Effect Of Different Concentrations Hypertonic Saline Resuscitation On Intestinal Injury In Severely Burned Rats

1. ObjectivesThis aim of this study was to investigate the effect of different concentrations hypertonic saline resuscitation on intestinal injury in severely burned rats by administration with 200mmol/L, 300mmol/L and 400mmol/L hypertonic salt solution(HS) versus lactated Ringer solution(LR). Inflammatory cytokines, oxidative stress and signal transduction pathway activation were detected to study the mechanism of HS resuscitation after thermal injury. 2. Materials and methodsOne hundred and four adult healthy SD rats(200-250 g weight) were randomly divided into five groups: control group, sodium lactate Ringer’s solution group(LR group), 200mmol/L hypertonic sodium solution group(HS200 group), 300mmol/L hypertonic sodium solution group(HS300 group) and 400mmol/L hypertonic sodium solution group(HS400 group). All the rats were shaven on back and scalded for 30% total body surface area(TBSA) except the control group. Tail vein catheterization was used on all rats, and burn resuscitation therapy was adopted in the rats of LR group, HS200 group, HS300 group and HS400 group. The abdominal aortic blood and intestinal tissue in rats were harvested from the control group and at 2h, 8h and 24 h post burn in the LR group and various HS groups after to be put death respectively. Part I:Effect of 200mmol/L, 300mmol/L and 400mmol/L hypertonic sodium fluid resuscitation on serum sodium concentration, moisture content of intestinal and blood inflammatory cytokines in severely burned rats.The tests were made respectively in the control group and at 2h, 8h, 24 h post burn after fluid resuscitation in the LR group, HS200 group, HS300 group and HS400 group which covers the following aspects such as serum sodium, moisture content of intestinal, blood inflammatory cytokines such as tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and high migration rate protein B1(HMGB1). The obtained data were statistically analyzed. Part II: Effect of 200mmol/L, 300mmol/L and 400mmol/L hypertonic sodium fluid resuscitation on tissue oxidative stress in severely burned ratsThe tests were made respectively in the control group and at 2h, 8h, 24 h post burn after fluid resuscitation in the LR group, HS200 group, HS300 group and HS400 group which covers the following aspects such as malondialdehyde(MDA) content, superoxide dismutase(SOD) and diamine oxidase(DAO) activity. The another intestinal tissue was fixed with 10% formalin and tested the von Willebrand factor(v WF) by using immunohistochemistry. Part III: Effect of 200mmol/L, 300mmol/L and 400mmol/L hypertonic sodium fluid resuscitation on the activity of p38 MAPK and JNK in the intestinal tissue of severely burned ratsThe p38 MAPK and JNK activity levels of intestinal tissue were measured respectively by using Western blotting(Western Blot) in the control group and at 2h, 8h, 24 h post burn after fluid resuscitation in the LR group, HS200 group, HS300 group and HS400 group. The obtained data were statistically analyzed. Results Part IThe concentrations of serum sodium at 2h, 8h, 24 h post burn in the LR group were significantly lower than those in the control group, relected HS200 group, HS300 group and HS400 group, the difference was statistically significant(P<0.01). The concentration of serum sodium in the control group was significantly lower than that at each time point post burn in HS300 group and HS400 group and at 2h, 24 h post burn in HS200 group, the difference was statistically significant(P<0.01). Compared with HS200 group,the concentrations of serum sodium at 2h, 8h, 24 h post burn in HS300 group were no significant difference(P>0.05), which in relected HS400 group were relatively higher, the difference was statistically significant(P<0.01). Compared with HS300 group,HS400 group was clearly increasrd only at 24 h post burn(P<0.05).The intestinal W/D ratios at 2h, 8h, 24 h post burn in the LR group, HS200 group and at 8h, 24 h in the HS300 group as well as at 8h in the HS400 group were higher than those in control group, the difference was statistically significant(P<0.05 or P<0.01). Compared with the LR group, the intestinal W/D ratios at 2h, 8h, 24 h post burn in HS400 group were relatively lower, the difference was statistically significant(P<0.05 or P<0.01), so were at 2h, 8h post burn in HS200 group and HS300 group. There were no significant difference in the intestinal W/D ratios at each time point post burn among HS200 group, HS300 group and HS400 group(P>0.05).Compared with the control group, the serum concentrations of TNF-α, IL-1β at 2h, 8h, 24 h post burn in LR group were obviously increased, so were the serum concentrations of TNF-α at 2h, 8h post burn in HS200 group and HS300 group as well as at 8h in HS400 group. The serum concentrations of IL-1β at each time post burn in three HS group were all clearly higher than those in the control group, the difference was statistically significant(P<0.05 or P<0.01). The serum concentrations of HMGB1 at 8h, 24 h post burn in the LR group, HS200 group and HS300 group and at 24 h post burn in HS400 group were significantly increased compared with the control group(P<0.05 or P<0.01), but the differences were no statistically significant at 2h post burn(P>0.05). Compared with LR group, the serum concentrations of TNF-α at 2h, 8h post burn in HS200 group, HS300 group and HS400 group were clearly decreased, which were relatively lower in the serum concentrations of HMGB1 at 8h, 24 h post burn and in the serum concentrations of IL-1β at each time point, the