Ginsenoside Rg1 Ameliorated Colitis by Regulating the Homeostasis of M1/M2 Macrophage Polarization and Intestinal Flora

Jian Long Jiangxi University of Traditional Chinese Medicine Xue-Ke Liu Jiangxi University of Traditional Chinese Medicine Zeng-Ping Kang Jiangxi University of Traditional Chinese Medicine Meng-Xue Wang Jiangxi University of Traditional Chinese Medicine Hai-Mei Zhao Jiangxi University of Traditional Chinese Medicine Duan-Yong Liu Jiangxi University of Traditional Chinese Medicine You-Bao Zhong (  zhong-youbao@foxmail.com ) Jiangxi University of Traditional Chinese Medicine https://orcid.org/0000-0003-2761-8639


Background
In ammatory bowel disease (IBD) is a chronic, non-speci c in ammatory disease affecting the gastrointestinal tract and can be classi ed as Crohn's disease (CD) and ulcerative colitis (UC). The incidence of ulcerative colitis has been increasing globally over the last half century [1]. The clinical manifestations of UC are mostly diarrhoea, mucus, pus and blood stools, and abdominal pain. UC has become one of the top 10 intractable diseases in the world due to its tendency to recur and di culty in complete cure [2]. It is now widely accepted that the development of UC is closely related to genetic ginsenoside Rg1 alleviated colonic in ammation by inducing NLRP12 expression and inhibiting IL-1β and TNF-α [10]. However, ulcerative colitis is dominated by chronic spontaneous intestinal in ammation.
Therefore, it is more reasonable to construct a model of chronic colitis is to go into the anti-in ammatory mechanisms of ginsenoside Rg1. In addition, the immunomodulatory effect of ginsenoside Rg1 is closely related to M1/M2 macrophage polarization. Ginsenoside Rg1 has been shown to treat a variety of diseases by regulating M1/M2 macrophage polarization, including acute lung injury [11], cardiovascular and cerebral-vascular diseases [12], autoimmune encephalomyelitis [13] etc. In vitro, ginsenoside Rg1 increased autophagic activity and anti-apoptosis in Raw264.7 macrophages [12]. However, whether ginsenoside Rg1 can act on macrophages in colitis has not been reported.
Accumulating evidence indicates that gut microbiota can promote or inhibit colonic in ammation [14]. Recently, research hotspots have shown that improving the composition of the intestinal ora is an emerging strategy to alleviate intestinal in ammation [15]. The addition of Arg to the feed prevents the development of colitis, accompanied by an improvement of the intestinal ora composition, with higher abundance of Bacteroidetes and lower Verrucomicrobia [16]. Hyaluronic acid-bilirubin nanomedicine (HABN) modulates the intestinal microbiota, increases overall diversity and the abundance of Akkermansia muciniphila and Clostridium XIVα to effectively restore intestinal injury [17]. However, the action mechanism of ginsenoside Rg1 in regulating intestinal ora composition and gut health is currently poorly understood. Here, the effects of ginsenoside Rg1 on mice with DSS-induced colitis and its action mechanism on macrophage activation, M1/M2 macrophage polarization and intestinal ora were investigated.

Mice
Male speci c pathogen-free (SPF) BALB/c mice (8 -9 weeks, 22 ± 2 g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (Changsha, China) (Animal Certi cate Number: SCXK (Xiang) 2019-0004). Reproduce freely with standard diet and tap water according to the Institutional Animal Care and Use Committee at the animal facility of Traditional Chinese Medicine (Nanchang, China). This protocol (license number: JZ2019-235) was approved by the Institutional Animal Care and Use Committee (IACUC) of Jiangxi University of Traditional Chinese Medicine.

DSS-induced colitis and treatment
All mice were divided randomly into 4 groups (10 mice per group): Control, DSS, DSS+Rg1 and DSS+Rg1 group. Male BALB/c mice of the DSS, DSS+Rg1, DSS+Y27632 groups received 3% (wet/vol) DSS in drinking water for 7 days, then normal water for 7 days, followed by administration of 2% (wet/vol) DSS for 7 days. Mice of the Control group received normal drinking water. At the beginning of the 8th day, mice in the DSS+Rg1 group were orally administrated with 200 mg/kg/day ginsenoside Rg1, mice in the DSS+Y27632 group were orally administrated with 10 mg/kg/day Y27632, and mice in the DSS and Control groups were treated with the equal volume of normal saline. Throughout the study, all mice were weighed once daily (09:00), and monitored daily for diarrhea, hematochezia, hunched posture and hair loss.

