Mechanism And Nursing Revelation Of Endoplasmic Reticulum Stress-autophagy Pathway In Intestinal Barrier Dysfunction Following Severe Burns

Background&AimEarly effective intestinal nursing interventions are extremely important for the treatment of patients with severe burn injury.It has been well recognized that intestinal mucosal barrier dysfunction and increased intestinal permeability are important factors contributing to the intestinal bacterial translocation,sepsis and death after severe burn or combined radiation-burn injury.Thus,recovery of intestinal mucosal barrier function is an important part of early intestinal nursing in patients with severe burns.Previous studies have shown that hypovolemic shock induces ischemic/hypoxic damage of the systemic tissues and organs in the early stage of severe burns,and that excessive and persistent ischemia/hypoxia damage causes intestinal mucosal barrier dysfunction.Tight junction（TJ）,located at the apical portion of the epithelial cell-cell contact,is one of the important components in the intestinal mucosal barrier.It plays a vital role in maintaining intestinal permeability.There are many influential factors affecting intestinal mucosal barrier dysfunction after severe burns,thus,the precise molecular mechanisms are still unclear.Previous studies have shown a possible interplay between ER stress and the activation of autophagy,and both of them are closely associated with the intestinal inflammation.Excessive and prolonged ER stress induces high level of autophagy or even autophagic cell death when intestinal ER homeostasis is not restored by UPR,and finally causes intestinal mucosa damage.Thus,we hypothesize that severe burn triggers ER stress in intestinal mucosa,which in turn,leads to the activation of autophagy,resulting in intestinal mucosa tight junction barrier dysfunction and increased intestinal permeability by causing the changes of both expression and localization of tight jucntion proteins.In this study,by utilizing a 30%total body surface area（TBSA）full-thicknes burn mice model and an intestinal monolayer cell model,respectively,we investigated the role of ER stress-autophagy axis in intestinal mucosa tight junction barrier dysfunction following severe burns,in order to further elucidate the mechanism of severe burn-induced intestinal mucosa tight junction barrier dysfunction,further clarifies the important role of intestinal mucosal barrier function in intestinal nursing,and provides a new guidance for intestinal nursing in severely burned and other critically ill patients,and provide new intervention targets and ideas for exploring accurate and effective medical and nursing measures.Methods1.The healthy female C57BL/6 mice were randomly divided into control group and burn group.The mice in burn group were treated with a 30%TBSA full-thicknes burn,and observed for 1h,2h,6h,12h,and 24h respectively.2.The healthy female C57BL/6 mice were randomly divided into control group,Tm group（1.0mg/kg）,Tg group（300ng/kg）,4-PBA group（80mg/kg）,burn group,burn+Tm group（1.0 mg/kg）,burn+Tg group（300 ng/kg）,burn+4-PBA group（80mg/kg）respectively.All burned mice were inflicted to a 30%TBSA full-thicknes burn,and observed for 6h.3.The healthy female C57BL/6 mice were randomly divided into control group,RAPA group（4mg/kg）,3-MA group（15mg/kg）,burn group,burn+RAPA group（4mg/kg）,burn+3-MA group（15mg/kg）respectively.All burned mice were inflicted to a 30%TBSA full-thicknes burn,and observed for 6h.4.Caco-2 monolayer cells were randomly divided into control group and hypoxia group.For hypoxia treatment,Caco-2 monolayers were cultured under hypoxic condition（1%O₂,5%CO₂,and 94%N₂）for 1h,2h,6h,12h,and 24h respectively.5.Caco-2 monolayer cells were randomly divided into normoxia group,normoxia+Tm group（5μg/ml）,normoxia+Tg group（1μmol/L）,normoxia+4-PBA group（2mmol/L）,hypoxia group,hypoxia+Tm group（5μg/ml）,hypoxia+Tg（1μmol/L）group,hypoxia+4-PBA group（2mmol/L）respectively.The monolayers were incubated under hypoxic or normoxic condition for 6h.6.Caco-2 monolayer cells were randomly divided into normoxia group,normoxia+RAPA group（50nmol/L）,normoxia+3-MA group（10mmol/L）,hypoxia group,hypoxia+RAPA group（50nmol/L）,hypoxia+3-MA group（10mmol/L）respectively.The monolayers were incubated under hypoxic or normoxic condition for 6h.7.The intestinal permeability of mice and permeability of Caco-2 monolayer cells were measured by FITC-dextran probe.8.The indirect immunofluorescence assay was used to detect the distribution of ZO-1and LC3B in intestinal mucosa,and TJ proteins ZO-1,occludin and claudin-1 in Caco-2monolayer cells,respectively.Finally,intestinal mucosa and Caco-2 monolayer cells are observed and imaged using a laser scanning fluorescence microscopy.9.The intestinal mucosa of mice was stained with HE assay,and were observed and imaged by light microscope.10.Autophagosomes in intestinal mucosa and Caco-2 monolayer cells were detected by transmission electron microscopy.11.The transepithelial electrical resistance（TER）of Caco-2 monolayer cells were measured by a volt-ohmmeter.12.The Western blot assay was utilized to analyze the proteins expressions of ZO-1,occludin,claudin-1,claudin-2,Bip,CHOP,XBP1,XBP1s,LC3-II/LC3-I,Beclin 1,Atg5and P62 in intestinal mucosa and Caco-2 monolayer cells,respectively.13.The formation of autophagosomes in Caco-2 monolayer cells were traced by GFP-LC3B fusion protein.Results1.Severe burn injury induced the increased intestinal permeability,the disruption of TJ barrier,and enhanced ER stress and autophagy.