naphthoquinones and Cicatrix--Hypertrophic

Pathological scarring is an intractable problem for both patients and clinicians. A major obstacle for the development of scar remediation therapies is the paucity of suitable in vivo and in vitro models. The "Scar-in-a-jar" model was previously established by our colleagues based on the principle of "Macromolecular crowding". This has been demonstrated to be an extracellular matrix-rich in vitro model offering a novel tool for studies related to the extracellular matrix. In the study reported herein, we have optimised this approach to model human dermal fibroblasts derived from hypertrophic tissues. This optimised in vitro model has been found to hold similar properties, such as increased collagen I, interleukins and transforming growth factor beta-1 expression, compared to that observed in hypertrophic scar tissue in vivo. In addition, Shikonin has been previously demonstrated to hold potential as a novel hypertrophic scar treatment due to its apoptosis-inducing property on hypertrophic scar fibroblasts. Other Shikonin analogues have also been reported to hold apoptosis-inducing properties in various cancer cell lines, however, the effects of these analogues on hypertrophic scar-related cells are unknown. We therefore evaluated the effects of Shikonin and its analogues on hypertrophic scar-derived human fibroblasts using the optimised "Macromolecular crowding" model. Our data indicates that Shikonin and Naphthazarin are the most effective molecules compared to related naphthoquinones. The data generated from the study offers a novel in vitro collagen-rich model of hypertrophic scar tissue. It also provides further evidences supporting the use of Shikonin and Naphthazarin as potential treatments for hypertrophic scars.

Topics: Animals; Apoptosis; Cell Line; Cicatrix; Cicatrix, Hypertrophic; Collagen; Extracellular Matrix; Fibroblasts; Humans; Models, Biological; Naphthoquinones; Skin

Hypertrophic scarring/hypertrophic scars (HS) is a highly prevalent condition following burns and trauma wounds. Numerous studies have demonstrated that transforming growth factor-β1 (TGF‑β1) plays an essential role in the wound healing process by regulating cell differentiation, collagen production and extracellular matrix degradation. The increased expression of TGF-β1 is believed to result in the formation of HS. Shikonin (SHI), an active component extracted from the Chinese herb, Radix Arnebiae, has previously been found to downregulate the expression of TGF-β1 in keratinocyte/fibroblast co-culture conditioned medium. In view of this, in this study, we aimed to further investigate the effects of SHI on TGF-β1-stimulated hypertrophic scar-derived human skin fibroblasts (HSFs) and examined the underlying mechanisms. Cell viability and proliferation were measured using alamarBlue and CyQUANT assays. The total amount of collagen and cell contraction were examined using Sirius red staining and the cell contraction assay kit. Gene expression and signalling pathway activation were detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Our results revealed that SHI reduced TGF-β1‑induced collagen production through the ERK/Smad signalling pathway and attenuated TGF-β1‑induced cell contraction by downregulating α-smooth muscle actin (αSMA) expression in the HSFs. The data from this study provide evidence supporting the potential use of SHI as a novel treatment for HS.

Topics: Actins; Cicatrix, Hypertrophic; Collagen; Down-Regulation; Fibroblasts; Humans; MAP Kinase Signaling System; Naphthoquinones; Skin; Transforming Growth Factor beta1

Hypertrophic scarring is a highly prevalent condition clinically and results from a decreased number of apoptotic fibroblasts and over-abundant production of collagen during scar formation following wound healing. Our previous studies indicated that Shikonin, an active component extracted from Radix Arnebiae, induces apoptosis and reduces collagen production in hypertrophic scar-derived fibroblasts. In the study reported here, we further evaluate the potential use of Shikonin as a novel scar remediation therapy by examining the effects of Shikonin on both keratinocytes and fibroblasts using Transwell® co-culture techniques. The underlying mechanisms were also revealed. In addition, effects of Shikonin on the expression of cytokines in Transwell co-culture "conditioned" medium were investigated.. Our results indicate that Shikonin preferentially inhibits cell proliferation and induces apoptosis in fibroblasts without affecting keratinocyte function. In addition, we found that the proliferation-inhibiting and apoptosis-inducing abilities of SHI might be triggered via MAPK and Bcl-2/Caspase 3 signalling pathways. Furthermore, SHI has been found to attenuate the expression of TGF-β1 in Transwell co-cultured "conditioned" medium.. The data generated from this study provides further evidence that supports the potential use of Shikonin as a novel scar remediation therapy.

Topics: Apoptosis; Cell Line; Cicatrix, Hypertrophic; Coculture Techniques; Collagen; Fibroblasts; Humans; Keratinocytes; MAP Kinase Signaling System; Naphthoquinones

The pathogenesis and therapy of hypertrophic scar have not yet been established. Our aim was to investigate the antiproliferative and antisecretory effects of lapachol, isolated from the stem bark of Avicennia rumphiana Hall. f., on hypertrophic scar fibroblasts.. The effects of lapachol on hypertrophic scar fibroblast proliferation were measured using the MTT assay, cell-cycle analyses and lactate dehydrogenase assays. The type I collagen α-chain (COL1A1), interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) mRNA and/or protein levels of hypertrophic scar-fibroblasts were quantitated by real-time PCR and ELISA.. Lapachol at 25 and 50 µm significantly inhibited the in vitro proliferation of hypertrophic scar fibroblasts, but not fibroblasts from non-lesional skin sites. In addition, lapachol had no apparent effect on cell cycle and lactate dehydrogenase activity in conditioned medium from lapachol-treated hypertrophic scar fibroblasts was nearly equal to that in medium from vehicle-treated cells. Lapachol treatment also inhibited COL1A1 and PAI-1 mRNA levels in hypertrophic scar fibroblasts, but did not affect IL-6 mRNA levels. The protein levels of IL-6 and PAI-1 in conditioned medium from hypertrophic scar fibroblasts treated with 50 µm lapachol were lower than those from vehicle-treated hypertrophic scar fibroblasts.. Lapachol decreased the proliferation rate of hypertrophic scar fibroblasts. As IL-6 and PAI-1 secretion was also lowered in lapachol-treated hypertrophic scar fibroblasts, our findings suggested that lapachol may have suppressed extracellular matrix hyperplasia in wound healing and possibly alleviated the formation of hypertrophic scar.

Topics: Avicennia; Biopsy; Cell Cycle; Cell Proliferation; Cells, Cultured; Cicatrix, Hypertrophic; Collagen Type I; Collagen Type I, alpha 1 Chain; Fibroblasts; Gene Expression Regulation; Humans; Interleukin-6; Lactate Dehydrogenases; Naphthoquinones; Osmolar Concentration; Plant Bark; Plant Stems; Plasminogen Activator Inhibitor 1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin