Oral, but not rectal, administration of EGCG alleviates DSS-induced colitis
To study the impact of EGCG on alleviation of DSS-induced colitis, an experimental colitis was induced in mice by administering 2.5% DSS in water continuously for seven days, followed by three days of daily oral (Fig. 1A) or rectal delivery (Fig. S1A) of 50 mg/kg body weight EGCG, as the optimal dose we have explored before (data not shown). To explore how oral EGCG or enteric delivery EGCG attenuates DSS-induced colitis, the symptoms of mice treated by two different exposure ways were measured. Oral administration of EGCG significantly alleviated colitis as measured by disease activity index (DAI, the combined score of body weight loss, stool consistency, and rectal bleeding [23]) (Fig. 1B), body weight loss (Fig. 1C), and colon length (Fig. 1D and 1E). However, rectal delivery of EGCG failed to improve any of the measurements above (Fig. S1B, S1C, S1D, and S1E). Histological analysis further showed obvious attenuation of inflammatory cell infiltration and mucosal damage and overall histology score in the colon in response to oral (Fig. 1F and 1G), but not rectal, administration of EGCG (Fig. S1F and S1G). Remarkably, during the whole experiment period, one mouse from DSS + Oral-PBS group was found dead on the tenth day, and one mouse from DSS + Rectal-PBS group and DSS + Rectal-EGCG group respectively were found dead on the ninth day.
To further assess the impact of EGCG on systemic and intestinal inflammatory response, myeloperoxidase (MPO) and pro-inflammatory cytokines in the plasma and colonic tissues were measured. Plasma concentrations of IL-1β (Fig. 1H), IL-6 (Fig. 1I), IL-8 (Fig. 1J), and TNF-α (Fig. 1K) were significantly decreased in DSS-treated mice in response to oral EGCG. In contrast, rectal EGCG failed to suppress the levels of any cytokine in the plasma (Fig. S1H, S1I, S1J, and S1K). MPO production is often increased in the colon of IBDs patients [24]. Similarly, DSS drastically elevated the MPO concentration in the colon of mice, but such an elevation was significantly dampened by oral EGCG (Fig. 1L). Furthermore, both oral and rectal EGCG significantly decreased the IL-6 level in the colon (Fig. 1M and S1M) with TNF-α showing a strong tendency to decrease as well (Fig. 1N and S1N). However, rectal administration of EGCG failed to suppress the levels of MPO (Fig. S1L) in the colon of DSS-treated mice.
Collectively, these results indicated that clinical colitis symptoms and colonic damage were ameliorated by oral, but not rectal administration of EGCG.
Oral administration of EGCG suppresses DSS-induced oxidative stress of the intestinal mucosa and restores barrier function
To assess the influence of EGCG on oxidative stress, total antioxidant capacity (T-AOC), total superoxide dismutases (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in the plasma and colon were measured. DSS significantly suppressed the T-SOD and CAT concentrations in the plasma, which, however, were largely restored by oral administration of EGCG (Fig. 2A and 2B). On the other hand, the MDA level in the plasma was significantly increased by DSS, but returned to normal by EGCG (Fig. 2C). Moreover, increased MDA in the plasma was also returned to normal by rectal EGCG (Fig. S2C). However, DSS-mediated suppression of the T-SOD and CAT concentrations in the plasma were not obviously impacted by rectal EGCG (Fig. S2). In the colon, T-AOC and T-SOD were significantly decreased by DSS, but restored to normal by oral EGCG (Fig. 2D and 2E). No significant differences in the levels of CAT (Fig. 2F) or GSH-px (Fig. 2G) were observed in the colon in response to DSS with or without oral EGCG. Colonic production of MDA was significantly increased by DSS, but reduced to normal by EGCG (Fig. 2H). However, the effect of DSS on T-AOC (Fig. S2D), T-SOD (Fig. S2E) or MDA (Fig. S2H) was not obviously impacted by rectal EGCG. Of note, the level of GSH-px in the colon was significantly increased by rectal EGCG even compared with the colon of the normal group (Fig. S2G). Furthermore, TUNEL assay revealed that DSS caused a significant increase in apoptotic in the colonic mucosa; however, both oral and rectal EGCG had no obvious beneficial effect on reducing apoptosis (Fig. 2I, 2K, S2I and S2K). To assess the effect of oral or rectal EGCG on the colonic mucosal barrier function, we counted mucin-secreting goblet cells in the colonic epithelia using Alcian blue staining. DSS significantly reduced the frequency of goblet cells and the thickness of colonic epithelial mucosa, and desirably, oral EGCG restored such damping to normal (Fig. 2J and 2L). Consistently, oral EGCG largely reversed DSS disruption of the fine structure of the brush border and tight junctions as revealed by transmission electron microscopy (TEM) (Fig. 2M). However, no obvious difference in the frequency of goblet cells or the thickness of colonic epithelial mucosa and fine structure was observed in response to DSS with or without rectal EGCG (Fig. S2J, S2L and S2M).
