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Investigating the potential of Shikonin as a novel hypertrophic scar treatment.

Fan C, Xie Y, Dong Y, Su Y, Upton Z - J. Biomed. Sci. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, 4059, Australia. c3.fan@connect.qut.edu.au.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: The data generated from this study provides further evidence that supports the potential use of Shikonin as a novel scar remediation therapy.

No MeSH data available.


Related in: MedlinePlus

Effects of SHI on cell proliferation and apoptosis. a Cell proliferation. Kc and HSF were co-cultured and treated with SHI (0.5, 1 and 3 μg/mL) for 72 h. Cell proliferation was measured using the CyQUANT assay. The data were expressed as the average percentage of the untreated control (0 μg/mL SHI) containing DMEM medium alone for 72 h and were pooled from the average data from five replicate experiments (with cells from 5 different patients) in which each treatment was tested independently in triplicate. Error bars indicate mean +/− SEM (n = 5). *p < 0.05 versus the untreated control. Statistical analysis was performed using One-way ANOVA and Tukey’s post-hoc test. b Representative images showing apoptosis induced by SHI treatment in Kc and HSF respectively using TUNEL assay. Kc and HSF were treated with SHI for 72 h at the concentrations indicated, and then stained with TUNEL reagent to detect apoptotic cells and DAPI to detect the nuclei of all cells. Cells were viewed and images were captured using a Nikon Eclipse TE2000-U system. Green indicates DNA fragments from apoptotic cells, whereas blue localises the nuclei of both live and apoptotic cells. Representative images from cells obtained from three patients are depicted. Scale bar: 0.2 mm. c Apoptotic rate (%) in Kc and HSF induced by SHI. Three randomly selected images were recorded and the numbers of green-staining apoptotic cells and blue nuclei for all cells were counted. The final data was the average cell number of nine different images from three different patients. Apoptotic rate = number of green cells / (number of green cells + number of blue cells). Error bars indicate SEM
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Fig1: Effects of SHI on cell proliferation and apoptosis. a Cell proliferation. Kc and HSF were co-cultured and treated with SHI (0.5, 1 and 3 μg/mL) for 72 h. Cell proliferation was measured using the CyQUANT assay. The data were expressed as the average percentage of the untreated control (0 μg/mL SHI) containing DMEM medium alone for 72 h and were pooled from the average data from five replicate experiments (with cells from 5 different patients) in which each treatment was tested independently in triplicate. Error bars indicate mean +/− SEM (n = 5). *p < 0.05 versus the untreated control. Statistical analysis was performed using One-way ANOVA and Tukey’s post-hoc test. b Representative images showing apoptosis induced by SHI treatment in Kc and HSF respectively using TUNEL assay. Kc and HSF were treated with SHI for 72 h at the concentrations indicated, and then stained with TUNEL reagent to detect apoptotic cells and DAPI to detect the nuclei of all cells. Cells were viewed and images were captured using a Nikon Eclipse TE2000-U system. Green indicates DNA fragments from apoptotic cells, whereas blue localises the nuclei of both live and apoptotic cells. Representative images from cells obtained from three patients are depicted. Scale bar: 0.2 mm. c Apoptotic rate (%) in Kc and HSF induced by SHI. Three randomly selected images were recorded and the numbers of green-staining apoptotic cells and blue nuclei for all cells were counted. The final data was the average cell number of nine different images from three different patients. Apoptotic rate = number of green cells / (number of green cells + number of blue cells). Error bars indicate SEM

Mentions: To identify the effects of SHI on cell proliferation, Kc and HSF were treated with different concentrations of SHI (0.5, 1 and 3 μg/mL) (Fig. 1a). SHI at 0.5 μg/mL showed no significant effects on both Kc and HSF proliferation compared to the untreated control (p < 0.05). SHI at 1 μg/mL decreased HSF proliferation by 21.5 % ± 3.7 % compared to the untreated control (p < 0.05), however, no inhibitory effect on Kc proliferation was detected at this concentration of SHI. Kc and HSF proliferation were 40.5 % ± 5.2 % and 50.7 % ± 7.6 % below the untreated control (p < 0.05) when exposed at SHI 3 μg/mL, respectively. Taken together, these data indicate SHI reduces both Kc and HSF proliferation in a dose-dependent manner with higher concentrations required, however, to elicit effects on Kc compared to HSF. Specially, 1 μg/mL SHI inhibits HSF but not Kc proliferation.Fig. 1


Investigating the potential of Shikonin as a novel hypertrophic scar treatment.

