Fig. 6
YYFZBJS inhibited tumor cell proliferation through regulating Treg in vitro.a Left: YYFZBJS extracts samples, 25.66 mg/ml; Right: the mix standard solution; Stationary phase: ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm); mobile phase: acetonitrile (a) and aqueous 0.1% formic acid (b) in gradient (time, min/B%: 0/95, 12/5,14/5,14.1/95,16/5); flow rate: 0.3 ml/min); column temperature:45 °C]. R:6.56 min liquiritigenin ([M-H]-,255.09518 m/z); 6.63 min, luteolin ([M-H]-,285.03936 m/z); 7.12 min mesaconine ([M + H]-,632.3065 m/z); 7.55 min, aconitine ([M + H]-,632.3065 m/z); hypaconitine 7.59 min ([M + H]-,632.3065 m/z). b Interaction network diagram between the active ingredients of YYFZBJS and their targets using prediction software of Cytoscape 3.6.1. c Experimental design indicating CD4 + CD25 + Foxp3+ T cells (Treg) were isolated from spleens of ApcMin/+ mice treated with or without ETBF and/or YYFSBJS in 62.5 μg/ml for 4 h. The ratio of cell to bacterial was 1:10. Then the primed Treg was collected and were assigned to MC-38 cells as 10:1 ratio. d Foxp3 mRNA expression was analyzed by real-time polymerase chain reaction analysis in Treg cells. The data are presented as the mean ± SD from at least three experiments. *P < 0.05, **P < 0.01 vs. ETBF group. e The decrease in ETBF count was observed in Treg incubated with ETBF and YYFZBJS (62.5 μg/ml) group. The representative gut bacteria also had higher colony-forming unit per milliliter as observed from agar plates. f MC-38 cells proliferation was assayed at 0, 12, 24, 36, and 48 h after co-culture with the primed Treg. The data are presented as the mean ± SD from at least three experiments. *P < 0.05, **P < 0.01 vs. MC-38 + Treg+ETBF. g The heatmap displays relative fold changes in expression levels normalized to the mean expression in the Control, Treg incubated with MC-38 cells (Treg: MC-38 = 10:1), ETBF primed Treg incubated with MC-38 cells, and YYFZBJS (62.5 μg/ml) combined with ETBF primed Treg incubated with MC-38 cells for each indicated mRNA of MC-38 cell. The color brightness of each unit is associated with differences. Blue color represents high expression and white color represents low expression. Not all the mRNAs in the figure were labeled. *P < 0.05, **P < 0.01 vs. MC-38 + Treg; ##P < 0.01 vs. MC-38 + Treg+ETBF. h Western blot and quantitative assay of β-catenin (nuclear, cytoplasm) in MC-38 cells. β-actin as loading control The data are presented as the mean ± SD from at least three experiments. **P < 0.01 vs. MC-38 + Treg; #P < 0.05, ##P < 0.01 vs. MC-38 + Treg+ETBF