1. TiO2-NPs mitigated TNBS-induced colitis symptoms
1.1. The General condition of animals
As shown in Figure 1A-1C, during the presensitization stage, appearance and social behavior of mice in all groups were normal, no difference in body weight changes or coefficient of feeding efficiency (CFE) was observed between groups, and animals were at slow growth status. After TNBS colorectal instillation at Day 7, we found that mice in TNBS group displayed the lowest body weight and CFE. However, the TNBS+VE group and TNBS+TiO2+VE group displayed significant increase in body weight when compared with the TNBS group at Day 8, which indicated that vitamin E and its co-administration with TiO2-NPs could mitigate colitis symptom in weight loss.
Recent clinic researches have reported the appearance of extraintestinal manifestations (EIMs) in crohn’s disease, and kidney, spleen, liver as well as biliary tract were the most frequently involved organs [24, 25]. As shown in Table 1, the organ weight coefficients of kidney and small intestine in TNBS group were significantly higher than the CT group, which indicated the potential organ injury during colitis inducement. Other experimental groups displayed significantly decreased kidney and small intestine organ weight coefficients when compared with TNBS group, which meant that the administration of TiO2-NPs and vitamin E as well as their co-administration could mitigate symptoms in organ injury. At last, no obvious difference was found in the coefficients of liver and spleen. As for pathological assessment, colitis mice in TNBS group and TNBS+TiO2 group exhibited obvious renal pathological changes shown as glomerular hyperemia and hemorrhage, which would be further improved in TNBS+VE group and TNBS+TiO2+VE group after colitis mice received vitamin E administration (Figure 2). Besides, no obvious pathological change could be observed in liver, spleen and mesenteric lymph nodes (Figure S1), which prompted the localized EIMs and no systemic inflammation was found. Especially for Jejunum, we noticed that TiO2 group and TNBS+TiO2 group displayed significantly decreased villi height and villi height / crypt depth ratio when compared with CT group and TNBS group (Figure S1 and Table S1), indicating that TiO2-NPs could disturb villi structure of heathy and colitis mice, which was also consistent with findings in our previous publications [16, 26].
1.2. Colitis activity of animals.
The DAI has been developed to assess whether or not the colitis is progressing in clinic [27]. As shown in Figure 1D, the TNBS group displayed significantly elevated DAI score after the TNBS colorectal instillation when compared with the CT group, which represented active status of disease in colitis mice. Moreover, it could be noticed that TNBS+TiO2 group, TNBS+VE group and TNBS+TiO2+VE group displayed significantly downregulated DAI scores when compared with the TNBS group from Day 8 to Day 10, though still higher than the CT group, indicating the potentially supportive mitigation of TiO2-NPs and vitamin E as well as their co-administration on colitis activity. At last, we also found that TiO2 group displayed higher DAI score than the CT group during the late experimental administration, which suggested that oral TiO2-NPs administration was potential to arouse colonic inflammation risks in heathy mice.
For further assess the mucosal injury status of colon, the gross examination was conducted to reflect macroscopic damage status. As shown in Table 2, the TNBS group displayed significant shortening of length and notably elevated colon mucosal damage index (CMDI) score when compared with CT group, furthermore, we noticed lower CMDI scores and larger colon length of TNBS+TiO2 group and TNBS+VE group than the TNBS group, which meant TiO2-NPs and vitamin E helped to mitigate the macroscopic structure damages of acute colitis. Moreover, TNBS+TiO2+VE group displayed larger colon length than the TNBS+TiO2 group, indicating that effect of TiO2-NPs could be further enhanced after the co-administration with Vitamin E. At last, we also noticed the organ weight coefficients of colon in TNBS+TiO2 group and TNBS+TiO2+VE group were significantly higher than that in TiO2 group.
