| IntroductionDigestive tract is the maximal immunizing tissue of body, thus it is very important of mucosal integrity and functional status of the gut to maintain the healthy organism. Meanwhile digestive tract is also the biggest bacteria pool of organism. The intestinal micro-ecological stability can be damaged when abrosion, administration of antiacid or antibiotics to heavy sick patient, which can result in alteration of intestinal bacterial flora. The alteration of intestinal flora is the main reason of bacteria translocation and enterogenic infection.The intestinal mucosal barrier includes mechanical barrier (mucus secreted by intestinal epithelium, enterocyte and the tight conjunction between them), biological barrier (bifidobacteria, bacillus acidi lactici, etc), chemical barrier (nitrogen monoxidum, power of hydrogen in enteric cavity, etc) and immunizing barrier (gut associated lymphoid tissue, phagocytic cell, secreted immunoglobulin, and defensins, etc). When organism suffers serious infection, trauma, burn, radiative or chemical therapy, or receives long-term traditional parenteral alimentation, mucosal permeability may be increased which may damage intestinal barrier function, and finally results in bacteria and endotoxin translocation. The translocation can stimulate inflammation cells cascade, release cytokine, produce active oxygen free radical group, and at last cause systemic inflammatory reaction syndrome (SIRS). Furthermore, multiple organ dysfunction syndromes (MODS) may be happened. Therefore, the intestinal tract is not only the target organ when MODS happen, but also the first affected organ. The intestinal mucosal impairment is the key reason of MODS. At present, there is no extremely effective treatment to MODS/SIRS, so it is especially important of how to diagnose earlier and prohibit its pathophysiologic proceeding.Endotoxin is the lipopolysaccharide (LPS) ingredient of Gram-negative bacterial cytoderm, and adipoid A, which belonged to exogenous mediator, is the virulent core of LPS. The activated complement system when endotoxemia, can provoke macrophage, neutrophil to release a great quantity of such inflammatory mediators as tumor necrosis factor (TNF-α), interleukin (IL-1, 6, 8), and platelet activating factor (PAF), and result in blood vessel endothelium damaged, platelet and leukocyte conglomeration, mitochondria dysfunction. At last, SIRS may be happened.Defensins are a kind of cationic polypeptide, and have the different area of rejecting to water and electrification in spatial structure, thus can insert themselves into phospholipids membrane of microorganism riched in negative electricity, and destroy their structure and function, then kill them. Some researches have indicated that defensins have broad-spectrum antimicrobial activity. They can not only kill bacteria, fungi and some enveloped virus, but also be the important signal material between innate and acquired defense.a-defensin is secreted by Paneth's cell of intestinal tract in human and rat, and defensin-5 of them has the strongest activity and is important in enteric natural defense. The recent study indicated the protection against lethal enteric salmonellosis in transgenic mice expressing a human intestinal defensin-5. Defensin-5 mRNA from Paneth's cell was increased protectively when intestinal barrier damaged because of hemorrhagic shock. Recombinate human defensin-5 (rHD-5) which is innate immunologic substance of intestinal mucosa can be not only bacteriostatic, but also resist to bacteria adhering in vitro experiment, thus has potential clinical application in preventing and curing enterogenic infection. Bifidobacteria are the predominant bacteria of biological barrier in intestinal mucosal barrier and one of the predominant probiotics of human intestinal microflora, participating in the digestion, nutrition, metabolism, absorption, immunity and anti-infection of host, especially important in keeping the integrity of intestinal mucosa barrier. As the main biological barrier of intestinal mucosa, bifidobacteria can enhance gastrointestinal defense, anti-infection and anti-tumor through immunological rejection, immune clearance and immunological regulation. At present, the study on its antibiosis mechanism includes three aspects as follows:①organic acid produced by bifidobacteria can significantly decrease intestinal tract pH value and inhibits the growth and multiplication of putrefying bacteria and pathogenic bacteria acid-non fast②the protein produced by bifidobacteria similar to bacteriocin have some bactericidal action.