腹部开放伤合并海水浸泡大鼠肠屏障功能损害的初步研究
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摘要
研究背景
肠屏障功能障碍(Intestine barrier functional disturbance,IBFD)是机体在各种临床危重病、外科手术及严重创伤后的常见并发症,具有较高的发病率,其发生机制尚未完全清楚。目前认为,应激状态下肠黏膜的急性病变涉及肠黏膜保护机制的削弱、损伤因素相对增强以及机体神经内分泌功能失调等诸多方面,是多因素综合作用的结果。肠黏膜屏障损伤及其介导的病理学改变使病情进一步加重。细菌或内毒素可经多途径发生易位,造成肠道细菌易位(bacterial translocation,BT)和肠源性内毒素血症(intestinal endotoxemia),进而启动全身炎症反应综合征(SIRS)并引起多器官功能障碍综合征(MODS)。研究发现,肠道血运障碍、肠道传输功能受损、炎症介质过度释放、氧自由基损伤、免疫功能损害以及肠黏膜上皮细胞凋亡等因素可能在肠屏障功能损伤机制中起着重要作用。保护肠屏障功能对改善预后具有重要意义。
海上作战时伤员落水并浸泡在碱性、高渗海水中是极常见的现象,这是一种严重的应激状态。前期的研究发现,战伤合并海水浸泡具有以下特点:血液出现高钠血症、高氯、高渗状态,以及严重的水、电解质紊乱,伴有代谢性及呼吸性酸中毒。严重的血流动力学紊乱、血液高凝状态、微循环障碍等,极易引发MODS。在海战伤救治的过程中,已经发现有海水浸泡对消化道屏障的破坏,是阻碍成功救治的难题之一。深入研究腹部开放伤合并海水浸泡对肠屏障功能的影响,为有效综合防治IBFD找出相应的对策,是降低早期伤后死亡率不可缺少的重要环节,也是本研究的目的。
目的
1.建立腹部开放伤合并海水浸泡大鼠模型,观察海水浸泡后大鼠血浆DAO、D-乳酸、LPS及肠道细菌易位的变化和肠黏膜组织形态学改变,确定大鼠肠屏障功能变化情况。
2.探讨腹部开放伤合并海水浸泡大鼠肠屏障功能损害的机制:观察肠蠕动速率的变化,肠组织MDA、SOD的含量,血浆TNF-α、IL-6的水平,肠道内容物SIgA、血浆IgA的变化及肠黏膜上皮细胞凋亡的情况。
方法
1.选择健康雄性Wistar大鼠50只,体重230±20g,清洁级,按照随机化原则分组,每组10只,分组情况如下:A组:腹部开放伤合并海水浸泡组(腹腔开放伤后浸泡于人工海水中);B组:单纯腹部开放伤组(单纯腹部开放伤后直接观察而不浸泡于海水或生理盐水);C组:单纯海水浸泡组(大鼠麻醉后未行任何手术,直接将腹部浸泡于人工海水中);D组:腹部开放伤合并生理盐水浸泡组(腹腔开放伤后浸泡于生理盐水中);E组:正常对照组(未行任何处理)。
2.在腹部开放性创伤和人工海水浸泡双重应激情况下,在应激1小时后,分别用分光光度法检测腹部开放伤合并海水浸泡大鼠血浆DAO和D-乳酸;动态浊度法检测血浆LPS;实验动物脏器细菌定量培养观察细菌易位情况。
3.按Haglund等评估小肠黏膜上皮损伤指数的方法,光学显微镜下观察肠黏膜组织病理学改变,透射电镜下观察肠黏膜组织超微结构变化。
4.采用活性炭标记法测定大鼠肠道蠕动速率,分光光度法检测肠组织MDA和SOD,酶联免疫吸附测试(ELISA)法检测血浆TNF-α、IL-6、IgA和肠道内容物SIgA。
结果
1.血浆D-乳酸活性检测:腹部开放伤合并海水浸泡组(A组)大鼠血浆D-乳酸活性(7.63±0.72 mg/L)、单纯腹部开放伤组(B组)血浆D-乳酸活性(5.46±0.57mg/L)、腹部开放伤合并生理盐水浸泡组(D组)血浆D-乳酸活性(4.97±0.95mg/L)较正常对照组(E组)大鼠血浆D-乳酸活性(4.06±0.39 mg/L)显著升高(P＜0.01),单纯海水浸泡组(C组)血浆D-乳酸活性(3.89±0.55mg/L)较E组无显著差异(P＞0.05);与B组比较,D组血浆D-乳酸活性无显著差异(P＞0.05),A、C组血浆D-乳酸活性有显著差异(P＜0.01)。
