Effects of drug-containing serum of Liuwei Dihuang Pill on Wnt/β-catenin pathway, migration and invasion of trophoblast cells induced by high glucose

doi:10.11958/20251508

Abstract

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

CLC Number:

R285

Figures/Tables 6

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

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

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

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

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^＊＊

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

References 20

[1]	KLEINWECHTER H J, WEBER K S, MINGERS N, et al. Gestational diabetes mellitus and COVID-19:results from the COVID-19-related obstetric and neonatal outcome study(CRONOS)[J]. Am J Obstet Gynecol, 2022, 227(4):631.e1-631.e19. doi:10.1016/j.ajog.2022.05.027.
[2]	GOU R, ZHANG X. Glycolysis:A fork in the path of normal and pathological pregnancy[J]. FASEB J, 2023, 37(12):e23263. doi:10.1096/fj.202301230R.
[3]	SUN L, SHI M, WANG J, et al. Overexpressed trophoblast glycoprotein contributes to preeclampsia development by inducing abnormal trophoblast migration and invasion toward the uterine spiral artery[J]. Hypertension, 2024, 81(7):1524-1536. doi:10.1161/HYPERTENSIONAHA.124.22923.
[4]	WANG J, WANG L, QIU H. High glucose regulates the cells dysfunction of human trophoblast HTR8/SVneo cells by downregulating GABRP expression[J]. Adv Clin Exp Med, 2024, 33(10):1123-1130. doi:10.17219/acem/174347.
[5]	JIANG L L, YANG D L, HAN Q, et al. LncRNA-NEAT1 blocks the Wnt/β-catenin signaling pathway by targeting miR-217 to inhibit trophoblast cell migration and invasion[J]. J Assist Reprod Genet, 2024, 41(8):2107-2115. doi:10.1007/s10815-024-03124-7.
[6]	ZHOU M, GAO Y, WU S, et al. Ubiquitin-specific protease 7 prevents recurrent spontaneous abortion by targeting enhancer of zeste homolog 2 to regulate trophoblast proliferation,apoptosis,migration,and invasion through the Wnt/β-catenin pathway[J]. Biol Reprod, 2023, 109(2):204-214. doi:10.1093/biolre/ioad053.
[7]	XIAO B, ZHANG W, JI N, et al. Knockdown of CCNB1 alleviates high glucose-triggered trophoblast dysfunction during gestational diabetes via Wnt/β-catenin signaling pathway[J]. Open Med(Wars), 2025, 20(1):20241119. doi:10.1515/med-2024-1119.
[8]	刘会中, 孙思怡, 李党召, 等. 六味地黄丸联合恩格列净治疗2型糖尿病的临床研究[J]. 现代药物与临床, 2023, 38(12):3091-3096.
	LIU H Z, SUN S Y, LI D Z, et al. Clinical study of Liu Wei Di Huang Wan combined with Empagliflozin in treating type 2 diabetes[J]. Mod Drug Clin, 2023, 38(12):3091-3096. doi:10.7501/j.issn.1674-5515.2023.12.032.
[9]	谭戬浩, 李红梅, 李辉, 等. 不同剂量六味地黄丸对妊娠期糖尿病模型大鼠脂糖代谢、脂联素及胰岛素抵抗的影响[J]. 中医学报, 2019, 34(8):1686-1690.
	TAN J H, LI H M, LI H, et al. Effects of different doses of Liuwei Dihuang Pills on lipid and glucose metabolism, adiponectin,and insulin resistance in a rat model of gestational diabetes mellitus[J]. Journal of Chinese Medicine, 2019, 34(8):1686-1690. doi:10.16368/j.issn.1674-89992019.08.398.
