Preliminary Study Of The Intestinal Barrier Function Injury In Rats With Open Celiac Seawater Immersion Wounds

BackgroundIntestine barrier functional disturbance(IBFD) is a usual complication when the organism is having various dangerous deseases,sugery operation or severe injury.It occurs very often.However,its precise pathogenesis is not very clear yet.Current opinion is that acute pathological changes of intestinal mucosa under the stress condition relate to such aspects as weakening of the protective mechanism of intestinal mucosa, relatively strengthening of injury factors and neuroendocrine functional disorder etc., which are the results of synthetic action of multiple factors.Intestinal mucosal injury and the pathological change that it mediates aggregate the condition of illness.Bacteria and endotoxin may translocate through multiple channels,causing BT and intestinal endotoxemia,thus start SIRS which causes MODS.Through study,it is found that the factors of microcirculation disturbance,lesion of enterokinesia function,excess release of mediators of inflammation,injury of oxygen-derived free radicals,lesion of immune function and apoptosis may play important roles in the mechanism of intestinal barrier function.Protection of intestinal barrier function has important significance to the improvement of prognosis.It is a usual scene that injured solders fall into and immerse in seawater of alky and hypertonicity,which is a severe stress condition.Previous study finds that seawater immersion wounds have the following characteristics:hypernatremia,hyperchloraemia, hypertonicity and severe electrolyte disturbances accompanied with metabolic and respiratory acidosis.Seawater immersion wounds may cause severe hemodynamics disorder,hypercoagulabale state(HCS) and microcirculation disturbance,which may easily cause MODS.During the remedy of the injured soldier,seawater immersion wounds' injury to intestinal barrier has been found and deemed to be one of the difficult problems to successful remedy.To deeply study the influence of seawater celiac immersion wounds to intestinal barrier function,to find correspondent solutions to effectively and integrally control and remedy IBFD,is a necessary and important component to reduce fatality of seawater immersion wounds,and also the objective of this study.Objective1.To build the model of rats with open celiac seawater immersion wounds,to observe DAO,D-lactate,level of LPS in plasma,change of BT and histomorphology of intestinal mucosa of rats with open celiac seawater immersion wounds,to ascertain the intestinal barrier function changes of rats.2.To explore the mechanism of intestinal barrier function injury of rats with open celiac seawater immersion wounds;to observe the enterokinesia speed change,the contents of MDA and SOD in the intestinal mucosa tissue,level of TNF and IL-6 in plasma,change of SIgA in the intestinal content and IgA in plasma,and the apoptosis of intestinal mucosa.Methods1.The rat model of open celiac wound was built with fifty male Wistar rats weighing 230±20g which were randomly divided into five equal groups;celiac seawater immersion group with open celiac wounds(Group A),open celiac wounds group without seawater immersion(Group B),abdominal seawater immersion group without open celiac wounds(Group C),celiac physiological saline immersion group with open celiac wounds(Group D) and normal control group(Group E).2.Under the dual stress conditions of open celiac wounds and man-made seawater immersion of rats,after immersion stress of 1 hour,to measure DAO,D-lactate in plasma by absorption spectrometry,LPS in plasma by dynamic nephelometry.To observe BT through fixed-quantity bacteria cultivation.3.To observe the pathologic change of intestinal mucosa tissue by light microscope and intestinal tissue ultramicrostructure change by TEM,in accordance with such methods as evaluation of intestinal mucosa epithelium tissue injury index.4.To measure rats' intestinal peristalsis speed by activated carbon marked method,to measure MDA and SOD in intestinal mucosa tissue by absorption spectrometry,to measure TNF and IL-6 in plasma by ELISA,and SIgA in intestinal content and IgA in plasma.Results1.Test of the activities of D-lactate in plasma of rats:Compared with that in Group E (4.06±0.39 mg/L),the activities of D-lactate in plasma of rats in Group A(7.63±0.72 mg/L),Group B(5.46±0.57 mg/L) and Group D(4.97±0.95 mg/L) are significantly higher(P＜0.01),and that in Group C(3.89±0.55 mg/L) is not significantly different (P＞0.05);compared with that in Group B,the activity of D-lactate in plasma of rats in Group D is not significantly different(P＞0.05),and those in Groups A and C are significantly different(P＜0.01).2.Test of the activities of DAO in