A Torilis japonica Extract-GHK-Cu Complex Attenuates Th2 Cytokines and Promotes Keratinocyte Recovery: A Potential Antioxidant Strategy for Atopic Dermatitis.

In vitro cell-based study using a TNF-α/IFN-γ-stimulated HaCaT keratinocyte model to evaluate the anti-atopic and regenerative properties of a Torilis japonica extract and GHK-Cu complex.

“Here, we formulated a complex of Torilis japonica extract (TJE) and GHK-Cu (Glycyl-L-histidyl-L-lysine copper(II)) complex and examined its anti-atopic and skin-regenerative properties using a TNF-α (Tumor necrosis factor-α)/IFN-γ (Interferon-γ)-stimulated HaCaT cell model.”

Human keratinocytes (HaCaT cells) and human dermal fibroblasts (HS68 cells).

“The cytotoxicity of TJE and the TJE–GHK-Cu complex was evaluated in human fibroblasts and HaCaT keratinocytes using the MTT assay.”

Technical triplicates were performed for each experimental condition.

“All assays were performed using technical triplicates for each experimental condition, unless otherwise indicated.”

Experimental treatments and measurements were conducted over periods ranging from 1 hour to 48 hours, with specific assays measuring outcomes at 24 hours and 48 hours.

“Cell migration was recorded at 0 h and 48 h using an optical microscope (CKX53, Olympus Corporation, Tokyo, Japan) at × 200 magnification.”

The TJE–GHK-Cu complex at a 6:4 ratio suppressed the expression of AD-related chemokines (TARC, CTACK), IgE production, and Th2 cytokines (IL-4, IL-5, IL-10, IL-13) more effectively than individual treatments.

“An optimized 6:4 ratio (TJE:GHK-Cu) yielded the highest efficacy compared to individual treatments, indicating a synergistic interaction.”

The TJE–GHK-Cu complex promoted keratinocyte migration and accelerated wound closure in scratch assays compared to TJE alone.

“However, the TJE–GHK-Cu complex accelerated the closure rate far beyond the TJE-only treatment (Figure 5).”

The formulation demonstrated strong radical scavenging activity and maintained high cell viability (approximately 94%) without significant cytotoxicity.

“The TJE–GHK-Cu complex exhibited approximately 94% cell viability, demonstrating good cellular compatibility under the experimental conditions.”

The study relied solely on in vitro models that do not fully replicate the complex immune microenvironment of atopic dermatitis, and it did not directly quantify intracellular ROS generation or evaluate barrier-associated proteins like filaggrin and loricrin.

“However, HaCaT cells cannot fully reproduce the complex immune microenvironment of AD because they lack interactions with immune cell populations such as T lymphocytes, dendritic cells, and Langerhans cells that contribute significantly to disease pathogenesis [1,2,3,4].”