2.血浆DAO活性检测:大鼠血浆DAO活性A组(6.24±1.54 KU/L)、B组(3.57±1.65 KU/L)、C组(0.42±0.04KU/L)、D组(3.40±1.58KU/L)较E组(0.36±0.04 KU/L)均显著升高(P＜0.01);而与B组比较,D组无显著差异(P＞0.05),A、C组有显著差异(P＜0.01)。
3.血浆内毒素水平检测:大鼠血浆内毒素水平A组(486.5±116.1 pg/ml)、B组(344.5±51.5 pg/ml)、D组(338.7±54.4 pg/ml)较E组(23.5+10.4 pg/ml)显著升高(P＜0.01),C组(23.0±11.7pg/ml)与E组无显著差异(P＞0.05);与B组比较,A、C组血浆内毒素有显著差异(P＜0.01),D组无显著差异(P＞0.05)。
4.实验动物脏器细菌定量培养:结果显示,正常动物在无菌条件下采集的血浆标本和其他脏器匀浆液标本经细菌培养后,没有菌落出现;A、B、D组血浆、肝脏及肠系膜淋巴结细菌培养后均可见大量菌落出现;与B组相比,A组和D组血培养及肝脏培养菌落数显著上升,且A组结果最高,肠系膜淋巴结培养,D组菌落数显著减少,A组菌落数无明显差异。
5.小肠黏膜组织形态学观察及评分:C、E组肠黏膜未见损伤,光镜下黏膜结构完整,小肠绒毛上皮完整、细胞排列整齐;A、B、D组均引起多部位的不同程度的肠道损伤,小肠绒毛顶端水肿、间隙增宽;部分区域上皮细胞萎缩,细胞核固缩、碎裂,胞浆嗜酸性变;固有膜毛细血管扩张,红细胞凝聚;淋巴管扩张,淋巴管内淋巴细胞聚集。小肠黏膜损伤评分值分别为:A组(5.10±0.74);B组(2.20±0.78);C组(0.20±0.42);D组(2.50±0.71);E组(0.20±0.42)。A、B、D组损伤程度评分显著高于C、E组(P＜0.01);与B组比较,A组肠黏膜组织损伤程度明显加重(P＜0.01),B组和D组间无显著差异(P＞0.05)。
6.小肠组织电镜观察:透射电镜下,C、E组大鼠肠上皮细胞微绒毛结构完整,排列整齐,胞浆内含大量线粒体,结构完整,嵴清晰;A、B、D组肠道损伤后肠上皮细胞微绒毛变短,排列不整齐,线粒体肿胀明显,部分嵴溶解消失,较大空泡形成;部分细胞核固缩,核膜表面凹凸不平,染色质集聚于核膜周边形成新月形或环形,呈凋亡的形态改变。
7.肠蠕动速率检测:检测肠蠕动速率活性炭标记法简单准确。大鼠肠道蠕动速率A组(15.36±1.63%)显著下降(P＜0.01),其蠕动速率只为E组(77.94±3.68%)的20%。B组(22.94±0.95%)、C组(32.34±3.58%)、D组(21.30±3.56%)肠蠕动速率显著低于E组(P＜0.01),与E组比较,腹部开放伤及海水浸泡能够非常显著地抑制肠道蠕动功能;与B组比较,D组无显著差异(P＞0.05),A、C组抑制肠道蠕动功能有显著差异(P＜0.01)。
8.血浆TNF-α浓度检测:大鼠血浆TNF-α浓度A组(105.20±21.17 pg/ml)、B组(68.43±20.09 pg/ml)、C组(67.77±16.57 pg/ml)、D组(57.25±14.21 pg/ml)较E组(40.24±10.29 pg/ml)显著升高(P＜0.05);而与B组比较,A组血浆TNF-α浓度显著升高(P＜0.01),C、D组无显著差异(P＞0.05)。
9.血浆IL-6浓度检测:大鼠血浆IL-6浓度A组(207.76±42.26 pg/ml)、B组(136.37±27.57 pg/ml)、D组(143.58±33.76 pg/ml)较E组(94.98±17.84 pg/ml)显著升高(P＜0.05),C组(107.02±26.09 pg/ml)较E组无显著差异(P＞0.05);而与B组比较,A、C组血浆IL-6浓度有显著差异(P＜0.05),D组无显著差异(P＞0.05)。
