Xiaotan Sanjie recipe (消痰散结方) suppressed the oncogenic potential of cancer-associated fibroblasts via the stromal-derived factor 1α/C-X-C chemokine receptor type 4 pathway in gastric cancer

doi:10.19852/j.cnki.jtcm.2026.02.003

Abstract

Abstract:

OBJECTIVE: To investigate the impact and mechanism of action of Xiaotan Sanjie (消痰散结方, XTSJ) on the invasiveness and metastasis of gastric cancer (GC) by modulating cancer-associated fibroblasts (CAFs). MKN-45 cells were used in in vitro experiments, and an in vivoxenograft model was established for further analysis.

METHODS: Two types of fibroblasts, CAFs and normal fibroblasts, were isolated from the tissue sample of two patients following GC surgery. The in vitro and in vivoexperiments were treated with XTSJ. Immun-ohistochemistry and enzyme-linked immunosorbent assay (ELISA) were used to assess levels of Vimentin, α-smooth muscle actin, and C-X-C chemokine receptor type 4 (CXCR4). The quantities of interleukin-6 (IL-6), stromal-derived factor 1α (SDF-1α), vascular endothelial growth factor (VEGF), and interleukin-1β (IL-1β) were measured using quantitative real-time polymerase chain reaction and ELISA.

RESULTS: The results indicated that CAFs significantly enhance the migration and invasion of GC cells compared to NFs. However, following treatment with XTSJ, CAF’s ability to promote GC was reduced. XTSJ treatment significantly inhibited CAF proliferation and downregulated the expression of CAF-associated cytokines IL-1β, IL-6, VEGF, and SDF-1α. The SDF-1α/ CXCR4 signaling pathway is a potential key mechanism through which CAFs impact GC invasion.

CONCLUSION: XTSJ treatment inhibited tumor-promoting capabilities in CAFs, suppressing GC progression, invasion, and metastasis. This study offers empirical support for the potential clinical application of XTSJ in treating and preventing GC metastasis.

Key words: cancer-associated fibroblast, gastric cancer, metastasis, stromal-cell-derived factor, chemokine (C-X-C motif) receptor 4, Xiaotan Sanjie recipe

ZHAO Ying, ZHU Zhenxin, YU Zhihong, XIU Lijuan, LIU Yongying, LU Ye, WEI Pinkang. Xiaotan Sanjie recipe (消痰散结方) suppressed the oncogenic potential of cancer-associated fibroblasts via the stromal-derived factor 1α/C-X-C chemokine receptor type 4 pathway in gastric cancer[J]. Journal of Traditional Chinese Medicine, 2026, 46(2): 294-305.

Figures/Tables 6

Figure 1 XTSJ inhibits the growth of GC tumors A: immunohistochemistry analysis for the expression of cytokines; A1: vimentin (control group); A2: α-SMA (control group); A3: CXCR4 (control group), A4: vimentin (XTSJ group); A5: α-SMA (XTSJ group); A6: CXCR4 (XTSJ group); B: tumor sizes; C: tumor weights; D: ELISA for vimentin, α-SMA, and CXCR4 detection. Control group: an orthotopic xenograft mouse model intragastrically administered 0.2 mL normal saline twice a day for 28 d; XTSJ group: an orthotopic xenograft mouse model intragastrically administered 0.2 mL XTSJ (1.39 g/kg) twice a day for 28 d. XTSJ: Xiaotan Sanjie; GC: gastric cancer; α-SMA: α-smooth muscle actin; CXCR4: C-X-C chemokine receptor type 4; ELISA: enzyme-linked immunosorbent assay. Statistical analyses were measured using one-way analysis of variance. Data were presented as mean ± standard deviation (n = 3). Compared with control group, aP < 0.05.