③the hydrogen dioxide (H2O2) produced by bifidobacteria can activate hydrogen peroxidase of organism and inhibit and kill Gram-negative bacteria.The ecological balance of intestinal tract microbial flora is important in maintaining the integrity of intestinal mucosal barrier. Exogenous bifidobacteria implanted in intestine can counteract the excess proliferation of pathogenic bacteria, reduce bacteria and endotoxin translocation, and synthesize glutamine with intestinal glutamic acid, which enhance impaired intestine recovery. Bifidobacteria can stimulate the expression ofβ-defensin-2 in intestinal epithelium cells. Bifidobacteria adhesion of secretion type can inhibit the harmfulness of LPS and H2O2 on enteric epithelium cells, thus keep the integrity of intestinal mucosal barrier.Although above-mentioned researches investigate the harm of gastrointestinal functional disturbance, the defense of organism itself and the protection of bifidobacteria, oxidation damage of intestine in endotoxin-induced infant rats, inflammatory reaction mediated by ICAM-1 and MPO and the protection of Defensin-5 and IGF-1 on endotoxin-induced intestine are still not reported in domestic and oversea records now, the further study on intestinal protective mechanism of bifidobacteria are needed in the future. Therefore, in this study, through molecular biology, histochemistry and pathomorphology method, we used endotoxin-demeged infant rats which intestine was damaged, observed morphological changes of ileum and investigated the oxygen free radical damage, inflammative mediators damage and non-specific immunizing protection of intestinal tract. Meanwhile, protective mechanism of exogenous bifidobacteria on ileum tissue was investigated. This study can provide new therapeutic tool and theoretical support for clinically preventing from MODS/SIRS when suffer gastrointestinal dysfunction.Materials and Methods1,Animal modle ProtocolWistar rats aged 18 days were divided into three groups, 40 infant rats for each. Control group(C group) were given an intraperitoneal(IP) injection of normal saline (1ml/kg); endotoxin group (E group) were given an IP injection of E coli O55:B5 endotoxin (LPS, 5 mg/kg); treatment group rats (T group) were intragastrically administrated with bifidobacteria suspension (2.0×109CFU/ml, 0.5ml every time, twice a day) 7days before injection of LPS, until the experiment completed.2, Tissue PreparationAll infant rats were decapitated respectively at 2, 6, 12, 24 and 72h after received injection of LPS or normal saline. Serum and ileum tissue were collected as following process:(1) The serum were collected and reserved at -20℃after centrifuging at 3500r/min for 15min at 4℃for measurement of histochemistry.(2) After clysis of iced normal saline, the gastrointestinal tissue was carefully separated, a 0.5-1cm segment of distal ileum 2-3cm proximal to the ileocecal valve from each rats was put into 4% polyoxymethylene disposed with 0.1mmol/L phosphate buffered solution(PBS) for measurement of immunohistochemistry and pathological histology observation after hematoxylin-eosine(H-E)staining.(3) A 2-3cm segment of distal ileum 3-4cm proximal to the ileocecal valve from each rats was put into Eppendorf tube removed ribonuclease, transferred into liquid nitrogen and then reserved at -80℃refrigerator for measurement of mRNA for RT-PCR research.(4) The rest ileum proximal to the ileocecal valve was homogenated after removing water with filter and weighing, the supernatants were reserved at -20℃after centrifuged at 3000r/min for 10min at 4℃for measurement of histochemistry.3. Methods and Evaluation3.1 Ileal morphological examination3.1.1 Macroscopic examinationAbnormal reaction of infant rats was observed after IP injection of LPS or normal saline. After the rats were killed and their abdominal cavity was open, the small intestine was carefully examined for such sign as intestinal color, edema, hemorrhage and distention.3.1.2 Light microscopic examinationThe fixed ileum were dehydrated, embedded in paraffin, microtome-sectioned continuously at 4-Sμm, and stained with hematoxylin and erosin(H-E) for histomorphomatric measurement.3.2 The content of superoxide dismutase(SOD),malondialdehyde (MDA) and myeloperoxidase(MPO) in serum and ileal tissue were determined respectively by histomedical method.3.3 The expression of insulin like growth factor-1(IGF-1) and intercellular adhesion molecule(ICAM-1) protein were evaluated by immunohistochemistry.3.4 The expression of Defensin-5,IGF-1 and ICAM-1 mRNA were determined respectively by reverse transcription-polymerase chain reaction(RT-PCR).4. Statistical AnalysisSoftware SPSS 10.0 for Windows was used in all statistical tests and measurement data were presented as mean((?))