目的 研究小槐花Desmodium caudatum抑制腫瘤細胞增殖和轉(zhuǎn)移的作用，并探討其對Wnt/β-連環(huán)蛋白（β-catenin）和腺苷酸活化蛋白激酶（AMPK）信號通路的調(diào)控作用。方法 采用不同條件制備8種小槐花提取物，MTT法檢測小槐花提取物1～8對HCT-116、SW480細胞增殖的影響；結(jié)晶紫染色檢測小槐花提取物2、3對結(jié)腸癌HCT-116、SW480細胞集落形成的影響；劃痕實驗檢測小槐花提取物2、3對HCT-116、SW480細胞遷移的影響；從活性較高的提取物2中提取分離出5個黃酮類化合物（1～5），MTT法檢測化合物1～5對HCT-116、SW480、HepG2、RAW264.7、LX-2細胞增殖的影響；結(jié)晶紫染色檢測化合物1 （8-異戊烯基槲皮素，0.5、1.0、2.0、5.0、10.0 μmol· L^-1）對HCT-116和SW480細胞集落形成的影響，劃痕實驗檢測8-異戊烯基槲皮素對HCT-116和SW480細胞轉(zhuǎn)移的影響。Western blotting實驗檢測8-異戊烯基槲皮素對HCT-116和SW480細胞中AMPK和Wnt/β-catenin信號通路相關(guān)蛋白表達的影響；熒光素酶報告基因檢測實驗觀察8-異戊烯基槲皮素對β-catenin與T細胞因子（TCF）結(jié)合能力的影響。結(jié)果 與對照組比較，小槐花根莖提取物2和3以及8-異戊烯基槲皮素能顯著抑制結(jié)腸癌細胞的增殖和遷移（P<0.05、0.01、0.001）。與對照組比較，8-異戊烯基槲皮素能夠顯著上調(diào)p-AMPK蛋白的表達（P<0.05、0.001），顯著下調(diào)AMPK、乙酰輔酶A羧化酶（ACC）、肉毒堿棕櫚?；D(zhuǎn)移酶1A （CPT-1A）和脂肪酸合成酶（FAS）蛋白的表達（P<0.05、0.01、0.001），表明其能夠促進癌細胞AMPK信號通路的激活。與對照組比較，8-異戊烯基槲皮素顯著下調(diào)β-catenin以及其下游糖原合酶激酶3β（GSK-3β）、核內(nèi)原癌基因（c-Myc）和G₁/S-特異性周期蛋白-D1 （Cyclin D1）的蛋白表達（P<0.05、0.01、0.001），同時顯著降低TCF報告基因結(jié)合位點（野生型）和熒光素酶開放閱讀框TOPflash（pGL3-OT）活性（P<0.05、0.01），但不影響FOPflash（含突變位點）活性，表明其抑制β-catenin的表達和β-catenin/TCF復合物的結(jié)合。結(jié)論 小槐花黃酮8-異戊烯基槲皮素通過調(diào)控Wnt/β-catenin和AMPK信號通路抑制結(jié)腸癌細胞增殖和轉(zhuǎn)移。

Objectives To investigate the mechanism of Desmodium caudatum in inhibiting the proliferation andmetastasis of tumor cells and its regulation on Wnt/β-catenin and AMPK signaling pathways. Methods Eight extracts of D. caudatum were prepared under different conditions. MTT assay was used to detect the effect of extract 1-8 on the proliferation of HCT-116 and SW480 cells. Crystal violet staining was used to detect the effect of extract 2 and 3 on colony formation of colon cancer HCT-116 and SW480 cells. Effects of extract 2 and 3 on HCT-116 and SW480 cells migration detected by scratch test. Western blotting assay was used to detect the effect of 8-prenylquercetin on AMPK and Wnt/β-catenin signaling pathway related protein expression in HCT-116 and SW480 cells. The effect of 8-prenylquercetin on the binding ability of β-catenin and T cell factor (TCF) by luciferase reporter gene test. Results Compared with control group, extracts 2 and 3 and 8-prenylquercetin significantly inhibited the proliferation and migration of colon cancer cells. Compared with control group, 8-prenylquercetin could significantly up-regulate the expression of p-AMPK protein (P < 0.05 and 0.001) and down-regulate the expression of AMPK, ACC, p-ACC, CPT-1A and FAS proteins (P < 0.05, 0.01 and 0.001), indicating that it could promote the activation of AMPK signaling pathway in cancer cells. In addition, compared with control group, 8-prenylquercetin downregulated the expression of β-catenin and its downstream proteins pGSK 3β, c-Myc and Cyclin D1 (P < 0.05, 0.01 and 0.001). Compared with control group, 8-prenylquercetin significantly reduced TOPflash activity in a concentration dependent manner, but not FOPflash activity, indicating that it inhibited β-catenin expression and β-catenin/TCF complex binding. Conclusion 8-prenylquercetin a flavonoid from D. caudatum, inhibits the proliferation and metastasis of tumor cells by regulating Wnt/β-catenin and AMPK signaling pathways.

R285.5

江蘇省雙創(chuàng)團隊項目（JSSCTD202133）；天津市自然科學基金項目（18JCYBJC94800）