Orally Administered Diosgenin Alleviates Colitis in Mice Induced by Dextran Sulfate Sodium through Gut Microbiota Modulation and Short-Chain Fatty Acid Generation

Abstract

Diosgenin (DIO) is a kind of steroid sapogenin derived from natural plants. It exerts strong anti-infection, antiallergy, antiviral, and antishock pharmacological properties. In this article, the protective effects of DIO against dextran sulfate sodium (DSS)-induced colitis in mice were researched. Compared with the 2.5% DSS treatment group, 15 mg/kg body weight of diosgenin alleviated colitis disease, evidenced by the increased body weight, the decrease in the disease activity index, and the histological scores. Furthermore, 16S rRNA high-throughput sequencing results demonstrated that DIO improved the colon homeostasis through modulating the gut microbiota, including increases in the relative abundance of several probiotic bacteria, such as Prevotellaceae (from 1.4% to 5.8%), Lactobacillus (from 12.3% to 29.7%), Mucispirillum (from 0.07% to 0.49%), and decreases in the pathogenic bacteria, such as Streptococcus (from 1.6% to 0.6%) and Pseudomonadaceae (from 0.004% to 0%).

In addition, the concentration of gut microbial metabolites, total short-chain fatty acids (SCFAs), acetic acid, and propionic acid were significantly increased after DIO supplementation. In conclusion, our findings suggested that DIO attenuates DSS-induced colitis in mice by means of modulating imbalanced gut microbiota and increases in SCFA generation.

Introduction

Inflammatory bowel disease (IBD), a chronic remitting and relapsing inflammatory disease of the intestinal tract, comprises two main clinical forms: Crohn's disease (CD) and ulcerative colitis (UC).¹ The typical symptoms of IBD include acute diarrhea, mucosa structure injury, and dysbiosis of microbial composition.^2,3 With westernized diet prevalence and lifestyle changes, IBD has been recognized as a worldwide health problem over the past decades. It was reported that the incidence rates for IBD, UC, and CD rise to 1.80, 1.33, and 0.46 per 10⁵ persons in 2018.⁴ Although the etiology of IBD still remains elusive, increasing evidence has revealed that intestinal microbiota dysbiosis, mucosal inflammatory damage, and oxidative stress are closely related to its occurrence.^5,6

The mainstream hypothesis regarding IBD pathogenesis suggests that it results from the combined interaction between environmental stress, genetic background, and intestinal immune disorders.⁷ Genetic research demonstrated that genes predisposing to IBD are mostly involved in the interaction between host and microbiome.⁸ The human gastrointestinal tract provides a suitable habitat for over 1000 kinds of bacteria, while microbiota and their metabolites protect the intestinal barrier and regulate immune system.^9,10 Furthermore, patients with IBD often present with a microbiota imbalance, including low microbial diversity, decreased numbers of beneficial bacteria, and a greater abundance of pathogenic bacteria.^11,12 Therefore, several studies have been conducted to investigate the regulation and recovery of microbiota, aiming to prevent and treat IBD.¹³

Short-chain fatty acids (SCFAs) are organic fatty acids with one to six carbon atoms. They are mainly produced by carbohydrate fermentation and protein degradation of intestinal microbiota.¹⁴ The hydrochloric acid attack on the intestine is adjusted by SCFAs, which are helpful for the proliferation of probiotics.¹⁵ In addition, the supplementation of SCFAs enhances the intestinal barrier through improved proliferation of healthy colonocytes and reduced infection by pathogenic bacteria.^16,17 It seems that SCFAs effectively regulate the intestinal microenvironment as a medium connected to host–microbiota interplay.

Currently, there is a limited number of treatment options for IBD because of its complex and unclear etiology. The commonly recommended medicine for IBD, such as 5-aminosalicylic acid, immunosuppressive drugs, and anti-TNF-α biological agents, have provided limited beneficial actions but serious side effects.¹⁸ Therefore, there is a great need to develop more effective therapies for IBD.

