由困難梭狀桿菌釋放的毒素所引起的腸道滲透性增加是導致困難梭狀桿菌感染時嚴重腹瀉的主因。柔嫩梭狀桿菌為健康成人腸道中佔比最多的菌種之一，並且已發現具有抗發炎以及恢復腸道屏障的能力。但柔嫩梭狀桿菌對於在困難梭狀桿菌感染下的腸道屏障功能有甚麼樣的影響還未知。有研究指出在感染症中使用益生菌所分泌出含有其代謝物的胞外體(EVs)可以減緩症狀。因此我們主要的研究方向是包含柔嫩梭狀桿菌代謝物的上清液或是胞外體是否能夠恢復困難梭狀桿菌毒素影響腸道屏障的功能。首先利用粒徑篩層析法來萃取嫩梭狀桿菌分泌之胞外體，並看到胞外體能夠與細胞株結合。並且發現柔嫩梭狀桿菌代謝物不只能恢復困難梭狀桿菌感染造成的緊密連接蛋白減少也能夠維持蛋白間連接，也可觀察到細胞間屏障功能的恢復。我們更進一步觀察柔嫩梭狀桿菌所影響的路徑，可以發現到其代謝物能夠抑制困難梭狀菌毒素的功能，並且也有活化能夠調控緊密連接蛋白Claudin-3表現的過氧化物酶體增殖物活化受體γ(PPARγ)的效果。最後在動物實驗結果中能夠看到柔嫩梭狀桿菌可以減輕CDI小鼠的症狀: 腹瀉、體重減輕以及大腸縮短，存活率也提高。綜合以上結果可以看出含有柔嫩縮狀桿菌代謝物的上清液以及胞外體可透過抑制毒素以及藉由活化PPARγ來恢復腸道屏障的機制來保護困難梭狀桿菌感染時腸道的屏障，因此達到減輕小鼠症狀的效果，這項研究顯示了柔嫩梭狀桿菌在未來能夠用於治療或輔助治療CDI的可能性。

Clostridium difficile-induced diarrhea have been associated with increased intestinal permeability which mediated by toxins. Faecalibacterium prausnitzii (F. prausnitzii) is a dominant member of human intestinal microbiota of healthy adults and its metabolites are suggested to have anti-inflammatory effects and restore the intestinal barrier function under certain disease conditions. However, the relationship between C. difficile infection (CDI) and F. prausnitzii has not been investigated. Probiotics have been shown to be effective against infectious diseases by probiotic-derived extracellular vesicles (EVs), which containing beneficial metabolites. Here, we proposed that the intestinal barrier function disturbed by C. difficile toxins will be resuscitated by the administration of F. prausnitzii metabolites, including cultured supernatant and derived-EVs. First, we purified F. prausnitzii-derived EVs from FPsup by size-exclusion chromatography and demonstrated F. prausnitzii-derived EVs can be internalized into colonic cells. To assess tight junction integrity of colonic cells, we found F. prausnitzii metabolites were able to restore tight junction integrity after C. difficile infection not only in tight-junction protein levels but also anatomic location, as well as cell adhesion or tight junction function. Further, we found F. prausnitzii metabolites protected C. difficile disrupted tight junction through the blockage of glucotransferase activity of C. difficile toxins. Meanwhile, the protection ability was associated with the activation of PPARγ, a nuclear receptor that can regulate claudin-3 tight junction protein. Gavage with F. prausnitzii metabolites in C. difficile infected mice resulted in a quick recovery of diarrhea, weight loss and extensive colonic shortening. Taken together, F. prausnitzii metabolites, including EVs, enhanced the intestinal barrier function by accelerating PPARγ regulated paracellular permeability, and blockade the effects of C. difficile toxins which attenuating the severity of C. difficile-induced colitis in mice. Therefore, our results suggest additional F. prausnitzii metabolites may blockade C. difficile toxins and restore the CDI-induced aberrant intestinal barrier structure and function through PPARγ activation, which pave a new therapeutic strategy for CDI in the future.

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