10.肠内容物SIgA含量检测:与E组(99.6±16.7 mg/L)比较,A组(32.26±7.57mg/L)、B组(65.08±10.07 mg/L)、D组(45.84±9.41 mg/L)肠道内容物SIgA含量显著减少(P＜0.01),C组(89.98±11.14 mg/L)无显著差异(P＞0.05);与B组比较,A、C、D组肠道内容物SIgA含量有显著差异(P＜0.01)。
11.血浆IgA含量检测:与E组(61.73±17.44 mg/L)比较,A组(32.10±10.84 mg/L)、B组(43.62±10.12 mg/L)、D组(38.68±12.98 mg/L)血浆IgA含量显著减少(P＜0.01),C组(65.84±8.18 mg/L)无显著差异(P＞0.05);与B组比较,A、D组无显著差异(P＞0.05),C组有显著差异(P＜0.01)。
12.大鼠肠组织MDA含量检测:腹部开放海水浸泡后,肠上皮细胞应激反应造成肠组织MDA含量明显增加;与E组(2.76±1.97 nmol/mg pro)比较,A组(4.70±1.17 nmol/mg pro)、B组(3.14±0.65 nmol/mg pro)、D组(4.37±1.32 nmol/mgpro)大鼠肠组织MDA含量显著升高(P＜0.01),C组(2.13±1.08 nmol/mg pro)大鼠肠组织MDA含量无显著差异(P＞0.05);与B组比较,A、C、D组大鼠肠组织MDA含量有显著差异(P＜0.05)。
13.大鼠肠组织SOD含量检测:腹部开放伤合并海水浸泡后,肠上皮细胞应激反应造成肠组织SOD含量明显减少;与E组(4.31±1.40 NU/mg pro)比较,A组(1.57±0.34 NU/mg pro)、D组(2.05±0.43 NU/mg pro)大鼠肠组织SOD含量显著减少(P＜0.01),B组(3.43±1.33 NU/mg pro)、C组(4.09±1.70 NU/mg pro)大鼠肠组织SOD含量无显著差异(P＞0.05);与B组比较,A、D组大鼠肠组织SOD含量显著减少(P＜0.01),C组大鼠肠组织SOD含量无显著差异(P＞0.05)。
14.小肠黏膜上皮细胞凋亡检测:TUNEL法显示,凋亡细胞主要位于小肠绒毛顶部,且随致伤程度的加重有由上向下发展的趋势。C、E组小肠黏膜绒毛顶部可见少量阳性凋亡细胞。A、B、D组小肠黏膜绒毛及隐窝上皮细胞凋亡明显增多。小肠黏膜上皮细胞凋亡指数的变化分别为:A组(45.60±7.0),B组(22.10±3.6),C组(5.10±0.6),D组(24.66±3.5),E组(4.51±0.7)。C、E组小肠黏膜上皮细胞凋亡指数呈现低水平;A、B、D组小肠黏膜上皮细胞凋亡指数均显著高于C、E组(P＜0.05);与B组比较,A组小肠黏膜上皮细胞凋亡指数显著增高(P＜0.01),D组无显著差异(P＞0.05)。
结论
1.大鼠腹部开放伤合并海水浸泡动物模型制作成功;腹部开放伤及腹腔海水浸泡双重应激可引起IBFD,肠黏膜通透性增高;出现肠源性内毒素血症和细菌易位,成为创伤后肠源性感染及多器官功能障碍综合征(MODs)发生的原因之一。
2.腹部开放伤合并海水浸泡大鼠肠道蠕动功能明显受到抑制,出现肠道动力障碍,肠道传输功能受损是肠源性内毒素血症和细菌易位的原因之一。
3.腹部开放伤合并海水浸泡可导致大鼠肠黏膜屏障功能受损,TNF-α、IL-6等炎症介质及氧自由基参与了腹部开放伤合并海水浸泡继发性炎症反应过程及肠道黏膜细胞损伤。
4.腹部开放伤合并海水浸泡大鼠肠道免疫功能下降。大量的肠黏膜上皮细胞凋亡是细菌易位和肠源性内毒素血症的重要机制之一。肠道局部免疫功能的紊乱可能是引起整个机体免疫功能紊乱的原因之一。
Background
Intestine 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.
Objective
1.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.
Methods
1.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.
