Tight junctions were altered during NEC pathogenesis.
To determine whether the intestinal epithelial barrier of NEC patients is altered, we first employed immunohistochemistry (IHC) to investigate the expression of tight junction components (claudin-1, occludin, and ZO-1) in patient tissues. Compared with the control group, which consisted of patients with congenital intestinal atresia, the NEC patients showed significantly decreased expression of occludin (Fig. 1a, b). Similar results were also observed in the neonatal mouse model of NEC. The expression of both claudin-1 and occludin significantly decreased in the small intestinal epithelial cells of NEC mice (Fig. 1c, d). Consistent with this result, a significant increase in serum FITC-dextran 4 kDa (FD4), which is a marker of gut permeability in vivo, was observed in NEC mice (Fig. 1e). Taken together, these results suggested that the intestinal barrier was impaired during NEC pathogenesis.
Probiotics lessened the severity of NEC and enhanced intestinal barrier function.
Since changes in the intestinal microbial community have already been observed in NEC patients and probiotics may relieve NEC injury by regulating intestinal flora8, we investigated whether the probiotic mixture used in this study could alleviate intestinal lesions in NEC mice. This probiotic mixture has been recommended in the clinic for the treatment of children with intestinal dysfunction but not for NEC patients. As the results showed, oral gavage of probiotics was found to effectively prevent weight loss in NEC mice, although the survival rate was not improved significantly (Fig. 2a, b, c). Hematoxylin and eosin staining of the terminal ileum, as well as histopathological scores, demonstrated a significant reduction in histological injury in probiotic-treated NEC mice (Fig. 2d, e). Furthermore, the probiotic mixture also significantly suppressed the expression of Tnf-α, Il-1β, and Il-6 in terminal ileac tissues, which were elevated in NEC patients20 (Fig. 2f). These results indicated that the probiotic mixture could ameliorate intestinal damage during NEC.
As the probiotic mixture could protect the intestine from NEC damage, we further examined whether the intestinal barrier and its functions were also restored. Two components of tight junctions, claudin-1 and occludin, which were reduced in NEC mice, were preserved in probiotic-treated mice (Fig. 3a, b). Consistent with this finding, similar results were confirmed by indirect immunofluorescence in the terminal ileum (Fig. 3c). We also employed transmission electron microscopy (TEM) to examine the ultrastructure of the cell-cell junctional complex in the terminal ileum. The images showed that the intercellular space in probiotic-treated mice exhibited less penetration of the electrodense dye ruthenium red than that in PBS-treated mice, suggesting a more complete structure of tight junctions (Fig. 3d, white arrows). To further confirm the improvement of barrier functions, gut permeability in vivo was assessed by measuring serum FD4 concentrations. Unsurprisingly, compared with PBS-treated mice, FD4 levels were reduced significantly in probiotic-treated mice, indicating that intestinal permeability was improved (Fig. 3e). Meanwhile, agar plates incubated with splenic lysates from NEC mice showed lower numbers of colony-forming units for probiotic-treated mice, further confirming that the probiotic mixture decreased gut permeability and prevented bacterial translocation to extraintestinal organs (Fig. 3f). Taken together, these results suggested that the probiotic mixture could enhance tight junction functions and prevent bacterial translocation.
Probiotics remodeled the microbiota composition of the small intestine.