Table 1 XTSJ inhibits IL-1β, IL-6, SDF-1α, and VEGF secretion by CAFs ($ \bar{x} \pm s$)

Cytokine	Protein concentration (ng/mL)			mRNA expression
Cytokine	NFs	CAFs	CAFs+XTSJ	NFs	CAFs	CAFs+XTSJ
IL-1β	27845.00±1318.00	40919.00±621.70	32700.00±1711.00^a	2.82±0.61	5.39±0.44	4.72±0.15^a
IL-6	19.34±0.91	26.13±0.04	21.51±0.37^a	1.58±0.57	1.74±0.16	1.27±0.51^a
VEGF	2597.00±866.40	3830±579.20	3387.00±624.50^a	3.03±1.46	6.33±1.84	4.98±0.61^a
SDF-1α	26.07±1.11	44.57±0.21	28.81±0.83^a	4.54±0.57	8.33±0.18	4.90±0.10^a

Table 2 XTSJ inhibits the secretion of IL-1β, VEGF, IL-6, and SDF-1α from GC tumors ($ \bar{x} \pm s$)

Cytokine	Protein concentration (ng/L)			mRNA expression
Cytokine	Control (n = 6)	XTSJ (n = 6)	P value	Control (n = 6)	XTSJ (n = 6)	P value
IL-1β	64.92±5.28	50.75±4.77	0.006	8.85±3.11	6.45±1.66	0.0067
IL-6	115.60±21.64	85.59±8.41	0.0101	2.94±0.96	1.86±1.08	0.0036
VEGF	404.80±45.24	262.30±30.65	0.001	1.57±0.56	0.92±0.43	0.0004
SDF-1α	787.10±72.38	448.20±35.22	0.001	1.24±0.41	0.71±0.24	0.0001

Figure 2 CAFs increase the migratory and invasive abilities of human GC MKN-45 cells A: cell invasion was assessed using the Transwell assay; A1: MKN-45 + 5% FBS-CM group; A2: MKN-45 + NFs-CM group; A3: MKN-45 + CAFs-CM group; B: quantitative analysis of cell invasion; C: cell migration was assessed using the wound healing assay; C1: MKN-45 + 5% FBS-CM group at 0 h; C2: MKN-45 + 5% FBS-CM group at 24 h; C3: MKN-45 + 5% FBS-CM group at 48 h; C4: MKN-45 + 5% FBS-CM group at 72 h; C5: MKN-45 + NFs-CM group at 0 h; C6: MKN-45 + NFs-CM group at 24 h; C7: MKN-45 + NFs-CM group at 48 h; C8: MKN-45 + NFs-CM group at 72 h; C9: MKN-45 + CAFs-CM group at 0 h; C10: MKN-45 + CAFs-CM group at 24 h; C11: MKN-45 + CAFs-CM group at 48 h; C12: MKN-45 + CAFs-CM group at 72 h; D: quantitative analysis of cell migration; E: analysis of SDF-1α levels using ELISA; F: expression of CXCR4 was examined by WB; G: relative protein expression levels of CXCR4. MKN-45 + 5% FBS-CM group: MKN-45 cells cultured with conditioned medium from 5% FBS-treated cells; MKN-45 + NFs-CM group: MKN-45 cells cultured with conditioned medium from normal fibroblasts; MKN-45 + CAFs-CM group: MKN-45 cells cultured with conditioned medium from cancer-associated fibroblasts. GC: gastric cancer; FBS: fetal bovine serum; CM: conditioned medium; NFs: normal fibroblasts; CAFs: cancer-associated fibroblasts; SDF-1α: stromal-derived factor 1α; CXCR4: C-X-C chemokine receptor type 4. Statistical analyses were measured using one-way analysis of variance. Data were presented as mean ± standard deviation (n = 3). Compared with MKN-45 + 5% FBS-CM group, aP < 0.01; compared with MKN-45 + NFs-CM group, bP < 0.05.