These results suggested that oral, but not rectal, EGCG suppressed DSS-induced oxidative stress, regulated the cell apoptosis of colonic epithelium, and restored mucosal barrier function.
Oral administration of EGCG alters the gut microbiota composition and metabolism
To further explore the impact of EGCG on the gut microbiota composition, 16S rDNA gene sequencing was performed with the fecal samples of DSS-treated mice receiving oral or rectal administration of EGCG. Neither oral (Fig. 3A) nor rectal EGCG (Fig. S3A) had a significant influence on richness of the fecal microbiota as indicated by the Sobs index. However, principal co-ordinates analysis (PCoA) showed a clear separation in the gut microbiota of DSS-treated mice between the DSS + Oral-EGCG group and DSS + Oral-PBS group (R = 0.1684, P = 0.0360) (Fig. 3B), indicating that the gut microbiota structure was significantly impacted by oral EGCG. The fecal microbiota structure was also partly influenced by rectal EGCG (R = 0.2177, P = 0.0110) (Fig. S3B).
To identify differentially enriched fecal bacterial taxa in DSS-treated mice in response to oral or rectal administration of EGCG, we explored the overall fecal bacterial composition of mice at the phylum and genus level by barplot and further performed differentia bacteria by LEfSe analysis. At the phylum level, Bacteroidetes, Firmicutes, and Proteobacteria were predominant phyla in the fecal microbiota (Fig. 3C and Fig. S3C). At the genus level, the fecal microbiota was dominated by Bacteroids, Lactobacillus, Escherichia-Shigella (Fig. 3D and Fig. S3D). Additionally, we found that two members of Akkermansia and UBA1819 were enriched by oral EGCG, while fourteen other taxa were enriched in the DSS only group (Fig. 3E). Seven bacterial taxa such as two members of Alloprevotella and Actinobacillus were particularly abundant in response to rectal EGCG, while eight other taxa such as members of Lactobacillus, Turicibacter, and Romboutsia were enriched in the DSS only group (Fig. S3E). Moreover, to assess how oral and rectal EGCG impacted the function of gut microbiota, we performed PICRUSt and revealed that multiple KEGG pathways such as metabolism of amino acids, energy, and lipids as well as membrane transport were enriched in response to oral EGCG administration (Fig. 3F and S3F).
To further explore the effect of oral or rectal EGCG on the synthesis of SCFAs, we measured the fecal concentrations of acetate, propionate, and butyrate. All three SCFAs were significantly diminished by DSS, but the productions of propionate and butyrate were significantly restored by oral (Fig. 2H and 2I), but not rectal (Fig. S2H and S2I), administration of EGCG. In fact, butyrate production was further significantly reduced in DSS-treated mice in response to rectal EGCG (Fig. S2I).
Alteration of the gut microbiota and increased production of SCFAs occurred in response to oral, but not rectal EGCG administration suggested that gut microbiota might play a critical role in alleviating DSS-induced colitis.