The 3β-Hydroxy-5-spirostene, also known as diosgenin (DIO), is a steroid sapogenin from herbal medicinal plants, derived from the hydrolysis of dioscin. DIO is not only used as an essential raw material for the production of steroid hormones, but also has wide applications in food development and cosmetic industries.^19,20 The various use of DIO owes to its remarkable biological activities and therapeutic efficacy, including strong antioxidant, anti-inflammatory, anti-infection, hypolipidemic, and hypoglycemic pharmacological effects.^21

–25 DIO has been shown to provide protection against systemic inflammatory response syndrome by inhibiting the NF-κB signaling pathway in mice.²⁶ However, the protective effect of DIO on intestinal inflammation has not been reported.

Here, we have designed a study to explore the effect of diosgenin on colitis induced by DSS, with particular emphasis on relieving intestinal inflammation and regulating the balance of gut microbiota.

Materials and Methods

Collection of chemicals

Diosgenin with purity over 98% was purchased from Nanjing Spring & Autumn Biological Engineering Co. Ltd. (Jiangsu, China). Dextran sulfate sodium (DSS, 36–50 kD) was purchased from Solarbio (Beijing, China). SCFAs (acetic, propionic, butyric, isobutyric, valeric, and isovaleric acids) were obtained from Shanghai Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). Assay kits of fecal occult blood was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Animals and treatment

Healthy male, 6-week-old C57BL/6 mice (body weight about 18.0 ± 1.5 g) were purchased from Dossy Experimental Animals Co. Ltd. (Chengdu, China). The mice were kept in a controlled room with a 12-h light–12-h dark cycle at room temperature (22°C ± 2°C) and constant humidity (50–60%). All mice were allowed ad libitum to food and water in special pathogen-free condition. All animal experiments were approved by the Animal Ethics Committee of Northwest A&F University, and Chengdu Dossy Experimental Animals Co. Ltd. (N0043155).

A total of 24 mice were randomly divided into 4 groups (6 mice per group) after acclimating for 1 week before the experiment. As shown in Figure 1A, colitis was induced in the mice by administration of 2.5% (w/w) DSS for 8 days. Specifically, the control group (CK) and the model group (DSS) were administered intragastrically with normal saline daily, while two diosgenin groups (DIO and DIO+DSS) were administered intragastrically with diosgenin that was emulsified with normal saline (15 mg/kg body weight) for 23 days. Then the mice in DSS and DIO+DSS groups were treated with 2.5% DSS in the last 8 days.

FIG. 1.

Effect of DIO on clinical symptoms of DSS-induced colitis in C57BL/6 mice. Animal model experimental design (A), body weight change (B), and the DAI score measured by body weight loss, rectal bleeding, and diarrhea of mice during the DSS treatment period (C). Animals supplemented orally with diosgenin and administered (DIO+DSS) or not (DIO) 2% DSS for 8 days were compared with nonsupplement animals treated (DSS) or not (CK) with DSS. Data are expressed as mean ± SDs (n = 6). *P < .050, **P < .010 compared with the control group; ^# P < .050, ^## P < .010 compared with the DSS group. DAI, disease activity index; DIO, diosgenin; DSS, dextran sulfate sodium; SD, standard deviation. Color images are available online.

During the experiments, body weight, solid fecal weight, blood in the stool, food, and water consumption were measured daily. The body weight change of each group was calculated as the percent difference between the average original body weight before DSS treatment and that on the observed day.²⁷ At the termination of the experiment, fresh feces were collected into aseptic tubes for 16S rRNA sequencing, and mice were anesthetized with 1% pentobarbital sodium and sacrificed. The cecal contents and half of the colon tissue were taken out, rinsed with cold saline, stored at −80°C for the following experiments, whereas the other half of colon was fixed in 4% poly paraformaldehyde for the histological observation.

Assessment of disease activity index

The grade and severity of intestinal inflammation were assessed by disease activity index (DAI). DAI was calculated as follows: change in stool blood (0: negative, 1: occult blood positive, 2: gross blood), stool consistency (0: normal, 1: loose stool, 2: diarrhea), and body weight loss (0: normal, 1: 1–5%, 2: 5–10%, 3: 10–15% 4: more than 15% weight loss).

