Study Of The Mechanisms Of Protective Effect Of Intestinal Trefoil Factor In Infant Rats With Intestinal Damage Caused By Endotoxemia

IntroductionGastrointestinal dysfunction is often regarded as originate factor, which pathogenesy is related with endotoxemia and impairment of gut mucosal barriar function. Intestinal mucosal barriar contain mechanical barriar, biologic barriar and immunologic barriar, which compose a protective coating and play an important role in maintaining homoeostasis. When the intestinal mucosal barriar is damaged and immune function decreased, a great quantity of bacterium and toxin get into blood to cause endotoxemia, which promote the release of cytokine and inflammation mediator. The results are to deteriorate intestinal mucosal barriar and accelerate the development process of severe cases. So gastrointestinal function disturbance is one of the important reasons that induce SIRS and MOSF.Severe infection is one of the common diseases in children, which may cause gastrointestinal function disturbance simultaneously in severe patients. The impairment of gut mucosal barriar function is a pathomechanism of gastrointestinal function failure and a common pathophysiological process in many diseases. The mechanism of impairment of gut function in children is unclear and the mortality is extremely high. When abdominal distention show up and bowel sounds decrease even to vanish and coffee - like matter appear, it usually indicate unfavourable prognosis. So it is significant to discover early and prevention to reverse the state of illness. The study of mechanism and explore effectively protective measure have become focal points.The damage of intestinal mucosal barriar is caused by hypoxia, inflammation mediator release and free radical increase, which result in multiple organ failure even death. A great quantity of TNF - a has an important effect in mediating sepsis shock. LPS,TNF -a,IL - lp can make iNOS gene expression obviously and induce the production of NO to produce inflammation and injury. Excess NO and 02~ are combined to form peroxynitrite, which can injury intestinal tissue by different mechanisms, including lipid peroxidation, degradation of ATP production, and disruption of the DNA. At the same time, peroxynitrite can also reinforce mucosal permeability, relax tight junction and lead to intestinal mucosal function disturbance. After the epithelial cells of intestinal mucosa are injured , intestinal epithelial cells release diamine oxidase ( DAO) and elevate the activity of DAO in plasm, which reflect the degree of ischemia and hypoxia in intestinal mucosa. Proliferating cell nuclear antigen (PCNA) , also called cyc-lin, generally distributes in enterocyte nucleus, and it is also an assistant protein of DNA that is necessary for cell proliferation. It is usually taken as a marker of cell proliferation and it takes part in the regulation of cell cycle itself. Cyclooxy-genase - 2 ( COX - 2) is called induced enzyme and its expression is increased by proinflammation cytokins factor such as IL - 1, IL — 6, TNF - a and endotox-in. The increased expression of COX -2 has been shown in inflammation conditions of the gastrointestinal tracts, but the effect of rITF to COX - 2 expression is not reported.Glutamine ( Gin) is nonessntial amino acid in plasm and cells, which is an important energy source of intestinal tract and it can promot the synthesis of protein and inhibit the degradation of protein. It is necessary for the maintenance of intestinl structure in both normal and stressed states as well as improvement of gut mucosal barriar function. It has no reported about whether Gin has effect on endogenous intestinal trefoil factor.Intestinal trefoil factor (ITF, also called TFF3) is a member of trefoil family. It is secreted by goblet cells of intestinal mucosa generally diatributed in gut. Because of its special structure, it has stable construction and cannot distinct by many kinds of digestive engyme. TFF3 may interact with the mucus;stablize the mucus gel by interacting with intestinal mucin and increasing the viscosity. Ex-pressed in early phase of injury, it can stimulate cell migrating and proliferating , promote epithelial cell repairment, and resist cellar apoptosis, so it is important in the self - protection mechanism of intestines.Recombonant intestinal trefoil factor (rITF) is production of yeast expression purification, which has been used on research of ulceration and neonatal necrotizing enterocolitis caused by hypoxia. It has been shown that rITF can decrease rats gastric ulcer index and inhibit the production of IL - 8, which has protective effect to gastrointestinal tract. TFF3 knockout rats have a higher susceptibility to gut related injury, but have obviously lessen of intestinal damage by applying rITF. It has no reported about the effect of rITF on infant rats caused by endotoxemia.The aims of this research are to study the mechanisms of ITF on intestinal injury By various research methods, we will testify that during endotoxemia, rITF may decrease DAO in plasm, enforce proliferation of cells, inhibit expression of COX - 2 and TNF - a, decrease production of NO. We also prove Gin can promote expression of TFF3. So we may be provide more information of pathophysiological mechanism and seek for prevention and treatment to gastrointestinal dysfunction.Materials and Methods1. Animal modelThe model of endotoxemia in 10 - day - old rats were made by intraper-itonael LPS injection. 