difference was statistically significant(P<0.05 or P<0.01). There were no significant difference in the serum concentrations of TNF-α, IL-1β at 2h, 8h, 24 h post burn and HMGB1 at 2h, 8h post burn among HS200 group, HS300 group and HS400 group(P>0.05),but the serum concentrations of HMGB1 at 24 h post burn in HS400 group was clearly decreased compared with the relected HS200 group(P<0.05). Part IIThe contents of intestinal tissue MDA at 2h, 8h, 24 h post burn in the LR group, HS200 group, HS300 group and HS400 group were higher than those in the control group, the difference was statistically significant(P<0.05 or P<0.01). The contents of intestinal tissue MDA at each time point post burn in LR group were higher than those in related HS300 group, HS400 group and HS200 group except at 24 h post burn, the difference was statistically significant(P<0.05). Compared with HS200 group, the contents of intestinal tissue MDA at each time point post burn in HS400 group were obviously decreased, the difference was statistically significant(P<0.05 or P<0.01). There were no significant difference in the contents of intestinal tissue MDA at 2h, 8h, 24 h post burn between HS200 group and HS300 group as well as between HS300 group and HS400 group(P> 0.05).The activities of intestinal tissue SOD at 2h, 8h, 24 h post burn in LR group were lower than those in the control group, so were at 8h post burn in HS200 group and HS300 group, the difference was statistically significant(P<0.05 or P<0.01). The activities of intestinal tissue SOD at 8h post burn in HS300 group and HS400 group were lower than those in relected LR group, the difference was statistically significant(P<0.05 or P<0.01). There were no significant difference in the activities of intestinal tissue SOD at 2h, 8h, 24 h post burn among HS200 group, HS300 group and HS400 group(P>0.05).The activities of intestinal tissue DAO at 2h, 8h, 24 h post burn in LR group, HS200 group, HS300 group and HS400 group were lower than those in the control group, the difference was statistically significant(P<0.05 or P<0.01). Compared with LR group, the activities of intestinal tissue DAO at 2h, 24 h post burn in HS300 group and at each time point post burn in HS400 group were obviously increased, the difference was statistically significant(P<0.05 or P<0.01). The activities of intestinal tissue DAO at 2h post burn in HS400 group and HS300 group were higher than those in related HS200 group(P<0.01). Except that, there were no significant difference in the activities of intestinal tissue DAO at another time point post burn among HS200 group, HS300 group and HS400 group(P>0.05).The activities of v WF immunohistochemistry in intestinal tissue show: compared with LR group, the expressions of intestinal tissue v WF in HS200 group, HS300 group and HS400 group were significantly increased, which was higher with the increase of HS concentration. Part IIIThe activity expressions of intestinal tissue p38 MAPK in LR group, HS200 group at 2h, 8h, 24 h post burn were higher than those in the control group, the difference was statistically significant(P<0.05 or P<0.01), so were at 8h, 24 h post burn in HS300 group and at 8h post burn in HS400 group(P<0.01). The activity expressions of intestinal tissue p38 MAPK at each time point post burn in LR group were higher than those in relected HS200 group, HS300 group and HS400 group, the difference was statistically significant(P<0.01). The activity expressions of intestinal tissue p38 MAPK at each time point post burn in HS400 group were lower than those in relected HS200 group, so were at 8h, 24 h post burn in HS300 group(P<0.01). The activity expression of intestinal tissue p38 MAPK at 2h, 8h post burn in HS400 group was clearly decreased compared with HS300 group(P<0.01).Compared with the control group, the activity expressions of intestinal tissue JNK at 2h, 8h, 24 h post burn in LR group and at 8h, 24 h post burn in HS200 group were significantly increased, the difference was statistically significant(P<0.05 or P<0.01), so were at 8h post burn in HS300 group and HS400 group. The activity expressions of intestinal tissue JNK at each time point post burn in LR group were higher than those in relected HS200 group, HS300 group and HS400 group, the difference was statistically significant(P<0.05 or P<0.01). Compared with HS200 group,the activity expressions of intestinal tissue JNK at 24 h post burn in HS300 group and at 8h, 24 h in HS400 group were significantly decreased(P<0.01). The activity expressions of intestinal tissue JNK at 2h, 8h post burn in HS400 group were lower than those in relected HS300 group, the difference was statistically significant(P<0.05 or P<0.01). Conclusion 1. Compared with LR, three different concentrations hypertonic saline resuscitation can prevent hyponatremia, decrease intestinal edema and inhbit the expression of inflammatory cytokines in severely burned rats. There were not significant differences in the curative effect among HS200 group, HS300 group and HS400 group. However, the concentration of serum sodium was significantly higher by using 400mmol/L HS. 2. Compared with LR, three different concentrations hypertonic saline resuscitation can attenuate oxidative stress of intestinal tissue and protect the intestinal endothelial cells in severely burned rats. Of all, the curative effect of 400mmol/L HS is the best one. 3. Compared with LR, three different concentrations hypertonic saline resuscitation can attenuate the activity expressions of p38 MAPK and JNK in intestinal tissue of severely burned rats. The curative effect of 400mmol/L HS is the best among three HS groups.