[Angelica sinensis promotes thymic cortical regeneration in mice with acute thymic involution induced by short-term rapamycin treatment].

Abstract

Objectives: To investigate the effect of Angelica sinensis on thymic cortical regeneration in rapamycin-treated mice and its underlying mechanisms.

Methods: Seventy-eight 6-8-week female BALB/c mice were randomly assigned by weight into six groups: blank control, model control, normal regeneration, and Angelica sinensis low-dose, medium-dose, and high-dose groups (the blank control group n=18, each of the other groups n=12). The blank control group received a daily intraperitoneal injection of isotonic saline (0.1 mL/10 g), while the other groups were intraperitoneally administered rapamycin (1 mg·kg^－¹·d^－¹) for three consecutive days to establish acute thymic involution models. After modeling, the Angelica sinensis groups received corresponding doses of Angelica sinensis (1, 2, 4 g/kg per day), while the normal regeneration group received an equal volume of saline. Body weight and dorsal hair growth were recorded daily. Forelimb grip strength was measured 2 hours after the last administration. Thymic tissue structure and the spatial distribution of thymic epithelial cells (TECs) and thymocytes were assessed by hematoxylin-eosin (HE) and immunofluorescence stainings. Development and homeostasis of T-cell subsets in the thymus and peripheral blood were analyzed by flow cytometry and rapid Wright-Giemsa staining. T-cell receptor excision circles in genomic DNA from peripheral blood mononuclear cells were detected by quantitative PCR (qPCR). The mRNA expression levels of thymic function-related genes, inflammatory factors, and Wnt signaling pathway-related genes were measured by quantitative reverse transcription PCR (qRT-PCR). Potential targets and signaling pathways were screened by integrating network pharmacology prediction and molecular docking.

Results: Rapamycin successfully induced acute thymic involution. The model control group showed a decrease in thymic index, weakened grip strength (both P<0.05), and no obvious hair regeneration. Histologically, the thymic cortical area was reduced, with a blurred corticomedullary junction, disrupted continuity of the cortical TEC cytoplasmic process network, early-stage blockade of thymocyte development, disturbed spatial distribution, and inhibited thymic output. Compared with the normal regeneration group, medium and high doses of Angelica sinensis increased the cortical area in a dose-dependent manner, and significantly enhanced the density of cortical CK8⁺ TECs and restored the continuity of their cytoplasmic process network (all P<0.01), while upregulating the gene expression of Foxn1 and its downstream target gene Dll4 (all P<0.05). Regarding thymocyte development, high dose Angelica sinensis increased the proportion of CD3⁺TCRβ⁺ thymocytes (P<0.05), promoted the balanced differentiation of CD4⁺CD8⁺ cells into mature single-positive thymocytes, and re-established normal spatial localization, manifested as increased density of CD8⁺ and CD4⁺CD8⁺ cells in the cortex and reaggregation of TCRβ⁺ cells in the medulla. Furthermore, medium dose Angelica sinensis significantly reduced the mRNA levels of thymic pro-inflammatory factors related gene TNF-α, TGF-β, and IGFBP5 (all P<0.05), thereby improving the local inflammatory microenvironment. For peripheral homeostasis, Angelica sinensis intervention maintained and increased the proportion of peripheral blood CD3⁺ T cells, elevated the percentage of CD34⁺ hematopoietic stem cells across all dosage groups (all P<0.05), and upregulated the expression of the Ccl25. Mechanistically, network pharmacology predicted the Wnt signaling pathway as a potential target of Angelica sinensis active components. Experimental validation revealed that medium dose Angelica sinensis significantly upregulated Wnt4 mRNA expression, inhibited Gsk3β, and increased CTNNB1 gene levels in the thymus (P<0.05).

Conclusions: Angelica sinensis promotes cortical regeneration and functional recovery after rapamycin-induced acute thymic involution by activating the Wnt/β-catenin/Foxn1 signaling pathway and improving the thymic inflammatory microenvironment, which collaboratively facilitate cortical thymic epithelial cell cytoskeletal repair and hematopoietic stem cell homing. This suggests its potential benefit for counteracting immune aging.