The Regulation Mechanism Of GPR109A On Intestinal Epithelial Barrier Integrity In CLP Mice

Intestinal epithelium is not only the leading place to absorption of nutrients,but also plays a critical barrier that protects against potentially harmful microbiota and pathogens.Under normal physiological state,the tight junctions,adherens junctions and desmosome is complete and stable of dynamic balance between adjacent cells,which form an effective barrier function.However,some pathological stimulus damage impair the integrity of the intestinal epithelial barrier and increase the intestinal permeability,prompting the intestinal contents and intestinal microbiota enter into the body,then leading to the multiple organ failure.Cecum ligation and puncture?CLP?is the most classical experiments to build the sepsis model of rodents,and is known as the"gold standard"model of sepsis.Previous study indicated that the integrity of intestinal barrier is broken during sepsis.Therefore,in this study,GPR109A^-/-and WT mice which were on a C57BL/6 background were underwent CLP to build sepsis model,then investigated the effects and mechanism of GPR109A on intestinal barrier in sepsis model.The results showed that the pathogenic manifestations in GPR109A^-/-mice were more serious than those in WT mice.These included more rapid loss of body weight,greater changes in body temperature,more severe clinical scores,lower survival rate,and more severe inflammatory response.The results indicated that GPR109A has a protective effects against CLP-induced sepsis model.We next measured the integrity of intestinal barrier in sepsis model and found that the serum of GPR109A^-/-mice had more FITC-dextran.The results indicated that the intestinal permeability is higher in GPR109A^-/-mice.The histoimmunofluorescence results showed that GPR109A^-/-mice had less MUC2 protein expression than WT mice.In this study,the tight junctions including claudin-1?Cldn1?,claudin-2?Cldn2?,Zo1,Zo2,and occludin?Ocln?in colonic tissue of WT mice were higher than GPR109A^-/-mice.The results of ileac tissue examination were consistent with that of colonic tissue.All the results indicated GPR109A provide a protective effect on the integrity of intestinal barrier during sepsis.In recent years,GPR109A was combined with metabolin of intestinal microbiota.Therefore,next,we measured the composition of intestinal microbiota in WT and GPR109A^-/-mice and found that there was a significant difference in the composition of intestinal microbiota between WT and GPR109A-/-mice,indicating that the GPR109A itself had the function of regulating the composition of intestinal microbiota.After CLP surgery,the intestinal microbiota in WT and GPR109A^-/-mice violently changed,suggesting that CLP surgery seriously disrupted the structure of intestinal microbiota in mice.To investigate the potential role of the microbiota in the sepsis model,we examined the effects of CLP surgery in mice with their microbiota removed via antibiotics.Surprisingly,all the experimental mice died within 48 h,suggesting that the gut microbiota is crucial for survival in the CLP-induced sepsis model.To further investigate the role of the gut microbiota in the CLP-induced sepsis model,we transplanted the intestinal microbiota of WT and GPR109A^-/-mice into each other and performed CLP surgery.The results showed that,transplanted intestinal microbiota of WT into the GPR109A^-/-mice dramatically improved the weight loss,the temperature change,clinical score,survival rate,and inflammatory response.The FITC-dextran content in serum of GPR109A^-/-mice is same as that in WT mice,indicating that there is no difference between WT and GPR109A^-/-mice intestinal peremeability.There were no difference MUC2 protein and tight junctions Cldn1,Cldn2,Cldn3,Zo1,Zo2 and Ocln of colonic tissue and ileac tissue in WT and GPR109A^-/-mice.In summary,GPR109A regulating the composition of intestinal microbiota which inhibiting the inflammatory response and maintains the intestinal epithelium barrier integrity in CLP-induced sepsis.