白藜芦醇调控TGFβ1/Smad信号通路抑制胃癌MGC803细胞增殖机制探讨

Resveratrol inhibits proliferation of human gastric cancer MGC803 cells via regulating TGFβ1/Smad signaling pathways

目的:探讨白藜芦醇对人胃癌MGC803细胞生长增殖的影响及其可能的作用机制。方法:用白藜芦醇溶液干预胃癌MGC803细胞,然后采用MTT法与台盼蓝染色实验检测白藜芦醇溶液对胃癌MGC803细胞生长增殖的影响。采用ELISA法检测MGC803细胞培养基中细胞外泌转化生长因子β1(TGFβ1)蛋白的含量。蛋白质印迹法检测白藜芦醇干预后MGC803细胞中p-Smad3、Smad4、Cyclin D1信号通路相关蛋白表达的变化。结果:MTT法检测结果显示:低、中、高剂量白藜芦醇组MGC803细胞的活力分别为对照组的0.937±0.046(t=1.266,P=0.274>0.05)、0.844±0.026(t=4.822,P=0.009<0.05)和0.540±0.038(t=10.997,P=3.89×10-4<0.001)倍,其中各剂量组间细胞活力存在明显差异(F=13.216,P=0.002)。同时台盼蓝染色计数结果也发现:对照组、低、中、高剂量白藜芦醇组MGC803细胞的死亡率分别为(4.83±3.00)%、(21.58±3.21)%、(34.84±2.87)%和(56.83±2.75)%,低、中、高剂量组MGC803细胞死亡率较对照组明显上升(P=0.016<0.05)。此外,对照组、低、中、高剂量白藜芦醇组MGC803细胞上清液中TGFβ1的含量分别为(1 332.32±39.81)、(1 264.80±11.58)、(1 170.54±15.51)和(1 148.12±24.01)pg/ml,各组间差异具有明显的统计学意义(F=11.520,P=0.003)。蛋白质印迹法检测结果显示:白藜芦醇干预后MGC803细胞中p-Smad3、Cyclin D1蛋白水平明显下降,并且随着白藜芦醇剂量的增加,抑制作用也明显增强(P<0.05)。Smad4蛋白的表达水平呈现出明显上调,且亦与白藜芦醇存在明显的量效关系(P<0.05)。结论:白藜芦醇可抑制胃癌MGC803细胞的增殖,并且其作用的发挥与白藜芦醇对TGFβ1/Smad信号通路的调控作用有关。

Objective： To investigate the effect of resveratrol on proliferation of human gastric cancer MGC803 cells and its possible machanism. Methods： Resveratrol was used to test human gastric cancer MGC803 cells. The proliferation of MGC803 cells was tested by MTT method and Typan blue. The expression level of TGFβ1 protein in culture medium of MGC803 cells infected with resveratrol was detected by ELISA. The expressions of Cyclin D1 were detected by Western blot. The content of phosphor- Smad3 and Smad4 related to transforming growth factor- beta1（ TGFβ1） /Smad signaling pathway were detected by Western blot and immunocytochemistry. Results： Cell viability of MGC8 0 3 cells was obviously inhibited by several doses of resveratrol which could be 0. 937 ± 0. 046（ t = 1. 266,P= 0. 274 0. 05）,0. 844 ± 0. 026（ t = 4. 822,P = 0. 009 0. 05） and 0. 540 ± 0. 038（ t = 10. 997,P = 3. 89 × 10- 40. 001） times than the control group,and the cell viability also have a huge difference among the three groups with resveratrol used（ F = 13. 216,P = 0. 002）. Similarly,the cell proliferation was obviously inhibited when several concentrations of resveratrol were used（ P = 0. 016 0. 05）. The expression of TGFβ 1 protein in culture medium of MGC 8 0 3 cells detected by ELISA were（ 1 332. 32 ± 39. 81）,（ 1 264. 80 ± 11. 58）,（ 1 170. 54 ± 15. 51） and（ 1 148. 12 ± 24. 01） pg/ml in control group,low,mid and high doses of resveratrol. Compared with the control group,the expression of TGFβ1 protein was decreased after the cells was treated with resveratrol（ F = 11. 520,P = 0. 003）,and the expression of p- Smad3 and Cyclin D1 protein in MGC803 cells detected by Western blotting was decreased after the cells was treated with resveratrol（ P〈0. 05）. In addition,the content of Smad4 protein in MGC803 was increased after the application of resveratrol（ P〈0. 05）. Conclusion： Reveals that resveratrol can inhibit proliferation of human gastric cancer MGC803 cells. This machanism may be related to the inhibition of TGFβ1 / Smad signaling transduction pathways.