International Immunopharmacology 117 (2023) 109839

Contents lists available at ScienceDirect

International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

Dioscin modulates macrophages polarization and MDSCs differentiation to

inhibit tumorigenesis of colitis-associated colorectal cancer
Jing Xun a, b, 1, Siying Zhou c, 1, Zongjing Lv c, Botao Wang d, Hai Luo c, Lanqiu Zhang a, b,
Lei Yang a, b, Aimin Zhang a, Xueliang Wu e, Zhenyu Wang a, b, Ximo Wang b, f, Xiangyang Yu a, b, *,
Qi Zhang a, b, *
a
Tianjin Key Laboratory of Acute Abdomen Disease Associated Organ Injury and ITCWM Repair, Tianjin Nankai Hospital, Tianjin Medical University, Tianjin 300100,
China
b
Integrated Chinese and Western Medicine Hospital, Tianjin University, Tianjin 300100, China
c
Graduate School, Tianjin Medical University, Tianjin 300100, China
d
Chongqing Traditional Chinese Medicine Hospital, Chongqing 400021, China
e
Department of General Surgery, The First Affiliated Hospital of Hebei North University, Zhangjiakou 075000, China
f
Institute of Integrative Medicine for Acute Abdominal Diseases, Tianjin Medical University, Tianjin 300100, China

A R T I C L E I N F O A B S T R A C T

Keywords: It has been reported that colitis is one of risk factors in colorectal cancer (CRC). Intervention of intestinal
Colitis-associated colorectal cancer inflammation and in the early stage of tumorigenesis is of great significance to control the incidence and mor­
Tumorigenesis tality of CRC. In recent years, natural active products of traditional Chinese medicine have been confirmed that
Dioscin
they had made great progress in disease prevention. Here, we showed that Dioscin, a natural active product of
Macrophage polarization
Dioscorea nipponica Makino, inhibited initiation and tumorigenesis of AOM/DSS-induced colitis-associated
Myeloid-derived suppressor cells
colon cancer (CAC), including alleviating colonic inflammation, improving intestinal barrier function and
decreasing tumor burden. In addition, we also explored the immunoregulatory effect of Dioscin on mice. The
results showed that Dioscin modulated M1/M2 macrophages phenotype in spleen and decreased monocytic
myeloid-derived suppressor cells (M− MDSCs) population in blood and spleen of mice. The in vitro assay
demonstrated that Dioscin promoted M1 as well as inhibited M2 macrophages phenotype in LPS- or IL-4-induced
bone marrow-derived macrophages (BMDMs) model. Based on the plasticity of MDSCs and its ability to differ­
entiate into M1/M2 macrophages, we here found that Dioscin increased M1- and decreased M2-like phenotype
during the process of MDSCs differentiation in vitro, suggesting Dioscin promoted MDSCs differentiate into M1 as
well as inhibited its differentiation into M2 macrophages. Taken together, our study indicated that Dioscin had
the inhibitory effect on the initial of tumorigenesis at early stage of CAC via the ant-inflammatory effect, which
provided a natural active candidate for effective prevention of CAC.

1. Introduction and individuals with chronic intestinal inflammation have a signifi­

cantly increased risk for developing colorectal cancer compared to ones
Colorectal cancer (CRC) has become the third most diagnosed cancer without colitis [5–7]. As colitis is one of the predisposing risk factors in
and the second most fatal cancer, which seriously threatens human CRC, colitis-associated colorectal cancer (CAC) accounts for approxi­
health [1]. Previous epidemiological studies have demonstrated that mately 5 % of CRC cases [8,9]. Therefore, intervention of intestinal
persistent inflammatory stimuli are frequently associated with the inflammation and in the early stage of tumorigenesis is of great signif­
development of cancer [2–4]. Inflammatory bowel diseases (IBDs) are a icance to control the incidence and mortality of CRC.
salient example of the link between chronic inflammation and cancer, It has been reported that CAC progresses through chronic

* Corresponding authors at: Tianjin Key Laboratory of Acute Abdomen Disease Associated Organ Injury and ITCWM Repair, Tianjin Nankai Hospital, Tianjin
Medical University, Tianjin 300100, China.
E-mail addresses: [email protected] (X. Yu), [email protected] (Q. Zhang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.intimp.2023.109839
Received 6 December 2022; Received in revised form 17 January 2023; Accepted 30 January 2023
Available online 19 February 2023
1567-5769/© 2023 Published by Elsevier B.V.
J. Xun et al. International Immunopharmacology 117 (2023) 109839

