Arnebin-1 promotes the angiogenesis of human umbilical vein endothelial cells and accelerates the wound healing process in diabetic rats.

Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Research Paper

Arnebin-1 promotes the angiogenesis of human umbilical vein endothelial cells and accelerates the wound healing process in diabetic rats Zhi Zeng, Bang-Hao Zhu n Department of Pharmacology, Sun Yat-sen University, Guangzhou, 510080, China

ar t ic l e i nf o

a b s t r a c t

Article history: Received 17 October 2013 Received in revised form 2 April 2014 Accepted 22 April 2014

Ethnopharmacological relevance: Zicao is a traditional wound healing herbal medicine that has been used for several hundred years in China. A survey of the published literatures revealed that arnebin-1, one of the naphthoquinone derivatives, played the most important role in wound healing property of this plant. However, whether arnebin-1 affects angiogenesis in vitro and has an effect on wound healing process in diabetic rats remains enigmatic. To investigate the effect of arnebin-1 with or without VEGF on proliferation, migration and tube formation of HUVECs in vitro and the effect of its topical application in the form of ointment on wound healing in a cutaneous punch wound model of alloxan-induced diabetic rats in vivo. Materials and methods: The pro-angiogenic functions of arnebin-1 on HUVECs including proliferation, migration and angiogenesis were evaluated through MTT assay, wound healing assay, transwell assay and tube formation assay in vitro. Male Sprague-Dawley rats were injected intraperitoneally with alloxan to induce type І diabetic rats. Three wounds were created in each rat on the dorsal surface, and then divided to be basement treated, arnebin-1 ointment treated and untreated group correspondingly. The indicators including wound closure rate and histological evaluation were investigated on day 4 and 7 post-wounding. Results: Without VEGF, arnebin-1 did not affect the proliferation of HUVECs significantly, but had a positive effect on cell migration and tube formation. However, in the presence of minimal VEGF, Arnebin1 could increase the proliferation, enhance the migration and promote the tube formation of HUVECs significantly. The wound closure rate was increased significantly in arnebin-1 treated group compared to that of untreated and basement treated groups in diabetic rats, and the histological evaluation also showed well organized dermal layer, reduced number of macrophages, increased number of fibroblasts, remarkable degree of neovascularization and epithelization in arnebin-1 treated group. Conclusion: These findings suggest that arnebin-1 has a pro-angiogenic effect, and a synergetic effect with VEGF promotes the wound healing process in diabetic rats. & 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Keywords: Arnebin-1 Angiogenesis Human umbilical vein endothelial cells Diabetes Wound healing Chemical compounds studied in this article: Arnebin-1 (PubChem CID: 32465) Beeswax (PubChem CID: 61429) Lard oil (PubChem CID: 3085226) Dimethyl Sulfoxide (PubChem CID: 679) L-glutamine (PubChem CID: 5961) Sodium Pyruvate (PubChem CID: 23662274) Pentothal Sodium (PubChem CID: 23665410) Alloxan (PubChem CID: 5781)

1. Introduction Chronic wound healing is a common and tiring complication of diabetes, causing ultimately important clinical morbidity, for example nonhealing ulcers, infection, gangrene and even amputation (Greenhalgh, 2003; Stadelmann et al., 1998; Lee et al., 2012). For the past few years, therapy of diabetic wounds has reinforced, but the amputation rate maintains high and is associated with increased morbidity and mortality (Galeano et al., 2008). In diabetes, the prevalence rate of diabetic foot ulcers are 4–10%, and the therapies of foot ulcers are comprehensive and costly

n

Corresponding author. Tel./fax: þ 86 20 87334786. E-mail address: [email protected] (B.-H. Zhu).