10 - day - old infant Wistar rats were divided into four groups. Group NS were served as the normal control group and injected with normal saline (1 ml/kg). Group LPS were treated with LPS (EcoliO55: B55mg/ kg, 5mg/ml) and group ITF were injected by (LPS5mg/kg + rITFO. lml/R) , group Gin were treated with ( LPS5mg/kg + GlnlOml/kg, the active ingredient of glutamine is 13. 46g/100ml). Followed by LPS administration, plasma and intestinal tissues were collected at 2, 6, 24 and 72h.2. Specimens collection and treatmentEach group of rats was sacrificed by decapitation at the time points of 2, 6,24 and 72h after injection. Plasma and intestinal tissues were collected and stored respectively according to the following protocols:2.1 Eight samples of mixed blood were collected with heparin anticoagula-tion in vessel and plasma were centrifugal seperation. The plasma were stored in-10X, ice - box in order to measure the activity of DAO.2.2 0. 5 ~ lcm intestinal tissue near ileocecal junction were fixed in 4% paraformaldehyde and 2. 5% glutaral for hematoxylin - eosin ( HE) staining, immunohistochemistry, transmission/scanning electron microscope.2.3 The other intestinal tissues were put in Rnase -free Eppendorf tubes, then put in liquid nitrogen immediately and stored at -70X..3. Experiment methods3. 1 The appearance and death information were monitored.3. 2 The change of the intestine pathology.3.2.1 Macroscopic changes of intestines were observed.3. 2. 2 Histological study of intestines was examined by light microscope.3.2.3 Ultramicrostructure changes and villi of intestinal tissues were examined by transmission electron microscope (TEM) and scanning electron microscope (SEM).3.4 The contents of NO of intestinal tissues and the activity of DAO were detected by biochemistry methods.3. 5 The protein expression of PCNA, TNF - a and TFF3 were examined by immunohistochemical methods.3. 6 RT - PCR was used in detection the mRNA of COX -2NTNF - ouiN-OS and TFF3.3.7 Western Blotting was used in the detection of the protein of COX - 2 > TFF3.4. Statistical analysisSPSS version 13. 0 was used to perform statistical analysis, with all dates expressed as (X ± S). Statistically significant differences in the mean values were analyzed with ANOVA, using the LSD for inter - group comparison. P < 0.05 is regarded as significant difference.Results1. General status of infant ratsAfter injection of LPS, the infant rats in LPS group showed diarrhea, abdominal distension and other gastrointestinal functional disturbance. The weight of rats did not increase and fatality was twenty percent. Whereas in ITF groups, the clinical symptom lessened compared with LPS group and fatality was ten percent. In NS group, the animals were survived well.2. The pathological findings of intestinal tissues2. 1 Macroscopic observation;2h after LPS injection, light congestion was found in intestinal truct and mesenterium. It had been seen obviously congestion, which accompanies with gastric retention and part of intestinal distention 6h after LPS injection but in 72h the symptoms mentioned above had improved. The change of intestine in ITF group lightened compared with LPS group. There was no change in intestine of NS group.2.2 The change of intestinal tissues in light microscope;There were no pathological changes in NS group and at 2h in LPS group. At 6h in LPS group, inflammatory cell infiltrate, congestion of villus interstitial substance, and cap-sular diastem in subepithelium could be seen. The congestion and edama increased and central chyle vessel distended in 24h LPS group. The change of intestine in ITF group lightened compared with LPS group.2.3 The change of intestinal tissues in TEM and SEM2. 3. 1 Transmission electron microscope (TEM);The intestinal villi of common enterocyte lined up in order, tight junction integrity, endocytoplasmic reticulum and mitochodrium clearly in the controls. In LPS group, it could be seen vacuole change of mitochodrium, cell nucleus condense, chromoplasm margin, break and depletion of part of microvilli, widen and disrupted tight junction. The change of intestinal tissues showed 2h after LPS injection, achieving the peak at 6h and 24h, while the injury improved in 72h. The change of intestine in ITF group was slightly compared with LPS group.2. 3. 2 Scanning electron microscope ( SEM) : The intestinal villi in LPSgroup were short, rarefaction and breaks partly, while in ITF group there were slightly changes.3. The dynamic changes of DAO in plasma;The activity of DAO in plasma was significantly higher in LPS group than that in NS group (P <0.05 or P <0.01) at every time after LPS administration. Contrasted with LPS group, in ITF group the DAO was decreased! P <0.01 or P <0.05) at every time points.4. The dynamic changes of PCNA at the protein level. Immunohistochemical staining showed, PCNA was expressed in enterocytenucleus. It decreased continually after LPS injection and reached the lowest point at 24 h point, in 72 h point it increased but could not attain normal level ( P <0.01 or P <0.05). In ITF group, the protein level of PCNA was significantly increased at 2, 6 and 24h, but had no change at 72h than that of in LPS group (P>0.05).5. The dynamic changes at COX - 2 mRNA and its protein level5. 