Effects Of Pyruvate–based Hydroxyethyl Starch On Intestinal Microvascular Permeability And Intestinal Barrier Function Of Burn Injury In Rats

Objective: Colloids combined with crystalloids are often used in treatment of burns, sepsis and various hypovolemic shock in clinical settings. The plasma and albumin as natural colloids are preferred. Due to the difficulty of obtaining blood products, hydroxyethyl starch(HES) and other artificial colloids have been commonly applied as the alternatives of plasma products because of their fast and powerful expansion effects. However, serious adverse reactions induced by the infusion of HES were found at home and abroad in recent years, thus widespread controversy was raised by the use of HES [1]. Several previous studies showed that sodium pyruvate had anti-acid, anti-inflammatory and anti-oxidant effects, and it could reverse the glucose metabolic disturbance and acid-base imbalance caused by variour types of shock and protect organ function [2]. The purpose of this study focuses the sodium pyruvate as a novel carrier solution in HES(pyruvate-bases HES) on effects on the reduction of vascular leakage and tissue edema and the protection of intestinal microvascular permeability and intestinal barrier function, compared with sodium chloride-HES and acetate Ringer-HES that are usually used in the clinical setting, in rats subjected with severe burns.Method:1 Animal Model:100 male SD rats, weighing 220g-270 g, were adaptive feeding for one week after purchase. Fasting 12 hours, but free access to water until 4 hours before surgery, rats were placed in an opened prefabricated container after intraperitoneal injection for anesthesia by pentobarbital sodium(50mg/kg) and cutting off hairs in the neck, back, buttocks and abdomen. The rats were emmerced in boiling water(100℃ water bath in boiling water) for 15 seconds on back and 8 seconds on abdomen to cause a 50% total body surface(TBSA) III degree burns. Rats in the sham control group were placed in warm water(37℃ water bath) for 15 seconds on back and 8 seconds on abdomen. Injured rats were then aseptically inserted with a catheter into the jugular vein for fluid infusion, fixed with a specific experimental vest after being connected to the infusion device and placed in the cages. The shaft assembly on the vest ensured the free movement of rats awaked from anesthesia in the cages during the infusion.2 Grouping and treatment: According to the treatment of burn, the rats were randomly divided into five groups(N=20, two subgroups each, n=10) :①the sham burn group(group SC, n=20);②the burn without infusion group(group N);③the burn with sodium pyruvate- HES group(Pyr-HES, group SP);④the burn with sodium acetate Ringer – HES group(Ace-HES, group SA);⑤the burn with sodium chloride- HES group(Na Cl-HES, group SN). The total amount of intravenous fluids were 0.75ml/kg×1% TBSA(1/2 colloidal rehydration formula) of various HES and the equal volume of Acetated Ringer’s solution(AR) with the colloid to crystalloid ratio of 1:1 witnin 12 h resuscitation. Rats in each fluid resuscitation group were made half injection with acetate Ringer’s solution in the first 4 hours after burns and one of various HES solutions was given for another half in the later 8 hours by a liquid micro-infusion pump according to the calculated volumes.3 Measurements and methods: Blood samples from abdominal aorta were drawn and intestinal tissues were harvested in-80°C liquid nitrogen for frozen storage after the rats were sacrificed at 8 hours and 24 hours after sacald injury in two subgroups, respectively. Just before rats were sacrificed, the followings were measured: ①intestinal mucosal blood flow(IMBF) ②intestinal microvas- cular permeability was detected by isocyanate FITC-dextran; the following indicators were also detected with samples: ③The changes of intestinal tissue water content were detected by the ratio of dry over wet method;④Activities of serum diamine oxidase(DAO) were detected by the UV spectrophotometer; ⑤The VEGF levels in intestinal tissues were detected by ELISA and immunohistochemical staining; ⑥The barrier protein(ZO-1) was detected by immunofluorescence(Western blot) and immunoblotResults:1 Intestinal mucosal blood flow(IMBF) in the N group and fluid resuscitation groups after burn injury were significantly lower than that in the SC group(P<0.05); IMBF was significantly higher in group SP than in group SA and group SN at 8 hours after injury(4 hours after colloid infusion)(P<0.05), while group SA showed higher blood flow compared to group SN(P<0.05). IMBF was higher in all three resuscitation groups than in group N. It had no significant difference between groups SP and SA but was higher in these two groups relative to the SN group at 24 hours. Rats in group N were all dead at 24 hours after injury.2 Fluorescein isothiocyanate-labeled dextran(FITC-dextran): Compare to the SC group, the FITC-dextran levels in the N group and the resuscitation groups at two time points after injury were significantly increased(P<0.05). Groups SP and SA at each time point had significantly lower FITC-dextran levels than groups N and SN(P<0.05) and the dextran level in the SN group was lower than that in the N group(P<0.05). The level was significantly lower in the SP group than in the SA group only at the time point of 8 hours(P<0.05) and there was no difference between these two groups at 24 hours. Rats in group N were all dead at 24 hours, when groups SP and SA had lower levels, compared to group SN.3 DAO activity in serum: The DAO activity in scald groups was significantly increased, compared with the SC group(P<0.05), the activities in the SP and SA groups were significantly lower than those in the N group(P<0.05). The activity was significantly lower in group SP than in group SA at 8 hours(P<0.05), but there was no statistically significant difference among three resuscitation groups at 24 hours after injury.4 VEGF in intestine: The VEGF expression in the scald groups was significantly increased, compared with the SC group(P<0.05). The expression was lower in the SP group than in the SN and SA groups(P<0.05) at 8 hours, when the SA group had a lower VEGF level, compared to the SN group(P<0.05). SP and SA groups had no statistically significant difference of the expressions at 24 hours after injury, but both of them showed a lower expression relative to the SN group.5 ZO-1 in intestine:(1)Western Blot analysis:Compared with the SC group, the ZO-1 expressions in scald groups were significantly reduced at two time points(P<0.05). The expression showed no statistically significant difference between SP and SA groups, but expressions in both groups were higher than in N and SN groups at 8 hours after scald. SN group was significantly better than N group. 24 hours after scald, compare to N group and other treatment groups the expression of ZO-1 of SP group was significantly increased;(2)The result from immunofluorescence technique was basically the same as that from Western Blot. Compared with the N group, the ZO-1 staining in the SC group displayed a continuous strong fluorescence signal; the ZO-1 signal in SP and SA groups was reduced, compared to the SC group, but higher than that in N and SN groups.6 Intestinal tissue water content: Intestinal tissue water contents in scald groups at two time points were significantly increased, compared with SC group(P<0.05). Intestinal tissue water contents in SP and SA groups were decreased at 8 hours after injury, compared with SN and N groups(P<0.05), when the content was significantly lower in group SP than in group SA(P<0.05) and was lower in group SN than in group N. There was no statistically significant difference of water contents in the intestine among three resuscitation groups at 24 hours after injury.Conclusion:1 Na Pyr-HES increased the IMBF, reduced the intestinal vascular leakage and tissue edema and protected intestinal function in burn rats;2 Na Pyr-HES was more beneficial than Na Cl-HES and more effective than Na Ace-HES at a considerable extent in the protection of intestine;3 Na Pyr-HES protected the intestinal microvascular endothelial and epith- elial barrier; the underlying mechanism might be closely associated with its effect on reducing VEGF expression and increasing ZO-1 expression and IMBF in intestine.