目的 基于Wnt信号通路探究表没食子酸儿茶素没食子酸酯（epigallocatechin gallate，EGCG）对人肝星状细胞LX-2活化的影响及其作用机制。方法 体外培养LX-2细胞，CCK-8法筛选EGCG的实验浓度；取对数生长期的LX-2细胞，设置对照组（正常培养）、模型组[10 ng/mL转化生长因子-β1（transforming growth factor-β1，TGF-β1）]、EGCG低剂量组（12.5 μmol/L EGCG＋10 ng/mL TGF-β1）、EGCG高剂量组（25.0 μmol/L EGCG＋10 ng/mL TGF-β1）、EGCG＋siRNA-NC组[小窝蛋白-1（Caveolin-1，Cav-1）-siRNA阴性对照＋25.0 μmol/L EGCG＋TGF-β1]和EGCG＋Cav-1-siRNA组（Cav-1-siRNA＋25.0 μmol/L EGCG＋TGF-β1），CCK-8法检测各组细胞存活率；流式细胞术检测细胞凋亡率和细胞周期分布情况；吖啶橙/溴乙锭（AO/EB）染色法观察细胞凋亡形态；qRT-PCR法检测细胞α-平滑肌肌动蛋白（α-smooth muscle actin，α‐SMA）、I型胶原蛋白（Collagen Ⅰ）和基质金属蛋白酶组织抑制剂-1（tissue inhibitors of matrix metalloproteinase-1，TIMP-1）mRNA表达情况；Western blotting检测细胞Cav-1和Wnt信号通路相关蛋白表达情况。结果 EGCG降低LX-2细胞存活率，呈剂量相关性。与对照组相比，模型组LX-2细胞存活率、S期和G₂/M期的细胞比例、细胞α‐SMA、Collagen I、TIMP-1 mRNA表达和Wnt1、Wnt5a、β-连环蛋白（β-catenin）、细胞周期蛋白Cyclin D1、原癌基因c-Myc蛋白表达水平均显著升高（P＜0.05），细胞凋亡率、G₀/G₁期的细胞比例、凋亡细胞比例、Cav-1蛋白表达水平均显著降低（P＜0.05）；与模型组相比，EGCG低、高剂量组LX-2细胞存活率、S期和G₂/M期的细胞比例、细胞α‐SMA、Collagen I、TIMP-1 mRNA表达和Wnt1、Wnt5a、β-catenin、Cyclin D1、c-Myc蛋白表达水平均显著降低（P＜0.05），细胞凋亡率、G₀/G₁期的细胞比例、凋亡细胞比例、Cav-1蛋白表达水平均显著升高（P＜0.05）；且在EGCG干预的基础上，沉默Cav-1的表达可显著上调Wnt1、Wnt5a蛋白表达，减弱EGCG对Wnt信号通路的抑制作用。结论 EGCG可能通过上调Cav-1表达，抑制Wnt信号通路激活，进而抑制LX-2细胞活化。

Objective To explore the effect and mechanism of epigallocatechin gallate (EGCG) on the activation of hepatic stellate cells LX-2 based on Wnt pathway. Methods LX-2 cells were cultured in vitro, and the experimental concentration of EGCG was screened by CCK-8 method. LX-2 cells in the logarithmic growth phase were divided into control group (normally cultured), model group [10 ng/mL transforming growth factor-β1 (TGF-β1)], low-dose EGCG group (12.5 μmol/L EGCG + 10 ng/mL TGF-β1), high-dose EGCG group (25.0 μmol/L EGCG + 10 ng/mL TGF-β1), EGCG + siRNA-NC group [Caveolin-1 (Cav-1)-siRNA negative control + 25.0 μmol/L EGCG + TGF-β1] and EGCG + Cav-1-siRNA group (Cav-1-siRNA + 25.0 μmol/L EGCG + TGF-β1), CCK-8 method was used to detect cell survival rate of each group; Flow cytometry was used to detect cell apoptosis and cell cycle distribution; Acridine orange/ethidium bromide (AO/EB) staining method was used to observe the morphology of cell apoptosis; qRT-PCR was used to detect α-smooth muscle actin (α-SMA), Collagen I and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) mRNA expressions of cells; Western blotting was used to detect the expressions of Cav-1 and Wnt signaling pathway related proteins of cells. Results EGCG reduced survival rate of LX-2 cells in a concentration-dependent manner. Compared with control group, LX-2 cells survival rate, ratios of S phase and G₂/M phase cells, mRNA expressions of α-SMA, Collagen I and TIMP-1, and protein expressions of Wnt1, Wnt5a, β-catenin, Cyclin D1, c-Myc were significantly increased in model group (P < 0.05), apoptosis rate, ratio of G₀/G₁ phase cells, ratio of apoptotic cells, and Cav-1 protein expression were significantly reduced (P < 0.05). Compared with model group, LX-2 cells survival rate, ratios of S phase and G₂/M phase cells, mRNA expression of α-SMA, Collagen I, TIMP-1, and protein expressions of Wnt1, Wnt5a, β-catenin, Cyclin D1, c-Myc in low-, high-dose EGCG groups were significantly decreased (P < 0.05), apoptosis rate, ratio of G₀/G₁ phase cells, ratio of apoptotic cells, and Cav-1 protein expression were significantly increased (P < 0.05); On the basis of EGCG intervention, silencing the expression of Cav-1 significantly up-regulated Wnt1 and Wnt5a protein expressions, and weakened the inhibitory effect of EGCG on Wnt signaling pathway. Conclusion EGCG may inhibit the activation of Wnt signaling pathway by up-regulating the expression of Cav-1, thereby inhibiting the activation of LX-2 cells.

R285.5

河南省2017年科技发展计划项目（172102310284）