Tissue regeneration effect of betulin via inhibition of ROS/MAPKs/NF-ĸB axis using zebrafish model

Betulin is the primary anti-inflammatory component of Betula platyphylla suk. cortex (birch bark), a time-honored Traditional Chinese Medicine (TCM) for healing trauma and tissue regeneration. However, the tissue regeneration effects and underlying molecular mechanism of betulin remain unknown. Therefore, it is necessary to investigate the wound repair effects and validate the mechanism of betulin in an appropriate model. In the present study, we evaluated the effects of tissue regeneration, melanin scavenging, and reactive oxygen species (ROS) inhibition of betulin using a zebrafish model. The mechanism of target genes and pathways were confirmed using quantitative polymerase chain reaction and western blotting in vivo, while molecular docking, absorption, distribution, metabolism, excretion, and toxicity investigations in-silico were conducted. Betulin significantly promoted the regeneration of zebrafish caudal fin length and area and alleviated melanin aggregation, as well as ROS generation. The relative mRNA expression of IL-1β, TNF-α, p38α, ERK1/2, and Caspase3, and the relative protein expression of p38α, ERK1/2, Caspase3, phosphorylated proteins of p-p38α, p-ERK1/2, and p-p65 were down-regulated following betulin administration. Meanwhile, the protein ratios of p-p38α/p38α, p-ERK/ERK, and p-p65/p65 were significantly decreased. In an in-silico study, binding affinities between betulin and P38α, ERK1, ERK2, and Caspase3, and the pharmacokinetic profile of betulin were predicted. The findings suggest that the tissue regeneration mechanism of betulin is based on the inhibition of excessive inflammatory responses, melanin aggregation, and the pro-apoptotic factor, Caspase3, during the proliferation phase via the ROS/MAPKs/NF-ĸB signaling axis. Our results suggest betulin as a potential candidate for tissue regeneration.