Role Of TLR4/NF-κB In Damage To Intestinal Mucosa Barrier Function And Bacterial Translocation In Rats Exposed To Hypoxia

Background and objectivesWhen bodys quickly access to plateau， the special geological and climaticenvironments at high altitude might lead to serious metabolic supply obstacles with obviousclinical manifestation. These phenomena most likely trigger acute severe mountainsickness(ASMS), which mainly includes high altitude pulmonary edema(HAPE) and highaltitude cerebral edema(HACE). ASMS caused by hypobaric hypoxia follows an unusuallyaggressive clinical course, with rapid deterioration and high fatality.Unfortunately, atpresent there are still many debates about ASMS unclear pathogenesis.Because of thebackground mentioned above, we chose the intestinal tract as a new incising point ofASMS.As a crucial organ, intestinal tract not only play a vital role in digestion and absorption,but also the biggest storage of bacteria and endotoxins in human body. The mucousmembrane barrier integrity can prevent intestinal toxin from invading tissues out of thelumen. Once intestinal bacteria and endotoxins enter the portal vein or lymphatic system,they can translocate to other tissues and organs, leading to a cascade response mediated byinflammatory mediators, wich may induce systemic inflammatory response syndrome oreven multiple organ dysfunction syndrome(MODS). High altitude is a special ecologicalenvironment for body because of the low pressure and hypoxia which is significant ingastrointestinal function.Recent studies have shown that hypoxia alone can damage the function of thegastrointestinal barrier and cause flora imbalance in rats. As a key transmembrane proteinclosely related to bacterial recognition, TLR4transfers extracellular antigen information into cells and induces inflammatory reactions. It provides a new research target for studiesof bacterial translocation and damage to the function of the intestinal barrier under hypoxicconditions.Toll-like receptor4(TLR4) is an important part of the innate intestinal immunesystem. It is the first line of the intestinal tract that recognizes bacteria. During thedevelopment of enteric septicemia, LPS, a strong activator of TLR4in the intestinalepithelium, specifically binds to TLR4which ultimately forms the signal wave thatactivates the final effector molecule nuclear factorκB (NF-κB). Because TLR4is expressedat high levels in the intestinal tract, and its specific ligand lipopolysaccharide (LPS) iswidely distributed among intestinal bacteria. For these reasons, we deduced that damage tothe function of the intestinal barrier and bacterial translocation under hypoxic conditionsmight be closely related to the TLR4/NF-κB signaling pathway.In this study, we used hypoxia to generate an intestinal bacterial translocation andbarrier damage model. To investigate the role and mechanism of TLR4/NF-κB in thefunctional damage in this model, we evaluate changes in the expression of TLR4, NF-κB,and related inflammatory mediators such as tumor necrosis factor-alpha (TNF-α) andinterleukin6(IL-6) after the application of the NF-κB inhibitor PDTC.In high-altitude environments, the incidence of gastrointestinal tract diseases is high.Because the gastrointestinal tract is often where MODS begins, intestinal infectionsinduced by damage to its barrier function are involved in the occurrence and development ofmany severe high-altitude diseases. Understanding the TLR4/NF-κB signaling pathway mayprovide a new target for the prevention of severe high-altitude diseases. therefore, it haspronounced clinical significance and high theoretical value.Materials and methods1. Establishment of the animal models: Sixty adult male Sprague-Dawley (SD)rats(200±20g)were purchased from the Research Animal Center of the Research Institute ofSurgery, Third Military Medical University. All procedures were approved by theExperimental Animals Committee of the Third Military Medical University, and the disposalof experimental animals is strict comply with the management requirements of experimentalanimals. Rats were randomly divided into3groups of20rats each: control (group C),hypoxia alone (group H), hypoxia+LPS (group HL). The animals in group C were placedin a normal environment, while those in groups H and HL were placed in a hypobaric chamber simulating an elevation of7000m for3days. The rats in group HL were given0.5mg/kg LPS via an abdominal injection at48h after reducing the pressure. The groups thatdid not receive drugs received abdominal injections of saline of the same volume at thesame time. Samples were collected from all groups after72h. Animals were subjected tointraperitoneal anesthesia by using10%urethane (1g/kg). For group C, the samples werecollected in the normal environment; for the other2groups, the animals were placed at asimulated altitude of4500m. The samples were collected quickly. Through model-relatedindicators we can estimate the animal model.2. Study