inflammation, dysplastic precursor lesions, low-grade dysplastic, high- the AOM/DSS-treated mice. Dioscin also modulated M1/M2 macro­
grade dysplastic, carcinoma [10]. Intestinal macrophages, the largest phages phenotype and decreased M− MDSCs population in mice. The in
number of mononuclear phagocytes in the intestine, playing an impor­ vitro assay demonstrated that Dioscin promoted M1 as well as inhibited
tant role in CAC progression. As we know, macrophages derived from M2 macrophages phenotype in LPS- or IL-4-induced bone marrow-
monocyte precursors have strong plasticity and undergo differentiation derived macrophages (BMDMs) model. In addition, Dioscin promoted
depending on the local tissue environment. LPS and IFN-γ polarize MDSCs differentiation into M1 as well as inhibit its differentiation into
macrophages toward M1 phenotype, which secrete pro-inflammatory M2 macrophages in vitro. These data suggested that Dioscin maybe
cytokines, such as TNF-α, IL-1β, IL-6 and IL-12. M1 macrophages pro­ regulated M1/M2 macrophages polarization and MDSCs differentiation
duce inducible nitric oxide synthase (iNOS) and promote Th1 responses during inhibiting CAC progression. Our research provided an efficiency
and inhibit tumor progression. M1 macrophages also express high level strategy for prevention of CAC.
of major histocompatibility complex II (MHC II), CD86, CD68. In
contrast, M2 macrophages with anti-inflammatory phenotype are acti­ 2. Materials and methods
vated by IL-4, IL-10 and IL-13. These cells express high level of IL-10,
arginase and CD206, playing a key role in promoting tumor progres­ 2.1. Drugs and reagents
sion [11,12]. Evidence shows that macrophages play a central role in the
dynamic processes of CAC progression. In the early stage of CAC, mac­ Azoxymethane (AOM) was purchased from Sigma-Aldrich Corp.(St.
rophages increase and mainly exhibit M1-like pro-inflammatory Louis, MO, USA). Dextran Sulfate Sodium Salt (DSS) (MW = 36 kDa-50
phenotype, which promote inflammatory injury. And M2-like macro­ kDa) was purchased from Yeasen Biotech (Shanghai, China). LPS was
phages inhibit tumor development by alleviating intestinal inflamma­ purchased from Solarbio Science & Technology (Beijing, China).
tion. While in the middle and late stage of CAC, M2-like macrophages M− CSF, GM-CSF, recombinant murine IL-4 and IL-6 were obtained from
are dominant and play a role in promoting tumor [13–15]. Therefore, PeproTech (Rocky Hill, NJ, USA). Dioscin was purchased from Med­
tumor-associated macrophages (TAMs) in tumor microenvironment ChemExpress (Princeton, NJ, USA).
(TME) have been thought to be composed of M1 and M2 two pheno­
types. But TAMs are generally characterized as M2-like macrophages 2.2. Animals studies
and correlate with poor prognosis of patients with cancer [16,17].
In addition to TAMs, myeloid-derived suppressor cells (MDSCs) also Male C57BL/6 mice (six-eight weeks old) were purchased from SPF
play important roles in creating an immunosuppressive TME [11,12]. (Beijing) Biotechnology Co., ltd. (Beijing, China), maintained in a
MDSCs are a mixed population of immature and highly immunosup­ specific-pathogen-free facility at Tianjin Nankai Hospital and randomly
pressive monocytic and granulocytic lineage cells in TME. MDSCs divided into five groups (n = 8). All in vivo procedures were performed in
commonly co-express CD11b and Gr1 (Ly6C and Ly6G) in mice. There accordance with protocols approved by the Animal Ethics Committee of
are two distinct subsets of MDSCs that have been identified, monocytic Tianjin Nankai Hospital (Approved NO. NKYY-DWLL-2022-106). To
MDSCs (M− MDSCs, CD11b+Ly6G-Ly6Chigh) and granulocytic MDSCs establish the CAC model, mice were intraperitoneally injected with 10
(G-MDSCs, CD11b+Ly6G+Ly6Clow). The expanded MDSCs inhibit pro­ mg/kg AOM and after one week, received drinking water containing 2.5
liferation and function of T cells by producing Arg1 and ROS [18]. More % DSS for one week. Mice then were given purified water for two weeks,
importantly, MDSCs differentiate M1- and M2-like TAMs under hypoxia followed by two additional DSS treatment cycles (Fig. 1A). During the
condition in tumor microenvironment [19–21]. Kitamura et al. whole time of the study, the control group was provided with normal
demonstrated that macrophage-committed precursors in tumors have water. For the treatment of CAC model, different dose of Dioscin (2.5, 5,
been shown to phenocopy M− MDSCs behavior [22]. TLR7/8 agonist 10 mg/kg) dissolved in 0.5 % CMC-Na were orally administered by
reverses oxaliplatin resistance via directing MDSCs to M1-lilke macro­ gastric gavage for every-two days. Vehicle were administered in control
phages in CRC [19]. These studies suggest that remodeling TAMs and mice. At the end of study, all animals were sacrificed by cervical dislo­
MDSCs are potential strategy for tumor therapy. cation after eyeball blood collection, and colon tissues were collected for
In recent years, a large number of pharmacological studies on natural subsequent analysis [30,31].
active products of traditional Chinese medicine have made great prog­
ress in disease prevention. Dioscin is one active compound extracted 2.3. Assessment of disease activity index (DAI) and histological analysis
from the roots of Dioscorea nipponica Makino. It has been reported that
Dioscin has anti-tumor, anti-inflammation, immunoregulation, anti- The distal colon tissues were fixed in 4 % paraformaldehyde for 24 h,
viral, anti-fungal and anti-allergic effects [23]. Dioscin could attenuate embedded in paraffin, sectioned and then stained with hematoxylin and
cisplatin-induced mucositis in rats by modulating the gut microflora eosin (H&E). The histological scores were determined using a previously
profile, enhancing intestinal barrier function and inhibiting the in­ described method [32]. The disease activity index scores were calcu­
flammatory response through the TLR4-MyD88-NF-κB pathway [24]. lated by the following method, based on the loss of body weight, stool
Besides, Dioscin could ameliorated the progress of osteoarthritis via consistency and degree of fecal bleeding: (a) body weight loss: 0 points
inhibiting Wnt/β-catenin pathway and upregulating PPAR-γ expression = none; 1 points = 1–5 %; 2 points = 5–10 %; 3 points = 10–15 %; 4
[25]. Moreover, Dioscin could alleviate crystalline silica-induced pul­ points = over 15 %; (b) stool consistency: 0 points = normal; 2 points =
monary inflammation and fibrosis [26,27]. More importantly, Dioscin loose stools; 4 points = diarrhea; (c) gross bleeding: 0 points = normal; 2
could inhibit inflammation and tumor progression by regulating mac­ points = stools with slightly blood; 4 points = gross bleeding [33,34].
rophages polarization. Studies have shown that Dioscin ameliorated UC
in DSS mice by inhibiting macrophage M1 polarization and promoting 2.4. Quantitative real-time polymerase chain reaction (qRT-PCR)
M2 polarization [28]. In addition, Dioscin inhibited melanoma pro­
gression by targeting Connexin 43 (a vital gap junction protein in tumor The total RNA of colon tissues and cells was extracted by using Trizol
microenvironment) and inducing M1 polarization [29]. Whether Dio­ (Invitrogen) reagent according to the protocol and then 2 ug RNA was
scin intervention have effects on the development of CAC remains to be reversely transcribed to cDNA using Hifair® III 1st Strand cDNA Syn­
determined. Therefore, we explored the effect of Dioscin on CAC pro­ thesis Kit (Yeasen Biotech) according to the manufacturer’s instructions.
gression and its immunomodulatory effects. The qPCR procedures were carried out using Hieff® qPCR SYBR Green
Here, our study revealed that Dioscin suppressed CAC initiation and Master Mix reagent (Yeasen Biotech) and the reaction was run under the
tumorigenesis, including alleviating colonic inflammation and conditions according to the manufacturer’s instructions. The relative
improving intestinal barrier function and decreasing tumor burden in mRNA expression used GAPDH as gene expression normalization and

