Immunological Anti-Tumor Activity of Icariside I by Regulating Gut Microbiota and Its Derived Metabolites in a Melanoma Mouse Model

gut microbiome plays important roles in antitumor by shaping systemic immune previously to enhance immunity and inhibit metabolites such as as molecules involved in signaling Gpr41:G protein-coupled receptor 41; Gpr43:G protein-coupled receptor 43; ILA:Indole-3-Lactic Acid; IA: Indole Acrylic Acid; IPA:Indole-3-Propionic Acid; IAA: Indole Acetic Acid; IAId:Indole-3-Aldehyde; PAS: Periodic acid-Schiff; Ptprh, protein tyrosine phosphatase, receptor type, H; CCL3:CC-chemokine ligand 3; GAPDH:Glyceraldehyde-3-phosphate dehydrogenase.


Results
Icariside I signi cantly inhibited B16F10 melanoma growth in vivo through regulation of gut microbiota and host immune. Oral administration of icariside I improved the altered gut microbiota community with marked restoration of Lactobacillus spp. and Bi dobacterium spp. abundance. Icariside I was also able to improve the levels of microbiota-derived metabolites such as short-chain fatty acids (SCFAs) and indole derivatives, consequently repairing intestinal barrier and systemic in ammation of tumor-bearing mice. Furthermore, icariside I up-regulated multiple lymphocyte subsets including CD4 + and CD8 + T cells or NK and NKT cells in peripheral blood.

Conclusions
These results suggested that icariside I may be developed as a novel and natural anticancer adjuvant via microbiome remodeling and host immune regulation.
Icariside I enhances host immunity with elevation of multiple lymphocyte subsets.

