Effect of Calcium-Sulphate-Bicarbonate Water in a Murine Model of Non-Alcoholic Fatty Liver Disease: A Histopathology Study

The progression of nonalcoholic fatty liver disease (NAFLD) is associated with alterations of the gut–liver axis. The activation of toll-like receptor 4 (TLR4) pathways by endotoxins, such as lipopolysaccharide (LPS), contributes to liver injury. The aim of the present study was to evaluate the possible beneficial effects of a calcium-sulphate-bicarbonate natural mineral water on the gut–liver axis by evaluating liver and terminal ileum histopathology in a murine model of NAFLD. NAFLD was induced in mice by administrating a methionine-choline-deficient (MCD) diet. The following experimental groups were evaluated: controls (N = 10); MCD+Tap water (MCD; N = 10); MCD+Calcium-sulphate-bicarbonate water (MCD/Wcsb; N = 10). Mice were euthanised after 4 and 8 weeks. Liver and terminal ileum samples were collected. Samples were studied by histomorphology, immunohistochemistry, and immunofluorescence. In mice subjected to the MCD diet, treatment with mineral water improved inflammation and fibrosis, and was associated with a reduced number of activated hepatic stellate cells when compared to MCD mice not treated with mineral water. Moreover, MCD/Wcsb mice showed lower liver LPS localization and less activation of TLR4 pathways compared to the MCD. Finally, Wcsb treatment was associated with improved histopathology and higher occludin positivity in intestinal mucosa. In conclusion, calcium-sulphate-bicarbonate water may exert modulatory activity on the gut–liver axis in MCD mice, suggesting potential beneficial effects on NAFLD.


Introduction
Nonalcoholic fatty liver disease (NAFLD), or metabolic-associated fatty liver disease (MAFLD) is a chronic liver disease defined by the presence of lipid accumulation within hepatocytes without excessive alcohol consumption [1,2]. NAFLD represents the most common cause of liver disease, affecting~25% of the population, with an increasing global prevalence [3,4]. NAFLD development results from a variety of metabolic, genetic, and environmental factors, which ultimately lead to fat overload in the liver and an inadequate handling of metabolic substrates by the hepatocytes [2]. In particular, liver steatosis drives hepatocellular injury due to an altered cellular energetic metabolism, abnormal lipid compounds, and reactive oxygen species, in a process that is overall defined as "lipotoxicity" [5]. In this context, inflammation and regenerative pathways occurring within the liver are at the basis of the progression from a fatty liver towards nonalcoholic steatohepatitis (NASH), which is characterized by the development of severe inflammation and, eventually, fibrosis progressing towards cirrhosis [6,7].

Effects of Calcium-Sulphate-Bicarbonate Water (Wcsb) on Liver Histopathology in MCD Mice
All MCD mice drinking calcium-sulphate-bicarbonate water (MCD/Wcsb) were characterized by a certain degree of NASH histopathological features (i.e., steatosis, lobular inflammation, and fibrosis; Figures 2 and 3). Among mice sacrificed after 4 weeks of treatment, MCD/Wcsb mice showed significantly lower inflammation (score= 0.25 ± 0.5), fibrosis (score= 0.25 ± 0.5) and collagen deposition (2.3 ± 0.4%) compared to MCD-4wk (a,b) Hematoxylin and eosin (H&E) stain on MCD and control (CTR) mouse livers. In MCD mice, steatosis (a) and lobular inflammation ((b), arrows) progressively increases after four weeks (4wk) and eight weeks (8wk) of MCD diet administration. I. Sirius red/fast green (SR/FG) stains in MCD and CTR mouse livers. Collagen deposition in mice livers increased after eight weeks of MCD diet administration. Scale bars: 100 µm (a,c) and 50 µm (b). In (a-c), histograms show means and standard deviations for steatosis and inflammation scores (a,b), and for the area percentage of the liver occupied by collagen fibers (c). Data from each animal are plotted as solid circles in the graph. * p < 0.05 vs. all other groups; • p < 0.05 vs. CTR.

