Dioscin ameliorates inflammatory bowel disease by up‐regulating miR‐125a‐5p to regulate macrophage polarization

Abstract Purpose Dioscin has been proven to have anti‐cancer, anti‐inflammatory, and anti‐infection roles. However, the role of Dioscin in inflammatory bowel disease (IBD) and its related mechanisms is unclear and needs further study. Methods The colitis model in mice was established. After Dioscin (20, 40, or 80 mg/kg) treatment, the colon length was measured by a ruler. Histopathology, inflammatory cytokines, gut permeability, tight junction proteins, macrophage infiltration, macrophage polarization, and miR‐125a‐5p level were detected by hematoxylin–eosin staining, enzyme‐linked immunosorbent assay, quantitative real‐time polymerase chain reaction (qRT‐PCR), FITC‐dextran, Western blot, and flow cytometry. In vitro experiments, after RAW264.7 cells induced by lipopolysaccharide (LPS)/interleukin‐4 (IL‐4), were treated with Dioscin and miR‐125a‐5p inhibitor, miR‐125a‐5p level, cell vitality, inflammatory cytokines, and M1/M2 marker genes were measured by qRT‐PCR and MTT assay. Results Dioscin (20, 40, or 80 mg/kg) relieved DSS‐triggered colitis and restrained the serum and colon of pro‐inflammatory cytokines expression. Meanwhile, different concentrations' Dioscin weakened M1 macrophage polarization but facilitated tight junction protein expressions, M2 macrophage polarization, and miR‐125a‐5p level in colitic mice. Moreover, miR‐125a‐5p inhibitor reversed the modulation of Dioscin on miR‐125a‐5p expression, cell vitality, and inflammatory cytokines in lipopolysaccharide (LPS)‐induced RAW264.7 cells. We further discovered that Dioscin restrained M1 marker gene (CD16) expression while intensifying M2 marker genes (CD206 and Arginase‐1) expressions in vitro, which was reversed by miR‐125a‐5p inhibitor. Conclusion Dioscin modulated macrophage polarization by increasing miR‐125a‐5p, thereby improving the intestinal epithelial barrier function and reducing IBD.


| INTRODUC TI ON
Inflammatory bowel disease (IBD) is an immune-mediated, chronic, and complex inflammatory disease of the digestive tract, including Chron's disease, ulcerative colitis, and uncertain colitis. 1 The main clinical manifestations of IBD are diarrhea, abdominal pain, bloody stools, loss of appetite, weight loss, and lack of trace elements. 2,3 IBD patients' condition is repeated and protracted, which brings a heavy burden to patients. At present, the pathogenesis of IBD is believed to be caused by multiple factors, including environmental factors and specific genetic factors, in which the intestinal immune system responds too strongly to the normal intestinal flora or the intestinal flora disorder induces abnormal immune responses. 4 Immune system disorder plays a pivotal role in the progression of IBD. 5 As an important innate immune cell, macrophages can be divided into classically activated macrophages (M1 type) and alternatively activated macrophages (M2 type) according to their activation status, which generates a critical role in the development of IBD. 6 In healthy intestines, the resident macrophages are M2-type macrophages with an anti-inflammatory effect, while the inflammatory M1-type macrophages are dominant in the inflammatory intestinal mucosa of IBD. 7,8 More and more scientific studies have shown that the regulation of macrophage activation and polarization has a certain effect on the treatment of IBD. 9,10 In recent years, botanicals have played a crucial function in the treatment of various diseases due to their extensive pharmacological activities, sufficient drug sources, safety, stability, and lasting efficacy. 11,12 Dioscin is a steroidal saponin compound, which can be extracted from the rhizomes of certain Dioscoreaceae plants. 13 Numerous studies have confirmed that Dioscin has the effects of protecting the liver, 14 regulating the body's immune function, 15 anti-inflammatory, 16 and anti-tumor. 17 Dioscin promotes antitumor immunity via inhibiting macrophage M2 polarization, 15 while reduces excessive inflammation via promoting macrophage M2 polarization. 18 Also, Dioscin attenuates murine ulcerative colitis by regulating macrophage polarization. 19 However, whether Dioscin can improve intestinal epithelial barrier dysfunction and inflammation by regulating macrophage polarization has not been reported yet.
Currently, various mouse models of IBD are indispensable tools for the preclinical validation of potential therapies. 20,21 Dextran sulfate sodium (DSS) method was used to induce severe colitis in mice after oral consumption of DSS, which was characterized by weight loss, diarrhea, bloody stool, ulcer formation, loss of epithelial cells, and infiltration of neutrophils. 22 Therefore, this research assessed the role of Dioscin on macrophage polarization in vitro and in vivo and its molecular mechanism using the DSS-induced colitis model in mice and RAW264.7 cells induced by lipopolysaccharide (LPS)/IL-4.

