The diseases of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) still keep a poor prognosis, which leads to high mortality every year worldwide. It is important to figure out the potential mechanisms of ALI and find better treatments for patients. LPS can regulate the lung injury involving biomacromolecule synthesis, hepatocyte apoptosis stimulation, free radical generation, lipid peroxidation and other various mechanisms. In this study, high levels of pro-inflammatory cytokines were found in serum and lung tissues from animal model of acute lung injury induced by LPS. When treated with calcitriol, the expression of these pro-inflammatory cytokines were decreased and anti-inflammatory cytokines were increased significantly.
The role of calcitriol on ALI has not been clear till now. It is generally accepted that vitamin D has an effective therapeutic role in sepsis and calcitriol, the active form of vitamin D, has been reported to induce host defense. Since ALI is characterized with the disruption of the alveolar endothelial and epithelial barrier[4, 5], vitamin D has been reported to attenuate lung edema via blocking the Ang-2-Tie-2-MLC kinase cascade and the renin-angiotensin system to repair alveolar endothelial and epithelial barrier. Meanwhile, several researches have demonstrated that 1,25(OH)2D3, the hormonal form of vitamin D, has been shown to reduce vascular permeability and ameliorate pulmonary edema[25–27]. Nie et al. showed that vitamin D augments transalveolar fluid clearance via unregulating the expression of a-ENaC. Moreover, vitamin D has been reported to alleviate seawater aspiration-induced lung injury via inhibiting nuclear translocation of NF-κB and membrane translocation of RhoA and protecting alveolar epithelial and pulmonary microvascular endothelial barrier.
In the current study, metabolomics was used to detect the changes of differential metabolites in the three groups. A total of 49 metabolites were identified as the differential metabolites and the trends of 16 metabolites were opposite in LPS group and LPS + VD group. These metabolites were selected to detect their target proteins via STITCH database. We integrated these target proteins with differential genes in GSE1871 and target proteins of VDR. Finally, a total of 25 proteins were selected and considered to be closely related to the mechanism of calcitriol ameliorates ALI. STRING database was used to demonstrate the GO analysis and KEGG pathway analysis.
From the KEGG pathways, TNF signaling pathway, PI3K-Akt signaling pathway and Toll-like receptor signaling pathway are closely related to the mechanism of calcitriol ameliorates ALI.
TNF signaling pathway is one of the critical pathways in the development and maintenance of inflammation and it has been found to provoke the release of inflammatory cytokines in ALI and mediate the following innate immune response and inflammatory process. Previous study demonstrated that TNF is initiated by TNF-α receptor 1 (TNFR1), its activation induces the increased expression of nuclear factor kappa B (NF-kB). Zhang et. al. has indicated that 1,25(OH)2D3 down-regulated TNF signaling pathway to alleviate osteoarthritis.
Toll-like receptor signaling pathway functions as molecular patterns in adaptive or innate immunity and the activation of toll-like receptor signaling pathway leads to the production of costimulatory factors and cytokines, which is one important reasons contributing to ALI. The normal activation of TLRs is critical to defense molecules in the recognition of pathogens, including M. tuberculosis[34–36]. In HIV-seropositive individuals, vitamin D rescues impaired Mycobacterium tuberculosis-mediated tumor necrosis factor release in macrophages by enhancing toll-like receptor signaling pathway. However, uncontrolled excessive TLR activation may lead to serious infection. Multiple lines of evidence showed that ARDS is associated with the vigorous activation of TLR2, TLR3, TLR4 and TLR9 and noninfectious ALI is associated with the activation of TLR2 and TLR4. In addition, the survival rates of ARDS patients were correlated with downregulation of TLR1, TLR4 and TLR5. One pharmacologic strategy to manage the excessive inflammation in ALI is to regulate over activation of Toll-like receptors (TLRs).
PI3K-Akt signaling pathway is expressed in eukaryotes widely, which plays critical roles in growth, differentiation, proliferation, and survival[43, 44]. Several groups have documented that 1,25(OH)2D3 activates PI3K/Akt in vitro in a variety of cell types, like squamous cell carcinoma cells, osteoblasts and podocytes[45–47]. Xiao et al. has demonstrated that 1,25(OH)2D3 activates PI3K-Akt signaling pathway to protect podocytes from apoptosis. Studies showed that the phosphorylation of Akt downregulates the downstream pro-apoptotic factor Bax and upregulates the anti-apoptotic factor Bcl-2, which inhibits cell apoptosis. LPS-induced ALI is highly associated with cell apoptosis and thus in the treatment of calcitriol, PI3K-Akt signaling pathway plays a critical role.
In conclusion, in this study, the pharmacologic effect of calcitriol against LPS-induced ALI was evaluated in vivo and with the combination of metabolomics, GEO database and network pharmacology, we found out 25 related proteins which were associated to the protective effects of calcitriol. GO and KEGG analysis were used to classify these identified proteins and we selected TNF signaling pathway, PI3K-Akt signaling pathway and Toll-like receptor signaling pathway to be the potential pathway which are closely related to the mechanism of calcitriol ameliorates ALI. Since the network pharmacology strategy produces many possible targets, these differential targets should be thoroughly tested in our further studies.