Histopathological analysis

Hematoxylin and Eosin (H&E) staining was used to observe pathological changes of the tissue. The colon tissues were fixed with 4% poly paraformaldehyde for 24 h, dehydrated in a graded ethanol series, then embedded in paraffin. The tissue was sectioned and stained with H&E. Histological score was evaluated by a standard grading system, which combined the conditions of intestinal inflammation, lesion depth, and colonic crypt destruction.

Measurement of contents of SCFAs in the colon

The content of SCFAs in feces, including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, pentanoic acid, and total acids, were determined by gas chromatography (GC). Five SCFAs were diluted with diethyl ether to configure standard samples. Samples of feces (0.200 g) were completely mixed with 1 mL ultrapure water by vortex mixer, then 50% H₂SO₄ (0.15 mL) and diethyl ether (1.6 mL) were added into the mixture. The mixtures were centrifuged for 10 min at 8000 g after shaking for 20 min. The upper ether solutions (1.0 mL) were taken and gently blown with a nitrogen blower to concentrate the solution to 0.2 mL. The samples were filtered and loaded onto a GC.

The column was a DB-FFAP, nitrogen was the carrier gas, and the flow rate was 2.0 mL/min. Gas phase conditions were, injection temperature 250°C, injection volume 2 μL, and split ratio 10:1. Heating program is as follows: maintained 50°C for 1 min, gradually increased the temperature to 120°C at a rate of 15°C/min, then increased the temperature to 170°C at a rate of 5°C/min, then increased the temperature to 220°C at 15°C/min, and maintained for 5 min. The detector was hydrogen flame detector FID, the detection temperature was 270°C.

Gut microbiota analysis by 16S rRNA gene sequencing

The microbial community DNA was extracted using the MagPure Stool DNA KF Kit B (Magen, China) following the manufacturer's instructions. DNA was quantified with a Qubit Fluorometer by using the Qubit dsDNA BR Assay Kit (Invitrogen, USA) and the quality was checked by running an aliquot on 1% agarose gel. Variable regions V3–V4 of bacterial 16S rRNA genes were amplified with degenerate PCR primers, 341F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTA AT-3′). The validated libraries were used for sequencing on lumina MiSeq platform (BGI, Shenzhen, China) following the standard pipelines of Illumina, and generating 300 bp paired-end reads.

Data were clustered into Operational Taxonomic Units (OTUs ) with a cutoff value of 97% using UPARSE software (v7.0.1090) and chimera sequences were compared with the Gold database using UCHIME (v4.2.40) for detection.

Statistical analyses

All data were expressed as the mean ± standard deviations. Microsoft Excel 2007 and SPSS 23.0 were used for statistical analyses. Significant differences between groups were analyzed by one-way factorial analysis of variance (ANOVA), and Duncan's multiple-range test was applied to identify group differences. P < .050 was considered statistically significant.

Results

DIO attenuated DSS-induced acute colitis symptoms

To explore the effect of diosgenin on DSS-induced mice, the body weight and DAI of mice in each group were analyzed. Compared with normal mice, DSS-induced mice all showed a decrease in body weight (P < .010). However, the weight loss of mice in the DIO+DSS group was less than that in DSS group (P < .050) (Fig. 1B). On the other hand, the intervention of diosgenin significantly reduced the DAI score of DSS-induced mice (P < .010), while there was no significant difference between the CK and DIO groups (Fig. 1C).

DIO reduced DSS-induced colon histopathological destruction

Compared with the normal architecture of colon sections of mice in the control group, those in the DSS group were obviously damaged, including the ulcerated tissues and incomplete goblet cell structure. However, the colon structure of DIO+DSS group had improved, evidenced by the decrease of inflammatory infiltration and recovery of goblet cell structure (Fig. 2A). The combined analysis of the histological structure in mice showed that DSS treatment increased the histological score of normal mice, while diosgenin had a significant alleviating effect on DSS-induced colon inflammation (P < .010) (Fig. 2B).