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J. Xun et al. International Immunopharmacology 117 (2023) 109839

Fig. 1. Dioscin alleviated tumorigenesis of AOM/ DSS-induced colitis-associated colon cancer. (A) A brief schematic of colitis-associated colon cancer model
induced by AOM/ DSS. (B) Body weight of mice during the entire study. (C-E) Disease activity index (DAI) scores during 3 cycles of AOM/DSS treatment. (F) The
statistical results of DAI during the entire study. (G) Representative picture of colon length. (H) The statistical results of colon length in all groups. (I) Representative
images of colon tissues with tumor burden. (J) The statistical results of tumor number and tumor sizes. (K) The statistical results of tumor burdens. Data are shown as
means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

was calculated by using the 2-△△Ct method. The primer sequences were containing protease and phosphatase inhibitors. The protein quantifi­
listed in Supplementary Table 1. cation used the Pierce™ BCA Protein Assay (Thermo Scientific, USA).
Western blotting analysis was performed according to published pro­
tocols. Briefly, protein samples were separated by 10 % SDS-PAGE and
2.5. Western blotting analysis transferred onto PVDF membranes. Membranes were blocked with 5 %
non-fat milk for 1 h at room temperature and then incubated at 4 ℃
The protein of tissues and cells was lysed with the RIPA lysis buffer