We further conducted pathological investigation to reflect the mucosal injury severity. As shown in Figure 3, H&E stained sections of colonic tissue presented mild mucosal inflammatory cell infiltrates with intact epithelium in TiO2 group, which indicated epithelial inflammation risks after healthy mice received TiO2-NPs oral administration. Moreover, the TNBS group, TNBS+TiO2 and TNBS+VE group exhibited obvious inflammatory cell infiltration as well as goblet cells and crypt structure loss, but these pathological changes seemed to be recovered in TNBS+TiO2+VE group after colitis mice received co-administration of TiO2-NPs and Vitamin E. Meanwhile, according to the result of pathological score assessment and mucosal height measurement (Table 2), the TNBS group and TNBS+TiO2 group exhibited significantly higher pathological score and mucosal height than CT group and other experimental groups, which was similar to the result of gross examination, however, no significant difference could be observed between these two groups.
2. TiO2-NPs downregulated NF-κB transcription and decreased cytokine levels in colitis mice
Generally, nuclear factor κB (NF-κB) controlled transcription of inflammation genes in colonic epithelium. Classic activation of NF‐κB could be initiated by a broad panel of different stimuli, including abnormal intestinal pathogen through TLRs or colonic tissue damage from oxidative stress [6]. On activation, NF-κB was rapidly released from cytoplasmic inhibitor (inhibitor of NF-κB, IκB) with the function of IKKs, transmigrated into the nucleus, and bound to DNA response elements in gene promoter regions [28, 29]. In the present study, we detected mRNA transcription levels of the NF-κB signal pathway in the colonic tissue of animals. As shown in Figure 4 and Figure S2, oral TiO2-NPs exposure significantly increased mRNA expression of P65 (subunit of NF-κB) in healthy mice. Meanwhile, the TNBS group showed significantly increased transcription levels of TLR-2, TLR-4, IKK2 and NF-κB P65 when compared with the CT group, which suspected an active NF-κB signal transcription in colon tissue of colitis mice. However, the transcription level of TLR-2, IKK2 and NF-κB P65 significantly decreased in the TNBS+TiO2 group after colitis mice received additional TiO2-NPs administration, and the transcription level of NF-κB P65 also downregulated in the TNBS+VE group, which meant that both TiO2-NPs and vitamin E administration could downregulated the NF-κB signal transcription level in colon tissue of colitis mice.
Among the downstream signaling events of NF-κB signal pathway, the inflammatory molecules and biomarkers transcription were believed to participate in immune-mediated injury in the gut and promote the progression of IBD [6]. In the present work, we found that healthy mice in TiO2 group displayed higher TNF-α mRNA transcription level than CT group. Meanwhile, colitis mice in the TNBS group showed significantly higher IL-1β, IL-10 and TNF-α transcription level in colon tissue when compared with healthy ones in the CT group, and the transcription level of HSP-90 also significantly decreased at the same time. Moreover, we noticed that the elevated IL-1β and TNF-α transcription level in TNBS group could be downregulated after colitis mice received TiO2-NPs administration, and the transcription level of anti-inflammatory cytokine IL-10 could also be further upregulated, indicating that TiO2-NPs administration could participate in reducing inflammation level of TNBS-induced acute colitis.
We also detected the expression level of typical inflammatory factors including TNF-α and IL-12 by Enzyme-linked immunosorbent assay (ELISA). As shown in Table 3, colitis mice in the TNBS group showed significantly higher TNF-α secretion level in colon tissue when compared with the CT group, and the TNBS+TiO2 group, TNBS+VE as well as TNBS+TiO2+VE group further displayed lower TNF-α level than the TNBS group, which was consistent with changes of TNF-α mRNA transcription level. Besides, the serum TNF-α and IL-12 contents were also detected to measure systemic inflammation levels of animals, no significant change was found in serum contents of cytokines in the TNBS group when compared with the CT group, which meant the localized inflammation after acute colitis inducement. However, we noticed that TiO2 group showed higher serum IL-12 level when compared with the CT group, which prompted TiO2-NPs administration could induce potential inflammation risk in healthy mice.
