Si-based agent showed efficacy in relieving the symptoms of UC
To examine whether the Si-based agent prevents the worsening of UC, we generated 5% DSS-induced UC model mice and performed a comparative analysis between the UC model mice fed with 2.5% Si-based agent-containing diets (Si-DSS group) and those fed with diets without the Si-based agent (Con-DSS group). In the Con-DSS group, the weight loss associated with UC was evident on the 2nd day of DSS administration; it further reduced to − 16.38 ± 0.008% on the 4th day (Fig. 1A). In contrast, in the Si-DSS group, the weight increased on the 1st day after DSS administration and then decreased after the 3rd day; the weight loss was -9.24 ± 0.007% on the 4th day (Fig. 1A). The weight loss in the Si-DSS group was significantly suppressed compared with that in the Con-DSS group.
Si-based agent alleviated the symptoms of UC. Time-dependent changes in the gross pathology, the intake of drinking and diet consumed per day, and survival rates of 5% DSS-treated mice. Line chart of weight loss rate (A), colitis score (B), the intake of drinking (C) and diet (D) consumed per day, and survival curve (E). White: Con-DSS group. Black: Si-DSS group. Each day of the horizontal axis is post-DSS dosing (A,B,E). Each diet treatment for a week (normal), DSS treatment for 3 days (DSS 3rd day) or 5 days (DSS 5th day) after each diet treatment for a week (C,D). Data are expressed as mean ± SEM of 25 mice (A,B,D), 4 cages (C,D: normal; 3 mice per cage) and 3 cages (C,D: DSS 3rd and 5th days) per group. †p < 0.09, **p < 0.01, ***p < 0.001 vs. Con-DSS, determined by Wilcoxon rank sum test (A,B) and Student’s paired t-test (C,D).
Subsequently, the degree of inflammatory symptoms was examined based on the colitis score. Inflammation in the Con-DSS group progressed from the 1st day of DSS administration; the colitis score was 3.41 ± 0.15 on the 4th day. Contrastingly, inflammation in the Si-DSS group was evident from the 2nd day, and the score was 1.92 ± 0.28, even on the 4th day. The inflammation symptoms in the Si-DSS group were significantly relieved compared with those in the Con-DSS group (Fig. 1B). Since the progression of colitis may also affect the intake of diet and drinking, the intake of diet and drinking consumed per day before and after administration of DSS was measured. In both groups, the intake of diet and drinking consumed per day gradually decreased after DSS administration increased (Fig. 1C,D). However, the decrease in intake tended to be mitigate in the Si-DSS group as compared with the Con-DSS group. No difference was observed between the two groups in the normal intake of diet and drinking consumed per day (Fig. 1C,D).
In addition, mortality associated with UC exacerbation was compared between the two groups. In the Con-DSS group, mice were observed for death from the 3rd day of DSS administration, and the survival rate was found to be 16% on the 6th day. In contrast, animals in the Si-DSS group were observed for death after the 4th day, and the survival rate was 40%, even on the 6th day (Fig. 1E). Taken together, these findings suggest that alleviation of the weight loss and inflammatory exacerbation associated with UC resulted in a significant decline in mortality in the Si-DSS group compared with that in the Con-DSS group (Table 1).
Administration of the Si-based agent significantly alleviated UC inflammation symptoms
To examine whether inflammation of the large intestine, the primary symptom of UC, was suppressed by administration of the Si-based agent, MRI of the UC model mice was performed on the 5th day after DSS administration. No pixels with high signal intensity were observed in either group under normal conditions (Fig. 2A). While some high signal intensity pixels indicating inflamed edematous regions surrounding the large intestine, were detected in the Con-DSS group, these pixels were scattered in the large intestine and slightly detected in the Si-DSS group (Fig. 2B). The inflammatory signal region in the Si-DSS group was significantly lower than that in the Con-DSS group (Fig. 2C). In addition, the blood vessels in the abdominal aorta, supplying blood to the large intestine and its branch arteries, were analyzed using MRA. Although swelling of the abdominal aorta, which is a characteristic feature of inflammation, was observed in the Con-DSS group on the 5th day after DSS administration, there were no significant changes in the aorta before and after DSS administration in the Si-DSS group (Fig. 2D,E). Imaging analyses demonstrated that the Si-based agent suppressed the inflammation of the large intestine.