Effects of Calcium-Sulphate-Bicarbonate Water (W csb ) on Liver Histopathology in MCD Mice
All MCD mice drinking calcium-sulphate-bicarbonate water (MCD/W csb ) were characterized by a certain degree of NASH histopathological features (i.e., steatosis, lobular inflammation, and fibrosis; Figures 2 and 3). Among mice sacrificed after 4 weeks of treatment, MCD/W csb mice showed significantly lower inflammation (score= 0.25 ± 0.5), fibrosis (score= 0.25 ± 0.5) and collagen deposition (2.3 ± 0.4%) compared to MCD-4wk mice (p < 0.05); no significant differences were observed in terms of steatosis (score= 1.3 ± 0.5) compared to MCD-4wk mice.  Histopathological changes in mice subjected to a methionine-choline-deficient (MCD) diet and treated with calcium-sulphate-bicarbonate water (Wcsb). (a,b). Hematoxylin and eosin (H&E) stain on mouse livers after four weeks (4wk, (a)) or eight weeks (8wk, (b)) of MCD diet administration with or without Wcsb. In MCD mice, no significant difference was observed in terms of liver steatosis (upper panels) after Wcsb administration. Lobular inflammation (lower panels, arrows) was reduced after Wcsb administration compared to untreated MCD mice. Scale bars: 100 μm (upper panels) and 50 μm (lower panels). Histograms show means and standard deviations for steatosis and inflammation scores. Data from each animal are plotted as solid circles in the graph. * p < 0.05 vs. MCD.

Figure 2.
Histopathological changes in mice subjected to a methionine-choline-deficient (MCD) diet and treated with calcium-sulphate-bicarbonate water (W csb ). (a,b). Hematoxylin and eosin (H&E) stain on mouse livers after four weeks (4wk, (a)) or eight weeks (8wk, (b)) of MCD diet administration with or without W csb . In MCD mice, no significant difference was observed in terms of liver steatosis (upper panels) after W csb administration. Lobular inflammation (lower panels, arrows) was reduced after W csb administration compared to untreated MCD mice. Scale bars: 100 µm (upper panels) and 50 µm (lower panels). Histograms show means and standard deviations for steatosis and inflammation scores. Data from each animal are plotted as solid circles in the graph. * p < 0.05 vs. MCD.   When the mice that had been euthanised after 8 weeks of treatment were analysed, MCD/W csb mice showed significantly less inflammation (score= 1.4 ± 0.9) compared to MCD-8wk mice (p = 0.034). No statistically significant differences were observed in terms of steatosis (score= 1.8 ± 0.8), fibrosis (score= 2.6 ± 0.5), and collagen deposition (5.4 ± 2.7%); however, 3 out of 5 MCD/W csb mice were characterized by lower steatosis and fibrosis scores compared to the MCD group.

Effects of W csb on Fibrogenetic Cells in MCD Mice Liver
The activation of hepatic stellate cells (HSCs) was studied using immunohistochemistry for alpha-smooth muscle actin (αSMA). Among the mice euthanised after 4 weeks of treatment ( Figure 3), MCD-4wk mice showed an increase in the number of αSMA+ HSCs (5.8 ± 0.9) compared to the control mice (1.0 ± 0.7; p < 0.001); moreover, MCD/W csb mice showed a lower number of αSMA+ cells (3.0 ± 2.0) compared to MCD-4wk mice (p = 0.041).

Effects of Wcsb on the Intestine in MCD Mice
LPS translocation induced by an MCD diet could be related to injury of the ileal mucosal barrier integrity. Therefore, we evaluated the terminal ileum of mice in our , phosphonuclear factor-κB (pNF-κB) (middle panels), and immunofluorescence for TLR4 (lower panels) in controls (CTR), and in MCD mouse livers, after four weeks (4wk) or eight weeks (8wk) of MCD diet administration, with or without W csb . W csb administration in MCD mice was associated with lower hepatocyte LPS localization, nuclear pNF-κB positivity (yellow arrows) and a reduced number of infiltrating TLR4+ macrophages (red arrows). Original magnification: 10× (immunohistochemistry) and 20× (immunofluorescence). In immunofluorescence, nuclei are in blue. (b) Heat maps report data from all animals for the percentage of LPS+ and pNF-κB+ hepatocytes, and for the number of TLR4+ macrophages. Each cell represents an individual animal.

