目的探讨绿茶提取物表没食子儿茶素没食子酸酯(EGCG)对人Tenon囊成纤维细胞(HTFs)活化的作用及其可能的作用机制。
方法收集9例9眼晚期原发性开角型青光眼患者小梁切除术中获取的结膜下Tenon囊组织，通过组织贴壁培养获得原代细胞，采用免疫荧光染色法(波形蛋白+角蛋白)进行细胞鉴定，取4~6代细胞进行后续实验。利用细胞计数试剂盒-8(CCK-8)法检测0~80 μmol/L EGCG干预条件下的细胞存活率。将细胞分为空白对照组、转化生长因子(TGF)-β1诱导组和EGCG干预组，分别于正常培养基、含10 ng/ml TGF-β1培养基以及含10 ng/ml TGF-β1+50 μmol/L EGCG培养基中培养。采用BrdU标记法检测各组细胞增生率，采用细胞划痕实验观察各组细胞迁移情况，采用免疫荧光法检测各组细胞中α-平滑肌肌动蛋白(α-SMA)的表达，采用Western blot法检测空白对照组、TGF-β1诱导组、10 μmol/L EGCG组和50 μmol/L EGCG组Smad2/3、p-Smad2/3、磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(Akt)及磷酸化Akt(p-Akt)的表达。
结果体外培养HTFs呈长梭形，生长规律，免疫荧光鉴定波形蛋白呈阳性。CCK-8检测显示，10、20、30、40、50 μmol/L EGCG处理细胞存活率与0 μmol/L EGCG处理细胞比较，差异均无统计学意义(均 P<0.05)。BrdU标记实验显示，TGF-β1诱导组细胞增生率为(66.37±12.65)%，高于EGCG组的(14.75±12.33)%，差异有统计学意义( P<0.05)。划痕实验第3天，TGF-β1诱导组相对划痕面积为(47.33±12.22)%，明显低于EGCG干预组的(92.67±4.04)%，差异有统计学意义( P<0.05)。免疫荧光染色结果显示，TGF-β1诱导组细胞α-SMA蛋白荧光较空白对照组显著增强，EGCG干预组α-SMA蛋白荧光则减弱至空白对照组水平。Western blot法检测结果显示，各组间细胞中p-Smad2/3、PI3K和p-Akt蛋白相对表达量总体比较差异均有统计学意义( F＝58.820、121.153、69.289，均 P<0.001)，其中TGF-β1诱导组p-Smad2/3、PI3K和p-Akt蛋白相对表达量明显高于空白对照组、10 μmol/L和50 μmol/L EGCG组，差异均有统计学意义(均 P<0.05)。
结论EGCG能够通过Smad通路及PI3K/Akt信号通路抑制TGF-β1诱导的HTFs活化。
ObjectiveTo investigate the effect of epigallocatechin gallate (EGCG) on the activation of human Tenon fibroblasts (HTFs) and its mechanism.
MethodsTenon capsule tissues from nine eyes of nine advanced primary open angle glaucoma patients during trabeculectomy were obtained for primary cell culture.HTFs harvested were identified by immunofluorescence staining for vimentin and keratin.Cells at passage 4-6 were used for experiment.Viability of HTFs treated with EGCG at 0, 10, 20, 30, 40, 50, 60, 70 and 80 μmol/L was detected by cell counting kit-8 (CCK-8) assay.The cells were divided into blank control group, transforming growth factor (TGF)-β1-induced group, and EGCG-treated group, which were cultured in normal medium, medium containing 10 ng/ml TGF-β, medium containing 10 ng/ml TGF-β+ 50 μmol/L EGCG, respectively.The proliferation rate of HTFs was detected by BrdU labeling assay.Cell migration was observed by scratch wound healing assay.The expression of α-smooth muscle actin (α-SMA) was measured by immunofluorescence staining.The protein relative expression levels of Smad2/3, phosphoinositide-3-kinase (PI3K), protein kinase B (Akt) as well as the phosphorylated Smad2/3 (p-Smad2/3) and phosphorylated Akt (p-Akt) were measured by western blot.This study was approved by the Ethics Committee of Guangdong Provincial People's Hospital (NO.GDREC2019331H[R1]).
ResultsThe HTFs harvested had spindle shape, grew regularly and were vimentin-positive.CCK-8 assay showed that there was no significant difference in the variability of HTFs treated with EGCG at 10, 20, 30, 40 and 50 μmol/L in comparison with 0 μmol/L EGCG treatment (all at P<0.05). BrdU labeling assay showed that cell proliferation in the TGF-β1-induced group was (66.37±12.65)%, which was significantly higher than (14.75±12.33)% in EGCG-treated group ( P<0.05). Three days after scratch, the relative scratch area in the TGF-β1-induced group was (47.33±12.22)%, which was significantly lower than (92.67±4.04)% in the EGCG-treated group ( P<0.05). Immunofluorescence assay showed that α-SMA fluorescence was significantly enhanced in the TGF-β1-induced group in comparison with the blank control group, which was reduced to blank control group level in EGCG-treated group.Western blot analysis showed that there were significant differences in the relative expression levels of p-Smad2/3, PI3K and p-Akt protein among the various groups ( F=58.820, 121.153, 69.289; all at P<0.001). The relative expressions of p-Smad2/3, PI3K and p-Akt in the TGF-β1-induced group were significantly higher than those in the blank control group, 10 μmol/L and 50 μmol/L EGCG-treated groups (all at P<0.05).
ConclusionsEGCG can suppress TGF-β1-induced HTFs activation through Smad and PI3K/Akt signaling pathways.
陈艳蕾,林宏亮,王盛,等. 表没食子儿茶素没食子酸酯对人Tenon囊成纤维细胞活化的抑制作用及其调控机制[J]. 中华实验眼科杂志,2022,40(11)：1023-1030.
DOI:10.3760/cma.j.cn115989-20211206-00672版权归中华医学会所有。
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陈艳蕾：参与研究设计、实施研究、数据分析，论文撰写及修改；林宏亮：参与研究设计、实施研究、数据分析、论文修改；王盛：参与部分实验和数据分析；覃泳杰：参与选题和研究设计、实施研究、数据分析；张洪洋：参与选题和研究设计、论文智力性内容修改和定稿
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