Si-based agent mitigated the inflammation of large intestine. Analysis of inflammation in the colon, using MRI and MRA. (A,B) Representative T2 weighted axial (left) and sagittal images (right) of the colon in the control (upper) and Si-based agent-treated groups (bottom) before treatment (A) and 5 days after 5% DSS treatment (B). Pre pretreatment, DSS 5 days after DSS treatment, Con control group, Si Si group. The colon of a control mouse (arrow) and an Si-based agent-treated mouse (arrowhead). (C) Average number of high signal intensity pixels in the axial image of the colon at the level of the pubis. White: Con-DSS group. Black: Si-DSS group. Data are expressed as mean ± SEM of five mice per group. *p < 0.05 vs. Con-DSS, determined by Student’s paired t-test. (D,E) Representative T2 weighted sagittal images of the colon (left) and images of blood vessels around the colon (right) in the control (upper) and Si-based agent-treated groups (bottom) before treatment (D) and 5 days after DSS treatment (E). (D,E) The colon of a control mouse (arrow) and an Si-based agent-treated mouse (arrowhead). Square: the subject of the blood vessel image around the colon. The abdominal aorta of a control mouse (arrow) and an Si-based agent-treated mouse (arrowhead).
Next, we examined whether the Si-based agent also affected the immune system involved in colitis. To analyze the RNA expression levels of pro-inflammatory cytokines and chemokines in the rectum, which is the primary UC lesion site, qRT-PCR was performed on the 3rd day after DSS administration. In the Con-DSS group, the expression levels of pro-inflammatory cytokines (TNF-α, IL-6, and IFN-γ) and those of neutrophil-attracting chemokine CXCL2 were increased by colitis. However, the above-mentioned increase in pro-inflammatory cytokine and chemokine RNA expression was significantly suppressed in the Si-DSS group (Fig. 3A–D). Since colon inflammation indicated aggravation of UC, pro-inflammatory cytokines in the colon were also investigated. Although there was no statistically significant difference between the control DSS and Si-DSS groups, it was found that the expression of inflammatory cytokines in the colon was also suppressed by the Si-based agent. In addition, the expression of CXCL2 was significantly suppressed in the colon (Fig. 3E–H).
Si-based agent suppressed the increase in pro-inflammatory cytokine and chemokine. mRNA expression of proinflammatory cytokines in the mouse large intestine 3 days after 5% DSS treatment, measured using quantitative reverse transcription-PCR analysis. Bar chart indicates the mean values (rectum: A–D; colon: E–H). TNF-α (A,E), IL-6 (B,F), IFN-γ (C,G) and CXCL2 (D,H). White: Con-DSS group. Black: Si-DSS group. Data are expressed as mean ± SEM of nine mice per group. ††p < 0.08, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con-DSS group, determined by Wilcoxon rank sum test.
These results suggest that administration of the Si-based agent significantly alleviated UC inflammation symptoms via the inhibition of pro-inflammatory humoral factor expression.
Administration of the Si-based agent significantly suppressed colonic atrophy and structural collapse associated with UC
Histological analysis was performed to examine whether administration of the Si-based agent is effective in suppressing the injury of the large intestine associated with colitis. In the UC model mice, it has been reported that the length of the large intestine is reduced due to inflammation; hence, we measured the length of the large intestine from the ascending colon to the anus at the rectum. Comparison of the results with the actual values of the large intestine of normal mice revealed that the large intestine of the Con-DSS group was significantly atrophied due to inflammation on the 3rd day after DSS administration (Fig. 4A,C). Moreover, hemorrhage was observed in the anus of the Con-DSS group, but not in that of the Si-DSS group. Atrophy of the large intestine was also observed in the Si-DSS group compared with that in normal mice, but the degree of atrophy was significantly suppressed compared with that in the Con-DSS group. Similarly, on the 5th day of DSS administration, atrophy of the large intestine in the Si-DSS group was suppressed compared with that in the Con-DSS group (Fig. 4B,D).
