Combined Rg1 and adipose-derived stem cells Alleviate DSS-induced colitis in a mouse model

Inammatory bowel diseases (IBDs) including Crohn's disease and ulcerative colitis are chronic inammatory disorders that can affect the entire gastrointestinal tract and the colonic mucosa, no medical or surgical cure for IBD, and all have side effects that limit their use, exhibit a high necessity for new therapeutic strategies. Adipose-derived stem cells (ADSC) therapy represents a promising option for the treatment of IBD. Rg1 Previous study indicated that ginsenoside (Rg1) can ameliorate inammatory disease such as colitis by inhibiting the binding of LPS to TLR4 on macrophages and restoring the Th17/Treg imbalance [1]. In this study, we investigated whether Rg1 can enhance the effect of ADSC on DSS-induced colitis in a mouse model.

colitis in a mouse model.

Background
The two major clinically de ned forms of in ammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC), are chronic remittent or progressive in ammatory conditions that can affect the entire gastrointestinal tract and the colonic mucosa [2]. Recently, increasing studies indicated that the interactions of gut microbiome, mucosal immune system and the manner in which environmental factors modify these relationships appear particularly relevant for the development of IBD [3]. IBD is associated with substantial morbidity, decreased quality of life, and colitis-correlated colorectal cancer (CAC) development, it has increasingly emerged as a public health challenge worldwide [4]. However, the drugs treated IBD are far from optimal, and patients have to face lifelong treatment and debility. So, it is urgent to develop treatment which could reduce side effects and improve long-term effect of IBD.
As an emerging therapy for patients with IBD, mesenchymal stem cells (MSCs) have promising future in restoring epithelial barrier integrity, homing to the damaged tissue, inhibiting in ammatory response, and regulating immunity [5][6][7] [8 -10]. Moreover, a lot of studies demonstrated that systemic administration of MSC by the intravenous or intraperitoneal injection could alleviate colitis of mouse model [11][12][13][14]. As a category of MSCs, We and other groups had reported that the ADSC was a feasible and effective treatment for Crohn's stula-in-ano, compared with traditional incision and thread-drawing, ADSC therapy could protect anal function of patients, relieve pain, allow quick recovery, be well-tolerated, and improve the quality of life during perioperative period [15].
Ginsenoside Rg1 is a traditional stem extract and is one of the main active ingredients of ginseng [16,17]. It has been reported that ginsenoside Rg1 can promote stem cell orientation transformation and induce stem cell proliferation [18,19]. For example, Rg1 could enhance the proliferation, differentiation, and soft tissue regeneration of human breast adipose ADSCs in collagen type I sponge scaffolds in vitro and in vivo, and a broad new organizational network was also formed [20]. Moreover, Zhu et al., reported that Rg1 markedly reduces proin ammatory cytokines that released from dendritic cells in a mouse DSSinduced colitis model [1]. To help to develop novel therapeutic procedures for IBD patients, in the current study, we investigate the role of the combination therapy of Rg1 and ADSC administration in a mouse DSS-induced colitis and explore the speci c mechanisms involved in this process.

Material And Method
Adipose Derived Stem Cell Derivation Human abdomen or buttock adipose tissues were collected with informed consent from patients receiving regenerative medicine using ADSC at Nanjing Hospital of Chinese Medicine A liated to Nanjing University of Chinese Medicine (Nanjing, China). Adipose tissues were collected from healthy adult male patient who provided informed consent at Nanjing Hospital of Chinese Medicine A liated to Nanjing University of Chinese Medicine (Nanjing, China). ADSC were isolated from samples, and stromal vascular fraction were cultured with serum-free culture medium, at 37℃ in an atmosphere containing 5% CO2. After reaching con uence, adherent cells were trypsinized and replated. Cells were passaged using serumfree culture medium three times, and the characteristics of ADSC were veri ed by analysis of the differentiation, proliferation, and immunologic phenotypes. The cells were frozen and delivered to Nanjing Medical University. After thawing, the cells were immediately washed, counted, suspended in phosphatebuffered saline (PBS), For ow cytometry analysis, ADSCs were harvested, washed, and incubated with speci c MSC marker antibodies CD90-FITC, CD44-PE, CD105-FITC, CD73-PE, CD34-PE, and CD45-FITC.