on the role of TLR4/NF-κB in damage to intestinal mucosa barrier function andbacterial translocation in rats exposed to hypoxia: One hundred rats treatment conditions asmentioned above. Rats were randomly divided into5groups of20rats each: control (groupC), hypoxia alone (group H), hypoxia+PDTC (group HP), hypoxia+LPS (group HL), andhypoxia+PDTC+LPS (group HPL). The animals in group C were placed in a normalenvironment, while those in groups H, HP, HL, and HPL were placed in a hypobaricchamber simulating an elevation of7000m for3days. The rats in groups HP and HPL weregiven100mg/kg PDTC via an abdominal injection at1h prior to reducing the pressure.The rats were injected once a day for3consecutive days. The rats in groups HL and HPLwere given0.5mg/kg LPS via an abdominal injection at48h after reducing the pressure.The groups that did not receive drugs received abdominal injections of saline of the samevolume at the same time. Samples were collected at72h after pressure reduction. Transmissionelectron microscopy was used to examine the ultrastructure of the intestinal tract, bacterialcultivation to observe translocation, kinetic turbidimetric assay to measure serum LPS, ELISA to detectTNF-α and IL-6serum concentrations, fluorescent quantitative RT-PCR to measure TLR4mRNA levels, and western blotting to detect changes in NF-κBp65and Occludin expression.Results1. The rats in group C were active, energetic, and lacked any inflammatory reactions inthe abdominal cavity that were visible to the naked eye. The rats in groups H and HL wereless active and were notably more tired. Prominent swelling was observed in their intestinalcanals, and the intestinal mucosa was congested. A small amount of ascites and mesentericlymph node swelling was observed in the rats from group HL. The rats in groups HP andHPL also showed reduced activity and appeared relatively tired. Mild swelling was observed in their intestinal cavities, but there was no notable mucosa congestion.2. Bacterial cultures of different organs collected from the rats in group C were allnegative. The rates of bacterial translocation in groups H and HL were significantlydifferent from those of group C (P <0.05). The concentration changing of LPS in differentgroups are same to bacterial cultures. Furthermore, in group H, La3+particles linearlydistributed in vascular endothelial tight junctions, and locally small amounts of La3+particles into the surrounding space by tight junctions. In group HL, La3+particles weredistributed diffusely in the capillary endothelium, basement membrane, and interstitialspace. The sub-epithelial TJs were open, a large number La3+particles were also foundinto the cytoplasm.3. After treatment with PDTC，the rate in HP and HPL group was significantly reducedcompared to H and HL group (p<0.05), respectively. The same situation can be found in theconcentration tendency of LPS. In group HP, La3+particles were localized on the surfacesof the vascular endothelial cells. No La3+sediment was found in the basement membraneor extravascular mesenchyme. In group HPL, a small number of La3+particles weredistributed in the capillary lumen. In some regions, a small amount of La3+particlesthrough the basement membrane, and no La3+particles were found distribution in theinterstitial space or cytoplasm.4. The serum concentrations of TNF-α, and IL-6in groups H and HL weresignificantly higher than those in group C. They were significantly higher in group HL(over2fold) than in group H (P <0.05). The concentrations of TNF-α, and IL-6in theserum of rats from group HP were significantly lower than the concentrations in those fromgroup H, and those in group HPL were significantly lower than in group HL (P <0.05).TLR4mRNA levels and NF-κBP65expression were consistent with the serum factorresults. Interestingly，Occludin expression is on the contrary.Conclusions1. animals placed in a hypobaric chamber set at7000m can establish stableexperimental intestinal injury model, which has laid foundation for further study on ASMS.2. Exposure to acute hypobaric hypoxia cause serious damage to intestinal ultramicrostructure， especiall to the small intestinal microvilli, enterocyte and vascularendothelial tight junctions. 3. Activation of TLR4/NF-κB signaling pathway is closely related to the damage to thefunction of the intestinal barrier and bacterial translocation induced by hypoxic conditions.Damage of intestinal ultra microstructure、down-regulated of TJ protein Occludin and thelarge release mediators of inflammation may participate in the pathological process. TheTLR4-mediated transcellular transfer of bacteria is probably involved in it.4. The damage to intestinal mucosa barrier function and bacterial translocation wassignificantly alleviated after blockage of the TLR4/NF-κB signaling pathway by PDTC.This result suggested that TLR4/NF-κB signaling pathway is an effective interventiontargets for treatment of intestinal injury in high altitude.