Figure 3 Role of CXCR4/SDF-1α signaling in CAF-induced invasive and migratory abilities in GC cells A: cell invasion was examined using the Transwell assay; A1: MKN-45 + 5% FBS-CM group; A2: MKN-45 + 5% FBS-CM + SDF-1α group; A3: MKN-45 + CAFs-CM group; A4: MKN-45 + CAFs-CM + AMD3100 group; B: quantitative analysis of cell invasion; C: cell migration was examined using the wound scratch assay; C1: MKN-45 + 5% FBS-CM group at 0 h; C2: MKN-45 + 5% FBS-CM + SDF-1α group at 0 h; C3: MKN-45 + CAFs-CM group at 0 h; C4: MKN-45 + CAFs-CM + AMD3100 group at 0 h; C5: MKN-45 + 5% FBS-CM group at 48 h; C6: MKN-45 + 5% FBS-CM + SDF-1α group at 48 h; C7: MKN-45 + CAFs-CM group at 48 h; C8: MKN-45 + CAFs-CM + AMD3100 group at 48 h; D: quantitative analysis of cell migration. MKN-45 + 5% FBS-CM group: MKN-45 cells cultured with 5% FBS as control; MKN-45 + 5% FBS-CM + SDF-1α group: MKN-45 cells treated with SDF-1α in 5% FBS medium; MKN-45 + CAFs-CM group: MKN-45 cells cultured with conditioned medium from cancer-associated fibroblasts; MKN-45 + CAFs-CM + AMD3100 group: MKN-45 cells cultured with CAFs-CM plus 100 nM CXCR4 inhibitor AMD3100. GC: gastric cancer; FBS: fetal bovine serum; CM: conditioned medium; SDF-1α: stromal-derived factor 1α; CAFs: cancer-associated fibroblasts. Statistical analyses were measured using one-way analysis of variance. Data were presented as mean ± standard deviation (n = 3). Compared with MKN-45 + 5% FBS-CM + SDF-1α, aP < 0.01; compared with MKN-45 + CAFs-CM, bP < 0.05.

Figure 4 XTSJ suppresses CAF-mediated invasion and metastasis in GC cells A: cell invasion was determined using the Transwell assay; A1: MKN-45 + 5% FBS-CM group; A2: MKN-45 + NFs-CM group; A3: MKN-45 + CAFs-CM group; A4: MKN-45 + XTSJ + CAFs-CM group; B: quantitative analysis of cell invasion; C: cell migration was examined using the wound scratch assay; C1: MKN-45 + 5% FBS-CM group at 0 h; C2: MKN-45 + NFs-CM group at 0 h; C3: MKN-45 + CAFs-CM group at 0 h; C4: MKN-45 + XTSJ + CAFs-CM group at 0 h; C5: MKN-45 + 5% FBS-CM group at 48 h; C6: MKN-45 + NFs-CM group at 48 h; C7: MKN-45 + CAFs-CM group at 48 h; C8: MKN-45 + XTSJ + CAFs-CM group at 48 h; D: quantitative analysis of cell migration; E: SDF-1α levels were analyzed using ELISA. MKN-45 + 5% FBS-CM group: MKN-45 cells cultured with conditioned medium from 5% FBS-treated cells; MKN-45 + NFs-CM group: MKN-45 cells cultured with conditioned medium from normal fibroblasts; MKN-45 + CAFs-CM group: MKN-45 cells cultured with conditioned medium from cancer-associated fibroblasts; MKN-45 + XTSJ + CAFs-CM group: MKN-45 cells cultured with conditioned medium from cancer-associated fibroblasts treated with 10% XTSJ for 24 h. GC: gastric cancer; FBS: fetal bovine serum; CM: conditioned medium; NFs: normal fibroblasts; CAFs: cancer-associated fibroblasts; XTSJ: Xiaotan Sanjie; SDF-1α: stromal-derived factor 1α; ELISA: enzyme-linked immunosorbent assay. Statistical analyses were measured using one-way analysis of variance. Data were presented as mean ± standard deviation (n = 3). Compared with MKN-45 + NFs-CM group, aP < 0.01, compared with MKN-45 + CAFs-CM group, bP < 0.05.

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