Animal
Mice six-to eight-week-old C57BL/6 male mice were purchased from experiment center of Nanjing Medical University (Nanjing, China) and housed under controlled temperature, humidity, and light cycle conditions. All animal experiments were conducted in compliance with regulations and approved by the Institutional Animal Care and committee the Nanjing Medical University.

Induction of experimental colitis and study design
Colitis was induced by providing drinking water containing 3% DSS (molecular weight 36,000-50,000; MP Biomedicals, Santa Ana, CA) for 7 days followed by regular water for 7 days. Mice were divided into ve groups, ADSC and ADSC + Rg1 groups were injected intravenously with 1*106 ADSC [21] on day 4th and 7th, whereas Control, Rg1 and DSS groups were injected with PBS. Moreover, Rg1 and ADSC + Rg1 groups were administered with Rg1 (at a dose of 20 mg/kg [24]) by gavage once daily from the rst day to the 14th day (Start with DSS treatment), whereas others were administered with PBS by gavage once daily. All were sacri ced on the 15th day after the start of the experiment.

Body Weight and Assessment of Colon Length
To evaluate the therapeutic effects of ADSC and Rg1, the body weight, and colon length were analyzed. Body weight was recorded daily, colon lengths were measured from the anus to the cecum soon after harvesting the colon. Samples were measured as an indirect assessment of in ammation.

Histopathological analysis
Histological score was calculated as follows. The colon was excised, xed in 10% formalin, embedded in para n wax, and sliced into 4-µm-thick sections. After hematoxylin and eosin (H&E) staining, histological evaluation was performed in a blinded manner according to a previously published scoring system. Histology was scored as described previously [22]. Simply, score 1: mild mucosal in ammatory cell in ltrates with intact epithelium; Score 2: in ammatory cell in ltrates into mucosa and submucosa with undamaged epithelium; Score 3: mucosal in ltrates with focal ulceration; Score 4: in ammatory cell in ltrates in mucosa and submucosa and focal ulceration; Score 5: moderate in ammatory cell in ltration into mucosa and submucosa with extensive ulcerations; Score 6: transmural in ammation and extensive ulceration.

Serum Parameter Analysis
We collected all the mice' blood serum and tested 6 immune cytokines, including IL-10, IL-6, IL-17A, IL-33, IL-1β, and TNF-α following the company's kit procedures. All of assay kits are purchased from R&D System. All immune cytokines were statistically analyzed by T-test included in GraphPad Prism software 8.0.

Flow Cytometry
Spleen were removed and mechanically dissociated into single cell suspensions. Splenocytes were incubated with Fc block (CD16/32) for 10 minutes to block non-speci c binding before stained with the conjugated antibodies. For surface staining, cells were incubated with speci c antibodies for 30 minutes on 4℃ followed by washing twice. For intracellular cytokine staining, cells were stimulated with phorbol myristate acetate and ionomycin and in the presence of monensin (eBioscience) for 5 h prior to staining with the BD Fixation/Permeabilization Solution kit. Treg cells were xed and permeabilized using the eBioscience Foxp3/transcription factor staining buffer kit (Invitrogen) according to manufacturer's instruction. Flow cytometry data were acquired on a BD FACSVerse ow cytometer (BD Bioscience) and analyzed using FlowJo version 10 software.

DNA isolation and 16S rRNA gene sequencing
In total, 100-mg stool samples were used to extract total bacteria genome DNA following the protocol of the DNA extraction kit (#DP328, Tiangen Company, Beijing, China). The concentration and purity of the extracted bacterial DNA were detected using the Qubit 2.0 Fluorometer (Thermo Scienti c, USA). The V3 and V4 regions of 16S rRNA genes were ampli ed using composite speci c primer. The 16S rRNA V3 and V4 speci c primers are 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The 16S rDNA data were analyzed using QIIME software package 1.9, all the analysis and calculation methods used in-house Perl scripts, with special reference to these two articles [23,24].

Statistical analysis
Student's t-test (unpaired, two-tailed) was used to gure out levels of signi cance for comparisons between two groups by using Graphpad Prism 8.0 software. Results are shown as mean ± SD, P value less than 0.05 was considered signi cant.

