The N-3PUFAs Modulates Intestinal Mucosa Immunity In A Mice Model Of Hemorrhagic Shock Resuscitation Injury

Trauma is a kind of stress when individual is subjecting to wound,burn,major operation massive blood loss or shock.7.5 patients out of 100 patients in urban hospital were attributable to trauma or intoxication and trauma has been the fourth in which cause urban residents illness.The treatment to Hemorrhagic shock which was due to insufficient blood circulation caused by trauma has been an challenge for surgeons.The intestinal mucosal is one of the most easily affected tissue by ischemia-reperfusion injury.The first hit of ischemia reperfusion(I/R)is tissue hypoperfusion,hypoxia and the following cell dysfunction,the activation of protease and phospholipase caused by ischemia.Then the restoration of intestinal blood circulation was followed by intestinal oxidative stress injury caused by reperfusion.The damage of intestinal epithelium is the second hit to the body.Then a systemic inflammatory response can also be triggered by translocation of pro-inflammatory compounds such as endotoxin and bacteria from the gut lumen which causes the third hit to the body.The three sequential hits were associated with poor clinical prognosis.The intestinal barrier is mainly composed of the tight junction between the intestinal epithelial cells(mechanical barrier),the intestinal mucosal lymphocyte cells,antimicrobial peptides secreted by paneth cells and mucin secreted by goblet cells(immune barrier),the hydrion and HCO3-in the digestive juice(chemical barrier)and the relative stable bacteria in the intestines(biological barriers).n-3 polyunsaturated fatty acids(n-3PUFAs)can reduce the pro-inflammatory cytokines and promote the synthesis of anti-inflammatory cytokines by regulating the ratio of n-3/n-6 PUFAs of cell membrane phospholipids.By this way,n-3PUFAs can regulate the immune function and protect the vital organs.In addition,n-3PUFAs can also project the intestinal mechanical barrier against I/R injury through improving the tight junction after hemorrhagic shock resuscitation.In this study,we firstly established a stable and repeatable mice controlled hemorrhagic shock resuscitation survival model by optimizing pre-existing models.It is the solid foundation for exploring the effects of I/R on mouse intestinal mucosa innate immunity and the effects of n-3PUFAs on intestinal mucosa innate immunity after I/R injury.Moreover,we attempted to study the effects of n-3PUFAs on the intestinal flora after hemorrhagic shock resuscitation.The results of this study showed that the intestinal mucosa innate immunity was damaged and the intestinal flora was influenced after hemorrhagic shock resuscitation.The injury was gradually aggravated over time.The most serious I/R injury appeared at 12 hours after hemorrhagic shock resuscitation and then the damage was reduced gradually.n-3PUFAs could decrease the intestinal I/R injury and improve the intestinal flora partly.This may provide reliable theoretical basis for n-3PUFAs improving intestinal barrier function in post-traumatic patient who suffered from I/R injury.It may guide to provide nutrition support for surgery patients more reasonably and effectively,so as to optimize the treatment.PART1The effects of hemorrhagic shock resuscitation on mouse intestinal mucosa innate immunityObjective:To explore the effects of hemorrhagic shock resuscitation on mouse intestinal mucosa innate immunity at different time.Methods:Forty eight C57BL/6J mouse were randomly assigned to 6 groups(1)control group(CON),(2)sham group(Sham),(3)4 hours after hemorrhagic shock resuscitation(HSR-4h),(4)12 hours after hemorrhagic shock resuscitation(HSR-12h),(5)24 hours after hemorrhagic shock resuscitation(HSR-24h),(6)72 hours after hemorrhagic shock resuscitation(HSR-72h).The left femoral artery was cannulated with a catheter under anesrthesia and the blood was withdrawn achieving a mean arterial pressure of 30 mmHg.The animals were remained in hemorrhagic shock for 90 minutes and then the animals were resuscitated.The mouse was sacrificed at different time and the terminal ileum tissue were harvested for lysozyme and mucin 2(MUC2)analysis.Results:Compared with control group,the decreases of lysozyme and MUC2 in intestinal tissue were initialed in HSR-4h group.The levels of lysozyme and MUC2 were decreased significantly in HSR-12h group(P<0.05,respectively)and the intestinal innate immunity was damaged.Then,the reduced lysozyme and MUC2 began to restore.Conclusions:The injury of intestinal mucosa innate immunity was the most serious in the HSR-12h group and this group was more suitable to explore the effects of I/R injury on intestinal barrier and intestinal microecology.PART 2The effects of n-3PUFAs on intestinal mucosa immunity in a mice model of hemorrhagic shock resuscitation injury.Objective:To explore the effects of n-3PUFAs on intestinal mucosa innate immunity in mouse after hemorrhagic shock resuscitation injury 12 hours.Methods:Forty C57BL/6J mouse were randomly assigned to 5 groups(1)controlgroup(cCON),(2)sham group(Sham),(3)12 hours after HSR(HSR),(4)HSR and n-3PUFAs group(n-3PUFAs),(5)HSR and n-6 PUFAs group(n-6 PUFAs).The mouse in HS group was operated as the description in part 1.The mouse in n-3PUFAs or n-6 PUFAs group were treated with n-3PUFAs or n-6 PUFAs in a dosage of 0.2g/kg(body weight)/day after resuscitation by vena caudalis respectively.The mouse was sacrificed at 12 hour after HSR and the liver,spleen and mesenteric lymph nodes(MLN)were collected aseptically for bacterial culture to evaluate the effects of n-3 PUFAs on intestinal barrier function.The terminal ileum tissue was also harvested for pathological detection,qRT-PCR and western blot analysis.The intestinal mucosa was scraped aseptically to assess the bacteria composition in the intestinal mucosa.Results:Compared with HSR group,n-3PUFAs could improved the levels of lysozyme and MUC2 in intestinal tissue(P<0.05,respectively)and increased the goblet cells obviously(P<0.05).Mouse in n-3PUFAs group had less bacteria-positive MLN(P<0.05)and the proportion of proteobacteria was reduced in intestinal mucosa compared with mouse in HSR group(P<0.05).The level of lysozyme in n-3PUFAs group was higher than n-6 PUFAs group(P<0.05),however the differences between n-3PUFAs group and control group were still significant(P<0.05).Conclusions:n-3PUFAs could decrease the intestinal I/R injury in mouse suffered from HSR.n-3PUFAs could also improve the intestinal barrier function and could help to stabilize the intestinal flora.