六味地黄丸含药血清对高糖诱导的滋养层细胞Wnt/β-catenin通路及迁移、侵袭的影响

doi:10.11958/20251508

摘要/Abstract

摘要：

目的探讨六味地黄丸含药血清对高糖诱导的滋养层细胞Wnt/β-链蛋白（β-catenin）通路及迁移、侵袭的影响。方法制备六味地黄丸含药血清，体外培养人绒毛膜滋养层HTR-8/Svneo细胞并分为：对照组（5 mmol/L葡萄糖+空白血清）、高糖组（30 mmol/L葡萄糖+空白血清）、低剂量组[30 mmol/L葡萄糖+（0.45 g/mL六味地黄丸药液）血清]、中剂量组[30 mmol/L葡萄糖+（0.90 g/mL六味地黄丸药液）血清]和高剂量组[30 mmol/L葡萄糖+（1.35 g/mL六味地黄丸药液）血清]。实时荧光定量PCR（qRT-PCR）法检测各组HTR-8/Svneo细胞中Wnt1、β-catenin、细胞周期蛋白D1（Cyclin D1）mRNA表达情况；蛋白免疫印迹（Western blot）法检测各组HTR-8/Svneo细胞中Wnt1、β-catenin、磷酸化糖原合成激酶-3β（p-GSK-3β）、Cyclin D1蛋白表达情况；Transwell法检测各组HTR-8/Svneo细胞迁移、侵袭情况。结果与对照组相比，高糖组HTR-8/Svneo细胞中Wnt1、β-catenin、Cyclin D1 mRNA表达水平，Wnt1、β-catenin、p-GSK-3β、Cyclin D1蛋白表达水平及迁移细胞数和侵袭细胞数降低（P<0.05）；与高糖组相比，低、中、高剂量组HTR-8/Svneo细胞中Wnt1、β-catenin、Cyclin D1 mRNA表达水平，Wnt1、β-catenin、p-GSK-3β、Cyclin D1蛋白表达水平及迁移细胞数和侵袭细胞数升高（P<0.05）；随着六味地黄丸含药血清剂量的升高，HTR-8/Svneo细胞中上述指标均逐次升高（P<0.05）。结论六味地黄丸含药血清可激活高糖诱导下HTR-8/Svneo细胞中的Wnt/β-catenin通路，促进HTR-8/Svneo细胞的迁移、侵袭过程。

关键词: 迁移, 侵袭, Wnt, β-catenin, 六味地黄丸含药血清, 高糖, 滋养层细胞

Abstract:

Objective To investigate the effect of drug-containing serum from Liuwei Dihuang Pill on Wnt/β-catenin pathway, migration and invasion of trophoblast cells induced by high glucose. Methods The drug-containing serum of Liuwei Dihuang Pill was prepared. Human chorionic trophoblast HTR-8/Svneo cells were cultured in vitro and divided into the control group (5 mmol/L glucose + blank serum), the high glucose group (30 mmol/L glucose + blank serum), the low-dose group (30 mmol/L glucose, serum medicine solution of Rehmannia Six Formula at 0.45 g/mL), the medium-dose group (30 mmol/L glucose, serum medicine solution of Rehmannia Six Formula at 0.90 g/mL) and the high-dose group (30 mmol/L glucose, serum medicine solution of Rehmannia Six Formula at 1.35 g/mL). The mRNA expressions of Wnt1, β-catenin and Cyclin D1 in HTR-8/Svneo cells were detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein expressions of Wnt1, β-catenin, phosphorylated glycogen synthesis kinase-3β (p-GSK-3β) and Cyclin D1 in HTR-8/Svneo cells were detected by Western blot assay. The migration and invasion of HTR-8/Svneo cells were detected by Transwell method. Results Compared with the control group, the protein and mRNA expression levels of Wnt1, β-catenin and Cyclin D1, the numbers of migration cells and invasive cells in HTR-8/Svneo cells were decreased in the high glucose group (P< 0.05). Compared with the high glucose group, the protein and mRNA expression levels of Wnt1, β-catenin and Cyclin D1, the numbers of migration cells and invasive cells were significantly increased in the low dose, the medium dose and the high dose groups of the HTR-8/Svneo cells (P < 0.05). In addition, with the increase of the drug containing serum dose of Liuwei Dihuang Pill, the above indexes in HTR-8/Svneo cells increased successively (P < 0.05). Conclusion The drug containing serum of Liuwei Dihuang Pil can activate Wnt/β-catenin pathway in HTR-8/Svneo cells induced by high glucose, and promote the migration and invasion of HTR-8/Svneo cells.

Key words: migration, invasion, Wnt, β-catenin, Liuwei Dihuang Pill containing serum, high glucose, trophoblast cells

中图分类号:

R285

图/表 6

图1 各组HTR-8/Svneo细胞中Wnt1、β-catenin、Cyclin D1 mRNA表达水平 n=6，FWnt1=48.333，Fβ-catenin=37.357，FCyclin D1=32.242，均P<0.01；a与对照组比较，b与高糖组比较，c与低剂量组比较，d与中剂量组比较，P<0.05。

Fig.1 The mRNA expression levels of Wnt1, β-catenin and Cyclin D1 in five groups of HTR-8/Svneo cells

图2 各组HTR-8/Svneo细胞中Wnt/β-catenin通路相关蛋白表达情况 A—E分别为对照组、高糖组、低剂量组、中剂量组及高剂量组。

Fig.2 Expression levels of Wnt/β-catenin pathway-related proteins in five groups of HTR-8/Svneo cells

图3 各组HTR-8/Svneo细胞中Wnt1、β-catenin、p-GSK-3β、Cyclin D1蛋白表达水平 n=6，FWnt1=57.610，Fβ-catenin=57.320；Fp-GSK-3β=71.879，FCyclin D1=57.439，均P<0.01；a与对照组比较，b与高糖组比较，c与低剂量组比较，d与中剂量组比较，P<0.05。

Fig.3 Protein expression levels of Wnt1, β-catenin, p-GSK-3β and Cyclin D1 in five groups of HTR-8/Svneo cells

图4 各组HTR-8/Svneo细胞迁移情况（×200）

Fig.4 Migration of HTR-8/Svneo cells in each group (×200)

表1 各组HTR-8/Svneo细胞迁移及细胞侵袭数比较（n=6，个/视野，$\bar{x}±s$）

Tab.1 Comparison of cell migration and cell invasion numbers between five groups of HTR-8/Svneo cells

组别	迁移细胞数	侵袭细胞数
对照组	68.49±4.65	85.55±5.61
高糖组	13.55±2.33^a	15.52±2.64^a
低剂量组	19.68±2.85^b	24.46±2.32^b
中剂量组	38.92±3.53^bc	48.02±3.38^bc
高剂量组	44.57±3.06^bcd	53.66±3.85^bcd
F	250.334^＊＊	323.073^＊＊

图5 各组HTR-8/Svneo细胞的侵袭情况（×200）

Fig.5 Invasion of HTR-8/Svneo cells in each group (×200)

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