Background
Melanoma is the most lethal form of skin cancer due to its high metastasis and drug resistance [1]. In the past, melanoma was mainly treated by surgery, radiotherapy and chemotherapy with short window period and high rate of recurrence [2]. In recent years, targeted therapeutic agents such as BRAF and MEK inhibitors or their combination were clinically applied for patients with metastatic melanoma and especially BRAF-mutant melanoma [3]. Currently, immunotherapy strategies have been developed from cytokine-based treatment to antibody-mediated blockade of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and the programmed cell-death protein 1 (PD-1) immune checkpoints for melanoma patients. Although a variety of cancers such as melanoma and non-small-cell lung carcinoma have been approved by the FDA for immunotherapy with increasing the survival rate [4], there are still many patients who are ineffective or partially respond at immunotherapy [5]. Furthermore, antibodies targeting CTLA-4 and PD-1 for tumor immunotherapy are required for intravenous antibody delivery with possibility of tumor recurrence and side effects.
Increasing evidence has shown that the gut microbiome is closely related to multiple pathophysiological processes and plays important roles in antitumor immunotherapy by shaping systemic immune responses [6,7]. Probiotics such as Lactobacillus spp. and Bi dobacterium spp. regarded as live microorganisms are bene cial for human health when given in su cient quantities [8]. Recent studies have shown that probiotics enhanced host immunity and attenuated tumor growth via tumor immunomodulation [9]. For example, oral administration of Bi dobacterium spp. effectively inhibited tumor growth of a melanoma mouse model by accumulating CD8 + T cells with promotion of antitumor immunity and anti-PD-L1 e cacy [6]. Bi dobacterium spp. can also activate natural killer (NK) cells and improve the defense against in uenza virus in a murine model [10]. Supplementation of Lactobacillus casei has been reported to enhance anti-tumor immunity in colon cancer model by promoting potent Th1 immune responses, activity of CD8 + T and NK cells in ltration in the tumor tissue [11]. Moreover, several cohort studies indicated that probiotics such as Bi dobacterium and Lactobacillus produced from longterm dietary ber and yogurt contributed to cancer prevention and treatment [12,13].
Microbiome-derived metabolites such as short chain fatty acids (SCFAs) are concurrently being recognized to be involved in mediating tumor pathogenesis and immunotherapy [14]. Faecalibaculum rodentiuma producing SCFAs has been identi ed as a novel anti-tumor bacterial strain mainly by inhibiting calcineurin and NAFTc3 activation, controlling protein acetylation and tumor cell proliferation [15]. Previous studies have shown that three butyrate-producing bacterial species were more abundant in better-responded melanoma patients who were treated with anti-CTLA-4 (ipilimumab) [16]. Butyrate was also reported to protect intestinal mucosa against in ammation and carcinogenesis by activating GPCRs (Gpr109a) and inhibiting in ammatory responses of macrophages and dendritic cells in the colon [17].
Tryptophan-derived indole derivatives from gut microbiota are key endogenous ligands with a high a nity of aryl hydrocarbon receptor (AhR), which is closely associated with multiple metabolic syndromes such as obesity, cancer, diabetes and high blood pressure [18,19]. A variety of intestinal microbes, especially Lactobacillus spp. and Bi dobacterium spp., can produce tryptophan-derived metabolites, such as indole-3-aldehyde (IAId), indole-3-propionic acid (IPA) and indoleacrylic acid (IA) [19][20][21], which were demonstrated to promote intestinal mucosal barrier integrity and inhibit in ammatory response by stimulating the expression of IL-22 and activating AhR [22]. Of particular note that Lactobacillus reuteri produces indole derivatives of tryptophan such as indole-3-lactic acid, which activate AhR and induce CD4 + CD8aa + T cells for regulation of intestinal mucosal functions in immunotherapy of in ammatory bowel diseases (IBD) [23]. Inulin naturally sourced from chicory root ber can produce prebiotics to inhibit melanoma growth by modulating the composition and diversity of gut microbiota and enriching CD4+, CD8 + T cells and plasmacytoid DCs in mouse models [24]. Sporoderm-breaking spores of G. lucidum was also found to signi cantly modulate gut microbiota structure and composition in tumor-bearing mice and thus increasing the population of CD8 + T cells [25].
Icariin and its derivatives including icaritin, icariside I and icariside II isolated from Herbal Epimedium are natural plant avonoids with a variety of biological activities such as anti-osteoporosis and anti-tumor effects and commonly used as Chinese traditional medicine [26,27]. In structure as shown in Fig. 1, icariin (A) is a prenylated avonol glycoside with rhamnosyl, glucosyl, and methoxy groups.
Deglycosylation of icariin will result in icaritin (B). Icariside I (C) and icariside II (D) can be formed when the rhamnose and glucose residues are removed, respectively. Previous studies have mainly focused on the activities of icariin, icaritin and icariside II and showed that these compounds exert their anticancer action through a number of cellular targets and pathways including apoptosis, cell-cycle modulation and immunomodulation [28]. For example, icaritin can effectively inhibit hepatocellular carcinoma (HCC) initiation and malignant growth through IL-6/JAK2/STAT3 pathway signaling [29]. Furthermore, icaritin can also exhibit anti-tumor immunity activity with increasing CD8 + T cells in ltration and reducing frequency of MDSCs [30]. Icariside II induced apoptosis in human PC-3 prostate cancer cells by initiating the inhibition of cyclooxygenase-2/prostaglandin E2 (COX-2/PGE2) pathway [31]. Less work, however, have been done on activities of icariside I probably owing to its too low content in Epimedium.
In this study, immunological anti-tumor activity of icariside I was investigated for the rst time in a B16F10 melanoma mouse model. Gut microbiota structure and composition in cecal content of mice was rstly analyzed by 16S rRNA gene sequence with the goal of screening those bacteria highly associated with tumor immunotherapy. Subsequently, targeted metabolomics approach was employed to quantitatively determine microbiota-derived metabolites such as SCFAs and indole derivatives. The population of multiple lymphocyte subsets in peripheral blood including CD4 + and CD8 + T cells, NK and NKT cells were measured with owcytometry. In addition, histopathological assessments, immuno uorescence quanti cation and biological assays were also used to verify the inhibitory effects of icariside I on the malignant B16F10 growth of melanoma. These ndings demonstrated that icariside I has strong immunological anti-tumor activity through regulation of gut microbiome and host immunity.

Reagents
Icariside I was obtained from icariin with rhamnosidase hydrolysis as follows: 2 g of icariin (98% content) was dissolved in phosphate buffer solution (pH=6.8) and uniformly mixed with 1 g of immobilized rhaminoglycoside enzyme following vertex for 24 hours at 60℃. After the crystals precipitated, the solid powders are ltered and collected, and nally recrystallized with methanol. A total of 1.3 g of icariin I (95% content) with a yield of 65% was obtained from icariin according to speci c reaction formula (Fig.  S1).