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overnight with different primary antibodies. Primary antibodies were as 2.10. Statistical analysis
follows: ZO-1 (#13663 s), occluding (#91131 s), p-NF-κB p65 (#3033),
NF-κB p65 (#8242) antibodies were obtained from Cell Signaling All data were presented as the mean ± standard error of the mean
Technology (Danvers, MA, USA). The GAPDH antibody (AB0037) was (SEM). Statistical analysis was performed on GraphPad Prism 9.0 soft­
purchased from Abways Technology (Shanghai, China). β-actin (AC026) ware. Statistical significance was analyzed by using one-way or two-way
and Arg1 (A4923) antibodies were obtained from ABclonal Technology analysis of variance test. P ≤ 0.05 was considered to be statistically
(Wuhan, China). The CD206 antibody (ab64693) was purchased from significant.
Abcam (Cambridge, UK). CD86 antibody (sc-28347) was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA). iNOS (80517-1- 3. Result
RR) antibody was obtained from Proteintech (Rosemont, IL, USA). After
that, membranes were incubated with horseradish-peroxidase conju­ 3.1. Dioscin suppressed the development of AOM/DSS-induced colitis-
gated secondary antibodies at room temperature for 1 h. Finally, associated colon cancer
immunocomplexes were detected using an ECL chemiluminescence kit
(Millipore). To determine the effect of Dioscin on CAC progression, we estab­
lished an AOM/DSS-induced mouse model (Fig. 1A). Mice were
2.6. Immunohistochemical staining randomly divided into five groups (n = 8): Control (Ctrl), Model (MOD),
Dioscin-Low (Dio-L, 2.5 mg/kg), Dioscin-Medium (Dio-M, 5 mg/kg) and
Formaldehyde-fixed, paraffin-embedded tissue sections were Dioscin-High (Dio-H, 10 mg/Kg). As expected, the AOM/DSS-treated
deparaffinized using xylene and alcohol and then pre-treated with an­ model mice showed obvious clinical characteristics of chronic colitis
tigen retrieval in citrate buffer (pH6.0, Solarbio Science & Technology, such as body weight loss, diarrhea, blood stool and increased DAI score
Beijing, China). After that, sections were incubated with 3 % H2O2 and during 3 cycles of DSS treatment. However, Dioscin treatment exhibited
followed by blocking with 5 % goat serum. The sections were then a less distinct weight loss, diarrhea and blood stool, as indicated by
incubated with primary antibodies at 4 ℃ overnight followed by incu­ significantly reduced DAI scores compared with model group. Although
bation with a biotinylated secondary antibody and then incubation with there was no dose effect of Dioscin (Fig. 1B-F). According to previous
an avidin-peroxidase complex. Immunocomplexes were detected using reports, colon length is an important indicator for evaluating the
diaminobenzidine, and the cells were counterstained with hematoxylin. severity of DSS induced colitis. Here, we found that compared with the
Images were acquired using an inverted microscope (Olympus Co., control group, the colon length was significantly shorter in the model
Tokyo, Japan). group, while treatment with Dioscin, especially high dose of Dioscin
significantly increased the colon length in AOM/DSS treated mice
2.7. Immunofluorescence staining (Fig. 1G, H). Furthermore, the colon tissue of mice was longitudinally
dissected and adenomas were observed in the distal colon of the model
Cells were fixed with methanol at 4℃ for 15 mins, and blocked with group, while the condition was improved in the Dioscin treated groups
1 % BSA for 1 h at room temperature. The cells were then incubated with (Fig. 1I). Then, the adenomas in the colon were removed for measure­
primary antibodies at 4 ◦ C overnight, followed by incubation with the ment and statistics. Compared with the model group, the number of
secondary antibody at room temperature for 1 h. Nuclear DNA was tumors and tumor burden in the Dioscin treatment groups were reduced
labelled with DAPI. Images were acquired with a fluorescence micro­ (Fig. 1J, K). These results indicate that Dioscin protects against colitis-
scope (Leica, Germany). associated tumorigenesis.

2.8. Macrophages preparation and polarization 3.2. Dioscin improved colonic inflammation and intestinal barrier
function in the AOM/DSS-treated mice
The bone marrow-derived macrophages (BMDMs) and myeloid-
derived suppressor cells (MDSCs) were obtained as previously To confirm the inhibitory effect of Dioscin on colitis-associated
described [35–37]. Briefly, for bone marrow-derived macrophages tumorigenesis, histopathology was used to further observe the pathol­
(BMDMs), 8-week-old C57BL/6 mice are sacrificed by cervical disloca­ ogy of colonic morphology and structure in AOM/DSS treated mice and
tion, femur and tibia bones were flushed by using sterile syringes. Bone Dioscin treated mice. The results showed that in the mice of model
marrow cells were cultured with Dulbecco’s Modified Eagle’s Medium groups, the structure of colon tissues was significantly damaged, in­
supplemented with 10 % fetal bovine serum (GIBCO, USA) and M− CSF flammatory infiltration occurred, crypts were damaged or even dis­
(10 ng/ml) for 6 days to differentiate into BMDMs. For BMDMs polari­ appeared, and colonic adenoma appeared. While these symptoms were
zation, BMDMs were treated with LPS (500 ng/ml) or IL-4 (20 ng/ml) significantly reduced after Dioscin administration (Fig. 2A). In addition,
alone or in combination with Dioscin (0.5, 1 μM). In the case of MDSCs, the histological scores of the colon were evaluated by the severity of
bone marrow cells were treated with GM-CSF (20 ng/ml) and IL-6 (20 inflammation, degree of inflammation infiltration, tissue regeneration,
ng/ml) for 4 days. and crypt damage. The results showed that the histological scores were
significantly reduced after Dioscin treatment compared to the model
2.9. Flow cytometry group (Fig. 2B). We further analyzed changes in colonic inflammation in
inflammatory tissue adjacent to cancer. Compared with the control
Single cells were resuspended in PBS containing 1 % BSA and stained group, the mRNA levels of pro-inflammatory factors TNF-α, IL-6 and IL-
with antibodies for 30 min at room temperature in the dark. The anti­ 1β were significantly increased in the model group, while the mRNA
bodies were as follows: CD45-APC (E-AB-F1136E, Elabscience Biotech­ level of anti-inflammatory factor IL-10 was decreased. Conversely, the
nology Co.,ltd. Wuhan, China), CD3-FITC (E-AB-F1013c), CD4-percp (E- levels of pro-inflammatory factors in the Dioscin groups were decreased
AB-F1097F), CD8-PE (E-AB-F1104D), CD11b-percp (E-AB-F1081F), F4/ and the mRNA level of the anti-inflammatory factor IL-10 was increased
80-PEcy7 (E-AB-F0995H), MHCII-PE (E-AB-F0990D), ly6C-PEcy7 (E- in a dose independent manner compared with the model group (Fig. 2C).
AB-F1121H), Ly6G-PE (E-AB-F1108D). CD86-APC (105012, BioLegend, Previous studies have shown that the activation of NF-ĸB pathway plays
San Diego, California, USA), CD206-APC (141708, BioLegend). Cells an important role in inflammation. Our data also showed that AOM/DSS
were then analyzed using a EXFLOW flow cytometer (Dakewe, Beijing, treatment significantly activated phosphorylated NF-κB p65 expression
China), and data analysis was performed using Flowjo software (Tree compared with control group, while Dioscin interventions, especially
Star, Inc., Ashland, OR). Dioscin-Medium group profoundly inhibited the phosphorylation of NF-