1 The dynamic changes at COX -2 mRN AThere was no expression of COX -2 mRN A in NS group. It could be seen the slightly expression of COX - 2 mRNA in 2h after LPS and reached the peak at 6h and decreased in 24, 72h group (P <0.01). The expression of COX -2 mRNA in ITF group decreased and have significant difference compared with LPS group at every time( P <0.01).5.2 The dynamic changes of COX -2 at protein levelWestern Blotting examination showed: There was no protein expression of COX - 2 in NS group. It could be seen the slightly expression of COX - 2 protein in 2h after LPS and reached the peak at 6h and decreased in 24, 72h group ( P < 0.01). The expression of COX - 2 protein in ITF group decreased and has significant difference compared with LPS group at 6, 24h( P <0.01). No protein expression could be seen in 2, 72 h group of ITF.6. The content of NO and expression of iNOS mRNA in intestinal tissues 6.1 The content of NO in intestinal tissuesThe NO content was significantly increased in LPS group and reached the maximum 24h after LPS administration (P <0.01). Contrasted with LPS group,the NO of ITF group were decreased and still sustained higher level than NS group at 6 and 24h (P <0.05 or P <0.01) .which attain normal at 72h.6.2 The expression of iNOS mRNA in intestinal tissuesThe weak expression of iNOS mRNA can be seen at every time in NS group. The obvious increased expression of iNOS mRNA at 6 and 24h in LPS group and significant higher than that in NS group ( P < 0. 01). In compared with LPS group, the expression of iNOS mRNA decreased at 6, 24h in ITF group (P < 0.01) and weak expression at 2 and 72h.7. The dynamic changes at TNF -a mRNA and its protein level7.1 The dynamic changes at TNF - a mRNAThere was no expression of TNF - a mRNA at every time in NS group. Theobvious increased expression of TNF - a mRNA at every time in LPS group andsignificant different with NS group ( P <0. 01). In compared with LPS group,the expression of TNF - a mRNA decreased at each time point of ITF group ( P<0.05 or P<0.01).7. 2 The dynamic changes at TNF - a protein levelThe IODT of TNF - a was significantly increased in group LPS and reached the maximum at 6h after LPS administration (P < 0. 01);Contrasted with LPS group, the expression of TNF - a in ITF group was decreased and still sustained high level at each time point than that in NS group ( P <0.01).8. The effect of Gin on endogenous TFF3 expression8.1 RT - PCR detection of the expression of TFF3 mRNAThere was expression of TFF3 mRNA at every time in NS group. TFF3 mRNA were slightly increased at 2h in LPS group, but obviously decreased at 6 and 24h. Although increased at 72h, it cannot attain normal, which has significant difference than that of in NS group (P<0.01or P < 0. 05). The expression of TFF3 mRNA in Gin group has obviously increased at 2, 6 and 24h( P <0. 01) but have no significant difference with LPS group at 72h.8.2 The protein expression of TFF3Immunohistochemical staining showed, TFF3 was expressed in goblet cell in intestinal tissues. There was slightly increase of dyeing at 2h in LPS group but decrease at 6 and 24h. In every time of Gin group, the dyeing of TFF3 elevatedthan that of in LPS group.8.3 Western blotting assay for detection of TFF3 protein expression There was expression of TFF3 protein at every time in NS group. TFF3 protein was slightly increased at 2h in LPS group, but obviously decreased at 6 and 24h and decreased to the lowest point at 24h. Although increased at 72h, it cannot attain normal, which was still lower than that in NS group (P < 0. OlorP < 0. 05). The expression of TFF3 protein in Gin group has obviously increased at 6 and 24h (P < 0.01) but .have no difference compared with LPS group at 2 and 72h (P>0.05).Conclusions1. Intestinal injury can be seen in infant rats with endotoxemia, especially in transmission electron microscope at 2, 6 and 24 hour, the damage can be improved by rITF.2. The activity of DAO in plasma elevated after LPS injection and play an important role in damaging intestinal mucosal barrier. The activity of DAO in plasma can be regarded as an index of intestinal injury. rITF can play a protective role by decreasing DAO in plasma.3. The decrease of PCNA in endotoxemia was obvious, while rITF can promote the expression of PCNA and bring into full play in cell proliferation.4. COX -2 joined in intestinal injury when exdotoxemia, rITF can down -regulated the expression both COX - 2 mRNA and COX - 2 protein level, which may decrease the effect of PG and TBX2.5. The production of NO caused by iNOS mRNA transcript has detrimental effect on intestinal tissues of infant rats, but rITF have marked protectively effects through decreasing the contents of NO and inhibit the expression of iNOS mRNA.6. As a proinflammatory cytokine, TNF - a plays an important role in the activation of LPS - induced exdotoxemia. It can insult intestinal tissues. rITF can decrease expression both TNF - a mRNA and its protein, which play a protective effect on intestinal injuries induced by LPS.7. Gin can promote the expression of endogenous TFF3 not only in TFF3 mRNA but also in protein level, which may reinforce the protective effect.