Melanoma cells culture
Murine melanoma B16F10-cell line was obtained from the cell bank of Chinese Academy of Sciences (Shanghai, China). B16F10 cells were cultured in RPMI 1640 medium (Gibco, USA) with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) at 37°C under an atmosphere of 5% CO 2 .  Mice were allowed to have free access to food and water.
After acclimation for one week, 40 mice were implanted with B16F10 tumor cells by subcutaneous injection at the right ank (0.1 mL/mouse, 1.0 × 10 6 cells/mouse) to generate melanoma animal models.
The remaining 10 mice were used as normal group. After tumors reached about 50 mm 3 , the B16F10 tumor-bearing mice were randomly divided into four groups (n = 10) as follows: tumor group, low dose (5 mg/kg body weight), medium dose (20 mg/kg body weight) and high dose of icariside I (80 mg/kg body weight) treated groups by gavage daily for seven days with corn oil. Meanwhile, normal mice were injected 0.1 mL of PBS at the similar site and received the same volume of corn oil by gavage. During 7day continuous treatment period, tumor growth was monitored every day with an electronic vernier caliper and tumor volume was calculated as V = 0.5 × a × b 2 , where a and b denote the longer and shorter diameter, respectively.
Mice were sacri ced by cervical dislocation after 8 h fasting when the tumor of tumor group reached about 1500 mm 3 . Fecal samples were collected before sacri ce. The tumor tissue was excised, weighed and photographed and part of the tumor tissue was xed in 10% formalin solution for histopathological assessment. Other major organs, including heart, liver, lung and kidney, were also collected for histopathological assessment. Peripheral blood was harvested for further analysis. All the samples including plasma, colon, ileum, cecal contents and tumor tissue were stored at -80 °C for later experiments.
Histopathological assessment Formalin-xed tumor biopsies were embedded in para n wax, sectioned (3-4 μm), and stained with H&Estaining and anti-Ki-67 staining. Tissues from heart, liver, spleen, lung and kidney were also embedded in para n wax and stained with hematoxylin and eosin (H&E). Colonic tissue was xed in 10% formalin solution for Periodic acid-Schiff (PAS) staining. All stained sections were observed and photographed under a light microscope (with 200× magni cation). Histopathological and immuno uorescence analyses were conducted by a quali ed pathologist from Wuhan servicebio technology CO., LTD as a paid service.

Gut microbiota analysis
For 16S rRNA gene sequencing analysis, total DNA of cecal contents (~100 mg) was extracted, 16S rRNA gene amplicon sequence library was prepared as described in the protocol of 16S Metagenomic Sequencing Library Preparation (Illumina, United States). Paired-end sequencing (2 × 300 bp) was performed using an Illumina MiSeq platform by Shanghai Majorbio Bio-pharm Technology Co., Ltd. The preparation of 16S rRNA gene amplicon sequence library, statistical analysis and data manipulation were described in the Supporting Information.
Quanti cation of short chain fatty acids Targeted analyses of short chain fatty acids (SCFAs) in the feces of mice were performed on a Shimadzu 2010 Plus GC-MS spectrometer (Shimadzu Scienti c Instruments) equipped with a ame ionization detector (FID) and a CP-FFAP CB capillary GC column (25 m × 0.32 mm, 0.3 μm, Agilent Technology). The procedure of sample preparation and SCFAs measurements was described previously [32] and in Supplementary Materials.

Quanti cation of indole metabolites
Quanti cation of indole metabolites in fecal, plasma and colon tissues was performed by multiple reaction monitoring (MRM) using an ultrahigh performance liquid chromatography (Agilent 1290) coupled with a 6460 triple quadrupole mass spectrometry (UHPLC-QQQ-MS, Agilent Technologies, Inc.).
The procedure of sample preparation and indole metabolites measurements are previously described [33] with some improvements and in Supplementary Materials.

ELISA analysis
The concentrations of lipopolysaccharide (LPS), CD14, proin ammatory cytokines including IL-1β and IL-6 in plasma were measured using ELISA kits (Shanghai Huyu biotechnology Co., Ltd) according to the manufacturer's instructions.

Quantitative real-time PCR (QPCR) and western blot analyses
The experimental procedure of samples preparation and data analyses was described in Supplementary Materials.