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Fig. 2. Dioscin improved colonic inflammation and intestinal barrier function in the AOM/DSS-treated mice. (A) Representative H&E staining of the fixed
sections of the colonic tissues. (B) Histological score of colons. (C) The relative expressions of inflammation-related genes in colon tissues. (D-E) IHC staining of
occludin expression and its statistical analysis. (F-G) IHC staining of ZO-1 expression and its statistical analysis. (H) Western blotting of the expressions of the NF-κB
signaling pathway and the expressions of intestinal barrier function-related proteins. (I)The relative expression of proteins after normalization against GAPDH. Data
are shown as means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

ĸB (Fig. 2H, I). It has been reported that intestinal integrity playing an Dioscin plays an important role in inhibiting AOM/DSS-induced colonic
important role in suppressing tumorigenesis. Therefore, we evaluated inflammatory responses and improving gut barrier function during
the expression of intestinal epithelial tight junction proteins, including tumorigenesis.
ZO-1 and occludin. Our data showed that, Dioscin treatment signifi­
cantly increased the expressions of ZO-1 and occludin compared with
the model group (Fig. 2D-I). Taken together, these results suggest that

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3.3. Dioscin regulated the ratio of macrophages and decreased MDSCs in data showed that the increased mRNA and protein expression of M2
the AOM/DSS-treated mice markers Arg1 and Mrc1 induced by IL-4 were significantly inhibited
after Dioscin treatment (Fig. 5B-D). Furthermore, flow cytometry and
We found that dioscin not only inhibited CAC progression, but also immunofluorescence assay demonstrated that Dioscin inhibited IL-4-
decreased the weight of spleen (Fig. S1A). Previous study has shown that induced BMDMs towards M2 phenotype (CD206+) (Fig. 5E-G). Taken
Dioscin could inhibit inflammation and tumor progression by regulating together, these results indicate that Dioscin inhibits M2 macrophages
macrophage polarization [28]. This suggests that Dioscin may regulate polarization of BMDM cells.
immune microenvironment of mice. Therefore, we further analyzed the
changes of immune cells in blood and spleen. Flow cytometry analysis 3.6. Dioscin promoted the differentiation of MDSCs into M1-lilke and
showed that the proportion of CD4+ T cells and CD+8 T cells in blood inhibited its differentiation into M2-like macrophages in vitro
decreased in AOM/DSS-treated mice compared with control. However,
these cells were recovered after Dioscin administration (Fig. 3A, B). The Since M− MDSCs subsets has potential to differentiated in macro­
similar results were obtained in spleen (Fig. S2A, B). We also found that phages and form M1- and M2-like TAMs in tumor site, and we found that
compared with the control group, percentage of M− MDSCs in the blood there was a significant decrease of M− MDSCs in spleen after Dioscin
and spleen of model group were increased, while Dioscin significantly treatment compared with AOM/DSS-induced mice (Fig. 3C, D), so we
reduced them. But there were no obvious changes of G-MDSCs among all speculated that Dioscin may promoted M− MDSCs differentiation into
groups (Fig. 3C, D). Furthermore, we assessed the proportion of M1 and M1-like macrophages. To confirm our suspicion, bone marrow cells were
M2 macrophages in spleen of mice. Interestingly, the ratio of M1 and M2 treated with GM-CSF and IL-6 for 4 days to differentiate into MDSCs or
in the AOM/DSS model group was increased compared to the control combination with Dioscin (Fig. 6A), then the proportion or markers of
group. On one hand, the proportion of M1 in Dioscin-medium group M1 and M2-like macrophages were detected. Flow cytometry analysis
were increased versus to model group, although there was no significant showed that the ratio of M1-like macrophages (CD86+) increased, while
difference in the low-dose and high-dose groups. On the other hand, M2-like macrophages (CD206+) decreased after Dioscin treatment
there was a significant decrease of M2 macrophages percentage after (Fig. 6B, C). In addition, Dioscin increased or decreased the mRNA levels
high dose of Dioscin treatment (Fig. 3E, F). Then, we examined the of M1-like macrophages markers TNF-α, IL-6 or M2-like markers Arg1
expression of iNOS and Arg1, a marker of M1 and M2 macrophages (Fig. 6D). Western blot analysis also exhibited an increase of M1-related
respectively, in colon tumor tissues. The results showed that Dioscin marker iNOS expression and a decrease of M2 marker Arg1 expression
