The Effects of Multiple Kidney-Tonifying Chinese Medicines on Thymus Regeneration in Rapamycin-Induced Mice

Abstract

Background: Rapamycin is universally recognized as a potential anti-aging drug, the important functional activities of which involve induction of reversible acute atrophy of the thymus and normal regeneration after discontinuation of treatment. Tonifying kidney is the core treatment principle of preventing aging in traditional Chinese medicine (TCM), and multiple kidney-tonifying Chinese medicines present notable anti-aging property. However, there is no research digging into the function of kidney-tonifying Chinese medicines in the process of thymus regeneration induced by rapamycin. Aims: This study explored the effects of 5 kinds of kidney-tonifying Chinese medicines on rapamycin-induced thymus regeneration, and whether the sequential use of Chinese medicines and rapamycin can potentiate the anti-aging effect. Methods: Initially, mice received intraperitoneal injection of 1 mg/kg rapamycin for 5 days; after the last administration, mice were fasted for 12 h. Cynomorium songaricum (CS), Cistanche deserticola (CD), Epimedium brevicornu (EB), Morindae officinalis (MO), and Drynaria fortunei (DF), the 5 kidney-tonifying Chinese medicines, were selected, and ethanol extract was prepared. 5 days after gavage administration, mice were sacrificed by cervical dislocation, after inhaling isoflurane gas. and the thymus samples were collected. The structure of thymus, as well as the morphology and distribution of thymic epithelial cells (TECs) was observed using hematoxylin-eosin (H&E) staining and immunofluorescent staining (CK5, CK8). Flow cytometry was conducted to detect the ratio of thymic cell subpopulation, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) was exploited to measure the mRNA level of Sirt6, P21, IL-1α, PTMα and Tα1. PCR method was utilized to determine T-cell receptor excision circle (TREC) level in the spleen. Results: CS, CD and EB were found to effectively promote thymus regeneration after rapamycin administration, of which CD had the strongest effect. Improving the morphology of TECs can facilitate negative and positive selection of cells, suppress compensatory increase in thymic output function during regeneration, elevate CD4⁺ T cell ratio, and improve immune function. CD enhanced expressions of thymus capsule and cortical epithelial cells, further expanded medulla, inhibited P21, IL-1α, IL-1β and Tα1 expressions, and augmented PTMα mRNA level. CS thickened thymus cortex area. EB promoted reticular differentiation of medullary epithelial cells, dampened P21, IL-1α, Sirt6 and Tβ4 mRNA levels, regulated thymosin expressions, boosted thymus regeneration and elevated physical capacity of mice. Conclusion: Kidney-tonifying herbs represented by Cistanche deserticola can significantly promote the process of thymic regeneration after treatment with rapamycin, the mechanism of which may be related to enhancing the thickness of thymic capsule and cortex, as well as accelerating the reticular differentiation of medullary epithelial cells.