Macroscopic evaluation
Mice were anesthetized deeply with 20% pentobarbital sodium , the abdominal cavity was quickly opened, the colon tissue was directly separated, the length and weight of the colon were measured, and the colon index = colon weight/mouse weight × 100%.

Pathological histology analysis
The proximal colon of mouse was xed in 4% polyformaldehyde solution for 24 hours. The tissue was dehydrated with gradient ethanol, transparent with xylene, embedded in para n, and nally cut into 4 μmthickness slices. The sections were stained with hematoxylin and eosin (Solarbio, Beijing, China), and and images were collected under the optical microscope (Lecia, Wetzlar, Germany) for pathological analysis.
The pathological injure of the colon were blindly assessed by two different pathologists, including in ammatory cell in ltration and tissue damage [18]. The scoring of in ammatory cell in ltration was evaluated as 0 (rare in ammatory cells in the lamina propria) to 3 (transmural extension of the in ltration of in ammatory cells), and tissue damage was evaluated ranging from 0 (no mucosal damage) to 3 (extensive mucosal damage and extension through deeper structures of the bowel wall).
Enzyme-linked immunosorbent assay (Elisa) The colon tissue (100 mg) was collected and lysised by RIPA (radio immunoprecipitation assay) solution at a ratio of 1:10, incubated at 4 ℃ for 1 hour, homogenized by ultrasonic homogenizer for 20 min, centrifuged at 4 ℃ for 15 min. Total protein in each sample was quanti ed by a total protein detection kit (Aidlab Biotechnlologies Co., Ltd., Beijing, China), and then diluted in 1× PBS to a nal concentration of 3000 ng/mL And then the supernatant was obstained and detected. The concentrations of IL-6, IL-33, CCL-2, TNF-α, IL-4 and IL-10 were measured by commercial Elisa kits (Invitrogen, Calif., USA) according to the manufacturer's protocol, and then the optical density (OD) values at 450 nm was detected using a microplate reader (Thermo, Varioskan, MA, United States). Then, each cytokine was quanti ed basally based on a standard curve established using an Elisa kit.

Microbial diversity analysis
The stool samples were collected on day 21 and immediately stored at −80℃ for bacterial DNA extraction. The microbial diversity analysis was entrusted to Majorbio Bio-Pharm Technology (Shanghai, China). Total bacterial DNA was extracted from fecal samples according to QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA) instructions. The V3-V4 region of the bacterial 16S rRNA gene was ampli ed with primers: 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR products were puri ed using the AxyPrep DNA gel extraction kit (Axygen, Union City, USA) and quanti ed by Qubit (Invitrogen, USA). Then, the quali ed libraries were sequenced using Illumina miseq platform. Operational taxonomic units (OTUs) were clustered with a 97% similarity cut-off using UPARSE (version 7.1). The taxonomy of each 16S rRNA gene sequence was assigned by the ribosomal database project (RDP) Classi er algorithm (http://rdp.cme.msu.edu/) against the SILVA (SSU123) 16S rRNA database using a con dence threshold of 70% [19]. Alpha diversity analysis (Mothur, version v.1.30.1) was used to evaluate the Chao1 abundance and the Shannon index. Principal coordinates analysis (PCoA) was performed using Mothur, and statistical analysis was performed based on the values of PC1 [20]. Linear discriminant analysis (LDA) coupled with effect size (LEfSe) measurements (based on nonparametric factorial Kruskal-Wallis sum-rank test and the Wilcoxon rank-sum test) was used to identify taxa that were signi cantly different (biomarkers) between groups, with P < 0.05 and an LDA score threshold of 4 [19]. Microbial difference analysis, correlation analysis, and co-occurrence network analysis were performed using I-sanger (Majorbio Bio-Pharm Technology Co. Ltd.; www.i-sanger.com). [21] Statistical analysis Data were expressed as the mean ± SEM (Standard error of mean). Statistical analyses were carried out using GraphPad Prism 7.0 software (San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by the Tukey test for multiple comparisons were performed to determine signi cance. All pvalues less than 0.05 were considered to be statistically signi cant.