increased AOM/DSS-induced the decrease of iNOS expression, while (Fig. 6E, F). Considering the cytotoxic effect of Dioscin on MDSCs
decreased AOM/DSS-induced the increase of Arg1 expression (Fig. 3G, reduction, CCK8 and PI/Annexin-VI assay were used to further analysis.
H). These data suggest that Dioscin has the immunomodulatory effect on The results showed that Dioscin treatment decreased cell viability and
CAC progression, including decreasing or increasing the proportion of promoted cells apoptosis (Fig. S4A-C). All above results indicate that
M− MDSCs and T cells, promoting M1 and inhibiting M2 macrophages. Dioscin not only promotes apoptosis of MDSCs, but also promotes bone
Previous studies have shown that Dioscin could induce M1 macrophages marrow (BM) cells differentiation into M1-like macrophages, while in­
polarization in TAMs-mediated melanoma metastasis [29]. Based on our hibits its differentiation into M2-like macrophages during the process of
results that the change of M1/M2 proportion in Dioscin treated AOM/ MDSCs differentiation.
DSS mouse model, suggesting that Dioscin may regulate macrophages To identify whether Dioscin regulates MDSCs differentiate into
polarization. We therefore using an LPS- or IL-4-induced bone marrow macrophages, we further treated MDSCs with Dioscin. Briefly, BM cells
derived macrophages (BMDMs) model to explore the effect of Dioscin on were induced to differentiate into MDSCs with GM-CSF and IL-6 for 4
macrophages polarization. days, then MDSCs were treated with Dioscin for 24 h to detect M1/M2
macrophages polarization (Fig. 6G). The results flow cytometry showed
3.4. Dioscin promoted M1 macrophages polarization in LPS-induced that Dioscin increased or decreased percentage of M1- or M2-like mac­
BMDMs model rophages (Fig. 6H, I). Consistently, the increase of M1-related and
decrease of M2-related markers were also observed in mRNA or protein
To identify the regulatory effect of Dioscin on M1 macrophages, bone levels (Fig. 6J-L). All these results demonstrate that Dioscin has the
marrow cells were cultured with M− CSF for 6 days to differentiate into ability to promote the differentiation of MDSCs into M1-like
BMDMs. According to previous study and the result of Raw264.7 with macrophages.
Dioscin treatment, we chose two doses of Dioscin (0.5 μM, 1 μM) which
had no cytotoxicity effect on BMDMs (Fig. S3A, B) [38]. Then, BMDMs 4. Discussion
were stimulated with LPS alone or in combination with Dioscin
(Fig. 4A), and M1-related markers were detected. The results showed The research and discussion surrounding inflammation and cancer
that the mRNA expressions of IL-6, IL-1β and iNOS were significantly has always been a hot topic. Based on previous evidences, inflammation
increased after Dioscin treatment compared with LPS alone group is causally related to cancer development. However, the role of
(Fig. 4B). Flow cytometry analysis demonstrated that Dioscin treatment inflammation in tumor development depends on whether the inflam­
increased percentage of CD86+ M1 macrophages compared with LPS mation is acute or chronic [39,40]. In general, chronic and persistent
group (Fig. 4C, D). Correspondingly, immunofluorescence staining also inflammation is considered to be a risk factor that triggers the devel­
showed similar results (Fig. 4E). Furthermore, we also found a dramatic opment and progression of tumors, such as gastric, colon, and hepatic
increase in the expression of NF-ĸB phosphorylation and iNOS at protein cancer. Besides, the development of chronic inflammation to tumor is a
levels upon Dioscin treatment than LPS (Fig. 4F, G). These results sug­ complex process, including chronic inflammation, dysplastic precursor
gest that Dioscin could promote BMDMs towards M1 polarization. lesions, low-grade dysplastic, high-grade dysplastic, carcinoma [10], in
which the research on immune cells and microenvironments has never
3.5. Dioscin inhibited M2 macrophages polarization in IL-4-induced stopped [41–43].
BMDMs model Previous studies have demonstrated Dioscin could reduce inflam­
mation of DSS-induced colitis by reversing the cytokines levels. At the
Next, we evaluated the effect of Dioscin on M2 macrophages polar­ same time, Dioscin could maintain the intestinal barrier function by
ization. Similarly, IL-4-stimulated cell model was employed. BMDMs increasing the expression of zonula occludens-1 (ZO-1), occludin and
were treated with IL-4 alone or in combination with Dioscin for 24 h mucin (Muc)-2. Consequently, Dioscin may become a competent
(Fig. 5A), then M2 macrophages-related markers were measured. Our candidate for ulcerative colitis (UC) therapy in the future [44].