±standard deviation(SD). Comparisons between 3 groups were calculated using independent sample t test, and the difference was considered to be significant if the P value was less than 0.05.Results1. The histopathological findings of ileal tissue in infant rats of each group1.1 Macroscopic examinationAfter LPS injected, the infant rats appeared lazy, dispirited, anorexia, and diarrhea and abdominal distention 1-2h later. The symptoms above were obvious 6h later, and such symptom as tachypnea, cyanosis in lip, less activity, mess body hairs without gloss appeared. The seriously sick rats showed spasm, cyanosis in four limbs, chilly body and even dead. The symptoms mentioned above of survivor after 24h were partly relieved and after 72h were recovered to normal. The infant rats administered intragastrically with bifidobacteria 7 days in advance appeared slight symptom, and recovered quickly. After the rats were killed and their abdominal cavities were opened, the intestinal tissues of C group were observed glossy with normal color. The rats in E group appeared intestinal hemorrhage with punctiform or lamellar, obviously at 6h. The symptoms in T group were better mitigated than E group.1.2 Light microscopic examination after H-E stainingThe intestinal villi of C group were integrated. The intestinal villi of E group were not significantly abnormal 2h later; and appeared shed and depletion of glandule in intestinal villi, thinness or depletion of striated border, different height and low density of intestinal villi, capillary congestion of the lamina propria and polymorphomuclear infiltration 6h later; and epithelial cell of intestinal villi in part were repaired 12h later, obviously repaired 24h later, nearly normal at 72h. The intestinal villi of bifidobacteria group were not significantly abnormal 2h later; and appeared damaged, repaired partly and accentuated secretion of goblet cell in intestine 6h later; obviously repaired 12h later, nearly repair or normal at 24h, and completely repair or normal at 72h.2. The content of serum and ileal tissue SOD, MDA and MPO in infant rats of each group2.1 The SOD activity of serum and ileal tissue in infant ratsThe SOD activity of serum in LPS group were significantly decreased at 6, 12, and 24h compared with C group (P＜0.05); and in bifidobacteria group were significantly increased at 2, 6, 12, and 24h compared with LPS group (P＜0.05).The SOD activity of ileal tissue in LPS group were significantly decreased at 6, 12, and 24h compared with C group (P＜0.05); and in bifidobacteria group were significantly increased at 6, 12, and 24h compared with LPS group (P＜0.05).2.2 The MDA content of serum and ileal tissue in infant ratsThe MDA content of serum in LPS group were significantly increased at 6, 12, and 24h compared with C group (P＜0.05); and in bifidobacteria group were significantly decreased at 6, 12, and 24h compared with LPS group (P＜0.05).The MDA content of ileal tissue in LPS group were significantly increased at 6, 12, and 24h compared with C group (P＜0.05); and in bifidobacteria group were significantly decreased at 12, and 24h compared with LPS group (P＜0.05).2.3 The MPO content of serum and ileal tissue in infant ratsThe MPO content of serum in LPS group were significantly increased at 6, 12, and 24h compared with C group (P＜0.05); and in bifidobacteria group were significantly decreased at 6, and 12h compared with LPS group (P＜0.05).The MDA content of ileal tissue in LPS group were significantly increased at 2, 6, and 12h compared with C group (P＜0.05); and in bifidobacteria group were significantly decreased at 6, and 12h compared with LPS group (P＜0.05).3. The molecular biological change of Defensin-5, IGF-1 and ICAM-1 in infant rats of each group3.1 The expression of Defensin-5 mRNA in ileal tissueThe expression of Defensin-5 mRNA in ileum tissue were positive in C group, and were significantly increased at 2h after LPS injected, the peak at 6h, and were gradually