Ginsenoside Rg1 ameliorated DSS-induced colitis
In this study, the rate of body weight change (Fig. 1A) in colitis mice decreased signi cantly from day 4 to day 21, and mouse weight (Fig. 1B) and colon length (Fig. 1D, E) decreased signi cantly, and the colon weight index increased signi cantly (Fig. 1F) at the end of the experiment. At the same time, the mucosal tissue structure of colitis mice was found to be disorganized under light microscopy, with epithelial detachment, ulcer formation and massive in ammatory cell in ltration (Fig. 1G), and their pathological damage scores were signi cantly higher than that of the Control group (Fig. 1H). After treatment with ginsenoside Rg1 and Y27632, the rate of weight change (Fig. 1A) in mice increased signi cantly from day 16 to day 21, mouse weight (Fig. 1B) and colon length (Fig. 1D, E) increased signi cantly, and colon weight (Fig. 1C) and colon weight index (Fig. 1E) decreased signi cantly at the end of the experiment; meanwhile, the pathological damage to the colon of mice in the DSS + Rg1 and DSS + Y27632 groups was effectively inhibited, less ulcer formation and in ammatory cell in ltration was occasionally observed under light microscopy (Fig. 1G), and its pathological damage scores were signi cantly lower than that of the DSS group (Fig. 1D). The above study showed that ginsenoside Rg1 and Y27632 was effective in alleviating DSS-induced experimental colitis.
Ginsenoside Rg1 regulated in ammatory cytokines in colon tissue Cytokines are key pathophysiological factors that govern the initiation, progression and resolution of IBD in ammation [22], including pro-in ammatory cytokines IL-6, IL-33, CCL-2 and TNF-α, and antiin ammatory cytokines IL-4 and IL-10. Therefore, in this study, the expression levels of tthese cytokines were detected in colonic tissue by Elisa assay. In the present study, the pro-in ammatory cytokines IL-6 ( Fig. 2A

Ginsenoside Rg1 improved intestinal ora composition
The fecal microbial populations of mice in the Control, DSS, DSS + Rg1 and DSS + Y27632 groups were analyzed using 16S gene sequencing. Distances were calculated for each sample group at the OTU level, and the rank of distances (Bray_Curtis) was smaller for the Control, DSS, DSS + Rg1, and DSS + Y27632 groups relative to the Between group (Fig. 6A), with between-group differences greater than within-group differences. Sequencing coverage was high for all four groups and sequencing results found that most of the diversity was captured in all samples (Fig. 6B). 378 OTUs overlapped between the groups: 433 OTUs in both the Control and DSS groups; 436 OTUs in the DSS and DSS + Rg1 groups; and 433 OTUs in the DSS and DSS + Y27632 groups (Fig. 6C). Compared to percent of community abundance genus level of the Control group, Lachnospiraceae of the DSS group was signi cantly down-regulated and Staphylococcus, Bacteroide and Ruminococcaceae_UCG_014 were signi cantly up-regulated (Fig. 6D). After ginsenoside Rg1 treatment, the percent of community abundance genus level of Lachnospiraceae were upregulated and Staphylococcus, Bacteroide and Ruminococcaceae_UCG_014 were downregulated (Fig. 6D). A comparative analysis of the samples showed that stress = 0.059, which allows us to assume that the order of the samples in this experiment was quite good (Fig. 6E). The 20 samples in this experiment had different community compositions, but there was some similarity within the groups. The DSS and DSS + Rg1 groups had separate gut microbiota from the Control group, while the distance between the DSS + Rg1 group and the Control group was smaller than the distance between the DSS group and the Control group (Fig. 6E). To determine the role of Rg1 in regulating the intestinal ora of colitis mice, differential analysis among these four groups at the species level (Fig. 6F) were analysed. A one-way ANOVA was performed on data from the top seven bacterial rankings of microbiota abundance in the samples to detect species differences in microbiota community abundance between groups, including Staphylococcus, Lachnospiraceae_NK4A136_group, unclassi ed_f_Lachnospiraceae, norank_f_Lachnospiraceae, Bacteroides, Prevotelaceae_UCG-001 and Eubacterium_ ssicatena (Fig. 6F). The above results showed that Ginsenoside Rg1 improved gut microbiota composition in DSS-induced colitis mice.
Ginsenoside Rg1 regulated the crosstalk between M1/M2 macrophage and intestinal ora The above results suggest that ginsenoside Rg1 is effective in regulating M1/M2 macrophage polarization and gut microbiota composition in colitis mice. However, the correlation or consistency between whether ginsenoside Rg1 regulates macrophages and intestinal ora has not been elucidated. Here, redundancy analysis/canonical correlation analysis (RDA/CCA) was used to analyze their correlation. The correlation analysis between macrophages and intestinal ora at the genus level revealed that the DSS group was closer to TLR2, while the Control and Rg1 groups were closer to CD206 and CD163 (Fig. 7A). TLR4 is known to be involved in M1 macrophage differentiation, while CD206 and CD163 are involved in M2 macrophage differentiation. TLR2 was found to play a key role in intestinal ora composition, with the DSS group having the smallest spacing to TLR2 and the Control group having the largest spacing to TLR2 (Fig. 7B); and TLR2 protein expression was signi cantly elevated in colonic tissues of colitis mice and down-regulated signi cantly after ginsenoside Rg1 treatment (Fig. 7C). The results revealed that ginsenoside Rg1 effectively regulated the crosstalk between M1/M2 macrophage and intestinal ora in DSS-induced colitis.