| Ethics statement
The protocol on the animal was allowed in compliance with the regulations of the China Animal Care and Use Committee. This research was authorized by the Committee of Laboratory Animals of Nanfang Hospital (2019030020). We make every effort to mitigate the suffering of animals.

| Dioscin
Dioscin was obtained from Sigma-Aldrich (SMB00576, USA). We fully dissolved Dioscin in 0.5% sodium carboxymethyl cellulose (CMC-Na, IS9000, Solarbio) solution in distilled water for in vivo experiments, and we fully dissolved Dioscin in 0.01% dimethyl sulfoxide (DMSO, D8371, Solarbio) for in vitro experiments. 23 23 On the 11th day, mice were euthanized by cervical dislocation. Next, the blood was obtained by enucleation of the eyeball. Then, mice were sacrificed by cervical dislocation, and colons of the mice were collected and measured using a ruler.
The colon tissues were dehydrated with gradient ethanol and embedded in paraffin. The paraffin-embedded tissues were sectioned, dewaxed, and hydrated. The slices were stained with hematoxylin solution (H8070, Solarbio). Then, we used 0.7% hydrochloric acid ethanol to differentiate for a few seconds and washed immediately.
The slices were stained with 1% eosin solution (G1105, Solarbio) and then dehydrated with gradient ethanol. After the slices were transparent and sealed, we used an optical microscope (Z735515, Sigma) at magnification 100× to observe the slices. The pathological changes in the experimental colitis were evaluated using histopathological score. 26 All the histological evaluations were performed and scored under blinded conditions.
The reaction wells were reacted with 100 μl of standard and test samples and then placed at 37°C for 90 min. Next, 100 μl of the biotinylated antibody working solution was appended to the reaction wells and placed at 37°C for 60 min. Then, 100 μl of enzyme conjugate working solution was appended to the reaction wells and placed at 37°C for 30 min. The reaction wells were then reacted with 100 μl of the chromogenic substrate and developed color at 37°C away from light for 15 min. Finally, 50 μl of stop solution was appended to the reaction wells and the absorbance (450 nm) was determined using a microplate reader (SpectraMax iD5, Molecular Devices).

| Intestinal epithelial permeability in vivo
The flux of FITC-dextran (4kDA, 46994, Sigma-Aldrich) is a classic biomarker for measuring intestinal epithelial permeability. After fasting for 12 h in each group, 20 mg/kg FITC-dextran was administered to the mice and the mice were treated for 4 h before sacrifice. After that, blood samples were collected after sacrifice and coagulated at room temperature for 1 h. Finally, the concentration of FITC-dextran was evaluated at the excitation wavelength (480 nm) and emission wavelength (520 nm) using the microplate reader.

| Isolation of colonic lamina propria mononuclear cells (LPMCs)
First, the colon tissues were washed with prepared phosphate buffer (PBS, D1040, Solarbio) and cut into small pieces. Next, the colonic mass was incubated with PBS without Ca 2+ and Mg 2+ supplemented with 5 mM EDTA (E6758, Sigma-Aldrich) and 1 mM dithiothreitol
MiR-125a-5p I and IC were transfected into RAW264.7 cells, and the transfection efficiency was determined by qRT-PCR after 48 h.

| Cell grouping
To observe the effect of Dioscin and miR-125a-5p I on LPS-induced RAW264.7 cells, the experiments were assigned into the Control group (untreated), LPS group (cells were exposed to 100 ng/ml LPS [L4391, Sigma-Aldrich] for 24 h), 28 LPS + Dioscin group (cells were pre-treated with 150 ng/ml Dioscin for 24 h, and then exposed to 100 ng/ml LPS for 24 h), 23   was determined using the microplate reader.  Figure 1A showed the chemical structure of Dioscin. Next, we meas-

| Dioscin protected the tight junction proteins of colitic mice
In this research, we determined the tight junction protein expressions of colitic mice and manifested that the expressions of Occludin, ZO-1, and Claudin1 were largely attenuated in the colonic tissues of the DSS group than the control group (Figure 2A,B, p < 0.001). On the contrary, Dioscin intensified the above protein expressions in the colonic tissues of DSS-triggered colitic mice in a dose-dependent manner (Figure 2A,B, p < 0.05).