Results
1.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).
Conclusion
1.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.
肠屏障功能障碍(Intestine barrier functional disturbance,IBFD)是机体在各种临床危重病、外科手术及严重创伤后的常见并发症,具有较高的发病率,其发生机制尚未完全清楚。目前认为,应激状态下肠黏膜的急性病变涉及肠黏膜保护机制的削弱、损伤因素相对增强以及机体神经内分泌功能失调等诸多方面,是多因素综合作用的结果。肠黏膜屏障损伤及其介导的病理学改变使病情进一步加重。细菌或内毒素可经多途径发生易位,造成肠道细菌易位(bacterial translocation,BT)和肠源性内毒素血症(intestinal endotoxemia),进而启动全身炎症反应综合征(SIRS)并引起多器官功能障碍综合征(MODS)。研究发现,肠道血运障碍、肠道传输功能受损、炎症介质过度释放、氧自由基损伤、免疫功能损害以及肠黏膜上皮细胞凋亡等因素可能在肠屏障功能损伤机制中起着重要作用。保护肠屏障功能对改善预后具有重要意义。
海上作战时伤员落水并浸泡在碱性、高渗海水中是极常见的现象,这是一种严重的应激状态。前期的研究发现,战伤合并海水浸泡具有以下特点:血液出现高钠血症、高氯、高渗状态,以及严重的水、电解质紊乱,伴有代谢性及呼吸性酸中毒。严重的血流动力学紊乱、血液高凝状态、微循环障碍等,极易引发MODS。在海战伤救治的过程中,已经发现有海水浸泡对消化道屏障的破坏,是阻碍成功救治的难题之一。深入研究腹部开放伤合并海水浸泡对肠屏障功能的影响,为有效综合防治IBFD找出相应的对策,是降低早期伤后死亡率不可缺少的重要环节,也是本研究的目的。
目的
1.建立腹部开放伤合并海水浸泡大鼠模型,观察海水浸泡后大鼠血浆DAO、D-乳酸、LPS及肠道细菌易位的变化和肠黏膜组织形态学改变,确定大鼠肠屏障功能变化情况。
2.探讨腹部开放伤合并海水浸泡大鼠肠屏障功能损害的机制:观察肠蠕动速率的变化,肠组织MDA、SOD的含量,血浆TNF-α、IL-6的水平,肠道内容物SIgA、血浆IgA的变化及肠黏膜上皮细胞凋亡的情况。
方法
1.选择健康雄性Wistar大鼠50只,体重230±20g,清洁级,按照随机化原则分组,每组10只,分组情况如下:A组:腹部开放伤合并海水浸泡组(腹腔开放伤后浸泡于人工海水中);B组:单纯腹部开放伤组(单纯腹部开放伤后直接观察而不浸泡于海水或生理盐水);C组:单纯海水浸泡组(大鼠麻醉后未行任何手术,直接将腹部浸泡于人工海水中);D组:腹部开放伤合并生理盐水浸泡组(腹腔开放伤后浸泡于生理盐水中);E组:正常对照组(未行任何处理)。
2.在腹部开放性创伤和人工海水浸泡双重应激情况下,在应激1小时后,分别用分光光度法检测腹部开放伤合并海水浸泡大鼠血浆DAO和D-乳酸;动态浊度法检测血浆LPS;实验动物脏器细菌定量培养观察细菌易位情况。
3.按Haglund等评估小肠黏膜上皮损伤指数的方法,光学显微镜下观察肠黏膜组织病理学改变,透射电镜下观察肠黏膜组织超微结构变化。
4.采用活性炭标记法测定大鼠肠道蠕动速率,分光光度法检测肠组织MDA和SOD,酶联免疫吸附测试(ELISA)法检测血浆TNF-α、IL-6、IgA和肠道内容物SIgA。
结果
1.血浆D-乳酸活性检测:腹部开放伤合并海水浸泡组(A组)大鼠血浆D-乳酸活性(7.63±0.72 mg/L)、单纯腹部开放伤组(B组)血浆D-乳酸活性(5.46±0.57mg/L)、腹部开放伤合并生理盐水浸泡组(D组)血浆D-乳酸活性(4.97±0.95mg/L)较正常对照组(E组)大鼠血浆D-乳酸活性(4.06±0.39 mg/L)显著升高(P＜0.01),单纯海水浸泡组(C组)血浆D-乳酸活性(3.89±0.55mg/L)较E组无显著差异(P＞0.05);与B组比较,D组血浆D-乳酸活性无显著差异(P＞0.05),A、C组血浆D-乳酸活性有显著差异(P＜0.01)。