In addition to enhancing intestinal barrier functions, the probiotic mixture might also remodel the microbiota composition of the gut, which might be directly or indirectly beneficial for the intestinal barrier21,22. To confirm this possibility, the composition and diversity of the microbiota in the small intestine were analyzed by 16S rRNA sequencing. As shown by the unclustering and abundance of the 30 most abundant operational taxonomic units (OTUs), the patterns of the NEC + PBS and NEC + probiotic groups were different from those of the control (Fig. 4a). To further characterize the bacterial community, we assessed the relative abundance at the phylum level. We found that the NEC + probiotic and control groups showed a similar relative abundance of Proteobacteria and Firmicutes which, on average, accounted for nearly 100% of all reads (Fig. 4b). The NEC + PBS group contained fewer Firmicutes and more Proteobacteria, which is consistent with the phenomena observed in NEC patients8. Further analysis of bacterial diversity by the Shannon index revealed the highest bacterial diversity in the NEC + probiotic group compared with the other groups, although no significant difference was observed among the three groups by calculation with the Chao 1 index (Fig. 4c, d). Both principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) demonstrated an apparent separation of the three groups, suggesting that they had different compositions of intestinal microbiota (Fig. 4e, f). The analysis of LEfSe, which is employed to identify the key bacterium affecting the discrimination between groups, identified 21 discriminatory taxa as key discriminants with an LDA score higher than 3.0 (Fig. 4g, h). The phyla Proteobacteria and class Gammaproteobacteria showed especially high LDA scores in the NEC + PBS group. Importantly, in the probiotic-treated group, the differentially abundant strains primarily included the genus Bifidobacterium, family Bifidobacteriaceae, order Lactobacillales, phyla Firmicutes, genus Enterococcus, and class Bacilli (Fig. 4g, h), and most of them were the components of the probiotic mixture that we used. Taken together, these results suggested that the probiotics colonized steadily in the small intestine of newborn mice and remodeled the microbiota community in NEC mice.
Probiotics induced PXR activation.
Since PXR has been demonstrated to play an important role in protecting the barrier in intestinal or liver disease17,23, we investigated whether PXR also plays a role in NEC pathogenesis. As shown in Fig. 5a and 5b, the expression of PXR was decreased significantly in NEC patients. Meanwhile, a reduction in PXR expression was also observed in the intestinal epithelial cells of NEC mice (Fig. 5c, d). Importantly, when mice were treated with a probiotic mixture during NEC modeling, the expression of PXR was significantly enhanced (Fig. 5e, f), and the transcription levels of PXR target genes, such as Cyp3a11 and Mdr1a, were also increased (Fig. 5g). These results indicated that the probiotic mixture might be an activator of PXR, which might help to relieve the pathological injury related to NEC.
Activation of PXR improved barrier function in Caco-2 cells.
To further confirm the role of PXR in enhancing barrier function and reveal its mechanism, we investigated it in Caco-2, a human-derived intestinal epithelial cell line that can be used to simulate the barrier in vitro24. To model this function, Caco-2 cells were seeded in Transwell inserts and cultured to form monolayer cells for the permeability assay. The results showed that transepithelial electrical resistance (TEER) decreased significantly when monolayer cells were treated with LPS at 5 or 10 µg/ml for 48 h (Fig. 6a), suggesting damage to the monolayer by LPS. Meanwhile, we measured changes in tight junction proteins. The results of this experiment showed that the expression of occludin was markedly depressed by LPS treatment at 5 µg/ml for 48 h (Fig. 6b, c). Meanwhile, we found that LPS did not have an effect on the PXR expression (Fig. 6d). To further investigate the role of PXR in regulation of the intestinal barrier, we established a stable PXR overexpression cell line (Fig. 6e). Interestingly, when Caco-2 cells overexpressed PXR, claudin-1 expression was elevated significantly, both in the absence and presence of LPS. Furthermore, PXR overexpression attenuated the reduction in occludin induced by LPS (Fig. 6f, g). Consistent with this finding, FD4 flux, an indicator of epithelial paracellular permeability to uncharged macromolecules, did not increase when the PXR-overexpressing Caco-2 monolayers were treated with LPS (Fig. 6h). Taken together, these results suggested that PXR could enhance barrier function by regulating the tight junction complex.
PXR could inhibit the phosphorylation of JNK.
As the MAPK pathway could affect tight junctions and cause dysfunction of the intestinal barrier23,25,26, we further examined whether PXR could regulate MAPK pathway signaling. After Caco-2 cells were treated with LPS at 5 µg/ml for 30 min, we observed increased phosphorylation of Erk and p38 and a notable increase in phospho-JNK levels (Fig. 7a, b, c). Interestingly, when Caco-2 cells overexpressed PXR, LPS treatment could not induce the phosphorylation of JNK, as shown by the results of flow cytometry and Western blot analysis (Fig. 7d, e, f, g). Furthermore, we found that the phosphorylation of JNK was inhibited in the terminal ileum when NEC mice were treated with probiotics (Fig. 7h), which caused PXR to be significantly enhanced (Fig. 5e, f). Taken together, these results indicated that PXR could inhibit the phosphorylation of JNK and thus protect the barrier.