Effects of W csb on the Intestine in MCD Mice
LPS translocation induced by an MCD diet could be related to injury of the ileal mucosal barrier integrity. Therefore, we evaluated the terminal ileum of mice in our experimental model ( Figure 5). For this set of experiments, we focused on mice treated for 8 weeks, given the higher degree of liver damage at this time point. H&E and PAS staining were used to observe the intestinal structure.
In MCD mice, the intestinal villi were shortened indicating structural damage induced by the diet; accordingly, the length of intestinal villi in MCD-8wk mice was lower (100.2 ± 39.8 µm) compared to control mice (155.5 ± 39.0 µm; p < 0.001).
Next, the positivity for intestinal occludin, a tight junction protein, was determined through immunofluorescence to evaluate the intestinal mucosal barrier integrity. The occludin positivity in enterocytes was significantly lower in MCD mice (score = 1.6 ± 0.5) compared to controls (score = 3.6 ± 0.5; p < 0.001); MCD/W csb mice showed a significantly higher enterocyte occludin positivity (score = 2.6 ± 0.5) compared to MCD mice (p < 0.05). experimental model ( Figure 5). For this set of experiments, we focused on mice treated for 8 weeks, given the higher degree of liver damage at this time point. H&E and PAS staining were used to observe the intestinal structure. In MCD mice, the intestinal villi were shortened indicating structural damage induced by the diet; accordingly, the length of intestinal villi in MCD-8wk mice was lower (100.2 ± 39.8 μm) compared to control mice (155.5 ± 39.0 μm; p < 0.001).

Discussion
In the present study, we demonstrated that, in MCD mice, treatment with calciumsulphate-bicarbonate water was associated with: (i) liver histopathology improvement, lower inflammation, fibrosis, collagen deposition, and a lower activation rate of fibrogenetic cells; (ii) lower hepatocyte localization of LPS; (iii) reduced TLR4 pathway activation, with less TLR4+ macrophages and nuclear pNF-κB positivity in hepatocytes; iv) a preserved terminal ileum histopathology, with longer villi and higher occludin positivity in enterocytes.
The term hydropinotherapy defines the act of drinking natural mineral waters, especially thermal waters, for therapeutic purposes [20]. This approach represents a useful tool in support of pharmacological therapies, especially in inflammatory diseases. Natural mineral waters, compared to tap water, are characterized by a high concentration of