3 TiO2-NPs triggered oxidative stress in colitis mice.
The body has antioxidant defenses mainly consisted of intracellular enzymatic antioxidants and nonenzymatic antioxidant glutathione to counteract the effects caused by ROS, and the uncontrolled oxidative stress was destructive to the GI tract and has been proved as an important pathogenic mechanism of IBD [3, 5]. As shown in Table 4, we found that the glutathione peroxidase (GSH-px) vitality, T-GSH, oxidized glutathione (GSSG) as well as GSH content, but not the GSH / GSSG ratio, were significantly decreased in colon tissue of TiO2 group after healthy mice received TiO2-NPs administration. Meanwhile, the significant ROS accumulation and decreased GSH-px vitality, GSH, T-GSH content and GSH/GSSG ratio could be observed in the TNBS group when compared with the CT group, indicating severe oxidative stress in colon tissue of colitis mice. Furthermore, the GSH and T-GSH content in TNBS+TiO2 group was lower than those in TNBS group, which meant that TiO2-NPs administration were potential to increase the antioxidant consumption and aggravate oxidative stress status in colon tissue of colitis mice. At last, the TNBS+VE group and TNBS+TiO2+VE group displayed significant decreased ROS accumulation in colon tissue when compared with colitis mice in TNBS group, but the GSH content and GSSG / GSH ratio were not obviously changed, which was in consistent with vitamin E’s capability to scavenge oxygen free radical and prompted its effect on balancing oxidative stress in TNBS-induced acute colitis [30].
On the other hand, we also detected the GSH-px vitality and glutathione content in serum to reflect the systemic oxidative stress status of animals. As shown in Table 5, except for the significantly decreased serum GSH-px vitality in the TNBS+TiO2 group when compared with CT group and other experimental groups, no significant change could be found in remaining indicators, which meant that acute colitis inducement would not lead to systemic oxidative stress in animals, and TiO2 administration was suspected to induce systemic oxidative stress risks in colitis mice.
4. TiO2-NPs repaired gut dysbacteriosis of colitis mice
4.1. Richness and evenness of gut microbiota in mice
Previous studies have pointed out that compositional and metabolic changes in the gut microbiota were major driver of IBD [31], thus microbial community profiles were created by clustering 16S rRNA sequences into operational taxonomic units. The rarefaction and rank abundance curves exhibited the plateau and indicated the vast majority of bacterial species in the samples had been covered (Figure S3). As shown in Table 6, we observed significant increase of the ACE and Chao1 richness estimator in the TNBS group when compared with the CT group, which indicated the imbalance of gut microbiome richness in colitis mice. Moreover, further downregulation of Chao1 index could be found in the TNBS+TiO2 group than TNBS group after colitis mice received administration of TiO2-NPs, indicating that imbalance of gut microbiome richness in colitis mice would be partly repaired by TiO2-NPs. Similar changes could also be observed on other richness estimators like Shannon, Simpson, observed species and PD whole tree index, though without significance.
Venn’s diagram in Figure S4 showed that all of the groups shared the compositional overlap of 377 core microbiota, these overlapping phylotypes contributed to majority of the CT group and TiO2 group, which shared proportion of 99.4% and 98.4% respectively. Meanwhile, more unique phylotypes were observed in colitis mice, among which the TNBS+TiO2 group displayed the highest proportion of 10.8%. As shown in Figure 5A, the histogram generated by clustering analysis at phylum level divided the mouse feces samples into four major groups, the CT group, the TiO2 group and TNBS group, the TNBS+VE and TNBS+TiO2+VE group, as well as the TNBS+TiO2 group. Noticeably, Firmicutes predominated in feces samples of the TiO2 group and TNBS group, while Bacteroidetes mainly distribute in faecal of other groups. And it could also be observed that the relative abundance of Proteobacteria in the TNBS+TiO2 group was higher when compared with other experimental groups. The Non-metric multidimensional scaling (NMDS) analysis showed that the samples of different groups were distinguished clearly (Figure 5B, stress = 0.191 < 0.200), suggesting a diverse gut microbiota composition among groups. Meanwhile, the results of 3D-principal component analysis (PCA) on evenness (Figure 5C) also showed significantly different microbial composition among groups (Amova, p <0.001). Compared with CT group, the obvious dissimilarities of gut microbiota composition were found in TNBS group, TNBS+VE and TNBS+TiO2+VE group. And after colitis mice received the single administration of TiO2-NPs or the co-administration of TiO2-NPs and vitamin E, obvious separations of gut microbiota composition could be further observed in the TNBS+TiO2 group and TNBS+TiO2+VE group when compared with the TNBS group.
