Radix Rehmanniae Praeparata promoted zebrafish fin regeneration through aryl hydrocarbon receptor-dependent autophagy

Abstract

Headings ethnopharmacological relevance: Rehmanniae Radix Praeparata (RRP), a staple in traditional Chinese medicine, is derived from Rehmannia glutinosa Libosch and is renowned for its wound-healing properties. Despite its clinical prevalence, the molecular mechanisms underlying RRP's wound-healing effects have not been fully elucidated. Aim of the study: This research endeavored to delineate the molecular and cellular mechanisms underlying the beneficial effects of RRP on wound healing, utilizing a zebrafish model. Materials and methods: Zebrafish larvae at 3 days post-fertilization were amputated at the fin and subsequently treated with RRP. The pro-wound healing and regenerative effects of RRP were evaluated through morphological analysis, assessment of cell proliferation and apoptosis, Additionally, mechanistic insights were gained through a comprehensive approach encompassing network pharmacology analysis, cell tracing, RNA-sequencing, CRISPR/Cas9 gene editing, and pharmacological inhibition. Results: Our findings demonstrate that RRP significantly accelerates caudal fin regeneration in zebrafish following injury by suppressing cell apoptosis, promoting cell proliferation, and upregulating the expression of regenerative-related genes. Furthermore, RRP triggers autophagy signals during the regenerative process, which is attenuated by the autophagy inhibitor chloroquine (CQ). Notably, the administration of RRP enhances the expression of ahr1 and ahr2 in the regenerating fin. Genetic knockout of ahr1a, ahr1b, or ahr2 using CRISPR/Cas9, or pharmacological blockade of AHR signals with the antagonist CH-223191, diminishes the regenerative potential of RRP. Remarkably, zebrafish lacking ahr2 completely lose their fin regeneration ability. Additionally, inhibition of AHR signaling suppresses autophagy signaling during fin regeneration. Conclusions: This study uncovers that RRP stimulates fin regeneration in zebrafish by inducing AHR signals and, at least partially, activating the autophagy process. These findings provide novel insights into the molecular mechanisms underlying the wound-healing effects of RRP and may pave the way for the development of novel therapeutic strategies. Graphical abstract: [Figure not available: see fulltext.]