FIG. 2.

DIO supplement reduced histological injury in DSS-induced colitis mice. H&E staining of representative colon sections shown under light microscopy (100 × ) (A), the histological scores assessed by colonic architecture condition (B). Data are expressed as mean ± SDs (n = 3). **P < .010 compared with the control group; ^## P < .010 compared with the DSS group. H&E, Hematoxylin and Eosin. Color images are available online.

DIO increased the content of SCFAs in feces

Results showed that the content of total organic acids in the DSS group were significantly higher compared with the control group, but the intervention of diosgenin restored the level (P < .010) (Fig. 3G). The treatment of DIO+DSS led to the increase of acetic acid (Fig. 3A), propionic acid (Fig. 3B), and isobutyric acid (Fig. 3D). Especially, the acetic acid increased from 0.68 to 1.18 mg/g, and propionic acid rose from 0.26 to 0.53 mg/g (P < .050). However, all of the changes of SCFAs between DIO and DIO+DSS group were not obvious.

FIG. 3.

DIO increased several main SCFAs in the feces of DSS-induced colitis mice. The content of acetic acid (A), propionic acid (B), butyric acid (C), isobutyric acid (D), pentanoic acid (E), isovaleric acid (F), and total acids (G). Data are expressed as mean ± SDs (n = 6). *P < .050, **P < .010 compared with the control group; ^# P < .050, ^## P < .010 compared with the DSS group. SCFAs, short-chain fatty acids. Color images are available online.

DIO ameliorated the structure of gut microbiome and regulated microbial community imbalance

To assess the change for species richness of gut microbiota induced by DSS treatment and diosgenin supplement, the similarity of OTUs among groups were described in the Venn diagram (Fig. 4A). The distinctive OTUs in CK, DSS, and DIO+DSS groups, respectively, accounted for 16.3%, 10.1%, 4.2% among the three groups. There was 20.9% of OTU difference between the CK and DSS groups, and there were 22.9% and 19.4% of OTU difference in DIO+DSS group compared with CK and DSS groups, which manifested different abundances of microbes among all groups.

FIG. 4.

The species richness and structure of the fecal microbiota after DIO treatment. The Venn diagram (A), species accumulation curve (B), PCA plot (C), and PLS-DA plot (D) based on OTU. OTUs, Operational Taxonomic Unit; PCA, principal component analysis; PLS-DA, partial least squares discrimination analysis. Color images are available online.

The species accumulation curve reached a plateau as samples increased, indicating that there was enough sequencing depth and species richness (Fig. 5B). Principal component analysis and partial least squares discrimination analysis (PLS-DA) showed the similarity of microbial community among groups visually.

FIG. 5.

The representative changes for the composition of the gut microbial community after DIO intervention at different taxa levels. The taxonomic distributions of the microbial communities at phylum (A) and family levels are described by bar plots (B); the relative ratio of Firmicutes to Bacteroidetes (C); changes for relative abundances of several pathogenic bacteria: Streptococcus (D), Coprobacillus (E), Pseudomonas (F), and probiotics at the genus level: Mucispirillum (G), Veillonella (H), Prevotella (I), Odoribacter (J). “Others” in the plot represents the taxa with relative abundance ≤0.5% in all samples. Data are expressed as mean ± SDs (n = 5). *P < .050 compared with the control group; ^# P < .050, ^## P < .010 compared with the DSS group. Color images are available online.

As is shown in Figure 4C, the CK and DSS groups evidently separated on the main component PC2. The DSS and DIO+DSS groups were not completely separated, but the DSS group approached the CK group. Moreover, PLS-DA suggested that the microbiota composition of mice in DSS group clustered separately from that in other groups, while diosgenin intervention in DIO+DSS group ameliorated the structure of microbiota more similar to the CK group (Fig. 4D). These results indicated that diosgenin supplementation had a positive effect on gut microbiota modulation in DSS-induced mice.