（1）Intestinal paracellular permeability was significantly increased after severe burns.（2）Expression of TJ proteins ZO-1,occludin,claudin-1 decreased significantly,whereas claudin-2 expression increased after severe burn injury.The localization of ZO-1was obviously altered after severe burns.（3）Under microscope,the histological damage of intestinal mucosa was observed after severe burns.（4）ER stress was significantly enhanced in intestinal mucosa after severe burns.（5）The level of autophagy was significantly increased after severe burn in intestinal mucosa.（6）Severe burn downregulated PI3K/AKT/mTOR pathway in intestine mucosa.2.The activation of ER stress-autophagy axis is involved in intestinal mucosa tight junction barrier dysfunction in severely burned mice.（1）ER stress-specific inducers Tm and Tg enhanced the ER stress and autophagy,and down-regulated of PI3K/AKT/mTOR signaling pathway in intestinal mucosa of burned mice.The ER stress-specific inhibitor 4-PBA significantly reduced the ER stress and autophagy,and up-regulated of PI3K/AKT/mTOR signaling pathway in intestinal mucosa of burned mice.（2）Both Tm and Tg further increased intestinal permeability and aggravated intestinal barrier dysfunction in burned mice,whereas 4-PBA significantly reduced intestinal permeability and alleviated intestinal barrier damage in burned mice.（3）The autophagy-specific inducer RAPA significantly increased intestinal autophagy in burned mice.The autophagy-specific inhibitor 3-MA effectively inhibited intestinal autophagy in burned mice.（4）RAPA further increased intestinal permeability and aggravated intestinal barrier dysfunction in burned mice.However,3-MA remarkably reduced intestinal permeability and significantly alleviated intestinal barrier damage in burned mice.3.The activaed ER stress-autophagy axis contributes to the hypoxia-induced intestinal epithelial tight junction barrier dysfunction（1）Hypoxia significantly decreased TER and increased permeability in Caco-2monolayer cells.（2）Hypoxia significantly reduced the expression of TJ proteins ZO-1,occludin and claudin-1 and obviously changed the localization of ZO-1,occludin and claudin-1 in monolayer Caco-2 cells.（3）Hypoxia remarkably induced the ER stress and autophagy in monolayer Caco-2cells.（4）Both Tm and Tg significantly enhanced ER stress and autophagy,and down-regulated PI3K/AKT/mTOR signaling pathway in hypoxic Caco-2 monolayer cells.However,4-PBA significantly inhibited the ER stress and autophagy,and up-regulated the PI3K/AKT/mTOR signaling pathway in hypoxic Caco-2 monolayer cells.（5）Both Tm and Tg further decreased TER,increased permeability and aggravated the barrier dysfunction of hypoxic Caco-2 monolayer cells,whereas 4-PBA significantly increased TER,decreased permeability and alleviated the barrier dysfunction of hypoxic Caco-2 monolayer cells.（6）RAPA significantly enhanced autophagy in hypoxic Caco-2 monolayer cells,while3-MA markedly inhibited autophagy in hypoxic Caco-2 monolayer cells.（7）RAPA further decreased TER,increased permeability and aggravated the barrier dysfunction in hypoxic monolayer Caco-2 cells.However,3-MA markedly increased TER,decreased permeability and alleviated the barrier dysfunction of hypoxic Caco-2 monolayer cells.Conclusions1.Severe burn induced intestinal permeability increase,intestinal mucosa TJ barrier dysfunction,and activation of both ER stress and autophagy in mice.2.Severe burn-induced intestinal epithelial ER stress promotes the activation of autophagy via inhibition of PI3K/AKT/mTOR pathway.And activation of ER stress and autophagy is required for intestinal TJ barrier dysfunction in severely burned mice.3.Hypoxia causes decreased TER,increased permeability and epithelial TJ barrier dysfunction in Caco-2 monolayer cells.The hypoxia-induced TJ barrier dysfunction is accompanied by the activation of both ER stress and autophagy in Caco-2 monolayer cells.4.Hypoxia-induced ER stress contributes to the activation of autophagy through inhibition of PI3K/AKT/mTOR pathway in Caco-2 cells.The activation of ER stress-autophagy axis is involved in the TJ barrier dysfunction induced by hypoxia in Caco-2 monolayers.5.This study further demonstrates the importance of early effective oxygen therapy and early enteral nutritional support in severe burn patients,and provides a new experimental basis for nurses to further study early effective nursing measures for severe burn patients.6.This study further clarifies the importance of intestinal mucosal barrier function and provides new ideas for intestinal nursing of severe burn patients,and provides new intervention targets for exploring accurate and effective medical and nursing measures in the future research.