Characterization of Human ADSC
ADSC expressed all speci c MSC markers CD73, CD105, CD90, CD44 and lacked expression of the hematopoietic markers CD34 and CD45. Differentiation to adipocytes, chondrocytes, and osteocytes was observed in ADSC (Fig. s1). ADSC and Rg1 administration ameliorated DSS-induced colitis As Fig. 1b shown, the body weights of the mice in the DSS groups were consistently reduced since the fth day, while the mice in the ADSC + Rg1 groups and Rg1 groups mice showed a signi cant improvement in weight loss, although there was an apparent decrease in the ADSC groups (P < 0.05). In the colitis model, disease severity is typically associated with colon length shortening due to intestinal in ammation [25]. The length of colon in the DSS groups were shorter than in the ADSC groups, Rg1 groups, and ADSC + Rg1 groups (p < 0.05).
Consistent with previous studies [11], H&E colon analysis (Fig. 1e) showed that DSS-induced colitis resulted in extended ulcerations, destroyed crypts and transmural in ammatory in ltration, with a barely complete mucosal structure. However, in mice treated with ADSC and Rg1, histological damage was ameliorated, as evidenced by a preserved mucosal architecture showing focal erosions and mild/moderate mucosa in ammatory in ltration. To evaluate the intestinal mucosal architecture and in ammatory in ltration, we used the histopathology score system [22] to quantity in ammatory severity degree, and the mice treated with ADSC and Rg1 had a signi cantly lower score than the mice treated with DSS only (P < 0.05) (Fig. 1f).

ADSC and Rg1 treatment could alleviate colitis by regulating pro/anti-in ammatory cytokines
Pro-in ammatory cytokines play a crucial role in the progression of DSS-induced colitis [26]. To explore whether ADSC and Rg1 treatment could alleviate colitis by regulating in ammation, we detected the cytokine expression in blood serum. As shown in Figure   .2a-h, the levels of in ammatory cytokines IL-6, IL-33, TNF-α, IL-1β and IL-17A in Rg1, ADSC, and ADSC + Rg1 groups decreased signi cantly and IL-10 increased compared with those in the DSS groups. Moreover, we found that the combined of ADSC + Rg1 groups can signi cantly inhibit the expression of in ammatory cytokines expression compared to Rg1 and ADSC alone groups (Fig. 2a, b, c, d, e, f, g, h). Therefore, Rg1 can enhance the effect of ADSC on DSS-induced colitis in a mouse model.

ADSC and Rg1 regulated Treg/Th17 balance to maintain intestinal homeostasis
Previous study has demonstrated that ADSC could inhibit Th17 response in T-cell-mediated autoimmune diseases [27]. It has also been reported that ADSC could inhibit Treg/Th17 differentiation in DSS-induced colitis model mice [15]. So we further compared the number of Th17 cells between groups. We found the number of Th17 cells were much higher in the DSS group than in the Rg1, ADSC, and ADSC + Rg1 groups (Fig. 3b, c, e). But rather, the percentage of Treg cells was remarkably higher in Rg1, ADSC, and ADSC + Rg1 groups compared with DSS groups. In summary, it indicated that Rg1 and ADSC administration selectively upregulated the frequency of Treg cells as well as downregulated the ratio of Th17 cells against DSS-induced colitis, improving the Treg/Th17 balance to maintain intestinal homeostasis. (Fig. 3a, d, f). Besides, the ADSC and Rg1 treated simultaneously showed better trends of recovering Treg/Th17 balance than ADSC and Rg1 groups alone.
ADSC and Rg1 treatment signi cantly altered the gut microbiota diversity and composition Gut microbiota is an important factor in regulate intestinal in ammation [28]. Therefore, we further compared the microbiota composition of ve groups. The alpha diversity of bacterial communities was evaluated according to Shannon's diversity index (Fig. 4a). The Shannon's diversity index of ADSC and Rg1 showed no statistical difference with DSS groups (Fig. 4a, P > 0.05). The PCoA scatterplot revealed the clear clustering of gut bacterial communities between ve groups (Fig. 4c). To compare the gut microbiota composition of the ve groups, we then conducted a statistical analysis of gut microbiota at the genus level using the Kruskal-Wallis test. At the genera level, compared to DSS groups, the gut microbiota of ADSC, Rg1, and ADSC + Rg1 groups was characterized by an increased Rikenellaceae RC9 and ruminococcaceae UCG-013 level (Fig. 4d) and a lower ratio of Erysipelatoclostridium and Escherichia-shigella (Fig. 4d). Interestingly, the ratio of Rikenellaceae RC9, Ruminococcaceae UCG-013, Erysipelatoclostridium and Escherichia-shigella lever in ADSC + Rg1 groups were more similar to control groups. In addition, we also found that the effect of ADSC on gut microbiota disturbance induced by DSS was not signi cantly improved, there was even a worsening trend. However, Rg1 can restore the disturbed gut microbiota induced by DSS treated by ADSC.
We also identi ed signi cant changes in the multiple biological pathways of ve groups (Fig. 4e). As shown in Fig. 4e, the fteen modules in the ve groups were involved in L-arginine degradation, superpathway of L-arginine, putrescine, and 4-aminobutanoate degradation, superpathway of methylglyoxal degradation, glyoxylate cycle, Citrate cycle (TCA cycle), L-methinonine biosynthesis, and heme biosynthesis, and methanogenesis from acetate. The overrepresentation of the L-arginine degradation pathway in DSS groups may be related to the high abundance of Escherichia-shigella, which has been shown to have a wide capacity for degrading polysaccharides in those samples [29]. In all, the predication analysis indicated that gut dysbiosis may induce a disease-linked state through the interference of physiological metabolic functions.

