Effects of a natural multi-component compound formulation on the growth, morphology and extracellular matrix production of human adult dermal fibroblasts

Abstract

The extracellular matrix (ECM) creates a tissue microenvironment able to regulate cellular signaling. The loss of ECM plasticity is associated with several pathologies, especially those involving chronic inflammation, therefore, the ECM represents a potential therapeutic target for certain conditions. The present study investigated the effects of a natural multi-component compound formulation, Galium-Heel^®, on the growth, morphology and ECM production of human dermal fibroblasts (HDF). The effects of the formulation on HDF growth and morphology were assessed by sulforhodamine B assay, trypan blue exclusion staining, FACS and ultrastructural analyses. The effect of the compound on reactive oxygen species production by HDF was performed by dichlorofluorescin diacetate assay. The expression of ECM components, matrix metalloproteinases (MMPs) and signaling molecules was analyzed by western blot analysis. The present results demonstrated that Galium-Heel^® did not significantly affect HDF growth, survival, cell cycle or morphology indicating the biocompatibility of the formulation. The formulation demonstrated antioxidant activity. Galium-Heel^® was able to modulate ECM by regulating collagens (type I and III) and MMPs-3 and -7 expression. In addition, the formulation was able to regulate molecules involved in TGF-β signalling, including mitogen activated kinase-like protein, GLI family zinc finger 2 and pro-survival proteins such as AKT. The present results demonstrating the effects of a natural multi-component compound on ECM composition, highlighted the possibility of pharmacologically modulating ECM molecules. The recovery and the maintenance of ECM homeostasis might be considered as a potential therapeutic goal to ameliorate pathological conditions.

Keywords: Galium-Heel®; collagen; extracellular matrix; fibroblasts; natural compound.

Figure 1.

Effects of Galium on HDF cell survival and death. (A) Survival and viability of HDF were assessed by the sulforhodamine B and (B) trypan blue exclusion assays 24, 48 and 72 h post-treatment with CTR or Galium in DMEM 0.2% BSA. The percentage of surviving cells treated with Galium was calculated by normalizing the optical density value to each control culture treated with sodium chloride at the same dilution (considered 100%). The percentage of cell death was compared with the total number of cells. Results are expressed as the mean ± SD of three independent experiments performed in triplicate. Galium, Galium-Heel^®; HDF, human dermal fibroblasts; CTR, sodium chloride.

Figure 2.

Effects of Galium on the expression of extracellular matrix molecules and MMPs. (A) Expression of collagen type I and type III, fibronectin, MMP-2, MMP-3 and MMP-7 in cell lysates derived from HDF cells treated for 48 h with CTR or Galium at the dilution of 1:2 in DMEM 0.2% BSA was assessed by western blot analysis. (B) Expression of collagen type I and III, fibronectin, MMP-2, MMP-3 and MMP-7 in the conditioned media derived from HDF-treated cells was assessed by western blot analysis. Equal loading of protein on the gel was confirmed by Coomassie blue staining of the gel. Each protein was normalized to total protein loading assessed by Coomassie blue staining. (C and D) Densitometric ratios and statistical analysis of the respective blots for sodium chloride- and Galium-treated cells. Data are expressed as the mean ± SD of two experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. CTR. Galium, Galium-Heel^®; MMP, matrix metalloproteinase; HDF, human dermal fibroblasts; CTR, sodium chloride.

Figure 3.

Effects of Galium on the expression and activation of pro-survival signaling pathway molecules. (A) Expression of MAPKs, AKT, GLI-2 was assessed by western blot analysis in cell lysates derived from HDF cells treated for 48 h with CTR or Galium at the dilution of 1:2 in DMEM 0.2% BSA. (B) Densitometric ratios and statistical analysis of the respective blots for CTR- and Galium-treated cells with phosphorylated data presented as a ratio to the total protein. Data are expressed as the mean ± SD of two experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. CTR. Galium, Galium-Heel^®; HDF, human dermal fibroblasts; p, phosphorylated; MAPK, mitogen-activated protein kinase; GLI-2, GLI family zinc finger 2; CTR, sodium chloride.

Figure 4.

Ultrastructural analysis of HDF cells treated with Galium or sodium chloride (CTRL). (A) Ultrastructural analysis of sodium chloride- and (B) Galium-treated HDF cells at ×7,000 magnification. (C) Ultrastructural analysis of sodium chloride- and (D) Galium-treated HDF cells at ×28,000 magnification. The arrow indicates an increase in the polymerization of intermediate filaments in Galium-treated cells. Galium, Galium-Heel^®; HDF, human dermal fibroblasts; N, nucleus; m, mitochondria; rer, rough endoplasmic reticulum.