2.血浆DAO活性检测:大鼠血浆DAO活性A组(6.24±1.54 KU/L)、B组(3.57±1.65 KU/L)、C组(0.42±0.04KU/L)、D组(3.40±1.58KU/L)较E组(0.36±0.04 KU/L)均显著升高(P＜0.01);而与B组比较,D组无显著差异(P＞0.05),A、C组有显著差异(P＜0.01)。
3.血浆内毒素水平检测:大鼠血浆内毒素水平A组(486.5±116.1 pg/ml)、B组(344.5±51.5 pg/ml)、D组(338.7±54.4 pg/ml)较E组(23.5+10.4 pg/ml)显著升高(P＜0.01),C组(23.0±11.7pg/ml)与E组无显著差异(P＞0.05);与B组比较,A、C组血浆内毒素有显著差异(P＜0.01),D组无显著差异(P＞0.05)。
4.实验动物脏器细菌定量培养:结果显示,正常动物在无菌条件下采集的血浆标本和其他脏器匀浆液标本经细菌培养后,没有菌落出现;A、B、D组血浆、肝脏及肠系膜淋巴结细菌培养后均可见大量菌落出现;与B组相比,A组和D组血培养及肝脏培养菌落数显著上升,且A组结果最高,肠系膜淋巴结培养,D组菌落数显著减少,A组菌落数无明显差异。
5.小肠黏膜组织形态学观察及评分:C、E组肠黏膜未见损伤,光镜下黏膜结构完整,小肠绒毛上皮完整、细胞排列整齐;A、B、D组均引起多部位的不同程度的肠道损伤,小肠绒毛顶端水肿、间隙增宽;部分区域上皮细胞萎缩,细胞核固缩、碎裂,胞浆嗜酸性变;固有膜毛细血管扩张,红细胞凝聚;淋巴管扩张,淋巴管内淋巴细胞聚集。小肠黏膜损伤评分值分别为:A组(5.10±0.74);B组(2.20±0.78);C组(0.20±0.42);D组(2.50±0.71);E组(0.20±0.42)。A、B、D组损伤程度评分显著高于C、E组(P＜0.01);与B组比较,A组肠黏膜组织损伤程度明显加重(P＜0.01),B组和D组间无显著差异(P＞0.05)。
6.小肠组织电镜观察:透射电镜下,C、E组大鼠肠上皮细胞微绒毛结构完整,排列整齐,胞浆内含大量线粒体,结构完整,嵴清晰;A、B、D组肠道损伤后肠上皮细胞微绒毛变短,排列不整齐,线粒体肿胀明显,部分嵴溶解消失,较大空泡形成;部分细胞核固缩,核膜表面凹凸不平,染色质集聚于核膜周边形成新月形或环形,呈凋亡的形态改变。
7.肠蠕动速率检测:检测肠蠕动速率活性炭标记法简单准确。大鼠肠道蠕动速率A组(15.36±1.63%)显著下降(P＜0.01),其蠕动速率只为E组(77.94±3.68%)的20%。B组(22.94±0.95%)、C组(32.34±3.58%)、D组(21.30±3.56%)肠蠕动速率显著低于E组(P＜0.01),与E组比较,腹部开放伤及海水浸泡能够非常显著地抑制肠道蠕动功能;与B组比较,D组无显著差异(P＞0.05),A、C组抑制肠道蠕动功能有显著差异(P＜0.01)。
8.血浆TNF-α浓度检测:大鼠血浆TNF-α浓度A组(105.20±21.17 pg/ml)、B组(68.43±20.09 pg/ml)、C组(67.77±16.57 pg/ml)、D组(57.25±14.21 pg/ml)较E组(40.24±10.29 pg/ml)显著升高(P＜0.05);而与B组比较,A组血浆TNF-α浓度显著升高(P＜0.01),C、D组无显著差异(P＞0.05)。
9.血浆IL-6浓度检测:大鼠血浆IL-6浓度A组(207.76±42.26 pg/ml)、B组(136.37±27.57 pg/ml)、D组(143.58±33.76 pg/ml)较E组(94.98±17.84 pg/ml)显著升高(P＜0.05),C组(107.02±26.09 pg/ml)较E组无显著差异(P＞0.05);而与B组比较,A、C组血浆IL-6浓度有显著差异(P＜0.05),D组无显著差异(P＞0.05)。
10.肠内容物SIgA含量检测:与E组(99.6±16.7 mg/L)比较,A组(32.26±7.57mg/L)、B组(65.08±10.07 mg/L)、D组(45.84±9.41 mg/L)肠道内容物SIgA含量显著减少(P＜0.01),C组(89.98±11.14 mg/L)无显著差异(P＞0.05);与B组比较,A、C、D组肠道内容物SIgA含量有显著差异(P＜0.01)。
