Lactic-Acid-Induced Zebrafish Skin Apoptosis as a Model for Evaluating Skin Barrier Function.

Abstract

Objective: The skin barrier function is essential for maintaining skin health. Consequently, it is crucial to evaluate the protective and restorative effects of topical medications and cosmetics on the skin barrier. However, methods that can directly assess the skin barrier function in zebrafish beyond their regenerative capabilities are limited. Therefore, we aimed to develop an in vivo, in situ, high-throughput screening model for assessing skin barrier function in zebrafish embryos.

Methods: Lactic acid was used to induce skin barrier damage and crystal violet was used for staining to observe skin barrier damage. Histopathology was assessed using H&E, Masson, and Victoria blue staining. Cellular apoptosis was evaluated using in situ acridine orange (AO) staining and flow cytometry with propidium iodide (PI) staining.

Results: A significant increase was noted in fluorescence in the trunk and tail regions of zebrafish embryos following lactic acid stimulation compared with those in the control group, whereas exposure to the positive control for 4 h significantly reduced the fluorescence area, showing 35%-45% inhibition. Histological sections revealed that lactic acid caused noticeable damage to the epidermis of zebrafish embryos, characterized by thinning of the epidermal layer and the presence of inflammatory cell infiltrates. An increase in apoptotic cells was observed through in situ AO and PI staining using flow cytometry, and RT-qPCR revealed elevated expression levels of apoptosis-related genes, including fas, bax, and caspase 3. Furthermore, six additional samples from various sources were screened using this model, which yielded reasonable outcomes for both positive and negative samples, indicating its potential application value in evaluating the skin barrier protective efficacy of drugs or compounds.

Conclusion: Our study supports the use of the lactic-acid-induced zebrafish skin injury model as a reliable in vivo and in situ method for evaluating skin barrier function.