Study Of The Mechanism Of Toll-Like Receptor 2 And 4 On The Intestinal Injury With Endotoxemia Infant Rats And Of Protective Effect Of Glutamine And Intestinal Trefoil Factor

Intestinal mucosal barrier contain mechanical barrier,biologic barrier and immunologic barrier,which compose a protective coating and play an important role in maintaining homoeostasis.When the body infected severe disease,immune function decreased,pro-inflammation resulted in intestinal mucosa injury.Furthermore, intestinal mucosal barrier is damaged,a great quantity of bacterium and toxin get into blood to cause endotoxemia,Which promote the release of cytokine and inflammation mediator,translocation of bacteria,infection of distal organs.The results are to leading to System Inflammation Reaction Syndrome(SIRS)and accelerate the development process of severe cases.So gastrointestinal function disturbance is one of the important reasons that induce SIRS and MOF(Multiple Organ Failure).Severe infection and septicemia is one of the common diseases in children,which may cause gastrointestinal function disturbance simultaneously in severe patients. When abdominal distention show up and bowel sounds decrease even to vanish,it usually indicate unfavorable prognosis and the mortality is extremely high.The impairment of gut mucosal barrier function is a pathomechanism of gastrointestinal function failure.The mechanism of impairment of gut function in children is unclear. 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 barrier is caused by hypoxia,inflammation mediator release and free radical increase,which result in translocation of bacteria, infection of distal organs,multiple organ failure even death.Endotoxemia may prompt inflammation mediator release,such as TNF-α,IL-1 and IL-6.Over-release of inflammation mediator result in SIRS,and activates endogenous antiinflammatory mediator such as IL-4,IL-10,release simultaneously.However,over-release of Anti-inflammatory may result in suppression of immune function and sensitivity to infection,which causes Compensary Ami-inflammatory Response Syndrome(CARS). When SIRS and CARS interact intensively each other,may cause Mixed Antagonists Response Syndrome(MARS),lead to sever organism damage.Thus,SIRS,CARS and MARS are all the fundament of MOF.Recently,Toll family of proteins,named as Toll-like receptor(TLR)was discovered in humane and mammalian.When microbe invading,TLR may recognize exogenous pathogens and activate innate immune responses to eliminate invading microbe.These TLRs are typeâ… transmembrane proteins,consists of Leucine-rich repeat(LRR)in extracellular domains.Each of TLRs contains the pool homologous in extracellular domains,which indicates different construct of TLR ligand.TLRs share significant similarities with the interleukin(IL)-1 receptor(IL-1R)and all contain the highly homologous Toll/IL-1R(TIL)cytoplasmic domain,named as TLR/IL-1R(TIR) homologous region.LRR is the construction fundamention of protein interaction, which recognize pathogen or product to activate innate immune responses.The homologous TIL cytoplasmic domain construct relates to activate of nuclear factor-B (NF-кB).TLRs mediate innate immune response and adaptive immune response through recognizing specific construction of microbe and microbe cell wall.A specific construct of conserved sequence in microbe named as Pathogen associated molecular patterns(PAMPs),including lipopolysaccharide(LPS),peptidoglycans and lipoteichoic. There are 11 known TLRs so far,each of which recognizes a different spectrum of Pathogen associated molecular patterns.TLRs interact with PAMPs and are activated, which activates immune cell to release cytokine through signaling and trigger innate immune response.Glutamine(Gln)is the most affluent condition-amino acid in human,which is an important energy source of intestinal epithelia cell and lymphocyte,and it can promote the synthesis of protein and inhibit the degradation of protein.It is necessary for the maintenance of intestinal structure in both normal