11.血浆IgA含量检测:与E组(61.73±17.44 mg/L)比较,A组(32.10±10.84 mg/L)、B组(43.62±10.12 mg/L)、D组(38.68±12.98 mg/L)血浆IgA含量显著减少(P＜0.01),C组(65.84±8.18 mg/L)无显著差异(P＞0.05);与B组比较,A、D组无显著差异(P＞0.05),C组有显著差异(P＜0.01)。
12.大鼠肠组织MDA含量检测:腹部开放海水浸泡后,肠上皮细胞应激反应造成肠组织MDA含量明显增加;与E组(2.76±1.97 nmol/mg pro)比较,A组(4.70±1.17 nmol/mg pro)、B组(3.14±0.65 nmol/mg pro)、D组(4.37±1.32 nmol/mgpro)大鼠肠组织MDA含量显著升高(P＜0.01),C组(2.13±1.08 nmol/mg pro)大鼠肠组织MDA含量无显著差异(P＞0.05);与B组比较,A、C、D组大鼠肠组织MDA含量有显著差异(P＜0.05)。
13.大鼠肠组织SOD含量检测:腹部开放伤合并海水浸泡后,肠上皮细胞应激反应造成肠组织SOD含量明显减少;与E组(4.31±1.40 NU/mg pro)比较,A组(1.57±0.34 NU/mg pro)、D组(2.05±0.43 NU/mg pro)大鼠肠组织SOD含量显著减少(P＜0.01),B组(3.43±1.33 NU/mg pro)、C组(4.09±1.70 NU/mg pro)大鼠肠组织SOD含量无显著差异(P＞0.05);与B组比较,A、D组大鼠肠组织SOD含量显著减少(P＜0.01),C组大鼠肠组织SOD含量无显著差异(P＞0.05)。
14.小肠黏膜上皮细胞凋亡检测:TUNEL法显示,凋亡细胞主要位于小肠绒毛顶部,且随致伤程度的加重有由上向下发展的趋势。C、E组小肠黏膜绒毛顶部可见少量阳性凋亡细胞。A、B、D组小肠黏膜绒毛及隐窝上皮细胞凋亡明显增多。小肠黏膜上皮细胞凋亡指数的变化分别为:A组(45.60±7.0),B组(22.10±3.6),C组(5.10±0.6),D组(24.66±3.5),E组(4.51±0.7)。C、E组小肠黏膜上皮细胞凋亡指数呈现低水平;A、B、D组小肠黏膜上皮细胞凋亡指数均显著高于C、E组(P＜0.05);与B组比较,A组小肠黏膜上皮细胞凋亡指数显著增高(P＜0.01),D组无显著差异(P＞0.05)。
结论
1.大鼠腹部开放伤合并海水浸泡动物模型制作成功;腹部开放伤及腹腔海水浸泡双重应激可引起IBFD,肠黏膜通透性增高;出现肠源性内毒素血症和细菌易位,成为创伤后肠源性感染及多器官功能障碍综合征(MODs)发生的原因之一。
2.腹部开放伤合并海水浸泡大鼠肠道蠕动功能明显受到抑制,出现肠道动力障碍,肠道传输功能受损是肠源性内毒素血症和细菌易位的原因之一。
3.腹部开放伤合并海水浸泡可导致大鼠肠黏膜屏障功能受损,TNF-α、IL-6等炎症介质及氧自由基参与了腹部开放伤合并海水浸泡继发性炎症反应过程及肠道黏膜细胞损伤。
4.腹部开放伤合并海水浸泡大鼠肠道免疫功能下降。大量的肠黏膜上皮细胞凋亡是细菌易位和肠源性内毒素血症的重要机制之一。肠道局部免疫功能的紊乱可能是引起整个机体免疫功能紊乱的原因之一。
Background
Intestine 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.
Objective
1.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.
Methods
1.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.
Results
1.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).
Conclusion
1.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.
引文
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