Histograms of species abundance at different taxa levels were constructed to analyze the specific difference in gut microbiota composition among experimental groups. At the phylum level, Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria were presented in all samples and Bacteroidetes and Firmicutes dominated the gut microbiota (Fig. 5A). DSS treatment increased the relative abundance of Firmicutes compared with the CK group, while diosgenin intervention caused a decrease of the Firmicutes proportion. The relative ratio of Firmicutes to Bacteroidetes was elevated in the DSS group when compared with the CK (P < .050) and DIO+DSS group (Fig. 5C).

At the family level, the microbial community influenced by DSS treatment had a higher proportion of Streptococcaceae and Peptostreptococcaceae, which differed from the CK and DIO+DSS group (Fig. 5B). Additionally, the abundance of Lactobacillaceae and Porphyromonadaceae that had decreased in DSS treatment mice, recovered to a higher level after diosgenin supplement.

At the genus level, to explore more information hiding behind the unclassified taxa in the family level, the Kruskal–Wallis test analysis was used to find communities with significant differences among groups. DSS treatment obviously elevated the relative abundance of Streptococcus (from 0.07% to 0.95%), Coprobacillus (from 0.02% to 0.40%), and Pseudomonas (from 0.0004% to 0.004%) compared with the CK group (P < .050), whereas diosgenin intervention decreased the proportion (P < .050) (Fig. 5D–F).

Moreover, diosgenin treatment caused much more Prevotella and Odoribacter in DSS-induced mice by an average of 4.2- and 46.4-fold (P < .050) in the DSS group (Fig. 5I, J). Diosgenin-supplemented mice had the highest abundance of Mucispirillum and Veillonella among three groups, which was an average of 7.1- and 20.6-fold (P < .050) to the DSS group.

Linear discriminant analysis and microbiota clustered heatmap analysis was performed to identify significantly altered bacterial phylotypes (Fig. 6). Compared with the CK group, the dominant microbiota in mice with DSS treatment included some pathogenic bacteria (Deltaproteobacteria, Desulfovibrionaceae, Turicibacteraceae, and Streptococcus). Mucispirillum in DSS-induced mice were increased after diosgenin intervention, which also contributed to a sharp increase in the Deferribacteraceae family.

FIG. 6.

Differential enrichment gut microbiota of animals in three groups. LDA effect (A) and the heatmap of gut microbiota at a genus level (B). LDA, linear discriminant analysis. Color images are available online.

Discussion

In this study, we described the effects of diosgenin intervention on body weight, colon structure, the content of SCFAs in feces, and intestinal microbiota composition of C57BL/6J mice with IBD induced by DSS. Diosgenin alleviated the inflammation in the colon of mice, increased the content of SCFAs in mice with IBD, and prevented the destruction of colon structure and the dysbiosis of intestinal microbiota induced by DSS.

In recent years, the incidence of IBD in China has been increasing year by year, and more and more studies have focused on the pathogenesis and treatment of IBD. Intestinal inflammation could easily cause macrophages to release free radicals, which change the redox state of the intestinal mucus layer.

DSS induced a significant increase in the concentration of reactive oxygen free radicals in the contents of the mouse colon and caused a decrease in total antioxidant capacity.²⁸ The normal oxidative homeostasis of the body needs to maintain the balance between free radicals and antioxidants. The antioxidant defense system is composed of many molecules that cooperate with each other, and can be roughly divided into three forms: superoxide dismutase (SOD), peroxidase, glutathione peroxidase, and other antioxidant enzymes; vitamin C, flavonoids, β-carotene, and other nonenzymatic antioxidant molecules; special proteins and hydrolytic enzymes that repair the oxidative damage of biological molecules are also an important part of the antioxidant system.²⁹

On the other hand, the secretion of proinflammatory cytokines regulated by inflammation-related signal pathways directly affects the development of intestinal epithelial tissue and IBD.³⁰ The NF-κB signaling pathway activated by the inflammatory process can regulate the expression of genes encoding cytokines (such as IL-6) and inflammatory enzymes (such as iNOS and COX-2), and upregulate the content of inflammatory proteins, affecting colonic mucosal integrity.^31,32 Moreover, the pathological process of IBD is also affected by the expression of antioxidant genes. Mice lacking the glutathione peroxidase gene will have symptoms and pathologies consistent with IBD. Compared with wild-type mice, Nrf-2-deficient mice are significantly more sensitive to chemically induced experimental IBD.³³

