Exploring the Wound Healing Potential of Piperine in Zebrafish (Danio rerio): Insights from Visual Observations and Gene Expression Analysis

Abstract

Introduction: A wound is a physical injury resulting in a break in the skin or underlying tissue, often caused by accidents, surgery, or disease. While medicinal plants have been recognized since time immemorial for their bioactive potential, evaluating their safety-to-efficacy ratio is critical. This study investigates the wound-healing potential and preclinical safety of piperine, the primary bioactive alkaloid in Piper nigrum, using a zebrafish (Danio rerio) model.

Objectives: The objective of this study was to evaluate the wound-healing efficacy of piperine in adult zebrafish through visual assessment of wound closure rates and to analyze the expression of angiogenic genes (Bax, HIF-1, VEGF-1, and MMP-2) to elucidate molecular mechanisms.

Methods: Adult zebrafish (n=24) were anesthetized with 0.2% Tricaine, and a single wound was created posterior to the abdominal region. The fish were divided into four groups (n=6): Group I (negative control/untreated), Group II (standard control/0.001 M doxycycline), and Groups III and IV treated with 0.001 M and 0.0001 M piperine, respectively.  Piperine concentrations (0.001 M and 0.0001 M) were selected based on a determined LD₅₀ of 60 μg/ml. Wound closure was monitored at 1, 4, and 7 days post-wounding (dpw) using a stereomicroscope and ImageJ software to calculate the wound healing percentage (WHP). On day 7, mRNA was isolated from muscle tissue for qRT-PCR analysis to quantify the relative expression of Bax, HIF-1, VEGF-1, and MMP-2. Statistical significance was determined using Student’s t-test.

Results: Piperine treatment demonstrated dose-dependent healing, with the 0.001 M group achieving near-complete closure (3 ± 1.8% residual area) by 7 dpw, representing a significant improvement over the negative control (68 ± 3%). Gene expression analysis revealed a marked upregulation of Bax, VEGF-1, and MMP-2, alongside a significant downregulation of HIF-1.

Conclusions: These findings highlight piperine as a promising and low-risk natural therapeutic agent for wound care acting through the modulation of angiogenesis-related gene expression.