The Role Of Insulin In Dysfunction Of Endothelial Cell Of Postburn And Its Intracellular Signal Transduction

BackgroundMultiple organ dysfunction syndrome has been reported to be the predominant cause of death in severely burned patients. Although the pathophysiology of multiple organ dysfunction syndrome involves a highly complex, integrated response that includes the activation of a number of cell types, the endothelial cell function has been demonstrated to be central to this process. Postburn endothelial“capillary leak”and subsequent interstitial edema constitutes one of the earliest stages in the development of endothelial cell dysfunction.Insulin resistance and hyperglycemia accompany critical illness, and the severity of this“diabetes of stress”reflects the risk of death in patients with severe trauma, burn injury, or sepsis. In a previous randomized, controlled study conducted in a surgical intensive care unit, strict control of blood glucose levels with insulin substantially reduced complications, such as severe infections and organ failure, and mortality. Some researchers believe that almost all of the benefits from intensive insulin therapy are due to the metabolic regulatory mechanism. However, in recent years, it has been proven that insulin can attenuate systemic inflammatory responses and modulate the proliferation, apoptosis, differentiation and immune functions of certain immune cells. That is to say that insulin has not only anabolic properties, but also glycemia-independent actions. Intensive insulin therapy was found to protect the endothelium of critically ill patients and therefore contributed to prevention of organ failure and death. To date, however, the precise mechanisms still remain obscure.In this study, we investigated the effects of insulin on endothelial cell dysfunction after severe burn injury. The first step was designed to investigate the effects of intensive insulin treatment on pulmonary damage after severe burn injury. We hypothesize that treatment with intensive insulin therapy immediately after burn injury will attenuate pulmonary edema, hemorrhage and inflammatory cell infiltration through decreasing pulmonary microvascular permeability. After that we designed to investigate the alterations in tight junction and apoptosis status as well as permeability of human umbilical vein vascular endothelial cells (HUVEC) in response to burn serum and insulin treatment, with special attention given to whether or not AKT signaling pathway participated in modulation of the process.Methods1. Seventy five male Sprague-Dawley rats were randomly divided into 3 groups: sham scald group, scald group ( subjected to 30% TBSA??scald ), scald + intensive insulin treatment group. The oxidase method was performed to measure the blood glucose contents of rats at 24 hours post-scald. Histological changes of the lung, the concentrations of myeloperoxidase (MPO) in lung tissue, superoxide dismutase (SOD) in serum and the protein contents in the bronchoalveolar lavage fluid (BALF) were measured in the rats at 12 posthurn hours. Technique of electron microscopy was performed to observe the changes of vascular endothelium cell structure and colorimetric method was used to determine the serum levels of nitric oxide (NO) and nitric oxide synthetases (NOS) at the same time.2. HUVEC were cultured in vitro and treated with 20% normal serum, 20% burn serum or 20% burn serum + insulin. Selected cultures were pretreated with phosphatidylinositol 3-kinase (PI3K)/AKT (protein kinase B) inhibitor (LY-294002). Permeability was assessed by migration of bovine serum albumin across cell monolayers. Cells were stained with rhodamine- phalloidin and examined. Cell extracts were obtained to assess zonula occludens-1 (ZO-1), occludin, phosphorylated AKT levels by immunoblotting.3. HUVEC were cultured in vitro and randomly divided into 4 groups: control, 20% normal serum, 20% burn serum and 20% burn serum + insulin. The transferase mediated nick end labeling (TUNEL) and flow cytometry were performed to measure the apoptosis of endothelial cells after 6 hours. Meanwhile, immunohistochemical technique and Western blotting were used to determine the protein expressions of bcl-2 and eNOS.4. HUVEC were cultured in vitro and randomly divided into 5 groups: 5% fetal bovine serum group, 20% healthy human serum group, 20% burn human serum group, 20% burn human serum followed by insulin ( 10-7 mol/L) group and pretreated with inhibitor (LY-294002) group. The serum IL-1?and TNF-?were observed by ELISA method. Meanwhile, the phosphorylation of inhibitor kappa B-?(I?B-?) in the cytoplasm and the content of NF-?B-p65 in the nucleus were detected after 6 hours by immunoblotting.Results1. Treatment with intensive insulin attenuated the pulmonary edema, hemorrhage and inflammatory cell infiltration of rats with severe burn injury. The protein content of BALF and MPO of lung tissue in intensive insulin group were lower than those in scald group (P <0.05), but SOD in serum in intensive insulin group was higher than that in scald group (P <0.01). The more favorable structures of endothelium cells in intensive insulin group were showed under electron microscope in comparison with the observation to scald group. Compared with the NO, NOS and cNOS serum contents in scald group, all of those in intensive insulin group increased significantly (P <0.01).2. Burn serum significantly enhanced monolayer permeability to albumin, whereas treatment with insulin (10-7 mol/L) limited this effect. Meanwhile, insulin (10-7 mol/L) reduced burn serum-induced F-actin stress fiber formation and decreased ZO-1 expressing. LY-294002 decreased cytoplasmic AKT phosphorylation and prevented the protection effects of insulin.3. Compared with the control group, burn serum could induce the apoptosis and down-regulate bcl-2 and p-eNOS protein expressions of HUVEC (P <0.01). Burn serum + insulin (10-7 mol/L) group could significantly decrease the apoptosis and up-regulate bcl-2 and p-eNOS protein expressions of HUVEC in comparison with the observation to burn serum group (P <0.01). eNOS showed no significant differences in three groups. 4. Compared with the fetal bovine serum group, burn serum could up-regulate the protein expressions of p-I?B-?in the cytoplasm and NF-?B-p65 in the nucleus (P <0.01). Compared with the burn serum group, the I?B-?phosphorylation and NF-?B-p65 nuclear translocation were apparently lowered (P <0.01). Specific inhibitor (LY294002) could abolish the inhibitory effects of insulin on NF-?B-p65 nuclear translocation of HUVECs induced by burn serum.Conclusions1. Intensive insulin treatment could effectively attenuate the acute lung injury caused by severely burned injury. The endothelium cell protection may play a key role in the potential mechanism of this insulin effect on the lung injury.2. Through PI3K/AKT pathway, insulin can attenuate increased endothelial permeability induced by burn serum. The effect is attributed to attenuating architectural disruption of protein components of the endothelial TJ, and may be very useful in inhibiting the multiple organ failures after burn injury.3. Insulin could markedly inhibit the apoptosis and up-regulate bcl-2 protein expression of HUVEC induced by burn serum, and its mechanism might involve the protein expression of phosphorylated eNOS.4. Through PI3K/AKT pathway, insulin could decrease the I?B phosphorylation, then inhibit NF-?B nuclear translocation induced by burn serum and improve the vascular endothelial cells function accordingly.