diminished later. The significant difference was at 2, 6, and 12h (P＜0.05) compared with C group. The expression of Defensin-5 mRNA of ileum tissue in bifidobacteria group were decreased at 2h compared with E group, and significantly diminished at 6, 12, and 24h(P＜0.05). The expression of Defensin-5 mRNA of ileum tissue in bifidobacteria group were increased at 2 and 6h compared with C group, the significant difference at 2h (P＜0.05), and gradually diminished to normal later.3.2 The expression of IGF-1 protein and mRNA in ileal tissue3.2.1 The expression of IGF-1 protein in ileal tissueThe result of immunohistochemistry staining for ileal IGF-1 showed that intestinal mucosa crypts and epithelial cells in C group were stained with more amounts of buffy grains, and the stain in LPS group were decreased, the average optical density value of immunohistochemistry in LPS group were significantly decreased at 12, 24, and 72h compared with C group(P＜0.05). The average optical density value of immunohistochemistry in bifidobacteria group were significantly increased at 12, 24 and 72h (P＜0.05).3.2.2 The expression of IGF-1 mRNA in ileal tissue The expression of ileal IGF-1 mRNA in C group were more; The expression of ileal IGF-1 mRNA in LPS group were significantly decreased at 6, 12, 24 and 72h (P＜0.05) compared with C control; the expression of ileal IGF-1 mRNA in bifidobacteria group were significantly increased since 6h compared with LPS group(P＜0.05).3.3 The expression of ICAM-1 protein and mRNA in ileal tissue3.3.1 The expression of ICAM-1 protein in ileal tissueThe result of immunohistochemistry staining for ileal ICAM-1 showed that periplast of intestinal mucosa in C group were stained with small amounts of buffy grains, and all the periplast of intestinal mucosa in LPS group were stained, the most obviously in 6h. The average optical density value of immunohistochemistry in LPS group were significantly increased at 6, 12, 24, and 72h compared with C group (P＜0.01). The average optical density value of immunohistochemistry in bifidobacteria group were significantly increased at 6 and 12h (P＜0.01), then gradually decreased later, and decreased at 72h compared with LPS group (P＜0.01).3.3.2 The expression of ICAM-1 mRNA in ileal tissueThe expression of ileal ICAM-1 mRNA in C group were little; The expression of ileal ICAM-1 mRNA in LPS group were significantly increased at 2h (P＜0.01) compared with C control, and gradually diminished later and had no significant difference in later time; The expression of ileal ICAM-1 mRNA in bifidobacteria group were significantly decreased at 2h compared with LPS group (P＜0.01), and had no difference at 2h compared with C group; Increased significantly at 6 and 12h compared with LPS group, decreased significantly at 24h, and normal at 72h.Conclusions1,Exogenous bifidobacteria supplementation can relieve the histopathological damage of ileum tissue in infant rats demeged by IP injection of LPS, and promote the reparation of damaged tissue.2,The SOD activity were decreased and the MDA content were increased in serum and ileal tissue of LPS-induced infant rats, and the above change could be improved by exogenous bifidobacteria supplementation, which showed that bifidobacteria could protect the intestinal tissue of infant rats through inhibition of lipid peroxidation damage.3,The MPO content of serum and ileal tissue and the expression of ICAM-1 protein and mRNA in ileal tissue were increased in LPS-induced infant rats, and the above change could be improved by exogenous bifidobacteria supplementation, which indicated that bifidobacteria could protect the intestinal tissue of infant rats through inhibiting the release of such inflammatory mediators as MPO and ICAM-1.4,The expression of IGF-1 protein and mRNA in ileal tissue were decreased in LPS-induced infant rats, and the above change could be inhibited by exogenous bifidobacteria supplementation, which indicated that bifidobacteria could protect the intestinal tissue of infant rats through elevating the IGF-1 expression. 5,The expression of Defensin-5 mRNA in ileal tissue was increased in LPS-induced infant rats, and the expression could not be increased after exogenous bifidobacteria supplementation, but diminished. The data indicated that bifidobacteria could protect the infant rats not through secreting defensin-5, the intestinal congenital mechanism.
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