The Changes Of Intercellular Adhesion Molecule-1, Oxidative Stress And Methallothionein On Intestinal Tissue In LPS-induced Intestinal Injury Neonatal Rats

IntroductionNecrotizing enterocolitis (NEC) is a common and devastating gastrointestinal condition in neonatal intensive care units ( NICU). The first comprehensive modern - day reports of neonatal NEC appeared in the literature in the mid -1960s. Over the past few decades there has been tremendous increase in the incidence of NEC cases, which has mainly been attributed to: (1) the increase in the number of premature births as a result of the more frequent application of caesarian section before 34 weeks of gestation for therapeutic reasons;and (2) the fact that in the modern era of NICUs and surfactant therapy, most premature babies are able to overcome a number of previously fatal health problems and survive, and thus render themselves susceptible to NEC. It is thought that NEC will eventually replace pulmonary insufficiency as the leading cause of death in premature infants.The incidence varies among countries as well as among different centres within the same country. In the United States , NEC occurs in 1 to 3 cases in 1000 live births, affecting 2000 to 4000 newborns eath year. Of these, 27% to 63% require surgical intervention. NEC - associated mortality increased from 11.5 to 12. 3/100 000 or 10% to 30% of all NEC cases across centers. Furthermore, the severity of NEC as per the modified Bell's classification as well as mortality has been suggested to be inversely related to birthweight and gestational age, with a report of even 100% mortality in infants with birthweights less than 1000 g and gestation ages less than 28 weeks.The etiology of NEC appears to be multifactorial. The prematurity is a major risk factor for NEC. No sigle aetiological factor accounts for NEC and it is likely that damage to the intestinal mucosa is the final common pathway of a number of noxious influences including enteral feeding of formula, intestinal hy-poxia and/or ischemia, and infection. These components probably act in concert to upset an already immature and delicate intestinal mucosal darrier, promote the inflammatory cascade that results in the pathology associated with this disease.In spite of extensive epidemiological, clinical, and basic research, the pathogenesis of NEC is not completely understood, and there is no effective pre-ventative treatment for this disease, At present accepted pathogenic mechanisms of NEC are mainly described as following: The initial event of NEC is probably polymorphonuclear leukocyte activation and adhesion to venules in the intestine, which intiates alocal inflammatory reaction invoving proinflammatory mediators including TNF, complement system, prostaglandins and leukotriene C4. Subsequent mesenteric vasoconstriction results in intestinal ischemia and reperfusion. Reactive oxygen sspecies ( ROS) produced by intestinal epithelial xanthine oxi-dase (XO) may be the final pathway for intestine injury. During tissue ischemi-a, there is intracellular accumulation of a large amount of hypoxathine and conversion of a large quantity of superoxide, and hypoxanthine is reconverted back to xanthine. This burst of ROS formation can cause severe tissue damage. Endogenous NO may help to maintain the integrity of the mncosal barrier and the microvasculature. The protective mechanism was decreased by mediators in intestine include nitric oxide produced by the constitutive (mainly neuronal) nitric oxide synthase that may cause gut barrier failure. The role of the growth factor on NEC. The development of intestinal tissue injury depends on the balance between injurios and protective mechanisms. An imbalance favoring of the mucosal barrier and bacterial entry, thereby launching a self - perpetuating vicious cycle , leading to shock, sepsis and, sometimes, death.However, extrapolation of these observation to human NEC must be made cautiously because these experiments were not generality conducted in newborn animal. These results led us to ask wether the mechanisms above - mentionedmight be associated with NEC in human preterm infants. To understand this disease , the foreign investigator established many animal models of NEC, including three;Animal model 1: Bowel necrosis nduced by platelet - activating factor, li-popolysaccharide, and tumor necrosis factor - a;Animal model 2: Hypoxia and LPS/hypoxia in experimental NEC;Animal model 3;Neonatal NEC - role of hypoxia, enteral feeding, and endogenous PAF.These models explain the development of NEC from different style, but lack some or all of the cardinal feature of the human condition. ROS may be the final pathway of NEC, the imbalance between oxidant and antioxidant is one of the pathogenesis of NEC, polymorphonuclear leukocyte (PMN) activation and PMN - endothelial adhesion is the initial event. Intercellular adhesion molecule - 1 (ICAM -1) play a crucial role in this procedure. The prevous study suggested LPS can induced the expression of ICAM - 1. P - selectin - deficient mice and fucoidin - treated ICAM - 1 deficient mice are also protected from the adverse effects of PAF, suggesting a possible role of adhesion molecule.Glutathione (GSH) is the body's most important water - soluble intracellu-lar antioxidant. It has crucial role in the maintenance of intracellular oxidation -redution balance and in cellular signalling to activate gene transcription. GSH detoxifies a number of ROS directly and also limits production of the peroxyni-trite ROS via its reation with nitric oxide. A relative deficiency of GSH may therefore predispose to disease mediated by ROS and increase ROS - induced tissue damage. Intestinal ischemia and reperfusion result in a dramatic decrease in GSH content of the bowel. During systemic inflammation and sepsis, the supply of the amino acids required for GSH synthesis is altered so that in septic infants and children GSH content and synthesis rates are decreased. There has been debatable about relationship between GSH levels and the development of NEC.Metallothione is a small intrcellular metal - binding protein, rich in cyste-ine, founctions as a scanvager of oxide free radical, its tole in intestinal injury remains unclear.The aim of the present study were to determine the dynamic ileum morphology change and to study wether or not the changes of intercellular adhesion molecule -1, glutathione antioxidative syetem , metallothione and their roles at the pathogenic mechanism in intestine injury with LPS - induced neonatal rat. We try to settle some trial foundation of providing more information of pathophysio-logical mechanism and seeking for prevention and treatment of NEC.Materials and Methods1. Animal modelWistar rats less 24h were given an intraperitoneal (IP) injection of 5 mg/ kg E coli O55;B5endotoxin or similar volume of saline.All pups were killed respectively at 1,3,6,12 and 24 hours after receiving LPS IP (n = 8) , the control pups (n = 8) were killed at 1 h after saline IP. The pups were excluded from the study were those that died before collection of the specimens.2. Specimens collection and NEC evaluationAll surviving animals were killed via decapitation. The gastrointinal tract was carfully removed. The small intestine was visually evaluated for typical signs of NEC. Results of macroscopic visual evaluation were recorded. The small intestine was than divided into two halves: jejunum and ileum. A 3cm segment of distal ileum 4 cm proximal to the ileocecal valve from each animal was frozen in - 80X1 for measurements of protein and mRNA . Histological changes in the ileum were scored by a blinded evaluator and were assigned a necortizing entero-colitis( NEC) score on a scale of 0 to 4 as follows;0 = normal,intact villous epithelium with normal histology;1 = mild villous edema , with epithelial sloughing confined to the tips of the villi;2 = mild midvillous necrosis;3 = moderate midvillous necrosis , with crypts still readily detectable;and 4 = severe necrosis of entire villi with complete absence of epithelial structures.3. Experimental methods and analysis marker3. 1 Macroscoppic exam: After the abdominal cavity was opened, the small intestine and colon was grossly examined for such the sign as intestinal discolora-tion, intestinal hemorrhage and distention.3. 2 Microscopic exam;The segment of distal ileum was harvested, fixed in 4% paraformadehyde, embedded in paraffin t microtome sectioned at 5 jxm, and counterstained with hematoxylin and eosin for histological evaluation of intestinal injury.3. 3 Biochemical methods.- The activities of GSH, GSSG, GSH - PX, GSH/GSSG, DAO, MPO, MDA and protein were measured.3.4 Immunohistochemistry;The localization of ICAM -1 and MT and their proteins expression were performed.3.5 Reverse transcription polymerase chain reaction ( RT PCR );The expression levels of ICAM - 1, MT1, MT2 were determined.4. Statistical analysisSoftware SPSS 13.0 for Windows was used in all statistical tests. Comparisons between the two groups were performed with test, from which all data were mean ± standard deviation ( SD). When P was less than 0. 05, the difference was considered statistically significant. The degree of correlation was described using the Spearman s rank - correlation test.Results1. The pathological findings of the ileum tissue1.1. At the organ level: The jejunum is ivory white and the ileum is pale yellow in control group. It has good elasticity. It is observed that the lesion site involves mainly in the ileocolic region following LPS treatment. The affected bowel is grossly distended, lusterless, and gray or greenish - gray, even it may be dark purple or black. The perforate can be seen in the fragile wall. The most deteriorated change is at 12h.1.2. At the microscope level;There is a normal, intact villous epithelium with normal histology of ileum tissue in the control group. It can be seen the mild villous edema, with epithelial sloughing confined to the tips of the lh villi after LPS injection and midvillous necrosis is deteriorated from top to the large part and the amount of the midvillous necrosis neonate rats is augmented with thetime goes on. The most deteriorated change is at 12h. There was severe necrosis of entire villi with complete absence of epithelial structures.1.3. The degree of ileal damage;The injury score of ileum tissue in the LPS group was also much significantly increased than the control group pups (P1.4. The changes of DAO activity: The level of DAO at lh after injection of LPS was significantly lower than control group (P < 0. 