Discussion
In In our study, we found that ADSC and Rg1 administrated could substantially ameliorate the colitis compared with ADSC and Rg1 treated alone, as indicated by lower body weight loss, colon length shortening, and better histological scores. Additionally, some of the potential mechanisms were presented, including (i) amelioration of colon mucosal barrier damage; (ii) modulation of the in ammatory response; (iii) reshaping the gut microbiota; and (iv) regulation of Treg/Th17 differentiation.
The excessive response of Th17 cells and insu cient function of Treg cells correlate with the onset of IBD. Previous study has demonstrated that ADSC could inhibit Th17 response in T-cell-mediated autoimmune diseases [27] and inhibit Treg/Th17 differentiation DSS-induced colitis model mice [30]. Consistent with previous study, we found ADSC treatment improved the Treg/Th17 balance. Th17 cells produce pro-in ammatory cytokines IL-17A, which contribute to the progression of IBD [31]. Tregs are derived from the thymus with the function of suppressing the innate immune response [32]. Previous study indicated that improving Treg/Th17 balance contributed to the re-establishment of intestinal immune homeostasis in IBD [33]. In this study, we also found the Rg1 can enhance the effect of ADSC of improving the Treg/Th17 balance.
We observed that the microbial community diversity and structure in ADSC, Rg1, and ADSC + Rg1 groups were signi cantly changed compared to those of DSS groups. For example, in DSS groups, we found that reduction of Rikenellaceae RC9 and Ruminococcaceae UCG-013 and increase of Erysipelatoclostridium and Escherichia-shigella compared with other groups. Rikenellaceae_RC9_gut_group is a dominant group of Bacteroidetes, which might affect intestinal permeability, oxidative stress and energy metabolism, and might contribute to the pathogenesis of acute myocardial ischemia [34] and in ammation [35]. The Ruminococcaceae family is a member of the Firmicutes phylum and comprises a broad spectrum of species with different functional properties. An underrepresentation of species belonging to the family has previously been reported in IBD [36]. Erysipelatoclostridium and Escherichia-shigella have been reported that play an important role in in ammatory response [37,38]. Besides, our results also showed that ADSC transplantation after combined with the ginsenoside Rg1 administration could signi cantly improve the ratio of Rikenellaceae RC9 and Ruminococcaceae UCG-013 and reduces the level of Erysipelatoclostridium and Escherichia-shigella. which was superior to the ADSC and Rg1 administration alone.
The complex interactions between biological pathways and gut microbiota are intense associated with host-microbe. Notably, gut microbiota plays an essential role in IBD through the pathways such as glyoxylate cycle, Citrate cycle (TCA cycle), and L-arginine degradation [39][40][41],consistent with this, the KEGG pathways in the ADSC, Rg1 and ADSC + Rg1group were different from DSS groups, such as glyoxylate cycle, Citrate cycle (TCA cycle), and L-arginine pathway reduced signi cantly, which implicated ADSC and Rg1 may modulate these pathway though restoring composition of gut microbiota.
Taken together, the study suggested the combination of ADSC and Rg1 administration may enhance clinical e cacy of IBD through restore the Treg/Th17 balance and gut microecological structure.

Conclusion
Overall, our results showed that Rg1 and ADSC treatment restore balance of pro/anti-in ammatory cytokines, Treg/Th17 balance, gut microecology. We con rmed that the combination of Rg1 and ADSC administration could alleviate DSS-induced colitis more e cient than that of Rg1 or ADSC treatment alone.