目的 研究白頭翁皂苷D對(duì)結(jié)腸癌上皮間質(zhì)轉(zhuǎn)化（epithelial-mesenchymal transition，EMT）的影響，并基于無(wú)翅型MMTV整合位點(diǎn)家族（wingless type MMTV integration site family，Wnt）/β-連環(huán)蛋白（β-catenin）信號(hào)通路研究其作用機(jī)制。方法 通過(guò)MTT細(xì)胞增殖實(shí)驗(yàn)研究白頭翁皂苷D對(duì)人結(jié)腸癌LoVo細(xì)胞的影響，細(xì)胞劃痕實(shí)驗(yàn)研究白頭翁皂苷D對(duì)LoVo細(xì)胞遷移的影響，Transwell細(xì)胞侵襲實(shí)驗(yàn)研究白頭翁皂苷D對(duì)LoVo細(xì)胞侵襲的影響，錨定非依賴(lài)性克隆形成實(shí)驗(yàn)研究白頭翁皂苷D對(duì)LoVo細(xì)胞惡性轉(zhuǎn)化的影響；采用實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)（real-time fluorescent quantitative polymerase chain reaction，qRT-PCR）檢測(cè)LoVo細(xì)胞EMT相關(guān)基因和Wnt/β-catenin信號(hào)通路基因的mRNA水平，采用Western blotting法檢測(cè)LoVo細(xì)胞EMT相關(guān)蛋白和Wnt/β-catenin信號(hào)通路蛋白的表達(dá)水平。結(jié)果 白頭翁皂苷D顯著抑制LoVo細(xì)胞增殖活性、遷移、侵襲和克隆形成，與對(duì)照組比較，白頭翁皂苷D可顯著降低EMT相關(guān)的波形蛋白（Vimentin）和蝸牛蛋白（Snail）的mRNA和蛋白表達(dá)（P＜0.05、0.01）、升高E-鈣黏蛋白（E-cadherin）的mRNA和蛋白的表達(dá)（P＜0.01），降低Wnt/β-catenin信號(hào)通路中Wnt3a、β-catenin、T細(xì)胞因子4（T-cell factor 4，TCF4）和原癌基因（myelocytomatosis viral oncogene homolog，c-Myc）的mRNA和蛋白表達(dá)（P＜0.05、0.01），升高糖原合成激酶-3β（glycogen synthase kinase-3β，GSK-3β）的mRNA和蛋白表達(dá)（P＜0.05、0.01）。白頭翁皂苷D抑制LoVo細(xì)胞遷移和調(diào)節(jié)E-cadherin與Vimentin表達(dá)的作用可被Wnt/β-catenin信號(hào)通路抑制劑IWR-1協(xié)同增強(qiáng)，也被Wnt/β-catenin信號(hào)通路激動(dòng)劑SKL2001拮抗減弱。結(jié)論 白頭翁皂苷D可抑制結(jié)腸癌的EMT，其機(jī)制在于抑制Wnt/β-catenin信號(hào)通路活性。

Objective To investigate the effects of Pulsatilla saponin D (PSD) on the epithelial-mesenchymal transition (EMT) of colorectal cancer, and explore its action mechanisms based on the wingless type MMTV integration site family (Wnt)/β-catenin signaling pathway. Methods Effect of PSD on the cell viability of LoVo cells was determined with the MTT assay. Effect of PSD on the migration of LoVo cells was assessed with the scratch wound healing assay. Effect of PSD on the invasion of LoVo cells was examined with the Transwell invasion assay. Effect of PSD on the malignant transformation of LoVo cells was appraised with the anchorage-independent colony formation assay. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) analysis was carried out to detect the effect of PSD on the mRNA levels of EMT-related genes and the genes of Wnt/β-catenin signaling pathway in LoVo cells. Western blotting was performed to measure the effect of PSD on the expression levels of EMT-related proteins and the proteins of Wnt/β-catenin signaling pathway in LoVo cells.Results PSD significantly inhibited the proliferation activity, migration, invasion and colony formation of LoVo cells. Compared with the control group, PSD could reduce the mRNA and protein levels of EMT-related Vimentin and Snail (P < 0.05, 0.01), increased the mRNA and protein levels of E-cadherin (P < 0.01). It decreased the mRNA and protein levels of Wnt3a, β-catenin, T-cell factor 4 (TCF4), and myelocytomatosis viral oncogene homolog (c-Myc) in Wnt/β-catenin signaling pathway (P < 0.05, 0.01), increased the mRNA and protein levels of glycogen synthase kinase-3β (GSK-3β) (P < 0.05, 0.01). Furthermore, the inhibition of cell migration and the regulation of the expressions of E-cadherin and vimentin in LoVo cells by PSD were synergized by Wnt/β-catenin signaling pathway inhibitor IWR-1, and antagonized by Wnt/β-catenin signaling pathway activator SKL2001. Conclusion PSD may suppress the EMT of colorectal cancer, potentially by inhibiting the activity of Wnt/β-catenin signaling pathway.

R285.5

國(guó)家自然科學(xué)基金資助項(xiàng)目（81573813）;四川省自然科學(xué)基金面上項(xiàng)目（2023NSFSC0653）