| Dioscin weakened macrophage infiltration and M1 macrophage polarization but facilitated M2 macrophage polarization in colitic mice
The flow cytometry assay manifested that the percentage of macrophages in the colon was largely elevated in the DSS group relative to the control group, while Dioscin obviously restrained the infiltra-

| Dioscin intensified the miR-125a-5p expression in colitic mice
As displayed in Figure 2I, the miR-125a-5p level was largely restrained in the colonic tissues of the DSS group than the control group (p < 0.001). Interestingly, Dioscin greatly elevated the miR-125a-5p level of the colonic tissues in a dose-dependent manner ( Figure 2I, p < 0.05).

| MiR-125a-5p I reversed the promotion of Dioscin on miR-125a-5p expression and cell vitality in LPS-mediated RAW264.7 cells
As exhibited in Figure 3A, we provided evidence to show that Dioscin notably upraised the level of miR-125a-5p in LPS-induced RAW264.7 cells, while miR-125a-5p I was the opposite (p < 0.05).
However, co-treatment of Dioscin and inhibitor reversed the regulation of Dioscin/inhibitor on miR-125a-5p expression ( Figure 3A,

| DISCUSS ION
At present, the non-biological treatment of IBD can relieve symptoms but does not affect the overall course of IBD. 30 In addition, although the biological therapy of monoclonal antibodies has a rapid response, it has many disadvantages and high costs, causing serious social and economic burden. 31 However, botanicals are more effective in the treatment of chronic inflammation due to their multi-component, multi-target, and multi-pathway characteristics. 32 Therefore, it is of practical significance to explore the role of Chinese herbal medicine in the treatment of IBD.
Many natural substances have been proven to have therapeutic effects on inflammatory diseases. For example, Bergenin restrained the activation of macrophages by modulating the PPARγ/SIRT1/ NF-κB-p65 pathway, thereby ameliorating DSS-triggered colitis in mice. 33 Berberine weakened macrophages polarized toward M1 by modulating the AKT1/SOCS1/NF-κB pathway, thus alleviating the incidence of DSS-triggered colitic mice. 34 In this study, we evaluated the effect of Dioscin on DSS-triggered colitis in mice and its underlying mechanism for the first time. The evidence clarified that after Dioscin treatment, the colon length of colitic mice was largely enhanced and Dioscin greatly reduced the inflammatory damage of the colon.
Based on the above results, we further detected the expression of inflammatory factors in serum and tissues of mice with colitis.
Our research results exhibited for the first time that IL-1β, TNFα, Ge et al.'s research has revealed that miR-125a-5p attenuated the IBD progression via targeting ETS-1. 41 The forced expression of miR-125a-5p weakened the expression of TNFα, IL-12, and iNOS triggered by LPS, while miR-125a-5p inhibitor repressed the expression of Arg1 triggered by IL-4. 42 In addition, only one study exhibited that Dioscin ameliorated the metabolic disorder of glucose and lipid in patients with type 2 diabetes by intensifying the inhibition of miR-125a-5p on STAT3. 43 Our study complemented the deficiencies of Dioscin and miR-125a-5p in IBD research. Dioscin upraised the miR-125a-5p level and cell vitality and restrained the IL-1β, TNFα, and IL-6 levels in LPS-induced RAW264.7 cells, while miR-125a-5p inhibitor was the opposite. Nevertheless, the above effects were reversed by co-treatment of Dioscin and miR-125a-5p inhibitor. We further observed that Dioscin greatly restrained the LPS-triggered CD16 expression but facilitated the CD206 and Arginase-1 expressions, which was reversed by co-treatment of Dioscin and miR-125a-5p inhibitor.
IL-4 has been proven to be a classic Th2 cytokine that can trigger M2 macrophage polarization. 44 We discovered that Dioscin further enhanced the expressions of CD206 and Arginase-1 after IL-4 exposure, while miR-125a-5p inhibitor was the opposite. More importantly, these effects were all overturned by co-treatment of Dioscin and miR-125a-5p inhibitor. All in all, Dioscin has a protective effect on DSS-triggered colitis and LPS/IL-4-triggered RAW264.7 cell injury, which is achieved by restraining the inflammatory response and colonic macrophage infiltration, especially by regulating the polarization of macrophages.

AUTH O R CO NTR I B UTI O N S
Lingyan Shi and Peichen Zhang substantially contributed to conception and design and involved in drafting the article or critically revising it for important intellectual content. Ruifang Jin, Xiaowei Chen, Lemei Dong, and Weichang Chen contributed to data acquisition, data analysis, and interpretation. All the authors contributed to the final approval of the version to be published and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.