Correlation between the gut microbiota and anti-oxidative and inflammatory parameters and SCFAs profiles
Spearman correlation analysis was further performed to understand the association between differentially enriched microbes and anti-oxidative, inflammatory parameters or SCFAs profiles. Correlation heatmap revealed that Akkermansia had a strong positive correlation (P < 0.05) with the T-AOC level in the colon and the butyrate level in the feces, but a significant negative correlation (P < 0.05) with IL-6 in the colon and IL-8 in the plasma (Fig. 4). Meanwhile, it revealed that UBA1819 showed a significantly positive correlation (P < 0.05) with the levels of T-SOD and CAT in the plasma and the production of propionate and butyrate. A significantly negative correlation (P < 0.05) between UBA1819 and the levels of IL-1β and IL-6 in the plasma and the levels of IL-6 and MDA in the colon was also observed (Fig. 4).
Above all, a strong correlation between the differentia microbiotas such as Akkermansia and UBA1819 strongly and the anti-oxidative and inflammatory parameters and SCFAs profiles might suggest a critical involvement of these bacteria in mediating EGCG alleviation of colitis.
Prophylactic EGCG alleviates DSS-induced colitis
Given the benefit of green tea consumption in reducing the risk of IBDs [10] and different impact on the gut microbiota by oral and rectal delivery of EGCG, we hypothesized that EGCG could alleviate DSS-induced colitis prophylactically. Mice were subjected to DSS in drinking water for five days to induce acute colitis after receiving daily oral gavage of 50 mg/kg body weight EGCG for 21 days (Fig. 5A), also as a simulation of about four to eight cups of tea per day for an adult [25]. Disease symptoms and body weight were monitored daily. Mice were then sacrificed and the colonic pathology was evaluated. The concentrations of several representative inflammatory mediators were also measured in both the plasma and colon. Apparently, prophylactic EGCG alleviated the symptoms of colitis in DSS-treated mice as indicated by significantly reduced daily DAI (Fig. 5B) and body weight loss (Fig. 5C), and increased length of colon (Fig. 5D and 5E). Histological analysis further revealed that prophylactic EGCG suppressed DSS-induced infiltration of inflammatory cells and damage to the colonic mucosa (Fig. 5F and 5G). Moreover, EGCG pre-supplementation tended to reduce the plasma levels of IL-1β (Fig. 5H), IL-6 (Fig. 5I), IL-8 (Fig. 5J), and TNF-α (Fig. 5K) in DSS mice. Strikingly, DSS-induced elevations of MPO (Fig. 5L), IL-6 (Fig. 5M), and TNF-α (Fig. 5N) in the colon were largely restored to normal levels by prophylactic EGCG. It is noted that the same dose of EGCG pre-supplementation caused essentially no alterations of any of the aforementioned parameters to normal mice, except for a significant reduction in the colonic levels of three mediators (MPO, IL-6, and TNF-α) (Fig. 5L, 5M, and 5N).
These results collectively indicated that prophylactic EGCG is capable of suppressing DSS-induced colitis symptoms, colonic injury, and inflammation.
Prophylactic EGCG reduces DSS-induced oxidative stress and apoptosis and improves the mucosal barrier function
Prophylactic EGCG largely restored the plasma levels of antioxidant enzymes, T-SOD and CAT that were significantly reduced by DSS (Fig. 6A and 6B). Consistently, MDA in the plasma was significantly increased by DSS, but reduced to normal in response to prophylactic EGCG (Fig. 6C). Similarly, the colonic levels of T-AOC (Fig. 6D), T-SOD (Fig. 6E), CAT (Fig. 6F), and GSH-px (Fig. 6G) were significantly reduced by DSS, but were largely restored to the normal levels by prophylactic EGCG. The colonic level of MDA was significantly increased by DSS, but returned below normal in response to prophylactic EGCG (Fig. 6H). Of note, EGCG pre-supplementation even elicited an obvious anti-oxidative response in both the plasma and colon in healthy mice (Fig. 6A-6H).
