical outcomes. However, the underlying mechanisms linking the brain-lung axis remain poorly defined. Here, we performed integrated transcriptomic, metabolomic, and 16 S rRNA microbiome analyses in patients with TBI-ALI before and after emergency treatment, and identified neuronal nitric oxide synthase (NOS1) as a key regulator associated with calcium signaling and cGMP-PKG pathway disorders. Functional validation in LPS-stimulated MLE-12 cells and a mouse TBI model demonstrated that NOS1 was markedly upregulated under pathological conditions, accompanied by excessive inflammation, oxidative stress, disrupted calcium homeostasis, and suppressed PKG1 signaling. NOS1 knockdown significantly reduced pro-inflammatory cytokine secretion, pathological NO production, and oxidative stress, while restoring cGMP levels, PKG1 activation, calcium homeostasis, and Nrf2/HO-1 antioxidant signaling, thereby reducing apoptosis and alleviating lung and brain tissue damage. Pharmacological inhibition of PKG1 with KT5823 markedly attenuated these protective effects, accompanied by reduced VASP phosphorylation and renewed calcium dysregulation, confirming the critical role of PKG1 signaling. Neurological assessment further showed that NOS1 silencing improved TBI-induced neurological deficits, whereas PKG1 inhibition partially reversed this benefit. In conclusion, our study demonstrates that NOS1 knockdown alleviates TBI-ALI, at least in part, by modulating the NO-cGMP-PKG1 signaling axis, which promotes antioxidant defense and restores calcium homeostasis. This study provides mechanistic insight into brain-lung axis injury and identifies the NOS1-PKG1 signaling axis as a potential therapeutic target.