Si-based agent suppressed the atrophy and structural disorder associated with intestinal inflammation. Measurement and pathological analysis of the large intestine of 5% DSS-treated mice. (A–D) The representative photograph (A,B) and the bar chart indicate the average of the actual values (C,D) 3 days (A,C) and 5 days after DSS treatment (B,D). (E,F) The representative HE-staining photographs obtained 3 days (E) and 5 days after 5% DSS treatment (F). Low-mag: low magnification and High-mag: high magnification. (G,H) The average bar chart of each histological score 3 days (G) and 5 days after DSS treatment (H). DT: DSS treatment. Colitis: DSS treatment. Gray: normal mice (4 mice), white: Con-DSS group (8 mice), and black: Si-DSS group (3 days: 8 mice, 5 days: 7 mice). Scale bar: 1 cm (A,B), 100 µm (C,D). (I,J) The representative F4/80-immunostaining photographs obtained 3 days (I) and 5 days after 5% DSS treatment (J). Upper panels: low magnification and bottom panels: high magnification. Scale bar: 100 µm (I), 50 µm (J). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs. normal mice or Con-DSS, determined by Student’s paired t-test.
Next, structural disorders of the large intestine were examined using HE-stained samples. In the Con-DSS group, the plications of the colon were partially damaged from the 3rd day after DSS administration, and were hardly observed on the 5th day (Fig. 4E,F). In addition, the crypts of the rectum were partially vacuolated on the 3rd day, and they almost disappeared on the 5th day. Conversely, the colon and rectum in the Si-DSS group did not show any injury on the 3rd day, and only a fraction of plications and crypts were damaged on the 5th day. In addition, we conducted comparative studies based on the histological score (Table 2). As the score value of the Si-DSS group was significantly lower than that of the Con-DSS group, it was confirmed that the injury of both the colon and rectum was suppressed in the Si-DSS group (Fig. 4G,H).
Furthermore, we performed immunostaining for F4/80 (mature macrophage marker protein) to confirm that the infiltration of immune cells into the large intestine of the Si-DSS group were less than that in the control group. In the Con-DSS group, more macrophage infiltration into the rectum was observed than in the colon (Fig. 4I,J). In the rectum, many macrophages were observed in the submucosal layer from the 3rd day after DSS treatment, and more macrophages were infiltrated in the mucosal layer in which the crypt structure was disrupted (Fig. 4I, rectum). On the 5th day, a large number of macrophage infiltrations were observed in the mucosal and submucosa layer where the epithelial cells were peeled off and the crypt structure was completely collapsed and became vacuolated (Fig. 4J, rectum). In the colon, only a few macrophages were observed in the submucosal layer on the 3rd day after DSS treatment, but on the 5th day, many macrophages were observed in the mucosal and the submucosal layer where the colon plicae had collapsed (Fig. 4I,J, colon).
On the other hand, in the Si-DSS group, only a few macrophages were observed in the submucosa of the rectum on the 3rd day after DSS treatment, and almost no macrophages were observed in the colon (Fig. 4I). On the 5th days after DSS treatment, macrophage infiltration was observed where some rectal crypt structures and colon plicae collapsed (Fig. 4J). Even on the 5th day of DSS administration, there were some areas where the rectal crypt structure and colon plicae did not collapse in the Si-DSS group, and no macrophages were observed in such areas. Therefore, the infiltration of macrophages into the large intestine of the Si-DSS group was significantly less than those of the Con-DSS group in which the colon plicae and the rectal crypt structure were almost destroyed.
From the above findings, it was clarified that administration of the Si-based agent significantly relieved damage to the large intestine by suppressing the progression of inflammation.
Si-based agent suppressed the increase in LPO levels associated with UC
Diacron-reactive oxygen metabolites test were performed to determine whether the Si-based agent also has an anti-oxidative effect. The dROMs value, an index of oxidative metabolites in blood, was higher after DSS administration than that in normal condition regardless of the Si-based agent administration, and it was higher on the 5th day than on the 3rd day (Fig. 5A). However, it was revealed that the increase in dROMs value of the Si-DSS group, was significantly alleviated from the 3rd day after DSS administration as compared with the Con-DSS group. No significant difference was observed between the two groups in normal dROMs value.
