ALI is a clinical disease with high mortality and high treatment burden. Despite great progress in diagnosis and treatment, disease prognosis is poor. ALI is characterized by the apoptosis and necrosis of vascular endothelial cells and impaired vascular integrity, leading to increased permeability and pulmonary edema. LPS is a glycolipid of the outermost membrane of Gram-negative bacteria. This glycolipid induces inflammatory reactions and cytokine secretion and has been shown to trigger ALI in animal models[14, 15]. In our study, the effect of LPS-induced ALI was assessed using a cell model. Some studies have found that TNF-α expression is increased in LPS-induced ALI. TNF-α reduces vascular permeability, leading to pulmonary vascular endothelial cell injury and pulmonary edema [16, 17]. MK promotes the release of MCP-1, MIP-2, and other chemokines, and the aggregation of macrophages and neutrophils[18]. MK expression is low in healthy organs and increases during oxidative stress, inflammation, and tissue repair[19, 20]. In this study, LPS increased the protein expression of MK in rat airway smooth muscle cells. However, the role of MK and related signaling pathways in ASMCs in LPS-induced ALI is incompletely understood.
MK promotes cell proliferation[21]. MK is expressed in many tumors and inflammatory diseases and has anti-apoptotic effects[22]. The present study assessed the effect of rMK on the proliferation and apoptosis of ASMCs. In a mouse model of acid- and ventilator-induced lung injury, an increase in collagen deposition and hydroxyproline levels and a decrease in lung compliance were attenuated in MK−/− mice vs. wild-type mice[25]. The inhibition of Nox1, MK, and Notch2 attenuated epithelial-to-mesenchymal transition (EMT), demonstrating that MK plays an important role in airway remodeling [10, 23]. MK enhances the hypoxia-induced proliferation and differentiation of human lung epithelial cells [24]. Our results showed that ASMC survival was lower in the MK siRNA group than in the LPS group, indicating that MK reduced LPS-induced ALI. In addition, LPS decreased the viability and rate of apoptosis of ASMCs, whereas rMK attenuated these effects. These results show that MK participates in the repair of inflammatory tissues, increases ASMC survival, and inhibits apoptosis, leading to airway remodeling, and these effects are reversed by MK inhibition.
Notch2 is an MK receptor and an evolutionarily conserved pathway that regulates many cell-fate decisions during development. Notch signaling activity is regulated by receptor proteolysis. Upon proteolysis, the Notch intracellular domain travels to the nucleus and interacts with a transcription factor complex to regulate gene expression [25, 26]. The γ-secretase inhibitor of Notch signaling LY411575 inhibits osteoclast differentiation and bone destruction through the Notch/HES1/MAPK/Akt pathway[27]. Notch2 causes tumor invasion and metastasis by regulating EMT and is downregulated by Notch inhibitors and endogenous compounds, resulting in mesenchymal-to-epithelial transition[28]. Notch2 interacts with MK in human lung epithelial cells, and MK-induced EMT is dictated by mechanical stretching. Notch2 signaling inhibition blocks EMT more effectively than endogenous MK. Silencing MK or Notch2 reduces pulmonary fibrosis[29]. Notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the human lung, and Notch2 inhibition can be a therapeutic strategy for preventing this pathological process in airway diseases[30]. Antisense oligonucleotides (ASOs) downregulated Jag1 and Notch2 in goblet cell metaplasia associated with allergen-induced asthma and upregulated the ciliated cell marker FoxJ1. Moreover, the ASO-mediated decrease in Jag1 and Notch 2 expression inhibited goblet cell metaplasia, mucus production, and airway remodeling [31]. Our results showed that LPS increased the mRNA and protein expression of MK and Notch2, consistent with previous studies. Tian et al. found that MK promoted the proliferation of gastric cancer cells through Notch signaling, whereas the cisplatin-induced apoptosis of these cells was reduced by rMK and enhanced by MK silencing. Apoptosis may be induced in MK siRNA-transfected cells by an apoptosome-dependent mechanism, with the downregulation of Bcl-2 expression, upregulation of Bax expression, reduction in mitochondrial membrane potential, release of cytochrome c, and the activation of caspases 3, 8, and 9[32, 33]. This study found similar results in airway smooth muscle cells. The inhibition of MK and Notch2 expression repressed the proliferation of ASMCs and promoted apoptosis, demonstrating that MK and Notch2 jointly participate in lung tissue remodeling.