## 4 Discussion
The DSS colitis model has been widely applied for studies examining anti-inflammatory and TJ permeability [52; 53], and we, therefore, believed it to be a suitable model our analysis of anti-inflammatory mechanisms associated with the intake of mineral water derived from DSW rather than the intraperitoneal in vivo TJ permeability model that uses LPS [54]. In this study, we provided mice with drinking water containing 5% DSS for seven days to establish an acute colitis model. DSS caused weight loss and colonic tissue damage, and it reduced colon length. Severe inflammation, high lesion depth, and glandular damage, which are typical of acute ulcerative colitis, were observed in the colons of these mice by microscopy [55]. Mice were provided HMLS and TM drinking water ad libitum for 21 days to investigate the effects of DSW-derived waters on colitis. Weight loss in response to colitis was not significantly inhibited. However, HMLS slightly improved colon length and the DAI score. Under intestinal inflammation, mucosal areas of the colon typically show spreading inflammation, damage to the glandular structures, and a reduction in goblet cells [33; 56]. Inflammatory cell infiltration and glandular damage were improved upon treatment with HMLS or TM, as indicated by hematoxylin and eosin staining. Similarly, in another study, Mg supplementation resulted in improved restoration of mucosal integrity and channel expression [57]. However, in the current study, due to the high DSS concentration, the pathological symptoms were not found to significantly improve. However, FD-4 permeability results reveal higher permeability in these groups. This can be explained by the fact that intestinal cells were disrupted by DSS before TJ permeability was confirmed. In line with this, we did not identify TJ-related DEG in DSS vs. DSW, likely because the high-level DSS treatment led to intestinal cell destruction. Similarly, in a previous study using 3% DSS, ZO-1 protein expression was not observed on Day 7 after DSS treatment [58]. RNA-seq results from DSS-induced colonic tissues revealed that the majority of DEG were involved in the immune system and inflammatory response. As _TNF_ and _NOS2_, which had the most protein nodes in the PPI network among the significant DEG between the DSS and HMLS groups,were significantly reduced, other genes related to inflammation were also significantly suppressed. This is in line with previous studies reporting that DSW reduced LPS-induced NO and iNOS expression in Raw 264.7 cells [26]. TNF-\(\alpha\) is one of the important inflammatory cytokines and strongly stimulates the production of cytokines such as IL-6 and IL-8, causing cytokine storms [59; 60]. TNF-\(\alpha\) -inhibitory drugs improve IBD [61]. We found that HMLS, which is high in magnesium, might suppress TNF-\(\alpha\) and thus mitigate IBD. In addition, magnesium reportedly inhibits NF-\(\kappa\)B and decreases the release of inflammatory cytokines by increasing intracellular basal IkB\(\alpha\) levels [62]. HMLS suppressed inflammatory gene expression more strongly than did TM. SLE was the most highly represented KEGG pathway in the DEG between DSS and HMLS. SLE is a chronic autoimmune disorder that involves immune dysregulation and antinuclear antibody and affects multiple tissues and organ systems [63]. Based on gene enrichment in the SLE pathway, HMLS promoted the generation of B cells and reduced the expression of _H2-Ab1_ and _Hbb-b1_, which are related to MHCII molecules involved in the immune response. This finding was consistent with a previous finding that a calcium-rich diet maintains intestinal permeability and protects from IBD-related intestinal inflammation in HLA-B27 transgenic mice [64]. Further, RNA-seq interestingly revealed that the expression of various histone genes that were downregulated by DSS were rescued upon HMLS water consumption. In general, when DNA damage occurs, histone gene expression decreases [65].