Si-based agent alleviated the oxidative stress associated with UC. Analysis of oxidative metabolite in mouse serum 5th day (A) and LPO in mouse serum 3rd day after 5% DSS treatment (B,C). Day-dependent changes in the mean of the dROMs value (A). Bar chart indicates the mean values. LPO lipoperoxide (A) and HEL Nε-(hexanoyl)lysine (B). The dot line in (B) indicates the detection limit for HEL (3.0). The positive rates for HEL were 29.4% (Con) and 17.6% (Si). White: Con or Con-DSS group. Black: Si or Si-DSS group. Data are expressed as mean ± SEM of 8 (A: normal), 9 (A: DSS 3rd and 5th day) and 17 mice (B,C) per group. *p < 0.05, *p < 0.05 vs. control, determined by Student’s paired t-test (A–C). Analysis of LPO accumulation on the 5th day after DSS administration using immunohistochemistry with anti-4-HNE antibodies (D). The representative photographs of the colon (upper) and the rectum (bottom) in the Con-DSS group (left) or Si-DSS group (right). Black arrows: epithelial cells. Scale bar: 50 µm.
Next, the blood levels of LPO were analyzed with enzyme-linked immunosorbent assay (ELISA) to determine whether the Si-based agent suppresses oxidative metabolites in the blood. It was found that the blood levels of lipid hydroperoxide (LPO) in the Si-DSS group were significantly lower than those in the Con-DSS group (Fig. 5B). Moreover, the blood levels of Hexanoyl-lysine (HEL; the early LPO) of the Si-DSS group were lower than those in the Con-DSS group (Fig. 5C).
In addition, immunocytochemistry for 4-hydoxy-2-nonenal (4-HNE), an indicator of increased lipid peroxidation chain reaction, was performed to confirm that the Si-based agent alleviated the production of LPO. In the Con-DSS group, strong positive signals were observed in the cytoplasm of epithelial cells and infiltrated immune cells in the colon and rectum on the 5th day of DSS administration (Fig. 5D left panels). On the other hand, in the Si-DSS group, few positive signals were observed in the colon or rectum (Fig. 5D right panels).
These findings demonstrated that the Si-based agent suppressed LPO production through its antioxidant ability.
Administration of the Si-based agent increased the amount of hydrogen generated in the large intestine
The results thus far revealed that the Si-based agent is effective in alleviating the symptoms of UC. Therefore, we investigated the mechanism of action of the Si-based agent in terms of antioxidant and anti-inflammatory effects. For Si-based agent to generate hydrogen, it is essential that the gastrointestinal environment is neutral or alkaline. Therefore, we measured the mouse gastrointestinal pH from the esophagus to the rectum. In the control group, the pH was neutral in the esophagus, but acidic in the stomach, weakly acidic in both the duodenum and jejunum, and finally neutral in the ileum (Fig. 6A,B). The pH of the large intestine was alkaline and that of the cecum was the most alkaline. Surprisingly, the gastrointestinal pH was generally inclined toward the alkaline side in the Si group compared with that in the control group.
Si-based agents increased the amount of hydrogen in the large intestine. Detection of GI pH and hydrogen content. (A) The line graph for GI pH of the control group (white circle and dotted line) and Si group (black circle and solid line). The sections used for pH detection were the esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum. Data are expressed as mean ± SEM of five mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control, determined by Student’s paired t-test. (B) Each pH variation is shown as color variation based on the scale. (C–G) The average bar graphs for gastrointestinal hydrogen content of the control or control DSS group (white bar) and the Si or Si-DSS group (black bar) in the normal state (C,D) and 3 days after 5% DSS treatment (E–G). (C) Total hydrogen content and (D–G) hydrogen content per weight. Data are expressed as mean ± SEM of nine mice and DSS-treated mice (Con: 8, Si: 7) per group.
Next, to examine whether hydrogen is actually generated in the intestinal tract (especially in the large intestine) of mice administered the Si-based agent, the levels of hydrogen in the stomach, small intestine, cecum, colon, and rectum were analyzed with gas chromatography. In the control group, hydrogen was detected abundantly in the intestinal tract, except in the cecum, where very slight amounts were detected (Fig. 6C). The total amount of retained hydrogen was the highest in the small intestine and similar in the large intestine and stomach. No difference was observed in the amount of retained hydrogen per body weight in the intestine other than in the cecum (Fig. 6D). In the Si-DSS group, the amount of retained hydrogen followed the same pattern as that in the Con-DSS group. Furthermore, the total amount of gastrointestinal retained hydrogen in the Si group, excluding the cecum, was higher than that in the control group. From the above-stated findings, it was clarified that the Si-based agent generated hydrogen in the intestinal tract. Interestingly, the mice affected with colitis had significantly reduced levels of hydrogen in the large intestine and small intestine, regardless of the Si-based agent treatment (Fig. 6E). Particularly, in the inflamed site, large intestine, the volume of hydrogen was significantly reduced compared with that in the small intestine (Fig. 6F,G). Surprisingly, the amount of residual hydrogen in the large intestine after the onset of colitis in the Si-DSS group was higher than that in the Con-DSS group (Fig. 6G). These results demonstrated that the decrease in the amount of hydrogen during inflammation was alleviated by the administration of Si-based agent.