(Majtan, 2011). Thus, care for diabetic wounds is still a great clinical problem and the evolution of therapies that improve wound healing in diabetic patients is mostly important (Zhang et al., 2011). Various kinds of growth factors provide the cellular and molecular signals necessary for the normal healing process but are insufficient in diabetic wounds (Wang et al., 2008). Delayed wound healing in diabetes mellitus is mainly associated with reducing the angiogenesis (Chithra et al., 1998), especially, attributed to downregulation of vascular endothelial growth factor (VEGF) expression (Frank et al., 1995). VEGF accelerates all steps in the cascade course of angiogenesis, and experiment research shows that a defect in VEGF regulation is mainly associated with wound healing disorders. Furthermore, the presence of a defect in VEGF regulation, characterized by an altered expression pattern of

http://dx.doi.org/10.1016/j.jep.2014.04.038 0378-8741/& 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Please cite this article as: Zeng, Z., Zhu, B.-H., Arnebin-1 promotes the angiogenesis of human umbilical vein endothelial cells and accelerates the wound healing.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.04.038i

Z. Zeng, B.-H. Zhu / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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VEGF mRNA and protein during skin repair in db/db mice, has been shown and thereby suggests that the impairment in VEGF regulation might be associated with wound healing abnormalities seen in these animals (Frank et al., 1995). Zicao is the common name for extracts from the roots of the Boraginaceae family. There are five species of Boraginaceae family, such as Lithospermum erythrorhizon Sieb. et Zucc, Arnebia euchroma (Royle) Johnst, Arnebia guttata Bunge, Onosma paniculatum Bur. et Franch. and Onosma hookeri Clarke var. longiflorum Duthie (Chen et al., 2002). These Boraginaceous plants are rich in naphthoquinone derivatives which are potent pharmaceutical substances with a wide spectrum of biological properties including wound healing, antibacterial, antifungal, antiviral, anti-inflammatory and anticancer (Akkol et al., 2009). The most important naphthoquinone derivatives in Zicao are shikonin, β,β-dimethylacrylshikonin (arnebin-1), deoxyshikonin, acetylshikonin, isovalerylalkannin and β-hydroxyisovaleryshikonin (Hsiao et al., 2012). Zicao is a traditional wound healing herbal medicine that has been used for several hundred years in China (Papageorgiou et al., 2008). A survey of the published literatures revealed that arnebin-1, one of the naphthoquinone derivatives, played the most important role in wound healing property of this plant. Arnebin-1 significantly accelerated both normal and hydrocortisone-impaired wound healing as revealed by advancing wound closure rate, promoting vessel formation, increasing synthesis of collagen to form a thick granulation tissue and re-epithelialization of the wounds compared with controls (Sidhu et al., 1999). Although arnebin-1 does improve wound healing, the mechanism still remains to be resolved. Moreover, the interesting problem whether the drug can have positive effects on the diabetic wound is unknown. Hence, in this experiment, we aimed to investigate the effect of arnebin-1 with or without VEGF on proliferation, migration and tube formation of HUVECs in vitro and evaluate the effect of its topical application in the form of ointment on wound healing in a cutaneous punch wound model of alloxan-induced diabetic rats in vivo.

2.3. Cell proliferation assay Cell proliferation was evaluated by the mitochondrial tetrazolium assay (MTT). HUVEC were plated at 3 103 cells/well in 96 well culture plates. Twenty-four hours later, the medium was replaced with the test medium supplemented with arnebin-1 at various concentrations (10 3, 10 2 or 0.1 μM) and with or without 1 ng/mL VEGF. After incubation for 24 h, the number of viable cells was determined using the MTT reagent according to the manufacturer's instructions. In brief, MTT reagent (10 μl) was added to the 100 μL medium, and incubated at 37 1C for 4 h. The supernatant was removed and dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. Absorbance (570 nm) of the medium was measured with Biotek Elx-800 plate reader. MTT is a tetrazolium salt that is converted into the insoluble formazan salt by succinate–tetrazolium reductase of the respiratory chain of active mitochondria of viable cells. 2.4. Wound healing assay Cell migration was measured using a scratch injury assay. After 24 h of assay media treatment, 90% confluent monolayer of HUVEC were wounded using a micropipette tip (10 ml white tip), gently rinsed with PBS, and then incubated in the same assay medium with 2% fetal bovine serum. HUVEC migration was photographed using a live cells imaging system (Zeiss Axio Observer Z1, Genmany) in 37 1C and 5% CO2 humidified atmosphere. Live cells can be photographed every 5 min and the results can be exported in way of movies which provided intuitive cell motion images. Individual cell migration was quantified from 3 separate experiments measuring 10 cells per treatment using Axio Vision microscope software (Zeiss, Germany) with an automatic and interactive tracking module as described in Starke et al. Parameters including total distance, straight distance, mean velocity, and tortuosity were analyzed. 2.5. Transwell assay