Discussion
In the present study, we demonstrated that, in MCD mice, treatment with calciumsulphate-bicarbonate water was associated with: (i) liver histopathology improvement, lower inflammation, fibrosis, collagen deposition, and a lower activation rate of fibrogenetic cells; (ii) lower hepatocyte localization of LPS; (iii) reduced TLR4 pathway activation, with less TLR4+ macrophages and nuclear pNF-κB positivity in hepatocytes; iv) a preserved terminal ileum histopathology, with longer villi and higher occludin positivity in enterocytes.
The term hydropinotherapy defines the act of drinking natural mineral waters, especially thermal waters, for therapeutic purposes [20]. This approach represents a useful tool in support of pharmacological therapies, especially in inflammatory diseases. Natural mineral waters, compared to tap water, are characterized by a high concentration of minerals and elements, such as calcium, carbonic metabolites, sodium chlorite, sulphates, and/or iron, in varying proportions according to the source [16]. Based on the composition, particularly with regard to the prevalent elements, different waters may have distinct beneficial effects on health. Interestingly, mineral waters have been empirically known for their beneficial effects on liver and biliary disorders. Recently, scientific evidence for the positive effects of mineral water compounds has been emerging, supporting their use for clinical purposes. The use of mineral water in patients affected by biliary disorders has been reported to modulate the severity of symptoms due to the altered bile flow [21] and was also associated with a reduced lithogenic risk and metabolic improvements from the regulation of bile composition [17]. Furthermore, mineral-rich waters can exert positive effects on fat and carbohydrate metabolisms in a murine model of metabolic syndrome [22].
In addition to the liver, mineral water administration was shown to have beneficial effects on the intestinal mucosa and gut microbiota, both in human and animal models.
Bicarbonates contribute to intestinal barrier constitution, and administration of bicarbonaterich water has been shown to have positive effects on intestinal histopathology [23,24]. Interestingly, sulphate water administration can modify gut microbiota, by favoring hydrogen sulfide production through sulphate-reducing bacteria (such as E. Coli) in the intestinal lumen, exerting local anti-inflammatory effects [23,25,26].
Indeed, a bidirectional relationship between the gut and the liver has been identified both in health and disease [8]; the so-called gut-liver axis is characterized by the direct afflux of nutrients and other substances from the intestine to the liver via the portal vein. However, pathological conditions affecting the gastrointestinal tract can cause a disruption to intestinal barrier integrity, thereby resulting in the translocation of potentially harmful compounds to the liver [8]. An emerging role of the gut-liver axis modification in the pathogenesis of NASH has been underlined. In particular, NASH is associated with an increased intestinal permeability, which is largely due to the alterations in gut microbiota composition, both in human subjects and in animal models. In this context, the altered nutritional regimen has a direct effect in determining the bacterial populations in the gut, favoring the eventual translocation of PAMPs to the liver [8]. Among PAMPs, LPS translocation to the liver can lead to the recruitment and activation of proinflammatory cells contributing to disease progression in human and experimental NASH [11].
In this scenario, in a murine model of NASH, we tested whether a natural mineral water (rich in calcium, sulphates, and bicarbonate) could have potential beneficial effects on liver histopathology through the modulation of liver LPS localization derived from gut translocation. Our investigation showed that mice with NASH, treated with W csb, were characterized by a slight but significant improvement in liver inflammation and fibrosis but not of hepatocyte steatosis compared to the untreated ones. In accordance with this, the increase in αSMA+ activated stellate cells was reduced in W csb treated mice. Furthermore, we reported LPS localization within hepatocytes in NASH mice, which was reduced in mice treated with mineral water administration. In a previous study [10], liver LPS localization was associated with the activation of TLR4/NF-κB pathways. TLR4 is a pattern recognition receptor (PRR) which can bind both to DAMPs and PAMPs; it is implied in the immune response to endotoxins from the activation of the transcriptional factor NF-κB both in macrophages and in hepatocytes [27]. Interestingly, the administration of W csb to NASH mice was associated with a lower number of TLR4+ macrophages within the liver and a reduction of pNF-κB in hepatocytes. Taken together with the reduced lobular inflammation, these data suggest that natural mineral water could potentially exert a modulatory effect on bacterial translocation and TLR4 pathway activation, leading to the observed improvements to liver injury.
To further support this hypothesis, we examined the histopathology of the terminal ileum and the integrity of the intestinal barrier. Bacterial translocation and endotoxemia in NASH are associated with an impairment of intestinal barrier integrity [28] and increased intestinal permeability [8]. In the MCD mouse model, intestinal dysbiosis was described together with alterations in intestinal mucosa [29,30]. Accordingly, we observed structural alterations of the intestinal mucosa of MCD mice, characterized by shorter villi and reduced occludin positivity on enteric epithelium compared to controls. Remarkably, in MCD mice treated with W csb , histological injury in the terminal ileum was less prominent compared to MCD mice alone; moreover, occludin localization between enterocytes was improved after mineral water treatment, suggesting that the positive effects of mineral water in these mice are also present on the terminal ileum and barrier integrity. Interestingly, it has been shown that bicarbonate-sulphate-calcium-magnesium water exerts positive effects on indirect markers of gut-liver axis activation and in modifying gut microbiota in patients with NAFLD in a prospective longitudinal interventional study [31].

Limitations of the Study
Our study represents a preliminary report of the positive effects of calcium-sulphatebicarbonate water in NASH mice and the gut-liver axis.
• NASH model. The MCD model does not replicate the NAFLD-related metabolic syndrome. In this model, the administration of a methionine-choline-deficient diet determines the development of hepatic steatosis, together with prominent inflammation and fibrosis; this is due to an impaired VLDL metabolism caused by methionine and choline deficiency. In turn, this leads to lipid accumulation in hepatocytes and subsequent lipotoxic injury, then progression towards NASH [32]. In the present study, we chose MCD instead of other models because it replicates the inflammatory and fibrogenetic injuries occurring in NASH more consistently than other models, thereby allowing us to better study the potential inflammatory response to LPS in the liver [29,30]. On the other hand, the model is not suitable for evaluating the potential beneficial effects of mineral water on glucose and lipid metabolisms [19,33]; accordingly, we did not observe significant effects on hepatic steatosis, and future studies are necessary to expand the investigations on metabolic features by using other disease models.