DIO, a phytochemical with multiple biological activities, has been demonstrated to have great potentiality on the treatment of diverse types of inflammatory diseases.³⁴ It has been pointed out that diosgenin treatment could attenuate oxidative stress through increasing the expression of antioxidant enzymes, such as SOD, HO-1, Nrf-2, and GSH-Px in vivo.^35,36 Acute neuroinflammation caused by endotoxemia could be mitigated by diosgenin treatment, while protecting roles of diosgenin against systemic inflammatory response syndrome has also been demonstrated.³⁷

The occurrence of IBD is affected by many factors, and is closely related to the interaction among environmental pressure, genetic background, and intestinal immune status, and its specific pathogenesis and treatment methods are still unclear.³⁸ However, numerous studies have shown that the composition of intestinal microbiota plays a vital role in the pathophysiology of IBD in experimental animals and human subjects.³⁹

DSS treatment alters the structure of the intestinal microbiota in CK group mice, and after the intervention of diosgenin, the mouse intestinal microbiota was close to the CK group mice. Specifically, the relative ratio of Firmicutes to Bacteroidetes, one of the more important indicators in the composition of intestinal microbiota, prominently increased in DSS group compared with the CK group, while diosgenin recovered the value. The intervention of diosgenin changed the specific microbiota in the stool of mice with colitis, and significantly increased the relative abundance of probiotic bacteria, such as Prevotella, Odoribacter, and Mucispirillum.

At the family level, DSS treatment significantly reduced the relative abundance of Lactobacillaceae and Porphyromonadaceae in the fecal microbiota of mice, which is consistent with previous reports.⁴⁰ Furthermore, it also caused a significant increase in the relative abundance of Streptococcaceae, Desulfovibrionaceae, and Turicibacteraceae. It has been pointed out that the increase of Streptococcaceae is related to metabolic syndrome and colon cancer, while Turicibacteraceae was positively related with colitis.^41,42 However, diosgenin can restore its relative abundance to normal.

SCFAs were produced by the metabolism of the intestinal microbiota, which were an important component for maintaining a normal intestinal environment, and played a positive role in the health of the human intestinal tract. Acetic acid improved the intestinal barrier function of mice with enteropathy;⁴³ butyric acid has also been proven to be the main energy source for colonic epithelial cells.⁴⁴ As is shown, diosgenin treatment can significantly increase the content of acetic acid and propionic acid in the stool of mice with colitis, and alleviate the decline in the total amount of SCFAs. At the same time, diosgenin can change the composition of the intestinal microbiota and specific bacteria in the feces of mice with DSS-induced inflammation, thereby effectively alleviating the disorder of the mouse colon environment and promoting intestinal health.

In a word, we described the effect of diosgenin on DSS-induced intestinal inflammation and intestinal microbiota imbalance in mice through the establishment of animal models and using SCFA analysis, and intestinal microbiota analysis. Diosgenin provides a theoretical basis for preventing IBD and protecting intestinal health, and provides new ideas for the development and application of diosgenin. However, its specific mechanism of protecting intestinal tissues from damage caused by IBD still needs to be further explored.

Footnotes

Authors' Contributions

X.P., Y.H., R.L., and M.X. contributed to the conception and design of the experiment. Material preparation and data collection were performed by Y.H., R.L., M.X., and J.L. Data analysis and the first draft of the article were finished by Y.H. Y.Y., F.M., R.J., and R.W. commented on previous versions of the article. All authors have read and agreed to the published version of the article.

Acknowledgment

The authors appreciate the College of Food Science and Engineering of Northwest A&F University for their technical assistance.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This research work was supported by the Science and Technology Plan Project of Shaanxi Province (2020NY-106), and Student's Platform for Innovation and Entrepreneurship Training Program (X202010712058, X201910712269).

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