[10]	贺景源. 基于Wnt/β-catenin信号通路探讨六味地黄丸对椎间盘软骨终板退变大鼠模型的影响[D]. 长沙: 湖南中医药大学, 2023:22-26.
	HE J Y. The effects of Liwai Dihuang Pill on the degeneration of intervertebral disc cartilage endplate in rat models based on the Wnt/β-catenin Signaling Pathway[D]. Changsha: Hunan University of Chinese Medicine, 2023:22-26.
[11]	DALDAL H, NAZIROĞLU M. Carvacrol protects the ARPE19 retinal pigment epithelial cells against high glucose-induced oxidative stress,apoptosis,and inflammation by suppressing the TRPM2 channel signaling pathways[J]. Graefes Arch Clin Exp Ophthalmol, 2022, 260(8):2567-2583. doi:10.1007/s00417-022-05731-5.
[12]	JIE Q, CHEN L, LIANG J, et al. Downregulated ETV4 inhibits the proliferation,migration,and invasion of trophoblast cells in preeclampsia[J]. Reproduction, 2023, 165(4):373-381. doi:10.1530/REP-22-0184.
[13]	BURTON G J, CINDROVA-DAVIES T, YUNG H W, et al. Hypoxia and reproductive health:oxygen and development of the human placenta[J]. Reproduction, 2021, 161(1):F53-F65. doi:10.1530/REP-20-0153.
[14]	CHEN M, SHI J L, ZHENG Z M, et al. An abnormal LPA/LPAR1-NHE1 axis leads to the autophagy deficiency of trophoblast cells in recurrent spontaneous abortion[J]. Reproduction, 2023, 166(5):357-368. doi:10.1530/REP-23-0224.
[15]	纪璐璐, 熊国平, 汪琳. 高糖环境下beclin2介导的自噬增强绒毛膜滋养层细胞凋亡[J]. 中国组织化学与细胞化学杂志, 2017, 26(2):109-114.
	JI L L, XIONG G P, WANG L. Enhanced autophagy mediated by Beclin-2 in trophoblast cells under high-glucose conditions promotes apoptosis[J]. Chin J Histochem Cytochem, 2017, 26(2):109-114. doi:10.16705/j.cnki.1004-1850.2017.02.003.
[16]	董得刚, 李红云, 徐聪, 等. 六味地黄汤联合健脾化湿汤治疗2型糖尿病(肾阴亏虚证)对中医证候积分及糖脂代谢的影响[J]. 四川中医, 2023, 41(11):150-152.
	DONG D G, LI H Y, XU C, et al. Effects of Liu Wei Di Huang Wan combined with Jian Pi Hua Shi Tang on TCM syndrome scores and glucose-lipid metabolism in the treatment of type 2 diabetes(Kidney Yin deficiency syndrome)[J]. Sichuan J Tradit Chin Med, 2023, 41(11):150-152.
[17]	陶乐维. 不同剂量六味地黄丸对胰岛素抵抗小鼠糖代谢及体重的影响[J]. 陕西中医, 2022, 43(3):275-278.
	TAO L W. Effects of different doses of Liu Wei Di Huang Wan on glucose metabolism and body weight in insulin-resistant mice[J]. Shanxi J Tradit Chin Med, 2022, 43(3):275-278. doi:10.3969/j.issn.1000-7369. 2022.03.001.
[18]	李晨, 李占恩, 苏宏伟, 等. KRT17调节Wnt/β-catenin信号通路对膀胱癌细胞增殖、凋亡及上皮间质转化的影响[J]. 天津医药, 2025, 53(5):462-467.
	LI C, LI Z E, SU H W, et al. KRT17 regulation of Wnt/β-catenin signaling pathway influences proliferation,apoptosis,and epithelial-mesenchymal transition in bladder cancer cells[J]. Tianjin Med J, 2025, 53(5):462-467. doi:10.11958/20241498.
[19]	LI Y, XING B X, WANG Y H, et al. CTHRC1 promotes growth,migration and invasion of trophoblasts via reciprocal Wnt/β-catenin regulation[J]. J Cell Commun Signal, 2022, 16(1):63-74. doi:10.1007/s12079-021-00625-3.
[20]	ZHANG X Y, QIN X Y, SHEN H H, et al. IL-27 deficiency inhibits proliferation and invasion of trophoblasts via the SFRP2/Wnt/β-catenin pathway in fetal growth restriction[J]. Int J Med Sci, 2023, 20(3):392-405. doi:10.7150/ijms.80684.