Abstract:

Objective To investigate the inhibitory effect of Codonopsis pilosula polysaccharides （CPPs） on colorectal cancer liver metastasis in the mice， and to clarify its regulatory effects on the transforming growth factor-β （TGF-β）/Smad signaling pathway， epithelial-mesenchymal transition （EMT）， and inflammatory cytokine profiles. Methods The mouse liver metastasis model was established by intrasplenic injection of green fluorescent protein （GFP）-labeled mouse colon cancer MC-38 cells. Fifty successul modeling mice were randomly divided into modeling group， 5-fluorouracil （5-FU） group， low dose of CPPs （CPPs-L） group， a medium dose of CPPs （CPPs-M） group， and high dose of CPPs （CPPs-H） group， with 10 mice in each group. Immunofluorescence staining was used to detect the proliferation activities of the tumor cells in the liver tumor tissue， and small-animal in vivo imaging was used to detect the degree of liver metastasis in colorectal cancer of the mice in various groups after treated with CPPs； enzyme-linked immunosorbent assay（ELISA） was used to detect the serum levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） of the mice in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of E-cadherin， N-cadherin， and Vimentin mRNA in liver metastatic focus tumor tissue of the mice in various groups； Western blotting method was used to detect the expression levels of TGF-β1， Smad2/3 and their phosphorylated forms， as well as E-cadherin， N-cadherin， and Vimentin proteins in liver metastatic focus tumor tissue of the mice in various groups. Results The HE staining results showed that compared with modeling group， the tumor cells in CPPs groups were arranged more sparsely， with reduced atypia and fewer mitotic figures. The immunofluorescence staining results showed that compared with modeling group， the proliferation activities of the cells in liver tumor tissue of the mice in the other groups was decreased（P<0.05）. In vivo imaging results showed that after 3 weeks of inhibition， compared with model group， the region of interest （ROI） in liver metastatic focus of the mice in CPPs-L， CPPs-M， and CPPs-H groups was gradually decreased in a dose-dependent manner （P<0.05）， and the ROI in liver tumor tissue in CPPs-H group was similar to that in 5-FU group. The ELISA results showed that compared with modeling group， the serum levels of TNF-α， IL-1β， and IL-6 of the mice in 5-FU， CPPs-L， CPPs-M， and CPPs-H groups were decreased （P<0.05）； the serum levels of IL-1β and IL-6 of the mice in CPPs-H group were higher than those in 5-FU group （P<0.05）. The Western blotting results showed that compared with modeling group， the expression level of TGF-β1 protein and the ratios of p-Smad2/Smad2 and p-Smad3/Smad3 in liver tumor tissue of the mice in 5-FU and different doses of CPPs groups were decreased； with the increasing of CPPs dose， the expression level of E-cadherin protein was gradually increased， while the expression levels of N-cadherin and Vimentin proteins were gradually decreased. The RT-qPCR results showed that compared with model group， the E-cadherin mRNA expression level in liver tumor tissue of the mice in different doses of CPPs groups were increased（P<0.05）， while the N-cadherin and Vimentin mRNA expression levels were decreased（P<0.05）. Conclusion CPPs can significantly inhibit colorectal cancer liver metastasis in the mice， and its mechanism may be related to blocking EMT by inhibiting the TGF-β/Smad signaling pathway and reducing the expression of inflammatory cytokines to alleviate the inflammatory response.

Key words: Codonopsis pilosula polysaccharides, Colorectal neoplasm, Liver metastasis, Epithelial-mesenchymal transition, Inflammatory cytokines