and stressed states as well as improvement of gut mucosal barrier function.Previously study of effect of Gln on intestinal mucosal function focused on nutrition aspect.Mechanism of Gln reduce pro-inflammation release is unclear.It has no reported about protect effect of Gln on intestinal mucosal function.Intestinal trefoil factor(ITF,also called TFF₃)is one of the trefoil family members.It is secreted by goblet cells of intestinal mucosa and generally distributed in gut.Because of its special structure,it has stable construction and can not destructed by many kinds of digestive enzyme.ITF may protect intestinal mucosa.It has demonstrated supplement of ITF relieves injury of intestinal mucosa.But,it is unclear weather ITF can relieves intestinal mucosa injury induced by endotoxin.The aims of this research are to study the mechanisms of TLR on intestinal injury induced by LPS and protection effect of Gln and ITF on intestinal injury.1.Expression of TLR mRNA and protein will be detected in normal intestine tissue.2.The level of TNF-α,IL-4,IL-10 and expression of TLR mRNA and protein in intestinal tissue will be tested during endotoxemia,whether expression of TLR mRNA and protein and level of TNF-α,IL-4 and IL-10 is increased in intestine,which relate to intestinal damage.3. whether Gln and ITF may inhibit expression of TLR mRNA and protein and decrease secrete level of TNF-α,IL-4 and IL-10 in intestinal tissue,which may relieve intestinal injury.Materials and Methods1.Animal model The model of endotoxemia in 10-day-old rats was made by intra-peritoneal LPS injection.10-dayold infant Wistar rats were divided into four groups.Group NS were served as the normal control group and injected with normal saline(1ml/kg);Group LPS were treated with LPS(EcoliO₅₅:B₅ 5mg/kg,5mg/ml);group Gln were treated with LPS 5mg/kg+Gln 10ml/kg(the active ingredient of glutamine is 13.46g/100ml); group ITF were injected by LPS 5mg/kg+rITF 0.1ml/kw.Followed by LPS adminis-tration,intestinal tissues were collected at 3,6,12,24,48 and 72h.2.Specimens collection and treatmentEach group of rats was anesthetize by intra-peritoneal injection of 10%chloral hydrate at the time points of 3,6,12,12,24,48 and 72h after injection.Intestinal tissues were collected and stored respectively according to the following protocols.(1)0.5-1cm intestinal tissues near ileocecal junction were fixed in 4% paraformaldehyde and 2.5%glutaral for hematoxylin-eosin(HE)staining,immuno-histochemistry,transmission/scanning electron microscope.(2)The other intestinal tissues were collected,then put in liquid nitrogen immediately and stored at-70℃until use.3.Experiment methods(1)The appearance of animals and death information were monitored.(2)The change of the intestine pathology.â‘ Macroscopic changes of intestines were observed.â‘¡Histological study of intestines was examined by light microscope.â‘¢Ultramicrostructure changes and villi of intestinal tissues were exam-ined by transmission electron microscope(TEM).(3)The contents of TNF-α,IL-4,IL-10 of intestinal tissues were detected by ELISA methods.(4)The protein expression of TLR2,TLR4 and NF-кB were examined by immunohistochemical methods. (5)RT-PCR was used in the detection of the mRNA of TLR2,TLR4 and NF-кB.(6)Western Blotting was used in the detection of the protein of TLR2,TLR4 and NF-кB.4.Statistical analysisSPSS version 13.0 was used to perform statistical analysis,with all dates expressed as((?)±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 abdominal distension and other gastrointestinal functional disturbance.The weight of rats did not increase and fatality was twenty percent.Whereas in Gln and ITF groups,the clinical symptom lessened compared with LPS group,In NS group,the animals were survived well.2.The pathological findings of intestinal tissues(1)Macroscopic observation:3h after LPS injection,light congestion was found in intestinal tract 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 Gln and ITF group lightened compared with LPS group.There was no change in intestine of NS group.