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Fig. 3. Dioscin regulated the ratio of macrophages and decreased MDSCs in the AOM/DSS-treated mice. (A, B) Representative flow cytometric plots and
frequency of CD4+T cells (CD3+CD4+) and CD8+T cells (CD3+CD8+) in blood. (C, D) Representative flow cytometric plots and frequency of M− MDSCs
(CD11b+Ly6G-Ly6Chigh) and G-MDSCs (CD11b+Ly6G+Ly6Clow) in blood and spleen. (E, F) Representative flow cytometric plots and frequency of M1 (CD11b+F4/
80+MHCII+) and M2 (CD11b+F4/80+CD206+) macrophages in spleen of mice. (G) Western blotting of the expressions of iNOS and Arg1. (H) The relative expression
of proteins after normalization against GAPDH. Data are shown as means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig. 4. Dioscin promoted M1 macrophages polarization in LPS-induced BMDMs model. (A) The schematic representation of bone marrow derived monocyte
differentiating into macrophages and M1 macrophages. (B) qRT-PCR analysis of mRNA expressions of M1 markers Il-1β, IL-6 and iNOS in cell lysates. (C, D)
Representative flow cytometric plots and percentage of CD86+ cells. (E) Immunofluorescence staining identified the proportion of CD86+ M1 macrophages. (F)
Western blotting of the expressions of the NF-κB signaling pathway and iNOS. (G) The relative expression of proteins after normalization against GAPDH. Data are
shown as means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

However, its regulatory effect on tumor development induced by Dioscin could alleviate inflammation of colon tissues and improve in­
chronic inflammation is unknown. Here, we used AOM/DSS model to testinal barrier function. In the early inflammatory stage of CAC, chronic
simulate the pathological process of CAC in mice, and evaluated the inflammation might be able to trigger tumor initiation in the presence of
therapeutic effects of Dioscin in this model. Our data showed that a mutagenic activation of oncogenes in colon epithelial cells

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J. Xun et al. International Immunopharmacology 117 (2023) 109839

Fig. 5. Dioscin inhibited M2 macrophages polarization in IL-4-induced BMDMs model. (A) The Schematic representation of bo ne marrow derived monocyte
differentiating into macrophages and M2 macrophages. (B)The mRNA expressions of M2 markers Arg1 and Mrc1 in cell lysates were detected by qRT-PCR. (C)
Western blotting of the expressions of Arg1 and Mrc1. (D) The relative expression of proteins after normalization against GAPDH. (E-F) Representative flow
cytometric plots. and percentage of CD206+ cells (G) Immunofluorescence staining identified the proportion of CD206+ M2 macrophages. Data are shown as means
± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

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J. Xun et al. International Immunopharmacology 117 (2023) 109839

Fig. 6. Dioscin promoted the differentiation of MDSCs to M1-lilke and inhibited its differentiation to M2-like macrophages in vitro. (A)The Schematic
representation of bone marrow derived monocyte differentiation into MDSCs and Dioscin treatment schedule. (B, C) The percentage of CD86 and CD206 was
detected by flow cytometry. (D) RT-qPCR analysis of M1- and M2-like macrophages markers mRNA expression. (E) Western blotting of the expressions of iNOS and
Arg1. (F) The relative expression of proteins after normalization against β-actin. (G) Bone marrow derived monocytes differentiated into MDSCs, and then MDSCs
were treatment with Dioscin, the schematic illustration of experiments was shown. (H, I) The percentage of CD86 + and CD206+ macrophages were detected by flow
cytometry. (J) The mRNA expressions of M1 markers (TNF-α, IL-6) and M2 markers (Arg-1, Mrc-1) were detected by RT-qPCR. (K) Western blotting of the expressions
of iNOS and Arg1. (L) The relative expression of proteins after normalization against β-actin. Data are shown as means ± SEM of three independent experiments. *P
< 0.05, **P < 0.01, ***P < 0.001.