plasma of rats:Compared with that in Group E (0.36±0.04 KU/L),the activities of DAO in plasma of rats in Group A(6.24±1.54 KU/L), Group B(3.57±1.65 KU/L),Group C(0.42±0.04 KU/L) and Group D(3.4±1.58 KU/L) are significantly higher(P＜0.01);while compared with that in Group B,the activity of DAO in plasma of rats in Group D is not significantly different(P＞0.05),those in Groups A and C are significantly different(P＜0.01).3.Test of the LPS levels in plasma of rats:Compared with that in Group E(23.5±10.4 pg/ml),the LPS levels in plasma of rats in Group A(486.5±116.1pg/ml),Group B (344.5±51.5pg/ml) and Group D(338.7±54.4 pg/ml) are significantly higher(P＜0.01),and that in Group C(23.0±11.7 pg/ml) is not significantly different(P＞0.05);compared with that in Group B,the LPS levels in plasma in Groups A and C are significantly different (P＜0.01),and that in Group D is not significantly different(P＞0.05).4.Bacteria quantization cultivation results of the tested rats' organs:The results manifest that,through bacteria cultivation,no colony occurs in the plasma samples and the homogenate samples of other organs of normal rats collected under the asepsis condition; through bacteria cultivation,large quantities of colonies occur in the plasma,liver and mesenteric lymph node(MLN) in Groups A,B and D;compared with that in Group B, through bacteria cultivation,the amount of colonies occurring in plasma and liver in Groups A and D significantly advances and that in Group A is the most,the amount of colonies occurring in MLN in Group D is significantly less,and that in Group A is not significantly different.5.Observation and scores of intestinal mucosa histomorphology:No damage to the intestinal mucosa is seen in Groups C and E,under the light microscope,the intestinal mucosa structures are complete,intestinal chorioepithelium is complete,the cells thereof line up in order.In Groups A,B and D,different degrees of intestinal track damages are seen,edema is seen on top of the intestinal villi,the gaps between the intestinal villi are widened;some chorioepithelium cells shrink;karyopyknosis,nuclear fragmentation, endochylema acidophilia occur;membrane propria mocrangium expansion and RBC agglomeration occur;lymphangiectasis and lymphocyte aggregation in lymphatic vessel occur.Injury scores of the intestinal mucosa are:Group A(5.10±0.74),Group B (2.20±0.78),Group C(0.20±0.42),Group D(2.50±0.71) and Group E(0.20±0.42). The injury degree scores in Groups A,B and D are significantly higher than those in Groups C and E(P＜0.01);compared with that in Group B,the degree of injury to intestinal mucosa in Group A is apparently severer(P＜0.01).There is no significant injury degree difference between Groups B and D.6.Observation of intestinal tissues by the TEM(transmission electron microscope): Under the TEM,the structures of the enterocyte microvilli are complete,and the enterocyte microvilli line up in order,there are large amount of mitochondria in the enterocyte endochylema,and the structures of the mitochondria are complete,the cristae therein are clear in Groups C and E.The enterocyte microvilli in Groups A,B and D, after intestinal track damages,become shorter,and do not line up in order,the mitochondria swell obviously,some cristae disappear,comparatively big vacuolations occur therein;some karyopyknosis occurs,the surface of the nuclear membrane is uneven,caryotin assembles around the nuclear membrane in the form of crescent or ring, presenting the transformation of apoptosis.7.Test of the enterokinesia speeds:The activated carbon marked method is easy and accurate to test the enterokinesia speed.The enterokinesia speed of Group A (15.36±1.63%) significantly descends,is only 20%of that of Group E(77.94±3.68%). The enterokinesia speeds of Group B(22.94±0.95%),Group C(32.34±3.58%) and Group D(21.30±3.56%) are significantly lower than that of Group E(P＜0.01). Comparing Group E with Group A,it can be seen that celiac seawater immersion and open celiac wounds can significantly inhibit enterokinesia speed.Compared with that of Group B(22.94±0.95%),the enterokinesia speed of Group D is not significantly different (P＞0.05),while those of Groups A and C are significantly lower(P＜0.01).8.Test of the levels of TNF-αin plasma of rats:Compared with that in Group E (40.24±10.29 pg/ml),the levels of TNF-αin plasma of rats in Group A(105.20±21.17 pg/ml),Group B(68.43±20.09 pg/ml),Group C(67.77±16.57 pg/ml) and Group D (57.25±14.21 pg/ml) are significantly higher(P＜0.05);while compared with that in Group B,the level of TNF-αin plasma of rats in Group A is significantly higher(P＜0.01), and those in Groups C and D are not significantly different(P＞0.05).9.Test of the levels of IL-6 in plasma of rats:Compared with that in Group E (94.98±17.84 pg/ml),the levels of IL-6 in plasma of rats in Group A(207.76±42.26 pg/ml),Group B(136.37±27.57 pg/ml) and Group D(143.58±33.76 pg/ml) are significantly higher(P＜0.01),and that in Group C(107.02±26.09 pg/ml) is not significantly different(P＞0.05);while compared with that in Group B,the levels of IL-6 in plasma of rats in Groups A and C are significantly different(P＜0.05),and that in Group D is not significantly different(P＞0.05).10.Test of the levels of SIgA in the intestinal content of rats:Compared with that in Group E(99.60±16.7 mg/L),the levels of SIgA in the intestinal content of rats in Group A(32.26±7.57 mg/L),Group B(65.08±10.07 mg/L) and Group D(45.84±9.41 mg/L) are significantly lower(P＜0.01),and that in Group C(89.98±11.14 mg/L) is not significantly different(P＞0.05);compared with that in Group B,the levels of SIgA in the intestinal content of rats in Groups A,C and D are significantly different(P＜0.01).11.Test of the levels of IgA in the plasma of rats:Compared with that in Group E (61.73±17.44 mg/L),the levels of IgA in the plasma of rats in Group A(32.1±10.84 mg/L),Group B(43.62±10.12 mg/L) and Group D(38.68±12.98 mg/L) are significantly lower(P＜0.01),and that in Group C(65.84±8.18 mg/L) is not significantly different (P＞0.05);compared with that in Group B,the levels of IgA in the plasma of rats in Groups A and D are not significantly different(P＞0.05),and that in Group C is significantly different(P＜0.01).12.Test of the levels of MDA in the intestinal tissues of rats:The stress reaction of intestinal enterocyte causes the level of MDA in the intestinal tissues of rats to advance significantly.Compared with that in Group E(2.76±1.97 nmol/mg pro),the levels of MDA in the intestinal tissues of rats in Group A(4.70±1.17 nmol/mg pro),Group B (3.14±0.65 nmol/mg pro) and Group D(4.37±1.32 nmol/mg pro) are significantly higher (P＜0.01),and that in Group C(2.13±1.08 nmol/mg pro) is not significantly different (P＞0.05).Compared with that in Group B,the levels of MDA in the intestinal tissues of rats in Groups A,C and D are significantly different(P＜0.05).13.Test of the levels of SOD in the intestinal tissues of rats:The stress reaction of intestinal enterocyte causes the level of SOD in the intestinal tissues of rats to descend significantly.Compared with that in Group E(4.31±1.40 NU/mg pro),the levels of SOD in the intestinal tissues of rats in Group A(1.57±0.34 NU/mg pro) and Group D (2.05±0.43 NU/mg pro) are significantly lower(P＜0.01),and those in Group B (3.43±1.33 NU/mg pro) and Group C(4.09±1.70NU/mg pro) are not significantly different(P＞0.05).Compared with that in Group B,the levels of SOD in the intestinal tissues of rats in Groups A and D are significantly lower(P＜0.01),and that in Group C is not significantly different.14.Test of the apoptosis cells of intestinal mucosa:It is revealed through TUNEL method that:apoptosis cells are mainly on top of the intestinal villi,and with the injury degree aggregating,the apoptosis cells develop downwards;small quantity of positive apoptosis cells are seen on top of the intestinal villi in Groups C and E;apoptosis cells apparently increase on the intestinal mucosa villi and crypto in Groups A,B and D.The variations of the apoptotic indexes(AIs) are:Group A(45.60±7.0),Group B(22.10±3.6),Group C (5.10±0.6),Group D(24.66±3.5) and Group E(4.51±0.7);the AIs of intestinal mucosa in Groups C and E are on low level,in Groups A,B and D are much higher than those in Groups C and E(P＜0.05);compared with that in Group B,the AI of intestinal mucosa in Group A apparently increases(P＜0.01).There is no significant AI difference between Groups B and D(P＞0.05).Conclusion1.The animal model building of rats with open celiac seawater immersion wounds is successful.Open celiac seawater immersion wounds may cause IBFD,increase of intestinal permeability,appearance of intestinal endotoxemia and bacterial translocation. Open celiac seawater immersion wounds become one of the causes of enterogenic infection and MODS.2.Open celiac seawater immersion wounds may inhibit intestinal transit,cause intestinal transit disfunction.Damage to intestinal transit function becomes one of the causes of gut origin endotoxemia and BT.3.Open celiac seawater immersion wounds may cause injuries to intestinal mucosal barrier function.TNF-α,IL-6 and oxygen-derived free radicals involve in the inflammatory reaction after the open celiac seawater immersion wounds occur.4.Inhibition of the immunological function of rats' intestinal mucosa occurs after the open celiac seawater immersion wounds.Massive apoptosis is one of the mechanisms that cause intestinal mucosal BT.Local immunity disorder of intestinal mucosa may be one of the reasons causing the entire organism immunity disorder.