Many patients with type 2 diabetes develop diabetic foot syndrome (DFS). In this condition, ulcers develop on the feet, and these ulcers tend to heal very slowly. Wound healing in patients with diabetes can be impaired by peripheral neuropathy, peripheral arterial disease, and high levels of inflammation found throughout the body. Treatment for DFS includes off-loading of pressure from the affected area, debridement, and antibiotic treatment to control infection. However, these treatments do not always directly speed the healing process. Birch (Betulaspp., Betulaceae) bark extracts have been found to speed wound healing after surgery. Birch bark contains pentacyclic triterpenes, the most common of which is betulin. Birch triterpenes have been found to increase the pro-inflammatory response and migration of keratinocytes. The goal of this study was to measure the effect of birch bark extract and isolated triterpenes on wound-healing gene expression, protein levels, and cytoskeletal structural changes in human keratinocytes and fibroblasts from healthy subjects and patients with diabetes.
Birch bark extract (Birken AG; Niefern-Öschelbronn, Germany) contained 86.9% betulin and 3.9% lupeol. Keratinocytes and fibroblasts obtained from healthy subjects and patients with diabetes were cultured with two different concentrations of birch extract (1 or 5 μg/ml), two different concentrations of betulin (0.87 or 4.35 μg/ml), or with lupeol. Expression of 48 genes and levels of related proteins involved in wound healing were measured. Each type of cell was exposed to birch bark extract, betulin, lupeol, or positive and negative control compounds known to affect the actin cytoskeleton. The cytoskeleton was then stained and viewed with fluorescent microscopy to measure the effect of birch bark extract on cytoskeleton structure. Lastly, Rho-GTPase concentration was quantified in those same cells. Rho-GTPase is responsible for the regulation of some aspects of the actin cytoskeleton structure. Data were analyzed with Student’s t-test, and analysis of variance was performed with Bonferroni’s post hoc tests.
The keratinocytes from both types of donors displayed similar increases in gene expression of the pro-inflammatory compounds interleukin (IL)-6 and tumor necrosis factor-α, and the chemokines interferon γ-induced protein 10 (IP-10) and IL-8, with exposure to birch bark extract and betulin. This response was dose-dependent and increased with both birch bark extract and betulin concentrations. Expression of the gene that produces fibrillin-1 decreased with exposure to birch bark extract and betulin in keratinocytes from nondiabetic subjects and increased in keratinocytes from patients with diabetes. In fibroblasts, expression of several wound-healing genes was increased by birch bark extract and betulin. These genes included those coding for pro-inflammations, chemokines, anti-inflammations, antioxidants, and growth factors. In keratinocytes, the concentrations of proteins associated with wound healing tended to increase with gene expression. Only a few differences in response to treatments were found between cells from healthy subjects and from patients with diabetes.
Two notable differences were monocyte chemoattractant protein-1 (MCP-1) and soluble intercellular adhesion molecule-1 (sICAM-1); MCP-1 increased significantly in the keratinocytes of healthy subjects and decreased, but not significantly, in cells from patients with diabetes. sICAM-1 also increased in keratinocytes of healthy subjects but remained unchanged in cells from patients with diabetes. Levels of several wound-healing proteins, including a chemokine, a necrosis factor, and a protein involved in macrophage migration, were increased in fibroblasts from healthy subjects and patients with diabetes, with betulin exposure. In fibroblasts from healthy subjects, the concentration of sICAM-1 increased, and IL-6 decreased when the cells were exposed to betulin. In fibroblasts from patients with diabetes, soluble vascular cell adhesion molecule-1 decreased with exposure to betulin. Exposure of keratinocytes from patients with diabetes to a positive control, birch bark extract, betulin, and lupeol resulted in the formation of cellular protrusions associated with the changes in the cytoskeleton. Exposure of fibroblasts to these same compounds resulted in elongation of the cells associated with modifications of the cytoskeleton structure. In addition, birch bark extract, betulin, and lupeol exposure resulted in the activation of p38 mitogen-activated protein kinase and Rho-GTPase in keratinocytes.
Birch bark extract and betulin significantly affected the expression of genes associated with wound healing in keratinocytes and fibroblasts. In keratinocytes, genes associated with inducing inflammation and cell signaling were increased. In fibroblasts, many of the genes tested showed increased expression with exposure to birch bark extract and betulin. Gene expression in these two cell types was rarely affected by the diabetic state of the donor, although there were some differences in baseline expression of genes that were dependent on the cell source. Changes in gene expression generally resulted in concomitant changes in cellular protein concentrations within the keratinocytes and fibroblasts. In addition, exposure to birch bark extract, betulin, and lupeol altered the cytoskeleton structure of the two cell types and changed the activation level of enzymes associated with cytoskeleton regulation. In keratinocytes, the effects included the production of cellular protrusions that are important to cellular migration. Fibroblasts became elongated upon exposure to birch bark extract, betulin, and lupeol. The authors conclude that birch bark extract or isolated triterpenoids may be helpful in the healing of wounds associated with DFS, though they caution against the use of birch bark in the pro-inflammatory phase of ulcer healing. Patients with diabetes often have high inflammation rates that may affect the healing of wounds by keeping wounds in a prolonged inflammatory state. Birch bark may further prolong this state. The authors recommend clinical trials in patients with DFS to elucidate the efficacy of birch bark extract and components in the healing of ulcers.
Wardecki T, Werner P, Thomas M, et al. Influence of birch bark triterpenes on keratinocytes and fibroblasts from diabetic and nondiabetic donors. J Nat Prod. April 2016;79(4):1112-1123.