The Study Of Dynamic Changes And Mechanisms Of Oxidative Stress, Nitric Oxide Synthase And Epithelial Growth Factor On Intestinal Injury In Neonatal Rats

IntroductionNecrotizing enterocolitis ( NEC) is a common and devastating gastrointestinal condition in neonatal intensive care units (NICUs). The first comprehensive modern - day reports of neonatal necrotizing enterocolitis ( NEC ) appeared in the literature in the mid - 1960s. Over the past few decades there has been a 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.The incidence varies among countries as well as among different centres within the same country. In the United States it has been reported to be approximately 10% of VLBW infants. In Hong Kong the incidence among VLBW infants has reached 28%. Although overall infant mortality has diminished, NEC -associated mortality increased from 11.5 to 12.3/100 000 or 10% -30% of all NEC cases across centres. Furthermore, the severity of NEC as per the modified Bells 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 birth weights less than 1000 g and gestational ages less than 28weeks.The etiology of NEC appears to be multifactorial. The prematurity is a major risk factor for NEC. No single 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 barrier - 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 a local inflammatory reaction involving proinflammatory mediators including TNF, complement system , prostaglandins and leukotriene C4. Subsequent mesenteric vasoconstriction results in intestinal ischemia and reperfusion. Reactive oxygen species (ROS) produced by intestinal epithelial xanthine oxi-dase ( XO) may be the final pathway for intestine injury. During tissue ischemia, there is intracellular accumulation of a large amount of hypoxathine and conversion of xanthine dehydrogenase to XO. Upon reperfusion, XO promotes formation of a large quantity of superoxide, and hypoxanthine is reconverted back to xanthine. This burst of reactive oxygen species formation can cause severe tissue damage. Endogenous NO may help to maintain the integrity of the mucosal barrier and the microvasculature. The protective mechanism was decreased by proinflammatory 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 injurious and protective mechanisms.However, extrapolation of these observations to human NEC must be made cautiously because these experiments were not generality conducted in newbornanimal. These results led us to ask whether the mechanisms above - mentioned might be associated with NEC in human preterm infants.The aims of the present study were to determine the dynamic ileum morphology change and to study whether or not the changes of XO, ROS, neuronal nitric oxide synthase (nNOS) , inducible nitric oxide synthase (iNOS) and epithelial growth factor (EGF) , epithelial growth factor receptor (EGFR) , transforming growth factor - a ( TGF - a ) and their roles at the pathogenic mechanisms in intestine injury with LPS - induced neonatal rat. We try to settle some trial foundation of providing more information of pathophysiological 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 lh 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 gastrointestinal tract was carefully 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 4cm proximal to the ileocecal valve from each animal was cut, fixed for histological evaluation of NEC. The rest of the ileum was snap frozen in — 80 °t for measurements of protein and mRNA . Histological changes in the ileum were scored by a blinded evaluator and were assigned a necrotizing en-terocolitis ( NEC ) score on a scale of 0 to 4 as follows: 0 = normal, intact vil-lous 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 Macroscopic exam: After the abdominal cavity was opened, the small intestine and colon was grossly examined for such the sign as intestinal discoloration, intestinal hemorrhage and distention.3. 2 Microscopic exam: The segment of distal ileum was harvested, fixed in 4% paraformadehyde, embedded in paraffin, microtome - sectioned at 5 ujm, and counterstained with hematoxylin and eosin for histological evaluation of intestinal injury.3. 3 W/D measurement; The fresh ileum tissue was weighted first wet weight (W) . Then put it into 80t incubator for 24 hours. Weight the tissue a-gain as dry weight ( D). Calculate the ratio of W/D.3.4 Biochemical methods; The activities of XO, ROS (OH ? and 02~ ) and SOD, the levels of H2O2, MDA and protein content were measured.3. 5 Immunohistochemistry: The localization of nNOS, iNOS and EGFR and their proteins expression were performed.3. 6 Enzyme linked immunosorbent assay ( ELISA) : The concentration of EGF in ileum was detected.3. 7 Reverse transcription polymerase chain reaction ( RT - PCR) : The expression levels of nNOS, iNOS, EGFR and TGF - a were determined.4. Statistical analysisSoftware SPSS 11.0 for Windows was used in all statistical tests. Comparisons between the two groups were performed with t test, from which all data are 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 paleyellow in control group. It has good elasticity. It is observed that the lesion site involves mainly in the ileocolic region following UPS 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 1 h 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 the time 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 ( P < 0.01).1.4 Intestine W/D ratio: The W/D ratio of ileum was significantly higher than control group during 24h after LPS injection (P <0. 05). The degree of intestinal injury was positively related to the W/D radio (7 =0.900, P <0.05).2. The changes of oxidative stress index2. IThe change of XO activity: The level of XO at lh after injection of LPS was significantly higher than control group (P <0.05). Its peak was at 12h. There was a significant positive correlation between the XO activities and the scoring of intestinal injury within 24h (7=0.900, P < 0. 05 ) .2. 2 The change of H2O2 level: The level of H2O2 during 24h after injection of LPS was significantly higher than control group ( P < 0. 05 ). There was no significant correlation between the H2O2 level and the scoring of intestinal injury within 24h (7 =0.700, P>0.05).2. 3The change of ROS (OH ? and O2r ): The activities of ROS at 3h, 6h, 12h, 24h after injection of LPS were significantly higher than control group (P < 0.05 ). There was no a significant correlation between the ROS levels and the scoring of intestinal injury within 24h (7=0.700, P > 0.05 ) .2.4 The change of MDA level: The level of MDA at lh after injection ofLPS 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 (7=0. 900, P < 0. 05 ) .2. 5 The change of SOD activity: The activity of SOD at 3h after injection of LPS was significantly lower than control group ( P <0.01) . There was a significant negative correlation between the SOD level and the scoring of intestinal injury within 24h (7 = -0.900, P<0.05).3. The expressions of nNOS and iNOS proteins and the expressions of nNOS mRNA and iNOS mRNA3.1 It is shown that the positive expression of nNOS is mainly in the nerve plexus and nerve fiber of the small intestinal wall by immunohistochemistry. The myenteric nerve plexus had relatively higher levels of nNOS expression in comparison with the submucous nerve plexus. The positive expression of nNOS was decreased after LPS injection. It is obviously statistical different at 6h -24h following LPS treatment ( P < 0. 01). The lowest level of its expression is at 12h (P < 0.001). There was a negative significant correlation between nNOS expression and the scoring of intestinal injury within 24h ( 7 = — 1. 000, P < 0.01).The expression of nNOS mRNA in ileum was gradually diminished after LPS injection. The level of its expression was significantly lower at 6h and 12 h ( P < 0.05).3.2 The immmunohistochemical analysis showed that iNOS protein was localized almost exclusively to the epithelial cells (enterocytes) in the surface vil-li, with some staining detected in the lamina propria. The positive expression of iNOS in LPS group only increased at 12h (P <0.05). There was no significant correlation between the expression of nNOS and the scoring of intestinal injury within 24h (7=0.400, P>0.05).The expression of iNOS mRNA was significantly increased at 6h, 12h following LPS injection ( P <0. 05 ).4. The EGP level, the expressions of EGFR protein and EGFR mRNA and the expression of TGF - a mRNA in the ileum tissue4. 1 Expressions of EGFR protein and EGFR mRNA:In ileum tissues the EGFR was present on the villi and crypt epithelial cells, localized in each rat on the basolateral and apical membranes of entero-cytes. There were low levels of EGFR signal in the control group. Its expression was no significant change at lh after LPS injection (P > 0.05). It was obviously statistically different at 3h, 6h, 12h and 24h following LPS injection (P < 0.01). There was no significant correlation between the EGFR protein and the scoring of intestinal injury within 24h (-y = 0. 800, P > 0. 05).The expression of EGFR mRNA is increased following LPS injection ( P < 0.05). Its expression peak was at 6h.4. 2 The concentration of EGF protein:EGF levels of the LPS - injected pups during 24h were significantly less than the control (P <0.05). There was a significant negative correlation between the EGF protein levels and the scoring of intestinal injury within 24h ( 7 = -1.000, P < 0.01).4. 3 Expression of TGF - a mRNA:The expression of TGF - a mRNA in the control group pups was observed. Its expression was increased in the initial stage following LPS injection ( P < 0.05). There were no significant difference at 6h, 12h and 24h after LPS injection (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 intraperitoneally in neonatal rats.2. The XO in intestine tissue may be a pivotal enzyme to result in intestinal inflammation and injury. Activation of XO with massive ROS production occurs as a consequence of ischemia and subsequent reperfusion. The oxidant - mediated lipid peroxidation was increased and the activity of enzyme of removing ROS was decreased in mediating LPS - induced bowel necrosis in the neonatal rats. LPS can make the imbalance of oxidant/antioxidant which may play a central role in mucosal injury and involve in the pathophysiological mechanism of the...