(2)The change of intestinal tissues in light microscope:Intestinal villi was integrity and epithelial cells was lined up in order.Chalice cell had been poorly seen in Intestinal villi.There were no pathological changes in NS group.At 3h in LPS group, inflammatory cell infiltrated,congestion of villus interstitial substance,and oedema could be obviously seen.The epithelial cell rank was derangement.Cellular edema could be obviously seen.The congestion was lighten and edema was absorbed in 6h LPS group.Chalice cell increased.The change of intestinal injury was peaked in 12h LPS group.The intestinal damage revived and epithelial hyperplasia could be obviously seen,Chalice cell increased obviously after 48h and 72h LPS group.The change of intestine in Gln and ITF group lightened compared with LPS group.Chalice cell increased markedly.(3)The change of intestinal tissues in TEM:Epithelial cells are pillar-like,cell nuclear is orbicular-ovate.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 seed cell nucleus condense.chromoplasm margin. break and depletion of part of microvilli,widen and disrupted tight junction.The change of intestinal tissues showed 3h after LPS injection,achieving the peak at 24h, while the injury improved in 72h.3.The dynamic changes of TNF-α,IL-4,IL-10 of intestinal tissues:The content of TNF-α,IL-4,IL-10 of intestinal tissues in 3h LPS group was significantly higher than that in NS group(P<0.05 or P<0.01).They all achieved peak at 6h after LPS administration,decreased at 12h.The content of TNF-αwas slightly higher in 24h LPS group than that in NS group,that of IL-4,I-10 of intestinal tissues in 24h LPS group was significantly higher than that in NS group(P<0.05 or P<0.01), lower than that in 3h LPS group.Then they all decreased at 72h.Contrasted with LPS group,in Gln and ITF group the content of TNF-α,IL-4,IL-10 was decreased(P<0.01 or P<0.05).4.The dynamic changes of TLR2.TLR4 and NF-кB mRNA:There was weak expression of TLR2 and NF-кB mRNA in NS group.while TLR4 mRNA obviously expressed.It could be seen the significantly expression of TLR2 mRNA at 3h after LPS and reached the peak at 72h.The expression of TLR4 and NF-кB mRNA was markedly increased and achieve the peak in 3h LPS group, decreased in 6,12h group(P<0.01),then increased in 24,48h group(P<0.01),and decreased in 72h group again.The expression of TLR2,TLR4 and NF-кB mRNA in Gin and ITF group decreased significantly compared with LPS group. 5.Western blotting assay for detection of TLR2,TLR4 and NF-кB protein expressionThere was weak expression of TLR2,TLR4 and NF-кB protein in NS group. TLR2,TLR4 and NF-кB protein was significantly increased in LPS group(P<0.01), but obviously decreased in Gln and ITF group compared with LPS group(P<0.01) The protein expression of TLR2,TLR4 and NF-кB Immunohistochemical staining showed that,TLR2 and TLR4 were expressed in cell membrane in intestinal epithelia cell.NF-кB was expressed in cytoplasm and nucleus in intestinal epithelia cell.There was significantly increase of the expression at of TLR2,TLR4 and NF-кB protein in LPS group(P<0.01),but decrease markedly in Gln and ITF group compared with LPS group(P<0.01).Conclusions1.Intestinal injury call be seen in infant rats with endotoxemia,especially in transmission electron microscope at 3 and 24 hour.Gln and rITF can relieve the intestinal damage.2.The content of TNF-α,IL-4,IL-10 of intestinal tissues elevated after LPS injection and increased TNF-αmay play an important role in damaging intestinal mucosal barrier.The content of TNF-α,IL-4,IL-10 of intestinal tissues can be decreased after Gln and ITF administration.3.There was weak expression of TLR2,TLR4 and NF-кB mRNA and protein in control group.The expression TLR2,TLR4 and NF-кB mRNA and protein elevated markedly in imestinal tissue injury by LPS-induced.Gln and ITF Call down regulate the expression of TLR2,TLR4 and NF-кB mRNA and protein.4.Gln and rITF can relieve the intestinal tissues injury by LPS-induced,through inhibiting the expression of TLR.