我们进行了两个实验,以使用体外跨上皮电阻(TEER)测定法评估新型基于细菌的直接饲喂微生物(DFM)对肠屏障完整性的影响。在实验1中,人类来源的Caco-2细胞以1×108CFU/transwell的速率接受或不接受(CON)含有动物小杆菌(以前的乳杆菌)506,Freudenrechiii507,副衣芽孢杆菌809和枯草芽孢杆菌597(BDP;BOVAMINEDEFEND®Plus)的DFM。与治疗应用(CON或BDP)同时进行,以2.8×107CFU/transwell的速率以2×2阶乘排列单独添加(PAT)或与BDP(PATBDP)一起添加A型产气荚膜梭菌的致病性攻击。在实验2中,Caco-2细胞也以2×2阶乘设计分配给CON或BDP,然后,治疗后两小时(CON和BDP),肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)的混合物单独(CYT)或与BDP(CYT+BDP)以10:1的比例加入,分别。在这两个实验中,TEER测量18小时。在实验1中,观察到TEER的DFM×病原体×小时相互作用(P<0.0001)。单独添加PAT最初倾向于增加TEER与CON从1.1到2.2h(P≤0.09),TEER在3.2h时增加(P<0.01),但TEER从5.4降低到实验结束时18.4h(P≤0.01)。另一方面,添加DFM,不管有没有致病的挑战,产生了更大的TEERvs.CON-CON和CON-PAT在大部分实验期间(P≤0.04)。在实验2中检测并报道了类似的相互作用(P<0.0001)。与所有其他治疗相比,从3.2小时到研究的剩余时间,CYT攻击降低了平均TEER(P≤0.03)。另一方面,与CON-CON相比,BDP-CYT能够在整个实验期间保持上皮细胞的完整性(P≤0.03),例外情况为3.2h(P=0.20)。此外,BDP-CON与CON-CON相比,TEER从3.2h增加到18.4h(P≤0.04),但与BDP-CYT相比,DFM和攻击后4.3至18.4h进入细胞。总之,产气荚膜梭菌A型和促炎细胞因子混合物在体外损害肠上皮细胞单层的完整性,而添加基于多物种细菌的DFM抵消了这些破坏性影响。
We conducted two experiments to evaluate the effects of a novel bacterial-based direct-fed microbial (DFM) on intestinal barrier integrity using the in vitro transepithelial electrical resistance (TEER) assay. In experiment 1, human-derived Caco-2 cells received or not (CON) a DFM containing Ligilactobacillus (formerly Lactobacillus) animalis 506, Propionibacterium freudenreichii 507, Bacillus paralicheniformis 809, and B. subtilis 597 (BDP; BOVAMINE DEFEND® Plus) at a rate of 1 × 108 CFU/transwell. Concurrently with treatment application (CON or BDP), a pathogenic challenge of Clostridium perfringens type A was added alone (PAT) or with BDP (PAT + BDP) at a rate of 2.8 × 107 CFU/transwell in a 2 × 2 factorial arrangement. In experiment 2, Caco-2 cells were also assigned in a 2 × 2 factorial design to CON or BDP and then, 2 h post-treatment administration (CON and BDP), a mixture of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) was added alone (CYT) or with BDP (CYT + BDP) at a 10:1 ratio, respectively. In both experiments, TEER was measured for 18 h. In experiment 1, a DFM × pathogen × hour interaction was observed for TEER (P < 0.0001). Adding the PAT alone initially tended to increase TEER vs. CON from 1.1 to 2.2 h (P ≤ 0.09), increased TEER at 3.2 h (P < 0.01), but reduced TEER from 5.4 to the end of the experimental period at 18.4 h (P ≤ 0.01). On the other hand, adding DFM, with or without the pathogenic challenge, yielded greater TEER vs. CON-CON and CON-PAT for most of the experimental period (P ≤ 0.04). A similar interaction was detected and reported in experiment 2 (P < 0.0001). The CYT challenge reduced mean TEER compared with all other treatments from 3.2 h to the remainder of the study (P ≤ 0.03). On the other hand, BDP-CYT was able to maintain the integrity of the epithelial cells when compared with CON-CON throughout the experimental period (P ≤ 0.03), the exception being at 3.2 h (P = 0.20). Moreover, BDP-CON increased (P ≤ 0.04) TEER when compared with CON-CON from 3.2 to 18.4 h, but also in comparison with BDP-CYT from 4.3 to 18.4 h post-DFM and challenge administration into the cells. In summary, C. perfringens type A and a pro-inflammatory cytokine cocktail compromised the integrity of intestinal epithelial cell monolayers in vitro, whereas adding a multispecies bacteria-based DFM counteracted these damaging effects.
Two experiments were designed to evaluate the effects of adding a bacterial-based direct-fed microbial (DFM) containing Lactobacillus animalis 506, Propionibacterium freudenreichii 507, Bacillus paralicheniformis 809, and Bacillus subtilis 597 on the integrity of intestinal epithelial cells challenged with Clostridium perfringens type A or a pro-inflammatory cytokine cocktail. Regardless of the challenge, the addition of the DFM maintained the integrity of the intestinal epithelial cells in vitro. These results help to elucidate the potential beneficial effects that the bacterial-based DFM containing L. animalis 506, P. freudenreichii 507, B. paralicheniformis 809, and B. subtilis 597 may bring to livestock species.