Inhibitory Effect and Mechanism of Yiqi Jianpi Huayu Recipe Combined with 5-FU on Gastric Cancer / 中国实验方剂学杂志

ABSTRACT

Objective:To observe the synergistic effect and mechanism of Yiqi Jianpi Huayu (YQJPHY) recipe combined with 5-fluorouracil (5-FU) on inhibiting gastric cancer growth, and investigate its possible mechanism through polarization direction of tumor-associated macrophages (TAMs) in tumor micro-environment. Method:A total of forty 615 mice were randomly divided into four groups including control group, YQJPHY(25 g·kg-1) group, 5-FU (25 mg·kg-1) group and combined group (YQJPHY plus 5-FU), with 10 mice in each group. The gastric cancer transplantation model was induced by axillary inoculation of MFC gastric cancer cells. Changes in tumor pathology were observed with hematoxylin-eosin staining (HE) method. The contents of M1 and M2 TAMs in tumor tissues were determined with flow cytometry, while the expression of CD206 in tumor was observed with immunohistochemistry. The mRNA expressions of TAM-related genes including interleukin-1β(IL-1β), interleukin-12(IL-12), tumor necrosis factor-α(TNF-α), arginase-1(Arg1), and chitinase 3-like 3(Ym1) were detected by using quantitative real-time polymerase chain reaction (Real-time PCR). Furthermore, the mRNA and protein expressions of epithelial-mesenchymal transition (EMT) related epithelial calcium adhesion protein(E-cadherin), nervous calcium adhesion protein(N-cadherin), Zinc finger transcription factor Slug, Snail and Waveform protein (Vimentin) were detected with Real-time PCR and Western blot, respectively. Result:The gastric cancer cells in tumors were distributed in nest and/or flaky shape, with fibroblastic connective tissue reaction and inflammatory cells infiltrated in interstitium. Coagulative necrosis of tumor was observed in various treatment groups. The amount of residual cancer cells in mouse was as follows from largest to smallest control group>YQJPHY group>5-FU group>combined group. Immunohistochemical studies showed that all treatment groups can reduce the expression of CD206 in the tumor, and the effect was most obvious in the combined group. The contents of M2-TAM in all treatment groups were lower than those in control group(P<0.05, P<0.01), and the most significant difference in the content of M2-TAMs was shown between the control group and the combined group. The contents of M1 TAMs were increased in YQJPHY and combined groups(P<0.05, P<0.01), with the largest incremental range in YQJPHY group. The expressions of Arg1 and Ym1 in YQJPHY and combined groups were lower, while IL-1β, TNF-α and IL-12 levels were higher than those in control group(P<0.05). The mRNA expressions of IL-1β, IL-12 and TNF-α in the combined group were significantly higher, while Arg1 and Ym1 mRNA expression levels were lower than those in the 5-FU group(P<0.05). As compared with the control group, the mRNA and protein expression levels of E-cadherin were up-regulated(P<0.05), while the mRNA expression levels of N-cadherin and Vimentin were down-regulation in the YQJPHY group (P<0.05), E-cadherin mRNA and protein expression levels were up-regulated, while N-cadherin, Slug, Snail, Vimentin mRNA expression as well as N-cadherin and Slug protein expression levels were down-regulated in the combined group(P<0.05). As compared with the 5-FU group, the mRNA and protein expression levels of E-cadherin were up-regulated(P<0.05), while the mRNA and protein expression levels of N-cadherin, Slug and Vimentin were down-regulated in the combined group(P<0.05). Conclusion:YQJPHY has an inhibitory effect on the growth of gastric cancer, and after being combined with 5-FU, it has a good synergistic effect on inhibiting the growth of gastric cancer and decreasing EMT. The mechanism may be related to promoting the conversion from M2-TAMs to M1-TAMs in gastric tumor micro-environment.