2. Materials and methods 2.1. Materials Arnebin-1 was purchased from Tokyo Chemical industry Co. Ltd. (Tokyo, Japan), and recombinant human vascular endothelial growth factor (VEGF) from PeproTech Inc. Growth factor-reduced Matrigel Basement Membrane Matrix was obtained from BD Biosciences (BD, Bedford, USA). Medium 199 was purchased from Gibco (USA). Fetal calf serum was purchased from PAA Co. (USA). All other reagents utilized were purchased from Sigma Chemical Co. USA unless otherwise specified.

Cell invasion assay was performed using Transwell chambers with 6.5 mm diameter polycarbonate membrane (8 μm-sized pores). The bottom chamber of the apparatus contained 600 μl of endothelial cell medium supplemented with arnebin-1 at various concentrations (10 3, 10 2 or 0.1 μM) and with or without 1 ng/mL VEGF. The HUVECs (200 μl) were added to the upper chamber (5 104 cells/well) and incubated in M199 medium with 2% fetal bovine serum. After 24 h incubation at 37 1C, non-invasive cells on the upper membrane surfaces were removed by wiping with cotton swabs. Invading cells were fixed with methanol and stained with 0.1% crystal violet. Cell invasion was quantified by counting cells on the lower surface using phase contrast microscopy. Then the membranes were washed with 30% glacial acetic acid. For HUVEC counting determination, the washing solution was detected at the wavelength of 540 nm.

2.2. Cell culture 2.6. Tube formation assay Human umbilical vein endothelial cells were isolated and cultured as previously described. Briefly, HUVECs were removed from human umbilical veins after 0.125% trypsin digestion, and cultured in medium 199 containing 20% fetal calf serum, penicillin (100 U/ml), streptomycin (100 U/ml), and heparin (50 U/ml), supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM), and endothelial cell growth factor (β-ECGF, 5 ng/ml), at 37 1C in 5% CO2 on 0.1% gelatin-coated culture flasks. Endothelial cells were identified by their morphology which appears “cobblestone” mosaic appearance after reaching confluence and the presence of von Willebrand factor. Passage 3–6 HUVECs were used for experiments.

To determine whether Arnebin-1 improved angiogenesis, we used the experimental in vitro Matrigel system. Growth factorreduced Matrigel Basement Membrane Matrix was thawed on ice at 4 1C overnight and all pipettes and 96-well flat bottom plate were pre-cooled before use. 96-well plates were coated with 50 ml Matrigel per well for 30 min at 37 1C. HUVEC were seeded at 4 104 cells per well in 100 ml assay media. After 16 h of incubation, tube-like structures were photographed using inverted microscope (IX71). The total tube length was quantified using Image J software (NIH, Bethesda, MD, USA).



2.7. In vivo diabetic wound healing study 2.7.1. Animals and induction of diabetes All animal procedures were approved by the Laboratory Animal Center of Sun Yatsen University. Male Sprague-Dawley rats weighing 250–300 g were housed in stainless steel wire cages under pathogen-free conditions. The rats were controlled at temperature 18–22 1C with a 12 h light-dark cycles and free to take food and water. After 1 week acclimatization, all the animals were allowed to be applied in the experiment. The rats were fast for 12 h and injected intraperitoneally with alloxan monohydrate dissolved in normal saline at a twice dose of 100 mg/kg every other day to induce diabetic model. Three days after alloxan injection, fasting blood glucose (FBG) levels of the rats were determined by a rapid glucometer. The rats showing FBG levels greater than 16.7 mmol/L were confirmed as diabetic rats for the research. FBG levels were monitored before and after the experiments.

2.7.2. Preparation of the ointment The ointment containing siritch (1.5 g), beeswax (5 g) and lard oil (0.15 g) was heated at 70–75 1C up to be solubilized, and then 6.65 mg arnebin-1 (0.1%) was added and mixed. Eventually, the mixture stirred until it cooled down to room temperature.