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J. Xun et al. International Immunopharmacology 117 (2023) 109839

[13,14,45,46]. This suggested that Dioscin may inhibit the initiation and inflammatory effect. Additional, Dioscin remodeled the immune
tumorigenesis by anti-inflammatory effects. We also found that Dioscin microenvironment, especially macrophages and MDSCs. Further study
decreased tumor numbers and burden, suggesting its inhibitory effect on showed that Dioscin promoted M1 as well as inhibited M2 macrophages
CAC progress after the initiation of neoplasia. polarization in LPS- or IL-4-induced bone marrow derived macrophages
During intestinal inflammation, such as UC, macrophages secrete (BMDMs) model. Meanwhile, Dioscin promoted the differentiation of
inflammatory cytokines and chemokines thus contributing to UC path­ MDSCs into M1-lilke and inhibited its differentiation into M2-like
ogenesis and progression to CAC. In addition, macrophages are among macrophages in vitro. Taken together, the current findings provide a
the most abundant cells in the colon tumor microenvironment, which potential and natural active candidate for effective prevention of CAC.
play a key role in tumor initiation, promotion and invasion [47–49].
Therefore, regulating macrophage phenotype to alleviate its adverse Data Availability
effects is an effective means to improve the development of CAC. In
recent years, Dioscin has been reported to play a role in colitis and All data during the study are available from the corresponding au­
melanoma by regulating macrophage polarization [28,29]. It is thors by request.
remarkable that both M1 and M2 macrophages all play different roles in
inflammation and tumor progression. Dioscin suppressed M1 polariza­ CRediT authorship contribution statement
tion and facilitated M2 polarization of Raw264.7 [28]. On the contrary,
Dioscin promoted M1 macrophages polarization in TAMs-mediated Jing Xun: Methodology, Investigation, Writing – original draft,
melanoma metastasis to suppress tumor progression [29]. Here, we Funding acquisition. Siying Zhou: Validation, Formal analysis, Writing
found that Dioscin promoted M1 and inhibited M2 phenotype in spleen – original draft. Zongjing Lv: Formal analysis, Resources, Visualization.
of mice during CAC progression, suggesting its regulatory effects on Botao Wang: Resources, Visualization. Hai Luo: Formal analysis,
macrophages polarization. Moreover, we certified the role of Dioscin on Visualization. Lanqiu Zhang: Project administration, Writing – review
macrophages polarization by using an LPS- or IL-4-induced bone & editing. Lei Yang: Resources, Project administration. Aimin Zhang:
marrow derived macrophages model. The results showed that Dioscin Resources, Supervision. Xueliang Wu: Resources, Supervision. Zhenyu
promoted M1 phenotype induced by LPS and inhibit M2 phenotype Wang: Supervision. Ximo Wang: Supervision, Funding acquisition.
induced by IL-4. These results seem to be contradictory to the study that Xiangyang Yu: Conceptualization, Supervision, Writing – review &
Dioscin inhibits M1 and promotes M2 polarization of Raw264.7 cells to editing, Funding acquisition. Qi Zhang: Conceptualization, Project
ameliorate UC mice [28]. This difference may be due to the different administration, Writing – review & editing, Funding acquisition.
sources and types of macrophages or the use of different doses of Dio­
scin. In addition, this also suggests that Dioscin may have different
Declaration of Competing Interest
regulatory effects on macrophages polarization during CAC progression,
which need to be studied in the further. This is also the limitation of this
The authors declare that they have no known competing financial
study that the effect of Dioscin on macrophages polarization in the
interests or personal relationships that could have appeared to influence
tumor microenvironment need to be further confirmed. In addition, the
the work reported in this paper.
regulatory mechanism of Dioscin on macrophages polarization need to
be explored.
Data availability
In our study, we also found the reduction of MDSCs in AOM/DSS-
induce mice after Dioscin treatment. Evidences confirm that MDSCs
Data will be made available on request.
play a key role in immune suppression in cancer and the accumulation of
MDSCs is related with poor prognosis of patients with tumor [50–52].
Acknowledgements
Moreover, M− MDSCs subset quickly differentiate into M1-like and M2-
like TAMs after entering the tumor environment [53]. Thus, targeted
This research was supported by the National Natural Science Foun­
therapy strategies for MDSCs generally can be divided into several cat­
dation of China (NO. 81978158), Natural Science Foundation of Tianjin
egories: promote the differentiation to mature cells, blockade of MDSCs
(No. 17ZXMFSY00220), The Key Areas of Traditional Chinese Medicine
expansion, inhibition of MDSCs suppressive activity or depletion of
in Tianjin (No. 2023008), Project of Tianjin Health Commission (No.
intratumoral MDSCs [54]. Research has shown that high-salt diet in­
2021043, No.2021008). We thank Tianjin KingMed Center for Clinical
hibits tumor growth in mice via mediating the differentiation of
Laboratory for the platform support.
M− MDSCs into M1-like macrophages, which consistent with our present
results that Dioscin could promote the differentiation MDSCs into M1-
Appendix A. Supplementary material
like macrophages [55]. This suggests that Dioscin plays an important
role in remodeling tumor immune microenvironment, especially MDSCs
Supplementary data to this article can be found online at https://doi.
and TAMs, thus inhibiting tumor progression. This also need to be
org/10.1016/j.intimp.2023.109839.
further confirmed in colon tumor tissues.
However, one of the limitations is that whether Dioscin inhibit the
functional activity of MDSCs needs to be investigated. As we know, References
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