Examination of the antioxidative mechanism of the Si-based agent in relieving the symptoms of UC
Si-based agent alleviate the symptoms of UC by replenishing lost hydrogen in the large intestine. It is possible that such hydrogen supplementation has anti-inflammatory and antioxidant effects. We focused on the metabolism of sulfur compounds, which are greatly involved in the redox action in the body, and conducted a comprehensive analysis of 87 types of sulfur metabolites (sulfur index analysis) in a total of four groups: the Con-DSS group, the Si-DSS group, the control group, and Si groups. As a result, 39 types of sulfur compounds were detected. The amount of glutathione-related sulfur compounds was significantly higher in the group administered the Si-based agent than in the non-administered group (Fig. 7A). Among these compounds, the expression levels of glutathione persulfide, which exhibits strong antioxidant effects, were remarkably increased. After performing multivariate analysis based on the detected sulfur-related compound data (Fig. 7B), similarity mapping analysis between groups was performed. In the control group, the normal large intestine was in the reduced state, but when colitis was induced, it shifted toward the oxidized state due to inflammation (Fig. 7C). In contrast, the large intestine of the Si group was in the reduced state similar to that of control, but the colitis-induced large intestine did not shift to the oxidized state (Fig. 7C). Taken together, we identified that the Si-based agent alleviated oxidation of the colon associated with inflammation due to colitis by inducing antioxidant sulfur compounds.
Si-based agent suppressed the intestinal oxidation associated UC via antioxidant sulfur compounds. Sulfur index analysis of the mouse large intestine. The average bar graphs for the expression of glutathione, oxidized glutathione, and each persulfide (A). White: control or con-DSS group; black: Si or Si-DSS group. (B) Contributory compounds in sulfur-index analysis. (C) The dot graph of individual values and the average for sulfur index analysis. Data are expressed as mean ± SEM of six mice per group. ♯p < 0.08, *p < 0.05, **p < 0.01, determined by Student’s paired t-test.
Si-based agent alleviated the visceral pain and discomfort associated with UC
Psychological stress is greatly involved in remission and relapse in patients with colitis. Therefore, we also examined the effects of colitis on the brain and the therapeutic effects of the Si-based agent on the brain. To examine whether the administration of the Si-based agent alleviates the visceral pain and discomfort associated with UC, we investigated the neuronal activities in the nuclei of the dorsal medulla oblongata [the nuclei of the solitary tract (NST) and the dorsal vagal nuclei (DVN)], and the central amygdala (CeA) using immunostaining for c-Fos, a neuronal active marker. Many positive cells were observed in all analyzed nuclei of the Con-DSS group, whereas positive signals were not almost detected in the Si-DSS group (Fig. 8).
Si-based agent alleviated the visceral pain and discomfort associated with UC. Analysis of the neuronal activity influenced by colitis, using immunofluorescence staining with anti-c-Fos antibodies. (A,B) The representative photographs of the dorsal medulla (A) and central amygdaloid nuclei (B) 5 days after 5% DSS treatment. Con Con-DSS group, Si Si-DSS group. The vagal dorsal motor nuclei and solitary tract nuclei (A), and central amygdaloid nuclei (B) of a control mouse (arrow) and an Si-based agent-treated mouse (arrowhead). Square in (B): the central amygdaloid nuclei, 12N: hypoglossal nerve nuclei, AP: area postrema, cc: central canal, and Ce: central amygdaloid nuclei. Scale bar: 200 µm.
These results demonstrated that the Si-based agent alleviated the visceral pain and discomfort associated with UC, as well as the symptoms of UC.