4.2. Microbial phylotype composition in mice
The classification of specific taxonomy groups of species was conducted and shown as Figure 6, Firmicute (27.50%), Bacteroidetes (16.09%), Proteobacteria (7.13%) and Deferribacteres (0.91%) were four major phylum in feces samples of mice. Meanwhile, Proteobacteria phylum was primarily composed of Gammaproteobacteria-Stenotrophomonas strain (7.13%), Deferribacteres phylum mainly consisted the Deferribacteres-Mucispirillum strain (0.91%), and the TNBS+TiO2 group shared the highest relative abundance proportion of these two strains. Moreover, Firmicutes phylum was primarily composed of Lactobacillaceae (15.69%), Enterococcaceae (1.32%), Ruminococcaceae (8.22%) and Lachnospiraceae (2.27%) at the family level, and it could be noticed that the TiO2 group, TNBS group as well as the TNBS+TiO2+VE group shared high relative abundance proportion of the first three taxonomy, respectively (Table S2). Then for other taxonomy at different levels, the classification of bacteria tended to be dispersed in groups and it would be hard to determine the dominant.
Furthermore, Ternary plot analysis was conducted to determine the predominant taxonomy among groups at the classification from phylum to genus. As shown in Figure 7, the Ruminococcaceae-Clostridium_papyrosolvens strain, Enterococcus_faecalis, Bacteroides_acidifaciens and Lachnospiraceae_bacterium_A4 from family to genus level dominantly distributed in the TNBS group when compared with the CT group. The Proteobacteria-Stenotrophomonas strain, Mucispirillum and Mucispirillum_schaedleri from phylum to species level dominantly distributed in the TNBS+TiO2 group when compared with the CT group and TNBS group. These results indicating the dominance of pathogenic flora in colitis mice and intervention effect of TiO2-NPs on phylotype composition of colitis mice. Moreover, we noticed that the co-administration with vitamin E would not markedly change the effect of TiO2-NPs on phylotype composition of colitis mice, except for the dominance of Ruminococcaceae-Clostridium_papyrosolvens stain in the TNBS+TiO2+VE group when compared with the TNBS+TiO2 group (Figure S5).
4.3. Gut-associated functional category of gut microbiota in mice
It was known that changes in gut microbiota structure could affect the gut-associated function by providing modifications to gut microbiota that would affect molecule, enzyme, metabolism, biosynthesis and signal pathway. Thus the Tax4Fun analysis as well as LDA effect size (LEfSe) analysis were used to predict the functional profiles of gut microbiota based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. When compared with the CT group, we observed that functional category “ovarian_Steroidogenesis” and “Secretion_systerm” were significantly enriched in the TiO2 group, while “Enzyme_families”, “amino_sugar_and_nucleotide_ sugar_metabolism” and “lipid metabolism” were also diminished at the same time (Figure 8A). Moreover, enhanced amino acid biosynthesis function (including “Arginine_biosynthesis”, “Phenylalanine_tyrosine_and_tryptophan_biosynthesis” and “Valine_leucine_and_isoleucine_biosynthesis”), “Butanoate_metabolism”, “Protein_folding_and_associated_processing”, “Methane_metabolism”, “Nitrotoluene_degradation” and “AMPK_signaling_pathway” could be observed in the TNBS group when compared with the CT group, but functional category “Galactose_metabolism” were diminished (Figure 8B and 8C). Meanwhile, oral TiO2-NPs administration significantly enriched the category “Phenylalanine_metabolism” of colitis mice in TNBS+TiO2 group (Figure 8B), while vitamin E administration enriched the category “Pathogenic_Escherichia_coli_infection” in TNBS+VE group (Figure 8C). At last, no functional category was significantly changed after the co-administration of TiO2-NPs and vitamin E.