TUNEL analysis further revealed that DSS significantly increased the frequency of apoptotic cells in the colonic mucosa, but prophylactic EGCG largely reversed the trend (Fig. 6I and 6K). Moreover, prophylactic EGCG also restored the number of Alcian blue-positive goblet cells in the colon that were reduced by DSS (Fig. 6J and 6L). TEM revealed prophylactic EGCG-mediated preservation of the brush border and tight junctions of the colonic mucosa that were damaged by DSS (Fig. 6M). Consistently, the mRNA expression levels of several major barrier function genes such as Claudin-1 (Fig. 6A), Occludin (Fig. 6B), Zona occludens 1 (ZO-1) (Fig. 7C), Mucin-1 (Fig. 7D), and Mucin-2 (Fig. 7E), were suppressed by DSS, but restored to or raised above their normal levels (as in the case of Claudin-1, Mucin-1 and Mucin-2) in response to prophylactic EGCG.
These results suggested that prophylactic EGCG is capable of suppressing DSS-triggered oxidative stress, regulating apoptosis and restoring mucosal barrier function in colonic epithelia.
Prophylactic EGCG shifts the gut microbiota composition and metabolism
Next, we investigated the impact of prophylactic EGCG on the gut microbiota composition of DSS-treated mice. Sobs Index was not affected by prophylactic EGCG in healthy or DSS-treated mice, although DSS caused a significant reduction (P < 0.001) in richness of the gut microbiota (Fig. 8A). PCoA analysis showed a clear separation between groups with and without DSS (R = 0.5160, P = 0.001, Fig. 8B). Additionally, prophylactic EGCG significantly shifted the gut microbiota composition in both healthy (R = 0.4277, P = 0.001, Fig. 8C) and DSS-treated mice (R = 0.6138, P = 0.001, Fig. 8D). Meanwhile, we explored the overall fecal bacterial composition of mice at the phylum and genus level by barplot. At the phylum level, Bacteroidetes, Firmicutes, and Proteobacteria were predominant in these groups (Fig. 8E). At the genus level, fecal microbiota was dominated by Bacteroids, Lachnospiraceae_NK4A136_group, Odoribacter and Lactobacillus (Fig. 8F). Indeed, LEfSe analysis revealed differential enrichment of fecal bacteria in DSS-treated mice in response to prophylactic EGCG (Fig. 8G). While ten bacterial genera such as Akkermansia, Faecalibaculum, and Bifidobacterium were enriched by EGCG, another ten genera such as Bacteroides were enriched in the DSS only group. Furthermore, PICRUSt analysis predicted an enrichment of multiple pathways such as amino acid metabolism, energy metabolism, and translation in DSS-treated mice in response to prophylactic EGCG (Fig. 8H). It is noteworthy that oral administration of EGCG to healthy mice caused an enrichment of 17 bacterial genera such as such as Akkermansia and UBA1819 (Fig. S4), which are associated with increased metabolism in carbohydrates and lipids and membrane transport (Fig. S5). Additionally, prophylactic EGCG completely restored the production of SCFAs such as acetate (Fig. 8I), propionate (Fig. 8J), and butyrate (Fig. 8K) in DSS-treated mice.
Correlation between differentially microbes induced by prophylactic EGCG and anti-oxidative, inflammatory parameters, and SCFAs profiles
Spearman correlation analysis of DSS-treated mice with and without receiving prophylactic EGCG revealed that 20 different fecal bacterial genera showed a significant correlation (P < 0.05) with the concentrations of at least one anti-oxidative or pro-inflammatory mediator in the plasma or SCFAs in the feces. For example, Akkermansia, Bifidobacterium and Faecalibaculum were positively correlated (P < 0.05) with CAT level in the plasma, T-AOC, T-SOD, CAT, GSH-px levels in the colon, and acetate, propionate, and butyrate in the feces, but with a negative correlation (P < 0.05) with the colonic levels of MDA, IL-6, TNF-α, and MPO (Fig. 9). Akkermansia and Faecalibaculum also showed a significantly positive correlation (P < 0.05) with T-SOD in plasma, but a negative correlation (P < 0.05) with the MDA level in plasma (Fig. 9). Additionally, a number of bacteria such as Akkermansia showed a significant correlation (P < 0.05) with the concentrations of anti-oxidative and inflammatory mediators as well as fecal SCFAs in healthy mice with or without receiving EGCG (Fig. S5).
Above, these results indicated that prophylactic EGCG regulates the gut microbiota composition and its metabolism, leading to a potential to restore the DSS-induced dysbiosis.