Effect Of Ghrelin On CLP-induced ALI In Mice And The Signal Pathways Involved In The Modulation

Objective:Acute lung injury (ALI) / Acute Respiratory Distress Syndrome (ARDS) is defined as the damages to the pulmonary capillary endothelial cells and alveolar epithelial cells, which led to diffuse interstitial and alveolar pulmonary edema that causes acute hypoxic respiratory insufficiency or failure, due to severe infections, shocks, traumas, burns and other non-cardiac diseases. Disordered coagulation and fibrinolysis is one of the important mechanisms in the inflammation cascade induced ALI. In early stages, procoagulant process is enhanced and the fibrinolytic process is inhibited, these would lead to systematic thrombosis and substantial deposition of fiber proteins, which is the crucial to the mechanism of ALI[1]. In past treatments, the protective ventilation strategy used although could reduce mortality in ALI/ARDS, the morbidity and mortality remained high. Therefore, in order to develop more scientific and effective treatments, more researchers started to focused their attentions on the abnormalities of cytokines and fibrinolytic process, which are important to the ALI/ARDS pathogenesis mechanism.Recent studies have further demonstrated the abnormal fibrinolysis activation and hyper inflammatory response are closely related to the pathogenesis of ALI. Takeshita et al[12]. found that in vitro tumor necrosis factor-α(TNF-α) activate nuclear factor-κB (NF-κB) and therefore mediated the secretion of plasminogen activator inhibitor -1 (PAI-1). In addition, other research showed[13] Ghrelin could inhibit the activation of NF-κB, which down-regulates PAI– 1 secretion. Foreign studies of sepsis have found treatments with Ghrelin can reduce NF-κB reduced plasma TNF-α, IL-1β, IL-6, other pro-inflammatory factors, and increased pulmonary blood flow in sepsis-induced ALI/ARDS mouse model. These treatments can also improve symptoms and decrease mortality of ALI/ARDS mouse. These findings have suggested a potential new treatment for ALI/ARDS.Ghrelin is a newly discovered 28 amino acid brain-gut peptide that is mainly found in the stomach[14]. It is the growth hormone secretagogue receptor. As we know, Ghrelin receptors are distributed all over body, including in the lung, which inspired many researchers interest in understanding the role Ghrelin played in sepsis– induced ALI. However, there haven't been any similar reports reported domestically. In our study, we used different doses of Ghrelin on the CLP-mediated lung injury to regulate the cytokine effects. By observing the ability to reduce pulmonary vascular permeability, improving the survival rate, and reducing the severity of lung injury, we try to identify an optimal therapeutic group. At the same time, we plan to observe how Ghrelin affects the expression of NF-κB on secretion of PAI-1and t-PA, explore the possible mechanism, and provide the theoretical basis for clinical treatmentContents and methods of study:Content and method of study:Part I: Ghrelin's protection on the CLP-induced mice1. Kunming mice were randomly divided into normal group (NS), sham-operated group (Sham), cecal ligation and perforation group (CLP) and Ghrelin intervention group (Ghrelin + CLP); then Ghrelin group is divided into intervention group 5nmol/kg, 10nmol/kg, 20nmol/kg, 40nmol/kg, 80nmol/kg five dose groups, respectively, with A, B, C, D, E said. Each dose group, respectively, after CLP at 5h, 10h, 15h time points times by intraperitoneal injection of Ghrelin.2. The mice of CLP group, Ghrelin intervention group were killed and sampling after 20h. The mice of normal group, sham-operated group were killed, sampling after given intraperitoneal injection of saline (2.5 ml/kg) and observe 20 hours, measurement and comparison of injury indicators in each group, including the lung wet/dry weight ratio, lung water content, water channel protein in lung tissue and serum inflammatory mediators, as well as histopathological score, to assess lung injury in mice, the survival rate calculated.Part II: the possible mechanisms of ghrelin on CLP-induced ALI: --- NF-κB signal transduction mechanism and the fibrinolytic system 1. Immunohistochemistry detected NF-κB in lung tissue of CLP-mice.2. ELISA detected plasma PAI-1 and t-PA level, and computed the ratio of t-PA/PAI-1.Results:Part I: Ghrelin's protection on acute lung injury of CLP mice1.Ghrelin impacts the pulmonary vascular permeability of CLP mice1.1. The lung water in the intervention group significantly lower than the CLP group (P<0.001), exceptionally group C. Ghrelin intervention group C lung wet / dry weight ratio was significantly lower than CLP group (P<0.001).1.2. Ghrelin intervention group C AQP1, AQP4 expression level was significantly higher than CLP group.1.3. CLP lung histology damage score was significantly higher than the normal group and sham operation group. Ghrelin intervention group C reduced tissue injury.2.Ghrelin intervention group impacted inflammatory factor in serum: ELISA results showed that ghrelin interfere with TNF-αserum levels. B group was statistically significant (P<0.05), C, D and E groups were statistically significant (P<0.001), Ghrelin-C group of IL-6, IL-1βwere significantly lower than the CLP group (IL-6,P<0.001,IL-1β,P<0.05).3.Ghrelin improved the survival rate of CLP-induced mice.CLP group at 7 days after the operation of all deaths, with the normal group, sham-operated group, Ghrelin-C group significant difference between the average survival time (P<0.001), Ghrelin-C group has three mice survived more than 10 days survival time using Kaplan-Meier Survival Analysis of Ghrelin on the impact of the survival rate of CLP mice, the results of the analysis, compared with the CLP Group Ghrelin interfere with the survival time of group C was significantly increased, significantly improve the survival rate of 20% (P <0.05). Ghrelin intervention CLP improved the survival rate of mice.Part II: Ghrelin-mediated intervention on the CLP-induced lung injury related to signal transduction mechanism --- NF-κB and its impact on the fibrinolytic system 1. Immunohistochemistry results showed that: Compared with normal group, sham operation group, CLP group of lung tissue expression of NF-κB markedly increased, Ghrelin intervention group C the expression of NF-κB weaker intensity than the CLP group, rather than alleviate the associated lung injury.2. ELISA results showed that: Compared with normal group, sham operation group, CLP group of t-PA levels slightly increased, PAI-1 levels were significantly increased, t-PA /PAI-1 ratio decreased fibrinolytic inhibition with ALI relevant.Compared with the CLP group, Ghrelin-C plasma levels of t-PA increased slightly (P<0.05), PAI-1 levels decreased significantly (P<0.001), t- PA /PAI-1 ratio increased significantly (P<0.001), and improve fibrinolytic activity of the Ghrelin .Conclusions:1. Ghrelin probably has the effect of anti-inflammatory and reducing pulmonaty vascular permeability, and improving the survival rate of CLP mice. the dose to achieve the best effect is Ghrelin intervention group C. Application of Ghrelin may help to improve the prognosis of ALI.2. Ghrelin maybe that reduces the inflammatory response of CLP-mediated acute lung injury via the NF -κB pathway, the enhance of fibrinolytic activity is another possible mechanism.