PMC

Comparison of LR and shikonin-induced differential expression of proteins in fibroblasts involved in wound healing processes. Only the differentially expressed proteins induced by either LR or shikonin are shown. Note that the proteins underlined with broken lines represent downregulated proteins, otherwise the proteins are upregulated. It is worth noting that the differentially expressed proteins induced by shikonin form a subset of all the differentially expressed proteins induced by LR. Thus, all differentially expressed proteins placed in the small circle represent the proteins induced by shikonin.

Detection of collagen secretion, TGF-β expression, and other related cell signals. LR5 and LR20 treatments are able to significantly upregulate collagen secretion by fibroblasts in a dose-dependent manner (a). It was also found that the upregulation of collagen was concomitant with the overexpression of TGF-β (b). It was further confirmed that p-Erk and p-p38 were also upregulated in cells after LR5 and LR20 treatments (c). In contrast, the expression of p-Erk and p-p38 was downregulated in S100-treated fibroblasts. The ratios of p-Erk and p-p38 were normalized against GAPDH and then compared with DMSO using the Fujifilm Multigauge system. The experiments were carried out in triplicate and showed the same results. *P < 0.01; ***P < 0.001.

The Boyden chamber migration test and the wound healing assay were used to detect the motility of fibroblasts after the various drug treatments. The migration assay showed that LR5-, LR20-, and S100-treated cells underwent less migrated through the chamber (a). The wound healing assay further demonstrated that treatment with the LR extracts was able to significantly decrease the migration of cells in a dose-dependent manner (b). Furthermore, the active component S100 was also able to create a similar effect on cell migration. Quantification of the chamber migration (c) indicated that LR20 significantly reduced cell migration and to the largest extent. Quantification of the wound area (d) showed that fewer cells migrated into the wound area when the cells were treated with LR20. The results are expressed as mean ± SD (n = 3). *P < 0.01; ***P < 0.001, compared with DMSO.

Immunoblotting of LEG-1 from the treated fibroblasts and the measurement of ROS within the treated fibroblasts. The immunoblot using LEG-1 antibody showed that LEG-1 (spot 17) was only significantly downregulated in cells that had undergone the LR20 treatment (a). On the other hand, GSTP-1 (B, spot 1, , cluster F) and PRDX-2 (C, spot 3, , cluster F) were upregulated in cells that underwent either LR treatment, which was confirmed by immunoblotting with specific antibodies. However, only overexpression of PRDX-2, but not overexpression of GSTP-1, was detected in cells after S100 treatment (c). The ROS assay showed that ROS production was significantly reduced in cells treated with LR20 (d). GAPDH was employed as the sample loading control. The related expression levels were detected by Fujifilm Multigauge ver. 2.0 and a Student's  t-test was used to evaluate the statistical significance. Results are from triplicate experiments and are presented as the mean ± SD (n = 3). *P < 0.05; **P < 0.01.

2D PAGE analysis of the protein differentially expressed in human fibroblast after treatment with the LR extracts or shikonin. Fibroblasts were treated with 5 μg/mL LR extract, 20 μg/mL LR extract, or 100 nM shikonin for 24 h, and then the whole cell lysates were collected and analyzed by 2D PAGE using a pH 4–7 IEF strips and 12.5% SDS-PAGE. The resulting gel was then visualized by silver staining. The treatments of the cells for the 2D PAGE are indicated below each gel and are (a) 0.5% of DMSO, (b) 5 μg/mL LR extract (LR5), (c) 20 μg/mL LR extract (LR20), and (d) 100 nM of shikonin (S100). It should be noted that there were 22 differentially expressed protein spots with ratio >1.5 or <0.8 fold compared to the DMSO control that were identified from LR5-, LR20-, and S100-treated cells. The corresponding protein spot identities are shown in .

SD rat experiment to verify the efficacy of the LR extracts in wound healing. The diameter of the wounds was used as a guideline for the rate of wound healing. Two wound circles were generated on the back of SD rats using an 8 mm biopsy punch. DMSO, LR5, and LR20 were used to treat the wounds and the diameter of the wounds was measured twice per day. It was found that the efficacy of wound closing rate of the wounds after LR5 and LR20 treatments was far much greater that when the wounds were treated with DMSO only (a). The wound healing rate was also recorded by digital camera at interval of 4 days (b). This demonstrated that the wound started to shrink after 2 days and continued to shrink further until 11 days. It is worth noting that wounds treated with LR20 showed the fastest wound closing rate compared to the other treatments (b). Results are triplicates and are presented as means ± SD (n = 6). *P < 0.05; **P < 0.01.

Hierarchical clustering and functional classification of protein expression induced in fibroblasts by the various drug treatments. The expression patterns of the identified proteins were categorized by UPGMA using Hierarchical Clustering Explorer 3.5 software, and the biological function classification was determined using BGSSJ and the SwissProt protein sequence database. Proteins with similar expression patterns were categorized into six different groups (clusters A, B, C, D, E, and F) in a tree-like diagram. Each row in the color mosaic map indicates one protein with a number matching the 2D-PAGE () and each column represents different groups of proteins identified from the fibroblasts treated with DMSO, LR5, LR20, and S100. A bright red color represents a high protein expression value and a bright green color represents a low protein expression value. Black color indicates that the protein was expressed at an average level. Further information on the differentially expressed protein spots is presented in .

Effects of LR extracts and shikonin on human fibroblast cell viability. The graphs represent the ratio of viability of fibroblast cells treated with various concentrations of LR extracts or shikonin using cells treated with DMSO as the control. (a) LR extract was toxic to fibroblasts in a dose-dependent manner at higher concentrations and reaches the IC50 at the concentration of 250 μg/mL. In contrast to the above toxicity, concentrations lower than 50 μg/mL promoted cell viability. It should be noted that 20 μg/mL of LR extracts were the optimal concentration for promoting cell viability, which gave an increase in the cell viability of more than 25%. Moreover, shikonin had a similar effect on cell viability (b), and the IC50 for fibroblasts was found to be about 3000 nM for shikonin. It was found the viability of fibroblasts was increased by >20% at 100 nM shikonin. Fibroblasts were seeded into a 24-well plate using 4 × 10⁴ cells per well. After the cells had attached, the designated drug concentration was used to treat the cells for 24 h. Cell viability was evaluated by the WST-1 assay, and the cell density was measured by spectrophotometer at OD450-OD690. It should be noted that fibroblasts treated with 0.5% of DMSO was used as the control. The Student's  t-test was used to evaluate the statistical significance of the results, which is presented as mean ± SD (n = 5). *P < 0.05; **P < 0.01, compared with the control.