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, 2017, 7932019

Hibiscus syriacus Extract From an Established Cell Culture Stimulates Skin Wound Healing

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Hibiscus syriacus Extract From an Established Cell Culture Stimulates Skin Wound Healing

O di Martino et al. Biomed Res Int.

Abstract

Higher plants are the source of a wide array of bioactive compounds that support skin integrity and health. Hibiscus syriacus, family Malvaceae, is a plant of Chinese origin known for its antipyretic, anthelmintic, and antifungal properties. The aim of this study was to assess the healing and hydration properties of H. syriacus ethanolic extract (HSEE). We established a cell culture from Hibiscus syriacus leaves and obtained an ethanol soluble extract from cultured cells. The properties of the extract were tested by gene expression and functional analyses on human fibroblast, keratinocytes, and skin explants. HSEE treatment increased the healing potential of fibroblasts and keratinocytes. Specifically, HSEE significantly stimulated fibronectin and collagen synthesis by 16 and 60%, respectively, while fibroblasts contractility was enhanced by 30%. These results were confirmed on skin explants, where HSEE accelerated the wound healing activity in terms of epithelium formation and fibronectin production. Moreover, HSEE increased the expression of genes involved in skin hydration and homeostasis. Specifically, aquaporin 3 and filaggrin genes were enhanced by 20 and 58%, respectively. Our data show that HSEE contains compounds capable of stimulating expression of biomarkers relevant to skin regeneration and hydration thereby counteracting molecular pathways leading to skin damage and aging.

Figures

Figure 1
Figure 1
Effect of Hibiscus syriacus ethanolic extract (HSEE) on wound healing. (a) Confluent HaCaT cells were wounded and then treated with HSEE at the indicated concentrations into the 0.5% FBS-containing medium. TGF β at concentration of 2.5 ng/ml was used as positive control. At times 0 and 24 h, phase-contrast pictures of the wounds at four different locations were taken. (b) The relative migration of the cells was expressed as % of wound repair compared to control. Each column value represents the average of three experiments and error bars indicate standard deviations. P value < 0.05 is represented by . (c) Confluent HDF cells were wounded and then treated with HSEE at indicated concentrations for 7 h in 0.5% FBS-containing medium. TGFβ at concentration of 2.5 ng/ml was used as positive control. Mitomycin C (10 μg/ml) was included in the media to prevent cell proliferation. At times 0 and 7 h, phase-contrast pictures of the wounds at four different locations were taken. (d) For each condition, the unclosed cell-free areas at time 0 were compared to those at 7 h by using the software Image J. The relative migration of the cells was expressed as % of wound repair compared to control. Each column value represents the average of three experiments and error bars indicate standard deviations. P value < 0.05 is represented by . P value < 0.001 is represented by ∗∗∗.
Figure 2
Figure 2
Effect of Hibiscus syriacus ethanolic extracts (HSEE) on pro-collagen I and fibronectin production. (a) HDF cells were seeded in 96-well plates at a density of 1.5 × 104 per well and treated with HSEE at the indicated concentrations for 24 h. Each experimental condition was done in quadruplicate. Ascorbic acid at concentration of 300 μM was used as positive control. Each column value represents the average of three experiments and error bars indicate standard deviations. P value < 0.05 is represented by . P value < 0.01 is represented by ∗∗. (b) HDF cells were seeded in 96-well plates at a density of 9 × 103 per well and treated with HSEE at the indicated concentrations for 72 h. Each experimental condition was done in quadruplicate. TGF β at concentration of 2.5 ng/mL was used as a positive control. Each column value represents the average of four experiments and error bars indicate standard deviations. P value < 0.05 is represented by . P value < 0.01 is represented by ∗∗.
Figure 3
Figure 3
Effect of Hibiscus syriacus ethanolic extract (HSEE) on collagen contraction. (a–c) 2 × 105 HDF cells were seeded in 24-well plates mixed with 2 mg/ml of collagen solution. The plates were incubated at 37°C for 30′ to induce collagen gel formation. TGFβ at concentration of 2.5 ng/ml was used as positive control. At the end of incubation the medium with treatment was added and the plates were incubated overnight. The day after the gel was released from the bottom of the well to induce collagen gel contraction. 24 h later, pictures were taken and gel area was estimated. Each column value of the plot represents the average of four experiments and error bars indicate standard deviations. P value < 0.05 is represented by . P value < 0.001 is represented by ∗∗∗. (b) Representative image of collagen floating gel after 24 hours of HSEE treatment. HDF cells treated with HSEE showed higher contractibility than untreated HDF cells.
Figure 4
Figure 4
AQP3 and FLG gene expression analysis in HaCaT cells treated with HSEE and HSEE effect on ex vivo skin explants. (a) HaCaT cells were seeded at concentration of 1.5 × 105 cells in 35 mm dishes. After 24 h, cells were treated for 6 h with HSEE at the indicated concentrations. Total RNA was prepared and reverse-transcribed. Obtained cDNAs were used to evaluate AQP3 and FLG gene expression by performing a semiquantitative RT-PCR. Retinoic acid at concentration of 1 μM was used as positive control. The values were normalized to rRNA 18S. Each column value represents the average of four experiments and error bars indicate standard deviations. P value < 0.05 is represented by ; P value < 0.01 is represented by ∗∗. P value < 0.001 is represented by ∗∗. (b) Representative experiment showing the effect of HSEE on ex vivo skin explants. The samples were wounded and treated with HSEE and TGFβ as indicated. After 3 days, the explants were processed to produce sections, which were then stained for morphology analysis. (c) Fibronectin was detected by immunostaining using a monoclonal primary antibody (Santa Cruz Biotechnology) and FITC-conjugated secondary antibody (green). The nuclei were poststained with propidium iodide (red). The length of the newly generated epidermis is indicated by the dotted lines. The leading edge is indicated by arrows.

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