2.7.3. Experimental wounding SD rats were anesthetized with pentothal sodium (35 mg/kg, intraperitoneal injection.). The hair on the dorsal side of each rat was shaved and the skin was sterilized with 70% ethanol. Full thickness cutaneous wound was made by an 8-mm skin biopsy punch (World Precision Instruments, WPI) under aseptic conditions. Three wounds were made in rats on the dorsal surface. The diameters of wound size were between 7.5 and 9 mm. Thereafter, the animals were individually caged.

2.7.4. Drug administration Wounds on the top of the dorsal side and those near the tail were treated with only the vehicle base (without the test compound) and untreated respectively, while those at the middle were treated with arnebin-1 (0.1% ointment). Thus, the wounds on the top side served as vehicle controls for the treated wounds. Having the control, vehicle control and test wounds on the same animal obviated the differences that may occur between groups due to animal-to-animal variation. The ointments of vehicle and test compound were applied every other day with separate spatulas, enough to cover the wounds with a thin layer of ointment. All the treatments were continued until the day of sacrifice.

2.7.5. Measurement of the wound closure Wound closure was examined every 48 or 72 h from the point of creation of wounds (2, 4 and 7 days post-wounding). Images were photographed with digital camera (DSC-W320 Sony; Sony Corp., Tokyo, Japan) at that time. Camera lens poses vertically to wounds and the distance was 3 cm. This position was fixed for every wound so the condition of image formation was identical for all images. After that, the chosen wound area of images was implemented using Image ProPlus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA). The number of pixels in each wound proportions was counted as proportions of the wound and used to estimate the percentage of wound closure. The rate of wound closure was counted with a formula as below: (proportions of original wound proportions of the rest of wound)/proportions of original wound 100.

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2.7.6. Tissue collection Rats were anesthetized by overdose of pentobarbital (200 mg/ kg, i.p.) on day 4 or 7 post-wounding, respectively. The whole wound with a margin of around 5 mm of ambient unwounded skin was excised. These wound tissues were used for histopathological examination and were fixed for 24 h in a 10% buffered formalin solution and embedded in paraffin.

2.7.7. Histomorphological analysis Five μm-thick sections were cut and stained with hematoxylin and eosin (HE) for histopathological examination. Three separate sections of each wound were inspected by light microscopy. The areas of the wounds were evaluated in all skin sections in 5 microscopic fields (100 to 400 magnification) by 2 observers blinded to the experimental protocol. The development of neovascularization, scar, epidermis, and granulation tissues was inspected.

2.7.8. Neovascularization assessment Images were photographed using inverted microscope (IX71, Olympus, Japan) per 400 magnification field and were transferred to the image J NIH, so the area of neovascularization was scaled. The numbers of pixels were acted as amount of neovascularization (Lau et al., 2009). For each wound six sections were selected and images were taken from three parts of each section.

2.7.9. Western blotting analysis Tissues were minced and homogenized in protein lysis buffer with protease inhibitor cocktail. The protein concentration of lysates was quantified by BCA Kit, and equal amounts of protein were separated by 10% SDS-PAGE and then transferred onto a PVDF membrane (Millipore, USA). The membranes were blocked in 5% non-fat dry milk diluted with TBST (in mmol/L: Tris–HCl 20, NaCl 150, pH 7.5, 0.1% Tween 20) at room temperature for 1 h and probed overnight at 4 1C with, a polyclonal rabbit anti-VEGF or a polyclonal rabbit anti-PCNA (Santa Cruz, USA), and then incubated for 1 h with a goat anti-rabbit IgG conjugated to horseradish peroxidase (Cell Signaling Technology, USA). Incubation with monoclonal mouse α-tubulin antibody (1:1000 dilution; Sigma, USA) was performed as the loading sample control. The proteins were visualized using ECLTM western blotting detection reagents (Amersham Biosciences Corp., USA). The densitometry of the bands was quantified using the Image J 1.38X software (USA). 2.8. Statistical analysis All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., USA). Data for each study parameter from each group were presented as mean 7standard error of the mean (S.E.M.). Data from each group were statistically analyzed by one-way analysis of variance (ANOVA). Differences were considered statistically significant at P o0.05.