Statistical data analysis
All experimental values are shown as mean ± standard deviation. Statistical data analyses and graphical illustrations were performed using GraphPad Prism software (version 7.0). All data between different groups were statistically analyzed using double-tailed Student's t test or Mann-Whitney test. P-values < 0.05 were considered as signi cance.

Results
Icariside I dose-dependently inhibits tumor growth Tumor bearing mice treated with different doses of icariside I (5 mg/kg, 20 mg/kg and 80 mg/kg body weight) by gavaging exhibited signi cant inhibition of tumor growth showing as tumor volume, weight and representative images for tumor ( Fig. 2A-C). Speci cally, relatively medium dose (20 mg/kg body weight) of icariside I treatment induced an average inhibition rate of 51.72% of tumor, which is higher than that of 31.03% and 37.93% induced by low and high dose of icariside I, respectively ( Fig. 2A-B).
Icariside I treatments signi cantly caused pathological changes including cell shrinkage, nuclear condensation and necrosis in tumor cells of tumor tissue in B16F10 bearing mice (Fig. 2D). Icariside I administration also inhibited tumor cell proliferation observed by anti-ki67 staining (Fig. 2D). In addition, safety assessments of icariside I in vivo with histopathological examination and body or tissue weight monitoring showed that icariside I is nontoxic or low toxicity to mice within the scope of doses used here by gavaging (Fig. S2).

Icariside I modulates gut microbiota with elevation of probiotics abundance
High-quality gut microbiota sequences were obtained from all the cecal contents samples of mice by 16S rRNA gene sequencing analysis. Microbial α-diversity analysis showed that tumor bearing mice exhibited higher OTU α-diversity indices than that of normal group. Nevertheless, icariside I treatment signi cantly recovered the unique OTU α-diversity indices in dose-dependent manner (Fig. 3A). The principal coordinates analysis (PCoA) based on Bray-Curtis distance indicated that an obvious shift of gut microbiota community structure occurred between tumor group and normal group (R 2 = 0.135, P = 0.089); whereas icariside I treatments restored the altered gut microbiota-induced by tumor growth (Fig. 3B, S2A-B). Within the intestinal microbiota, Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Tenericutes were observed as the dominant bacteria at the phylum level (Fig. 3C). Of particular note that icariside I treatments signi cantly recovered the abundance of Actinobacteria and Firmicutes, which were relatively reduced by tumor xenograft (Fig. 3E-F). At genus level, hierarchical clustering analysis represented by heatmap demonstrated that normal and icariside I treatments groups exhibited gut microbiota community shift in contrast to those of tumor group (Fig. 3D). In detail, the most striking changes of genera were typical probiotics such as Lactobacillus and Bi dobacterium, which were signi cantly higher in both normal and icariside I treatments groups than those in tumor group (Fig. 3G-H). Icariside I treatments also induced higher levels of Firmicutes/Bacteroidetes, Adlercreutzia, Allobaculum,Turicibacter and Clostridium and lower level of Bacteroidetes,Ruminococcus and Bacteroides in cecal contents of mice than those in tumor groups (Fig. S3C-J). In addition, the two typical probiotics showed a negative correlation with tumor weight, especially Lactobacillus (Fig. 3I, S3K).

Icariside I improves intestinal short chain fatty acid metabolism
Short-chain fatty acids (SCFAs), the most abundant microbial metabolites in the intestine, plays an important role in regulation of host immune system by activating G protein-coupled receptors such as Gpr41 and Gpr43 on intestinal epithelial cells. Here targeted metabolomics analyses showed that icariside I dose-dependently improved the abundance of short-chain fatty acids (SCFAs) including acetate, propionate and butyrate as well as total SCFAs in the feces of mice, which were signi cantly down-regulated by tumor growth in tumor-bearing mice compared with normal mice (Fig. 4A-B). Spearman's rank correlation analysis indicated that SCFAs positively correlated to probiotics (Lactobacillus and Bi dobacterium) (Fig. 4C-E), which was previously reported to produce SCFAs in the feces of mice [34]. Meanwhile, icariside I administration signi cantly improved the mRNA levels of Gpr41 and Gpr43 in the colon, further con rmed signi cant elevation of SCFAs produced from bacterial fermentation in intestine (Fig. 4F-G).

