| Quorum sensing(QS)is a process by which bacteria sense their own population density and regulate their behavior Quorum sensing inhibitor(QSI)disrupts QS signals and can be used as an alternative to antibiotics to reduce bacterial virulence It has a wide range of applications in medical and agricultural fields It has been reported that QSI can be used as a feed additive to significantly improve growth traits in pigs,but its mechanism o has not been elucidated The intestinal mucosal barrier is an important protective barrier against external aggressions,and QSI may play a role in protecting the intestinal cells and intestinal barrier in pigs by degrading the potentially harmful QS signals of intestinal flora in pigs;however,the mechanism of QSI action and the mechanism of QS signal damage to the intestine of pigs have not been elucidated In this study,AiiA was selected as the QSI for follow-up study Using the QS signal molecule OdDHL orally fed to ICR mice,it was observed that the small intestinal villi were wrinkled,sparsely arranged and shortened in length,and the peripheral blood DAO and D-lactic acid concentrations of mice were significantly increased,which demonstrated that the intestinal mucosal barrier of mice was damaged under the action of OdDHL Subsequently,in this study,we demonstrated for the first time that OdDHL could directly damage the intestinal cells of weaned piglets and destroy the intestinal barrier through the OdDHL piglet attack protection test,while QSI could greatly antagonize the damage of OdDHL and protect the piglet intestine from the QS signal Thus,it is proved that pig intestine is the target of QSI,and QSI can protect pig intestine by degrading the harmful QS signal of intestinal flora,which provides a basis for the potential application of QSI in the farming industry.In the above study,it was confirmed that OdDHL could damage the intestinal barrier in pigs,and QSI could antagonize the process of intestinal damage,but the specific mechanism has not been elucidated Therefore,in this study,the concentration of OdDHL was selected by MTT assay,and the apoptosis rate of piglet intestinal epithelial cells IPEC-J2 induced by OdDHL were determined by flow cytometry,and it was found that OdDHL directly stimulated the apoptosis of IPEC-J2 cells in a concentration-dependent manner The piglet intestinal cells could sense the bacterial QS signal through population sensing and up-regulate the expression level of matrix metalloproteinase 2/9;increase their own Caspase3/8/9 activity,reduce the expression of apoptotic protein Bcl2,and increase the expression of apoptosis-related proteins Bax,Bid,Cytc,FASCD95 This demonstrated that OdDHL could induce apoptosis in pig intestinal cells through both mitochondrial and death receptor pathways,and QSI could antagonize this apoptosis-inducing process by degrading QS signals After weaned piglets were attacked,the TUNEL test confirmed the synergism between the damage of piglet intestinal barrier and the disorder of apoptosis regulation in space and time,and proved that the disorder of apoptosis regulation induced by QS signal was the main cause of the damage of piglet intestinal barrier,and QSI could antagonize the apoptosis-inducing function by degrading the population-sensing signal,thus protecting the integrity of piglet intestinal barrier.As an important component of cell membranes,lipid rafts are involved in many biological functions,which can activate apoptosis-related proteins and participate in endogenous and exogenous pathways to induce apoptosis And whether lipid rafts are involved in the apoptosis-inducing process of porcine intestinal cells IPEC-J2 by OdDHL molecules needs to be further investigated After removing cholesterol from IPEC-J2 cells and disrupting the structure of the lipid receptor,a 50%decrease in apoptosis stimulated by OdDHL was observed OdDHL was coupled with FITC green fluorescent group,and the interactions between OdDHL and porcine intestinal cells were observed qualitatively and quantitatively by fluorescence microscopy and flow cytometry,respectively,and it was found that lipid rafts mediated the adsorption of OdDHL to cells,and the number of OdDHL in adsorbed cells was significantly reduced after the removal of lipid rafts The disruption of the lipid-rich structure resulted in a significant decrease in the activity of apoptosis-related proteins Caspase3/8/9,a decrease in the expression of FASCD95,Bax,Bid,and Cytc,and an increase in the expression of anti-apoptotic Bcl2 protein OdDHL-FITC was incubated separately with many lipids associated with lipid raft structure,and the affinity of OdDHL to these lipids was observed It was found that OdDHL did not have high affinity to cholesterol,but high affinity to lipid raft mimics(sphingolipids,cholesterol,phosphorylcholine mixture),demonstrating that the lipid raft-mediated adsorption of OdDHL to cells originated from the intact lipid raft structure,rather than from OdDHL specific adsorption to cholesterol.The present study provides preliminary evidence that lipid-raft-mediated adsorption of OdDHL to porcine intestinal cells subsequently activates the mitochondrial apoptotic pathway and the death receptor apoptotic pathway,resulting in disrupted IPEC-J2 apoptosis and damaged to the intestinal barrier in pigs The above data provide a theoretical basis and a new perspective for the widespread application of QSI in the farming industry. |
PDF Full Text Request