Ginsenoside Rg1 regulated Nogo-B/RhoA signal pathway
The ATP-competitive ROCK-I and ROCK-II inhibitor Y27632 is effective in alleviating the development of UC [25]. Therefore, we further investigated whether ginsenoside Rg1 could play a critical role in the treatment of ulcerative colitis by regulating the Rock signaling pathway. In this study, the levels of Rock1 (Fig. 8A, B) and RhoA (Fig. 8A, C) proteins were signi cantly increased in the colonic tissues of colitis mice. After treatment with ginsenoside Rg1 and Y27632, the levels of Rock1 (Fig. 8A, B), RhoA (Fig. 8A, C) and Nogo-B (Fig. 8A, D) proteins in colonic tissues of colitis mice decreased signi cantly. These results suggest that ginsenoside Rg1 inhibit the activation of Nogo-B/RhoA signaling pathway in colitis mice.

Discussion
DSS-induced colitis model can mimic the pathogenesis of human colitis [26] and is commonly used in the study of colitis pathogenesis and development of new drugs [27]. In the present study, the colitis model was successfully replicated with lower body weight, shorter colonic length, larger colonic weight index, more ulcer formation and massive in ammatory cell in ltration in the colonic mucosa, and higher pathological damage scores. Ginsenoside Rg1, one of the main components of Panax ginseng, has potent anti-in ammatory properties [10]. In this study, the changes of body weight, colon length, colonic weight index and colonic mucosal injury of colitis mice were effectively improved after treatment of ginsenoside Rg1. Our study demonstrated that ginsenoside Rg1 was also e cacious in DSS-induced chronic colitis.
Nogo-B is a member of the reticulohistone family of proteins, mainly located in the endoplasmic reticulum (ER), and is involved in a variety of cell biological processes. Nogo-B is required for macrophage homing, and Nogo-B de ciency impairs macrophage migration [46]. In vivo experiments, Nogo-B is involved in in ammatory responses and macrophage in ltration and macrophage-mediated tissue remodelling were found in Nogo-B −/− mice [47]. In vitro experiments, LPS stimulated macrophage activation and Nogo-B protein was downregulated in a time-and dose-dependent manner [48]. In addition, Nogo-B is required for the immune response and its over-expression induces the expression of the proin ammatory cytokines CCL-1, TNF-α, IL-1β and TGF-β, and enhances the migratory activity of macrophages and recruits macrophages to move chemotactically towards local in ammation sites [48]. Impaired RhoA function leads to over-expression of CD4 + T cell integrin α4β7 and colonic localization, in ammatory cytokine storm and the development of colitis [49]. The Rho kinase (ROCK) inhibitor Y27632 reduced intestinal permeability and inhibited the Rock-MLC and Rock-NF-κB pathways, effectively attenuating TNBS-induced colitis in mice [50]. In our study, both of ginsenoside Rg1 and Y27632 inhibited the expression of Rock 1, RhoA, and Nogo-B proteins in colonic tissues of colitis mice. Importantly, the trends of Nogo-signaling in regulating pro-in ammatory cytokines and macrophages of colitis mice were largely consistent with ginsenosides Rg1. These ndings suggest that the key target of ginsenoside Rg1 in regulating macrophage polarization in colitis mice is closely related to Nogo-B signaling, laying the foundation for future studies.
The interaction between TLR and intestinal ora can maintain immune system homeostasis [51], and the underlying mechanisms may be closely related to macrophage homeostasis. The disruption of intestinal ora leads to the activation of immune cells by TLR [52], which further leads to in ammation. In TLR2 gene knockout mice, expression of M1 macrophages was reduced and expression of M2 macrophages was increased [53]. Compared to wild-type mice, levels of IL-6 and TNF-α were signi cantly reduced in M1 macrophage culture supernatants from TLR2 −/− mice, while levels of IL-10 were signi cantly increased in M2 macrophage culture supernatants [53]. Inhibition of TLR2 expression reduces the secretion of in ammatory factors by macrophages for the treatment of osteoarthritis [54]. Interestingly, we found that changes in macrophage activation or polarization proteins TLR2, TLR4, CD206, and CD163 were closely associated with intestinal ora composition. It implies that macrophage polarization during the course of colitis affects the structure of the intestinal ora. The correlation among colitis, macrophages, and intestinal ora needs to be further investigated, which is the focus of the next study.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used in the current study are available from the corresponding author on reasonable request.

Competing interests
All authors declare that they have no con icts of interest.