Fan C, Xie Y, Dong Y, Su Y, Upton Z - J. Biomed. Sci. (2015)

Effects of SHI on cell proliferation and apoptosis. a Cell proliferation. Kc and HSF were co-cultured and treated with SHI (0.5, 1 and 3 μg/mL) for 72 h. Cell proliferation was measured using the CyQUANT assay. The data were expressed as the average percentage of the untreated control (0 μg/mL SHI) containing DMEM medium alone for 72 h and were pooled from the average data from five replicate experiments (with cells from 5 different patients) in which each treatment was tested independently in triplicate. Error bars indicate mean +/− SEM (n = 5). *p < 0.05 versus the untreated control. Statistical analysis was performed using One-way ANOVA and Tukey’s post-hoc test. b Representative images showing apoptosis induced by SHI treatment in Kc and HSF respectively using TUNEL assay. Kc and HSF were treated with SHI for 72 h at the concentrations indicated, and then stained with TUNEL reagent to detect apoptotic cells and DAPI to detect the nuclei of all cells. Cells were viewed and images were captured using a Nikon Eclipse TE2000-U system. Green indicates DNA fragments from apoptotic cells, whereas blue localises the nuclei of both live and apoptotic cells. Representative images from cells obtained from three patients are depicted. Scale bar: 0.2 mm. c Apoptotic rate (%) in Kc and HSF induced by SHI. Three randomly selected images were recorded and the numbers of green-staining apoptotic cells and blue nuclei for all cells were counted. The final data was the average cell number of nine different images from three different patients. Apoptotic rate = number of green cells / (number of green cells + number of blue cells). Error bars indicate SEM
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Related In: Results  -  Collection

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Fig1: Effects of SHI on cell proliferation and apoptosis. a Cell proliferation. Kc and HSF were co-cultured and treated with SHI (0.5, 1 and 3 μg/mL) for 72 h. Cell proliferation was measured using the CyQUANT assay. The data were expressed as the average percentage of the untreated control (0 μg/mL SHI) containing DMEM medium alone for 72 h and were pooled from the average data from five replicate experiments (with cells from 5 different patients) in which each treatment was tested independently in triplicate. Error bars indicate mean +/− SEM (n = 5). *p < 0.05 versus the untreated control. Statistical analysis was performed using One-way ANOVA and Tukey’s post-hoc test. b Representative images showing apoptosis induced by SHI treatment in Kc and HSF respectively using TUNEL assay. Kc and HSF were treated with SHI for 72 h at the concentrations indicated, and then stained with TUNEL reagent to detect apoptotic cells and DAPI to detect the nuclei of all cells. Cells were viewed and images were captured using a Nikon Eclipse TE2000-U system. Green indicates DNA fragments from apoptotic cells, whereas blue localises the nuclei of both live and apoptotic cells. Representative images from cells obtained from three patients are depicted. Scale bar: 0.2 mm. c Apoptotic rate (%) in Kc and HSF induced by SHI. Three randomly selected images were recorded and the numbers of green-staining apoptotic cells and blue nuclei for all cells were counted. The final data was the average cell number of nine different images from three different patients. Apoptotic rate = number of green cells / (number of green cells + number of blue cells). Error bars indicate SEM
Mentions: To identify the effects of SHI on cell proliferation, Kc and HSF were treated with different concentrations of SHI (0.5, 1 and 3 μg/mL) (Fig. 1a). SHI at 0.5 μg/mL showed no significant effects on both Kc and HSF proliferation compared to the untreated control (p < 0.05). SHI at 1 μg/mL decreased HSF proliferation by 21.5 % ± 3.7 % compared to the untreated control (p < 0.05), however, no inhibitory effect on Kc proliferation was detected at this concentration of SHI. Kc and HSF proliferation were 40.5 % ± 5.2 % and 50.7 % ± 7.6 % below the untreated control (p < 0.05) when exposed at SHI 3 μg/mL, respectively. Taken together, these data indicate SHI reduces both Kc and HSF proliferation in a dose-dependent manner with higher concentrations required, however, to elicit effects on Kc compared to HSF. Specially, 1 μg/mL SHI inhibits HSF but not Kc proliferation.Fig. 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, 4059, Australia. c3.fan@connect.qut.edu.au.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: The data generated from this study provides further evidence that supports the potential use of Shikonin as a novel scar remediation therapy.

No MeSH data available.


Related in: MedlinePlus