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 filtered and collected, and finally 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 specific reaction formula (Fig. S1).
Sodium chloride, Heparin sodium, diethyl ether, isoflurane anesthesia, hydrochloric acid (HCl), NaH2PO4·2H2O, and K2HPO4·3H2O were purchased from Sinopharm Chemical Co. Ltd. (Shanghai, China). HPLC-grade water, formic acid (FA), acetonitrile (MeCN), methanol (MeOH) and standards of tryptophan metabolites and short chain fatty acids were obtained from Sigma-Aldrich (Shanghai, China).
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% CO2. B16F10 cells were collected by dissociating the cells in trypsin and washed and resuspended in PBS for tumor inoculation.
B16F10 melanoma-bearing mouse model
The animal experiment was performed according to Chinese National Guidelines and approved by the animal ethics committee of Wuhan Institute of Physics and Mathematics (WIPM, China). A total of 50 female C57BL/6 mice (18–22 g, aged 6–7 weeks) were purchased from Charles River Co. Ltd (Beijing, China) and housed together in groups of three or four mice per cage with a 12 h light/dark cycle and constant temperature (22 ± 1°C) and humidity (50 ± 10%) at Wuhan Institute of Virology (Hubei, China). 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 flank (0.1 mL/mouse, 1.0 × 106 cells/mouse) to generate melanoma animal models. The remaining 10 mice were used as normal group. After tumors reached about 50 mm3, 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 7-day continuous treatment period, tumor growth was monitored every day with an electronic vernier caliper and tumor volume was calculated as V = 0.5 × a × b2, where a and b denote the longer and shorter diameter, respectively.
Mice were sacrificed by cervical dislocation after 8 h fasting when the tumor of tumor group reached about 1500 mm3. Fecal samples were collected before sacrifice. The tumor tissue was excised, weighed and photographed and part of the tumor tissue was fixed 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-fixed tumor biopsies were embedded in paraffin wax, sectioned (3-4 μm), and stained with H&E-staining and anti-Ki-67 staining. Tissues from heart, liver, spleen, lung and kidney were also embedded in paraffin wax and stained with hematoxylin and eosin (H&E). Colonic tissue was fixed in 10% formalin solution for Periodic acid-Schiff (PAS) staining. All stained sections were observed and photographed under a light microscope (with 200× magnification). Histopathological and immunofluorescence analyses were conducted by a qualified 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.
Quantification 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 Scientific Instruments) equipped with a flame 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.
Quantification of indole metabolites
Quantification 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.
Quantification of peripheral blood lymphocyte by flowcytometry
Peripheral blood was collected from the orbital vein plexus with EDTA-Li micro-anticoagulant tube. Blood sample (100 µL) was stained with BV421 anti-mouse CD3e (BD Horizon, NY, USA), FITC anti-mouse CD49b (BD Pharmingen, NY, USA), PE anti-mouse CD4 (BD Pharmingen, NY, USA), PE-Cy7 anti-mouse CD8a (BD Pharmingen, NY, USA) at 4°C in dark for 30 min, and then erythrocytes were lysed in Lysing Buffer (BD Pharm Lyse, NY, USA) for 5 min. After washing with pre-cold PBS for two times, T cell subsets in the peripheral blood were accounted with a FACS Canto II cytometer (BD, NY, USA), and the quantitative data were analyzed by FlowJo software (version 7.6.1).
ELISA analysis
The concentrations of lipopolysaccharide (LPS), CD14, proinflammatory 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 significance.