Effects of the PI3K/Akt/HO-1 pathway on autophagy in a sepsis-induced acute lung injury mouse model

Sepsis-induced lung injury is a condition of acute hypoxic respiratory insufficiency caused by systemic infectious factors, resulting in damage to alveolar epithelial and capillary endothelial cells, diffuse pulmonary interstitial edema, and alveolar edema. Heme oxygenase (HO)-1, typically associated with inflammation, exhibits anti-inflammatory properties. Autophagy, a pathway for cellular waste degradation, plays a crucial protective role during stress. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway is pivotal in mediating cellular responses to inflammatory stimuli. Therefore, we hypothesized that HO-1 interacts with autophagy and is regulated by the PI3K/Akt pathway in mice experiencing sepsis-induced lung injury.

To induce sepsis-induced lung injury, mice underwent cecal ligation and puncture (CLP). Prior to surgery, hemin or Sn-protoporphyrin IX (SnPP) was administered via intraperitoneal injection. Survival rates were monitored from days 1 to 7 post-surgery, and lung histology was assessed 24 hours post-surgery. Pro-inflammatory and anti-inflammatory factors in plasma and lung tissue were quantified using enzyme-linked immunosorbent assay (ELISA). Protein expression levels of HO-1, Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, p62, and lysosome-associated membrane protein (LAMP)2 were measured 24 hours post-surgery. Immunofluorescence was used to observe HO-1 and LC3B-II protein expression levels, and autophagosomes were detected using electron microscopy at the same time point.

Additionally, administration of PI3K inhibitors LY294002, PI3K activators Recilisib, and hemin before surgery allowed measurement of Akt, p-Akt, HO-1, and LC3-II levels 24 hours post-surgery. Our findings indicate that HO-1 overexpression increased survival rates and mitigated sepsis-induced lung injury. HO-1 overexpression reduced levels of pro-inflammatory cytokines (TNF-α, IL-1β) and increased anti-inflammatory cytokines (IL-10, HO-1). Furthermore, HO-1 overexpression correlated with elevated expression of Beclin-1, LC3B-II, and LAMP2 proteins, decreased p62 protein expression, and significantly enhanced autophagosome formation. Conversely, downregulation of HO-1 yielded contrasting results.

LY294002 inhibited p-Akt/Akt, HO-1, and LC3B-II protein expression, whereas hemin reversed the inhibitory effect of LY294002. The protective effects of HO-1 were mediated through autophagy, regulated by the PI3K/Akt signaling pathway during sepsis-induced lung injury in mice.