The Effect And Mechanism Of Insulin On Systemic Inflammatory Reaction Syndrome After Severe Burn Injury

Objective:The uncontrolled inflammation induced by severe burn is one of the important causes which lead to MODS and mortality. At present, it is presumed the uncontrolled inflammation happened because of the unbalance between the pro-inflammatory cytokines and the anti-inflammatory cytokines. Recent researches showed that insulin could improve the inflammation after severe burn by regulating the balance of pro-inflammatory cytokines and the anti-inflammatory cytokines. Macrophage is one kind of key inflammation cells. Macrophage can secrete many kinds of pro-inflammatory and anti-inflammatory cytokines. So, investigate the mechanism of regulating function of secreting inflammation cytokines and signal transduction is very important to elucidate the anti-inflammatory mechanism of insulin. Our reasearch is going to determine the possible effect of different doses of insulin on the pro-inflammatory cytokine IL-1 and the anti-inflammatory cytokine IL-10 and the protective effect of insulin on hepatic injury of oxyradical in early stage of severe burn. In vitro, we will determine the quantity and function of PI3K-Akt of the insulin signal transduction and the expression of TNF-α, IL-1βand IL-10. At the same time, to determine the quantity and function of IκB and NF-κB, to elucidate the signal transducion mechanism of insulin. It may supply the theoretic basis for applying insulin to control the uncontrolled infalmmation after severe burn and increase the cure rate of severe burn patients, it may also suppy new way to treat uncontrolled inflammation.Methods:1. Sprague-Dawly rats (30% TBSA, full thickness burn) were randomly divided into two groups to receive either normal saline only (n = 14, with intra-peritoneal injection of 40ml/kg normal saline, group A) or normal saline plus different doses of insulin (n = 42, with intra-peritoneal injection of 40ml/kg normal saline plus subcutaneous injection of 1 IU/kg, 3 IU/kg or 5 IU/kg insulin respectively, group B1, B2, B3). In addition, 7 rats without thermal injury were set up as normal control group (group N). The outcome measures encompassed the serum glucose, blood pressure, serum IL-1βand IL-10 in rats at 6 hours and 24 hours after scald and different treatments.2. Sprague-Dawly rats (30% TBSA, full thickness burn) were randomly divided into two groups to receive either normal saline only (n = 28, with intra-peritoneal injection of 40ml/kg normal saline, group A) or normal saline plus different doses of insulin (n = 28, with intra- peritoneal injection of 40ml/kg normal saline plus subcutaneous injection of 3 IU/kg insulin respectively, group B1). In addition, 28 rats without thermal injury were set up as normal control group (group N). The outcome measures encompassed the total antioxygen capability (T-AOC), superoxide dismutase (SOD), reactive oxygen species (ROS) and intercellular adhesion molecule (ICAM-1) in hepatic tissue, and serum alanine transaminase (ALT) in rats at 6, 12, 24, 48 hours after scald and different treatments.3. The U937 cell was cultured in 1640 medium and divided into six groups as 10% normal rat serum (group A),10% normal rat serum and 10U/L INS (group B), 10% normal rat serum and 10U/L INS and LY-294002 (group C), 10% scalded rat serum (group D), 10% scalded rat serum and 10U/L INS (group E), 10% scalded rat serum and 10U/L INS and LY-294002 (group F). The nuclear and cytoplasmic protein was extracted to determine the p-Akt, IκB and NF-κB.at 6 and 24 hours. The supernatant liquids were collected to determine the TNF-α,IL-1βand IL-10 at 24 hours.Results:1. The serum glucose increased quickly in rats after scald, which was demonstrated in group A to a range of 10.72±1.76 mmol/L by 6 hours post-scald. Insulin decreased the serum glucose by a dose-dependent pattern in rats of groups B1, B2 and B3. And insulin at a dose of 3 IU/kg body weight reduced the serum glucose to a range of 5.97±0.76 mmol/L in rats at 6 hours after thermal injury, which was almost eqeal to the normal range of 5.13±0.62 mmol/L (p < 0.05). Meanwhile, insulin at this dose improved the blood pressure of scalded rats by keeping the blood pressure at a stable condition similar to the normal. Insulin significantly decreased the serum IL-1βby a dose-dependent pattern in scalded rats. It was partially showed after administration of insulin of 3 IU/kg by serum IL-1βmeasurements as 54.04±8.64 ng/L and 46.83±6.77 ng/L much lower than that without insulin administration in rats at 6 hours and 24 hours after thermal injury (p < 0.01). Insulin could increase the serum IL-10 of scalded rats. In comparison with group A, the most significant increase of serum IL-10 was seen by application of 3 IU/kg insulin as 45.46±5.20 ng/L in rats 24 hours post scald (p < 0.01).2. The hepatic T-AOC and SOD decreased quickly in group A by 6 hours post-scald (P < 0.05), While the ROS increased rapidly at the same time (P < 0.05). Later on, the ICAM-1 and serum ALT increased from 12 to 48 hours post-scald (P < 0.05). The tendency become more obviously in the followed time points after 6 hours (P < 0.01). The hepatic T-AOC and SOD were 368.3±60.7 U/g and 210.7±29.1 U/g by 24 hours post-scald in group B much higher than those in group A (P < 0.01). At the same time, the ROS was demonstrated to a range of 154.3±28.6 U/g much lower than that in group A (P < 0.01). The significantly decreased ICAM-1 and serum ALT were shown by insulin treatment in scalded rats, which were demonstrated to ranges of 244.8±54.4 mmol/L and 78.9±15.8 ng/L by 48 hours post-scald (P < 0.01). Meanwhile, insulin improving liver morphology of scalded rats was confirmed by pathological section.3. Insulin significantly increased TNF-α, IL-1βand decreased IL-10 at the same time by increasing p-Akt and NF-κB in U937 cell cultured by RPMI 1640 medium and 10% normal rat serum. When U937 cell was stimulated by 10% scalded rat serum, the decreased TNF-α, IL-1βand increased IL-10 were shown by ELISA, and the significantly decreased p-Akt and increased NF-κB were demonstrated by Western Blotting. Insulin attenuated the effect of scalded rat serum on U937 cell by increased p-Akt. Meanwhile, LY-294002, the blocking agent of PI3K, interrupted the insulin effect. Conclusions:Our data provided an insight that insulin might attenuate the inflammatory cascade response by decreasing the pro-inflammatory cytokine IL-1βand increasing the anti-inflammatory cytokine IL-10, thereby keep or restore the systemic homeostasis after severe burn. The best administration dose of insulin in early burn stage should be determined by serum glucose normalizing. At the same time, insulin might attenuate the hepatic injury in severely scalded rats by decreasing the ROS and increasing the T-AOC and SOD.Insulin attenuated the effect of scalded rat serum on macrophage and depressed the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokines, which were mediated partly by PI3K-Akt pathway.