The pathogenesis of ALI is complex and has made rapid progress. Gram-negative bacteria infection is the main pathogenic element, which induces lung inflammatory cell infiltration and oxidative stress response, leading to pulmonary interstitial edema and alveolar cavity exudation[15]. After LPS injection, ALI model will be established, which will lay the foundation for animal experiment[16, 17]. In this study, the mouse model of lung tissue injury induced by lipopolysaccharide showed destroyed alveolar structure, inflammatory infiltration of alveolar cavity. Therefore, the ALI mouse model could be successfully established by induction with lipopolysaccharide. The pathological score of lung tissue and the W/D ratio of lung tissue in the ORI treatment group and Dex group were lower than ALI group. The results confirmed that dexamethasone and ORI could reduce the pathological damage of lung tissue and alleviate alveolar interstitial edema. Therefore, ORI had a certain protective effect on lung tissue.
At the early stage of inflammatory reaction, the total quantity of inflammatory cells in peripheral blood decrease rapidly and then increase gradually[18]. Therefore, peripheral blood WBC becomes a key marker of acute infection. The quantity of WBC in the blood after LPS injection is decreased significantly[19]. This study showed that the total white cells in bronchoalveolar lavage fluid of ALI group were higher than those of other groups, and the proportion of various types of inflammatory cells was raised to different degrees. At the same time, it proved that ORI played an anti-inflammatory affect by regulating the ratio of white blood cells in peripheral blood to restrain the accumulation of inflammatory cells in lung tissue, thereby inhibiting the occurrence of ALI and protecting lung tissue.
ALI is an organ injury caused by systemic inflammatory response of the body[20]. The overexpression of TNF-α advance the secretion of MIP-2, enhances the accumulation of WBC and the activation and infiltration of neutrophils [21, 22]. The stimulation of inflammation up-regulates the level of ICAM-1 in pulmonary vascular endothelial cells, and increases the neutrophils infiltration to the inflammatory sites, resulting in aggravation of tissue damage. The immune dysfunction of the body further triggers the apoptosis of alveolar epithelial cells in the lung tissue[23]. This study showed that the levels of MIP-2, ICAM-1 and TNF-α in the lung tissue of the ALI group were higher than the Control group, indicating that the lung tissue produced a serious inflammatory response, which triggered an inflammatory chain reaction, local neutrophil infiltration, inflammatory exudation and the aggravation of inflammatory response. ALI often produces a great number of oxygen free radicals with the activation of macrophages and neutrophils and respiratory burst[24]. Excessive free radicals can make the unsaturated fatty acids in the biofilm peroxidation to form a lipid peroxidation product, MDA, which is increased[25]. The antioxidant SOD in the body is decreased, and the antioxidant capacity is decreased[26]. Among them, MPO is used as a marker enzyme for neutrophil activation, and the change in its activity reflects the degree of neutrophil activation[27]. SOD, as an important oxygen free radical scavenger in the body, has extremely strong antioxidant effect, capable of scavenging superoxide anion and free radicals to defend cells from damage[28]. This study showed that ORI had good antioxidant and anti-inflammatory effects, reduced the expression of cytokine-induced neutrophil chemokine MIP-2, inhibited the migration of neutrophils and the release of leukocytes, and reduced the expression of ICAM-1, thereby inhibiting the adhesion of neutrophils to tissues and improving the degree of pulmonary edema. It could advance the expression of SOD, ameliorate the ability of scavenging free radicals in mice, reduce the release of lipid metabolites such as MDA and MPO, reduce the generation of pro-inflammatory factor TNF-α, and control the cascade reaction of inflammation, thereby improving the lung tissue function.
In the research on the treatment of ALI, VIP not only inhibits inflammation of lung tissue, but also improves pulmonary edema, and it is considered one of the potential therapeutic targets[29]. Study have shown that VIP, as an immune neuropeptide, can bind to G protein-coupled transmembrane receptors, activate adenylate cyclase (ACs) and promote cAMP release. cAMP is a critical material for histiocytes to maintain physiological function[30]. PKA is one of that main target proteins of cAMP. cAMP can bind to the regulatory subunit of PKA to promote phosphorylation of PKA, and a complete VIP/cAMP/PKA activation pathway is formed[31]. AQPs, which are closely related to the water and liquid metabolism of tissue cells, are one of the main downstream target proteins in this pathway[32]. VIP/cAMP/PKA/AQPs play a critical role in the regulation of water and liquid metabolism of lung histiocytes[33]. Thus, VIP can dominate the level of AQPs through cAMP/PKA, thereby affecting the water and liquid metabolism of lung histiocytes and improving pulmonary edema. AQPs are a family of receptor proteins distributed on cell membranes that control the transmembrane transport of water molecules[34]. AQP1 is confirmed to be closely related to ALI pulmonary edema. AQP1 is mainly distributed in the capillaries, lymphatic vessel and mesothelial cells beside the respiratory tract, and is mainly involved in the reabsorption of water and liquid in the pulmonary interstitium[35]. AQP1 is highly dependent on the phosphorylated PKA regulatory pathway. p-PKA regulates AQP1 by binding to phosphorylation sites in AQP protein, affecting water-liquid transport and cell endocytosis and exocytosis[32]. VIP/cAMP is also the activator of p-PKA, which will increase the water and liquid transport capacity of AQP1, and enhance the reflux absorption of the lymphatic and venous system, thereby reducing the interstitial lung and alveolar edema[36]. Therefore, the activation of VIP/cAMP/PKA pathway could promote the activated expression of AQP1. This study suggested that the level of tissue AQP1 protein in the lung in the ALI group was reduced during the development process of ALI. After treatment with oridonin, the expression level of AQP1 in the lung tissue in each group was significantly higher than that in the ALI group, and each protein in the VIP/cAMP/PKA signaling pathway was in a high expression state. This result further indicated that the VIP/cAMP/PKA/AQPs signaling pathway might be restrain in the lung tissue with acute lung injury, and oridonin could exert its protective effect on the lung of mice with ALI by activating the VIP/cAMP/PKA/AQPs signaling pathway.
In summary, oridonin reduces LPS-induced ALI by targeting VIP/cAMP/PKA/AQPs signaling pathway, as shown in the flow chart illustrated in Fig. 11. This study will provide a theoretical basis for the clinical application of oridonin in the treatment of acute lung injury.