•
Mechanistic study. Here, we reported an association between thermal water administration, liver and intestine histologic improvement, as well as LPS hepatocyte localization. However, future analyses should be aimed at performing an in-depth assessment of the mechanisms responsible for the modulation of inflammatory and fibrogenetic injuries of the liver, in addition to confirming the modulatory properties on the intestinal barrier and microbiota [18,34].

Conclusions and Future Perspectives
In conclusion, our study describes the effects of natural mineral water rich in calcium, sulphates, and bicarbonates on liver and terminal ileum histopathology, suggesting a possible modulation of the gut-liver axis and of inflammatory injury as mediated by LPS and TLR4 pathways ( Figure 6). The gut-liver axis represents a key mechanism in nonalcoholic fatty liver disease (NAFLD) progression towards nonalcoholic steatohepatitis (NASH), triggering the activation of toll-like receptor (TLR) 4 and nuclear factor-k B (NF-kB) pathways in the liver via translocation of endotoxins (e.g., lipopolysaccharide: LPS). This is due to altered intestina permeability and gut microbiota composition. In the present study, we investigated the effects o calcium-sulphate-bicarbonate natural mineral water on the methionine-choline-deficient diet mode of NASH. Mineral water administration was associated with improvements in liver and termina ileum histopathology. Further studies should be performed in order to confirm the findings of othe NAFLD models; clarify the mechanisms at the basis of these beneficial effects; and test the eventua possible application of mineral water administration to patients as a supporting therapy fo NAFLD/NASH.
Future studies in humans may allow us to evaluate whether the administration o Wcsb could represent a helpful addition to diet and physical activity in NAFLD/NASH o MAFLD subjects. Particularly, the absorption of nutrients from water can be affected by food; therefore, future studies translating these findings in a human setting should take into account dietary regimen and diet composition [14,15]. Moreover, clinical research on Figure 6. Framework figure. The gut-liver axis represents a key mechanism in nonalcoholic fatty liver disease (NAFLD) progression towards nonalcoholic steatohepatitis (NASH), triggering the activation of toll-like receptor (TLR) 4 and nuclear factor-k B (NF-kB) pathways in the liver via translocation of endotoxins (e.g., lipopolysaccharide: LPS). This is due to altered intestinal permeability and gut microbiota composition. In the present study, we investigated the effects of calcium-sulphatebicarbonate natural mineral water on the methionine-choline-deficient diet model of NASH. Mineral water administration was associated with improvements in liver and terminal ileum histopathology. Further studies should be performed in order to confirm the findings of other NAFLD models; clarify the mechanisms at the basis of these beneficial effects; and test the eventual possible application of mineral water administration to patients as a supporting therapy for NAFLD/NASH. Future studies in humans may allow us to evaluate whether the administration of W csb could represent a helpful addition to diet and physical activity in NAFLD/NASH or MAFLD subjects. Particularly, the absorption of nutrients from water can be affected by food; therefore, future studies translating these findings in a human setting should take into account dietary regimen and diet composition [14,15]. Moreover, clinical research on therapeutic applications of calcium-sulphate-bicarbonate water in NAFLD should include a detailed assessment of patient behavior, lifestyle intervention and physical activity, in order to obtain reproducible results and definitive evidence of mineral water efficacy [18,35]. Finally, future mechanistic studies in experimental models should be focused on the effects of natural mineral waters on the entero-hepatic circulation of bile acids; this is a pivotal mechanism at the basis of the gut-liver axis that influences NASH development [8].