Icariside Iimproves microbiota-derived indole metabolism and intestinal AhR
Targeted analysis of microbiota-derived indole metabolites involved in tryptophan metabolism showed that tumor growth obviously impaired indole metabolic pathway (Fig. 5A-D), mainly manifested by signi cant down-regulation of indole metabolites including indole acetic acid (IAA), indole-3-propionic acid (IPA) and indole in the colon and feces of tumor-bearing mice, which are commonly recognized as endogenous AhR ligands [19,35]. Icariside I administration markedly improved the levels of these indole metabolites in the colon and feces of tumor-bearing mice (Fig. 5A-D), accompanied with the recovery of the abundance of indole metabolites in plasma (Fig. S4A-B). Consistently, both mRNA and protein levels of AhR were restored in the colon of tumor-bearing mice after icariside I treatment (Fig. 5E), as well as the mRNA level of colonic IL-22 (Fig. S4D), which has been reported to be associated with intestinal homeostasis [22]. Further Spearman's rank correlation analyses showed that both Lactobacillus and Bi dobacterium were positively correlated with multiple microbial-derived indole metabolites (Fig. S4C) and AhR agonists (Fig. 5F-G) in feces of mice. On the contrary, tumor weight negatively correlated with microbiota-derived AhR ligands in feces, colon and plasma of tumor-bearing mice (Fig. S4E-G).

Icariside I improves gut permeability, systemic in ammation and immunity
Periodic acid-Schiff (PAS) staining of colon showed that tumor-bearing mice exhibited lower number of mucin-producing goblet cells than normal animals (Fig. 6A-B). Consequently, gut permeability was altered due to tumor growth, manifested by signi cant up-regulation of mRNA levels of markers of gut permeability such as Myosin and Ptprh in the ileum and plasma CD14 of tumor-bearing mice, leading to disruption of intestinal barrier integrity and gut leakiness [19] (Fig. 6C-E). Tumor-bearing mice also exhibited high level of bacteria endotoxin LPS in plasma and proin ammatory cytokines including Il-6 and Il-1β ( Given close relationship between gut microbiota and derived metabolites and host immunity, we next analyzed the impact of icariside I administration on systemic immunity including multiple lymphocyte subsets by flowcytometry. The results showed that icariside I treatments markedly enhanced the percentage of CD4+ T cell and CD8+ T cell in peripheral blood of tumor-bearing mice, who exhibited lower levels of CD4+ T cell and CD8+ T cell than normal mice (Fig. 7B, E-F). Furthermore, owcytometry analysis showed that icariside I treatments also enhanced the percentage of natural killer (NK) cells and natural killer T (NKT) cells in peripheral blood of tumor-bearing mice (Fig. 7A, C-D), suggesting that icariside I has noteworthy immunological anti-tumor activity by regulating multiple lymphocyte subsets in the circulatory system.

