Zuogui Wan alleviated maternal kidney-yin deficiency-induced thymic epithelial cell dysfunction in newborn rats through Wnt/β-catenin signaling pathway

Abstract

Ethnopharmacological relevance: Kidney-yin deficiency (KYD) during pregnancy is common and associated with possibility of thymus hypoplasia in neonates. Zuogui Wan (ZGW) is a classic traditional medicine to treat KYD.

Aim of study: The Wnt/β-catenin signaling pathway is essential for thymic epithelial cell (TEC) viability, function and for thymus integrity. We evaluated whether maternal diets with ZGW in KYD rats ameliorates epithelial cell dysfunction in the fetal thymus, and investigated its underlying mechanism in which the Wnt/β-catenin signaling pathway is involved.

Materials and methods: Rats were randomly assigned to four groups (n = 8). Two experimental groups received KYD induction with or without ZGW supplementation. The other 2 vehicle groups were sham operated and administrated with normal saline or ZGW. KYD was established using periodically chronic shaken stimulus and threaten stress. Success of the model induction was evaluated by the general observation, changing of the body weight and plasma thyroxine level. Then, pregnant of vehicle and KYD rats were fed with or without ZGW-supplemented diet throughout the F1 gestation. Postnatal thymi samples were obtained after delivery for histological examination. In vitro, TECs of the newborn rats whose mother suffered KYD were isolated, and cultured using the serum containing ZGW with or without the supplement of Wnt4/β-catenin pathway inhibitor ICG-001. Cell viability was evaluated by CCK-8 assay. Meanwhile, the thymi tissues and TECs were collected for biochemical analysis. Levels of thymosin β4 (TMSβ4) and thymosin α1 (Tα1) were detected by ELISA assay. The mRNA and protein expression of Wnt4, β-catenin, and Foxn1 were determined by RT-qPCR and Western blot respectively.

Results: In vivo, KYD resulted in significantly increased apoptosis of TECs and atrophy of the thymi, especially in the medullary zone. The morphological changes observed in KYD rats were ameliorated by ZGW treatment. Meanwhile, the decreased TMSβ4, Tα1, Wnt4, β-catenin, and Foxn1 levels in KYD rats were also significantly alleviated by ZGW administration. In vitro, elevated TMSβ4 and Tα1 levels accompanied with upregulated Wnt4, β-catenin, and Foxn1 expressions in the TECs were observed after ZGW intervention, however, which were significantly downregulated by ICG-001 supplement.

Conclusions: Maternal kidney-yin deficiency could result in TEC dysfunction in newborn rats. ZGW was able to improve the growth and development of TEC, potentially by regulating the Wnt/β-catenin pathway.

Keywords: ICG-001; Kidney-yin deficiency; Thymic epithelial cell; Thymus; Wnt/β-catenin pathway; Zuogui Wan.