Xiaoyaosan, a traditional Chinese medicine, inhibits the chronic restraint stress-induced liver metastasis of colon cancer in vivo

Figures & data

Table 1. The contents of each crude medicinal component in 10 g granulated XYS.

Herbal name	Chinese name	Latin binomial	Family	Amount
Chinese Thorowax Root	Chaihu	Bupleurum chinense DC.	Umbelliferae	8.1 g
Radix Angelicae Sinensis	Danggui	Angelica sinensis (Oliv.) Diels.	Umbelliferae	8.1 g
Radix Paeoniae Alba	Shaoyao	Paeonia lactiflora Pall.	Paeoniaceae	8.1 g
Rhizoma Atractylodis Macrocephalae	Baizhu	Atractylodes macrocephala Koidz	Compositae	8.1 g
Poria	Fuling	Poria cocos Wolf.	Polyporaceae	8.1 g
Radix Glycyrrhizae	Gancao	Glycyrrhiza uralensis Fisch	Leguminosae	8.1 g
Herba Menthae	Bohe	Mentha haplocalyx Briq.	Lamiaceae	3.9 g
Rhizoma Zingiberis Recens	Shengjiang	Zingiber officinale Rosc.	Zingiberaceae	3.9 g

Table 2. Sequences of the primers used in the real-time PCR analysis.

Gene	Primer sequence (5′–3′)
IL-6	F: TAGTCCTTCCTACCCCAATTTCC
	R: TTGGTCCTTAGCCACTCCTTC
VEGF	F: GCCAGACAGGGTTGCCATAC
	R: GGAGTGGGATGGATGATGTCAG
MMP-9	F: CTGGACAGCCAGACACTAAAG
	R: CTCGCGGCAAGTCTTCAGAG
TGF-β	F: AGACCACATCAGCATTGAGTG
	R: GGTGGCAACGAATGTAGCTGT
GAPDH	F: AGGTCGGTGTGAACGGATTTG
	R: TGTAGACCATGTAGTTGAGGTCA

Figure 1. Effect of chronic stress and XYS on liver metastasis in a CRC model. (a) Representative images of the metastatic liver taken on day 28 after tumour cell injection. (b) Representative images of the spleen taken on day 28 after tumour cell injection. (c) Data represent average liver (left) and spleen (right) weights for the four groups. *p< 0.05, ***p< 0.001. BC: blank-control; BS: blank-stress; XC: XYS-control; XS: XYS-stress.

Figure 2. Effects of chronic restraint stress and XYS on the tumour microenvironment. (a) CD11b⁺F4/80⁺ cells were quantified by flow cytometry in disaggregated HT-29 primary spleen tumour harvested 28 days after cell injection. (b) Data represent the average CD11b⁺F4/80⁺ composition. (c) CD11b⁺Gr^loLy6C^hi and CD11b⁺Gr^hiLy6C^lo cells were quantified by flow cytometry in disaggregated HT-29 primary spleen tumour harvested 28 days after cell injection. (d) Data represent the average CD11b⁺Gr^loLy6C^hi composition. *p< 0.05, **p< 0.01 and ***p< 0.001. BC: blank-control; BS: blank-stress; XC: XYS-control; XS: XYS-stress.

Figure 3. Effects of chronic restraint stress and XYS on liver metastasis in a CRC model. (a) mRNA expression of TGF-β, IL-6, MMP-9 and VEGF in spleen tumours relative to mice in the BC group. (b) Protein expression of TGF-β, IL-6, MMP-9 and VEGF in spleen tumours. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (c) Relative protein expression levels. GAPDH was used as an internal standard. (d) Immunohistochemical detection of VEGF protein expression in metastatic liver tumour tissue. (e) Immunohistochemical detection of CD31 protein expression in metastatic liver tumour tissue (magnification ×200). (f) IHC positive rate of VEGF and CD31 in metastatic liver tumours (magnification ×200). *p< 0.05, **p< 0.01 and ***p< 0.001. BC: blank-control; BS: blank-stress; XC: XYS-control; XS: XYS-stress.