Discussion
Epimedium known as Yin Yang Huo or Horny Goat Weed is widely used as a traditional herbal formula in many Asian countries such as China, Japan, and Korea [36]. The main bioactive compounds isolated from Herbal Epimedii include icariin, icaritin, icariside I and icariside II with similar molecular structure.
Recently, a large number of studies have focused on the anti-cancer activity of three compounds including icariin [37], icaritin [38], and icariside II [39] in a variety of cancers, such as hepatocellular cancer [29], prostatic cancer [38], and multiple myeloma [40]. Less work, however, have been done on the antitumor activity of icariside I probably due to its too low content in Epimedium. In this study, the underlying mechanisms and the role of the gut microbiota in the immunological anti-tumor activity of icariside I in a B16F10 melanoma model were investigated for the rst time using a combination of 16S rRNA gene sequencing, targeted metabolomics and biological assays.
Emerging evidences suggest that the gut microbiota plays an important role in cancer immunotherapy by modulating host immune system [6,7]. Here icariside I treatment signi cantly improved tumor xenograftinduced microbiota diversity and composition alterations including Firmicutes, Actinobacteria and Bacteroidetes at phyla levels. It is particularly noteworthy that oral administration of icariside I markedly increased the abundance of Bi dobacterium and Lactobacillus, typical probiotics bene cial for human healthy with immune enhancement, in the cecal content of tumor-bearing mice. Consequently, gut permeability, intestinal barrier integrity, systemic endotoxin LPS and in ammation induced by tumorgrowth were also improved after icariside I treatment. Signi cant inhibition of tumor growth as showing decreased tumor weight and volume as well as histopathological improvement were expectedly observed.
In agreement with our ndings, previous investigations found that oral administration of Bi dobacterium alone effectively improved tumor growth control to the same degree as PD-L1 antibody immunotherapy [6]. Oral Lactobacillus can inhibit tumor growth by promoting antitumor immunity and enhancing activity of multiple lymphocyte subsets [41]. Previous studies also revealed that both Bi dobacterium and Lactobacillus have ability to maintain integrity of colonic mucus layer and intestinal permeability for antiin ammation of mice [42,43].
In addition to gut microbiota itself, its derived metabolites such as SCFAs and indole derivatives have also been shown to have immunoregulation function through metabolic signaling pathway. Our results revealed that the levels of SCFAs and indole derivatives in the gut of tumor-bearing mice were signi cantly recovered after icariside I treatment. Many studies have shown that SCFAs especially butyrate are able to protect the intestinal mucosa from in ammation, improve host immune responses and reduce liver metastasis of colon cancer cells through metabolic signaling regulation [17,44]. Here signi cant elevation of SCFAs indicated activation of intestinal bacterial fermentation process by icariside I, which further con rmed with up-regulation of colonic Gpr41 and Gpr43 mRNAs. Previous studies also showed that microbiota-derived SCFAs can enhance cellular metabolism and memory potential of antigen-activated CD8 + T cells in participating in the immune response against intracellular pathogens and tumors [45], as well as promoting activity and differentiation of CD4 + T cells [46]. Most of microbiota-derived indole derivatives are endogenous ligands of AhR, which is involved in host immunometabolism and multiple pathophysiological processes of metabolic diseases such as obesity and cancer [47]. Signi cant down-regulation of AhR activity with lower levels of gut microbiota-derived AhR agonists such as indole derivatives were previously observed in metabolic syndromes such as obesity, diabetes and blood high pressure (BHP) of humans and animal models [19]. Furthermore, AhR activation by tryptophan derivatives (e.g. 6-formylindolo [3,2-b]carbazole) has been reported to enhance NK cells control on tumor growth and maintain NK cells in the liver of mice [48,49]. In this study, icariside I markedly up-regulated the levels of indole derivatives in fecal and colonic samples of tumor-bearing mice. Concomitantly, colonic AhR expression at both mRNAs and protein levels was also improved after icariside I treatments in a dose-dependent manner. Interestingly, our results also revealed a signi cant positive correlation between microbiota-derived SCFAs and indole derivatives and Lactobacillus and Bi dobacterium. Actually, numerous studies have reported that microbiota-derived SCFAs and indole derivatives could be produced from probiotics such as Lactobacillus and Bi dobacterium [19,20,34].
Immunological anti-tumor activity of icariside I was further directly supported by signi cant elevation of multiple lymphocytes including CD4 + T, CD8 + T, NK and NKT cells in peripheral-blood of tumor-bearing mice after icariside I treatment, indicating enhancement of host immune function by icariside I. It is well known that adaptive T cells including CD4 + T and CD8 + T cells can mediate cytotoxic responses for antitumor immunotherapy [50]. Speci cally, CD8 + T cells are currently regarded as the most effective elements for tumor destruction, which can be activated by the tumor cell-expressing surface molecules leading to tumor cell apoptosis [51]. Melanoma patients with higher number of CD8 + T cells are more likely to gain better outcomes concerning immunotherapy and therefore are often valued as immunotherapeutic targets in tumor cell lysis [52]. NK and NKT cells are able to recognize and spontaneously kill tumor cells without prior sensitization by secreting multiple cytokines and chemokines such as IFNγ and CC-chemokine ligand 3 (CCL3) and activating the caspases of tumor cells [53]. In recent years, CD4 + T cells have received increasing attention due to their functional diversity in directly or indirectly regulating anti-tumor immunity, such as promoting the activation and differentiation of CD8 + T cells and enhancing the activity of killing tumor cells [54,55].
Collectively, this study revealed that icariside I as a novel compound isolated from Epimedium signi cantly improves host intestinal barrier and immunity through regulation of gut microbiota and its derived metabolites. Speci cally, icariside I treatment markedly upregulated levels of probiotics such as Lactobacillus and Bi dobacterium and their metabolites including SCFAs and indole derivatives, which participate in host metabolic regulation through colonic Gpr41/Gpr43 and AhR signaling. These ndings suggested that icariside I (5, 20 and 80 mg/kg body weight) exhibits low toxicity and immunological anti-