05 ). Its peak was at 12h. There was a significant negative correlation between the DAO activities and the scoring of intestinal injury within 24h (-y = —0.447 ,P <0.05).2. The changes of oxidative stress index2. IThe change of GSH content: The level of GSH at 3h after injection of LPS was significantly lower than control group (P < 0.05). Its peak was at 12h. There was a significant negative correlation between the GSH content and the scoring of intestinal injury within 24h(y = - 0. 339,P < 0.01).2.2. The change of GSSG content: The level of GSSG during 24h after injection of LPS was significantly higher than control group (P <0.05). There was no significant correlation between the GSSG level and the scoring of intestinal injury within 24h (y = 0. 257, P > 0.05).2.3. The change of GSH/GSSG: The ratio of GSH/GSSG during 24h after injection of LPS were significant lower than control group (P <0.05). There was a significant negative correlation between the GSH/GSSG ratio and the scoring of intestinal injury within 24h( -y = -0. 390,P <0.01).2.4. The change of GSH - PX level: The level of GSH - PX at 6h and 12h after injection of LPS was significantly lower than control group ( P <0.05). Its peak was at 6h. There was no significant correlation between the GSH - PX levels and the scoring of intestinal injury within 24h (y = -0.046, P >0.05).2.5. The change of MDA level;The level of MDA at lh after Injection of LPS was significantly higher than control group ( P < 0. 01). Its peak was at 12h. There was a significant positive correlation between the MDA levels and the scoring of intestinal injury within 24h (y = 0.556, P > 0.01}.2.6. The change of MPO activity: The activity of MPO at lh after injection of LPS was significantly higher than control group ( P <0.01). there was a sig-nifycant correlation between the MPO level and the scoring of intestinal injury within24h (7 = -0.420,P<0.01).3. The expressions of ICAM - 1 proteins and the expressions of ICAM - 1 mRNA3.1. It is shown that the positive expression of ICAM - 1 is mainly in the intestinal epithelial cells and endothelial cell by immunohistochemistry. The positive expression of ICAM - 1 in LPS group was increased at lh (P <0.05). Its peak was at 12h. There was a significant correlation between the expression of ICAM -1 and the scoring of intestinal injury within 24h (7=0.471, P < 0. 01).3.2. The expression of ICAM - lmRNA in ileum was gradually increased after LPS injetion (P <0.05). Its peak was at 12h. There was a significant positive correlation between the ICAM - lmRNA and the scoring of intestinal injury within 24h (7 =0.472, P <0.01).4. Expressions of MT protein and MT1 ,MT2mRNA:In ileum tissues the MT was present on the villi and crypt epithelial cells, localized in each rat on the basolateal and apical membranes of entero - cytes. There were positive expression in the control group. Its expression was significant higher at lh after LPS injection ( P <0. 05). Its peak was at 6h, and decreased dramatic at 12h ( P < 0. 05). There was no significant correlation between the MT protein and the scoring of intestinal injury within 24h (7 = - 0. 219,P>0.05).The expression of MT1, MT2 mRNA is increased following LPS injection (P <0.05). Their expression peaks was at 6h. and decreased dramatic at 12h (P < 0.05). There was no significant correlation between the MT1, MT2mRNA levels and the scoring of intestinal injury within 24h (7=0.045, P > 0.05;r = 0. 009,P>0.05).Conclusions1. The feature of intestinal injury was consistent with NEC at the organ level and the microscope level while LPS was injected intraperitonrally in neonatal rats2. The decrease of GSH anti -oxidative founction, induced imbalance between oxidative and antioxidative. intestinal inflammation and injury. Activation of XO with massive ROS production occurs as a consequence of ischemia and subsequent rperfusion. The oxidant - mediated lipid peroxidation was increased and the activity of enzyme of removing ROS was decrased in mediating LPS - induced bowel mecrosis in the meonatal rats. LPS can make the imbalance of oxi-dant/amtioxidant which may play a central role in mucosal injury and involve in the pathophysiological mechanism of the development of NEC3. The expression of ICAM - lmRNA and protein was significantly increased in the peak of intestinal injury.PMN activation and adhesion to venular endothelium, increase in vascular permeability , complement activation, release of ROS and flammatory mediators, lead damage to the intestinal wall.4. LPS -induced intestinal injury, oxidative stress increased, induced expression of antioxidant MT, acts as a scavenger of toxic radicals.