Experimental Setting
Eight-week-old male C57BL/6 mice were purchased from Charles River Laboratories (Charles River UK Ltd., Margate, UK). Mice were divided into the following experimental groups: Mice were housed in a dedicated facility at Sapienza University of Rome in compliance with Italian regulations. Mice were kept in a room maintained at a temperature of 23 ± 1 • C and 50 ± 10% relative humidity, with food and water available ad libitum. The animal room was on a 12:12 h light:dark cycle. Mice were individually identified by ear punching.
Control mice were fed with a normal chow diet and were randomly divided into mice drinking tap or calcium-sulphate-bicarbonate water at room temperature. No differences were revealed in terms of weight, serum analysis, or histopathology; therefore, these mice were grouped and considered altogether as controls.
Steatohepatitis was induced in mice through a methionine-choline deficient (MCD) diet as previously reported. The amount of water consumed by the animals was measured daily. Drinking bottles were replaced once per day. Weight was measured weekly.
Mice were euthanised after 4 weeks (N = 5 per group) or 8 weeks (N = 5 per group) of treatment. At the time of euthanasia, mice were anaesthetized using saturated diethyl ether vapor and euthanized by cervical dislocation. Blood samples were drawn from a cardiac puncture. The liver and terminal ileum were harvested and processed for histology.
The study protocol (n • 553/2019-PR) was approved by the Italian Ministry of Health and by the university commission for animal care following the criteria of the Italian National Research Council. Animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health.

Histopathology, Immunohistochemistry and Immunofluorescence
Tissue samples were fixed in formalin and embedded in paraffin. Three-micron sections were obtained, and routine histological stains were performed, including hematoxylineosin (H&E) and Sirius Red/Fast Green (SR/FG).
For immunohistochemistry, endogenous peroxidase activity was blocked by a 30 min incubation in methanolic hydrogen peroxide (2.5%). Antigens were retrieved, as indicated by the vendor, by applying Proteinase K (Dako, Glostrup, Denmark, code S3020) for 10 min at room temperature. Sections were then incubated overnight at 4 • C with primary antibodies (Table 1). All primary antibodies are of commercial use; have been tested and validated by manufacturers; and have been previously used and published [11]. For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included [36,37]. Then, samples were rinsed twice with phosphate buffered saline (PBS) for 5 min, incubated for 20 min at room temperature with a secondary biotinylated antibody, and then with Streptavidin-horseradish peroxidase (LSAB+, Dako, Glostrup, Denmark code K0690). Diaminobenzidine (Dako, Glostrup, Denmark code K3468) was used as a substrate, and sections were counterstained with hematoxylin.
For immunofluorescence, nonspecific protein binding was blocked using specimen incubation with 5% normal goat serum and then with primary antibodies overnight. Sections were incubated for 1 h at room temperature with labelled isotype-specific secondary antibodies (AlexaFluor ® , Invitrogen, Life Technologies Ltd., Paisley, UK) and counterstained with 4,6-diamidino-2-phenylindole (DAPI) for visualization of cell nuclei [36,37].
Sections were examined in a coded fashion with a Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Weltzlar, Germany), equipped with a video-camera (Jenoptik Prog Res C10 Plus Videocam, Jena, Germany) by two independent researchers. Slides were further scanned with a digital scanner (Aperio ScanScope ® CS and FL Systems; Aperio Digital Pathology, Leica Biosystems, Milan, Italy) and processed using ImageScope.
Histopathological evaluation of the liver was performed according to previous indications and included scoring of hepatocyte steatosis, hepatocyte hypertrophy, lobular inflammation, and fibrosis [43]. Moreover, on SR/FG-stained slides, collagen deposition was automatically calculated with an algorithm and expressed as a positive area percentage [11]. Terminal ileum samples were radially cut; mean villus length was quantified from ten nonoverlapping 20× fields per mouse on H&E-stained slides [44,45].
For LPS positivity, the percentage of positive hepatocytes was automatically calculated by an algorithm for the entire section. The nuclear localization of pNF-κB by hepatocytes was automatically calculated using specific algorithms for the entire section [11].

Statistical Analysis
Continuous variables were expressed as mean ± standard deviation. Mann-Whitney U-test was used to study differences among groups. All tests were two-tailed, and a statistical significance was set at a p-value of less than 0.05. Analyses were performed using computer software packages (IBM SPSS Statistics v20.0, Armonk, NY, USA).  Institutional Review Board Statement: The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Sapienza University of Rome.
Data Availability Statement: Data is available from the Authors upon reasonable request.