3. Results 3.1. The effect of arnebin-1 on HUVECs viability As shown in Fig. 1A, arnebin-1 at concentration range from 1 10 3 μM to 10 1 μM did not present significant effect on HUVECs viability, however, a higher concentration (1 μM) of arnebin-1 significantly inhibited the cell viability to 80.8 70.06% compared with control after 24 h treatment (P o0.05).


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Fig. 1. Effects of arnebin-1 on HUVECs proliferation in the absence (A) or presence (B) of VEGF. Cells (3000 per well) were incubated with M199 containing 2% FBS and treated with or without arnebin-1 at different concentrations (10 3, 10 2, 10 1 or 1 μM) in the absence or presence of VEGF (1 ng/ml) for 24 h, respectively. Cell proliferation was assessed by MTT assay. Data are expressed as percentage of vehicle (0.1% DMSO) control. Values are means7 SEM. nPo 0.05, nnPo 0.01 vs control; ##Po 0.01 vs VEGF (n¼6).

Fig. 2. Effect of arnebin-1 on HUVECs migration in the absence (A and B) or presence (C and D) of VEGF in wound healing assay. HUVECs were incubated with the assay media (control), arnebin-1 (10 3, 10 2, 10 1 μM) in the absence or presence of VEGF (1 ng/ml) for 12 h and wounded by scratching with a micropipette tip. Cell migration was monitored for the next 12 h using a live cells imaging system. Migration was estimated by measuring the cell numbers within the wounded region. Data are expressed as percentage of vehicle (0.1% DMSO) control. Data represent means 7 SEM. nP o0.05, nnPo 0.01 vs control; #Po 0.05 vs VEGF (n¼ 3).



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Fig. 3. Arnebin-1 improved HUVECs migration in the absence (A and B) or presence (C and D) of VEGF in Transwell assay. A total of 2 104 HUVECs were seeded in the top chamber and treated with different concentrations of arnebin-1 (10 3, 10 2, 10 1 μM) in the absence or presence of VEGF (1 ng/ml). After 8 h, the HUVECs that invaded through the membrane were stained and quantified. Data are expressed as percentage of vehicle (0.1% DMSO) control. Data represent means 7 SEM. nPo 0.05, nnPo 0.01 vs control; ##P o 0.01 vs VEGF (n ¼4).

3.2. Arnebin-1 promoted the proliferation of HUVECs in the presence of VEGF In the minimal VEGF (1 ng/ml) treated alone group, the proliferation of cells was increased about 1.15 fold (vs the control group). However, in the presence of VEGF (1 ng/ml), arnebin-1 increased the proliferation of HUVECs in a concentrationdependent manner compared with the VEGF alone group, with a maximal effect at 10 1 μM reaching about 1.33 fold (vs the control group, Fig. 1B). 3.3. The effect of arnebin-1 on HUVECs migration in the absence or presence of VEGF Migration of endothelial cells is a critical initiating event in the formation of new blood vessels and the repair of injured vessels. To determine the effect of arnebin-1 on HUVECs migration, we adopted wound healing assays and transwell migration assays. To compare the differences in migratory behavior, images were taken at the same size, and the number of cells that had migrated into the wound region was determined after 12 h compared to that of control. Confluent scrape-wounded HUVECs monolayers were incubated with arnebin-1 at various concentrations (10–3, 10 2 and 10 1 μM) in the absence or presence of VEGF (1 ng/ml). In the groups treated with drug alone, the number of migrated cells was increased 1.3–1.6 fold (vs the control group) with the strongest effect at 10 2 μM (Fig. 2A and B). In the groups treated with VEGF alone, the number of migrated cells was also increased about

1.4 fold compared with that of control (Fig. 2C and D). In the presence of VEGF, arnebin-1 at 10 2 and 10 1 μM significantly enhanced the cell motility which reached 1.8 fold (vs the control group). The transwells migration assay also demonstrated the similar results of the wound healing assay (Fig. 3). 3.4. Arnebin-1 promoted the tube formation of HUVECs in the absence or presence of VEGF HUVECs were plated on Matrigel to form tubular structures. Compared with control group, tube formation was significantly induced by both VEGF and arnebin-1. In the presence of VEGF, the treatment of arnebin-1 at 10 2 and 10 1 μM enhanced the formation of capillary-like structures more significantly than that of VEGF treated alone group (Fig. 4). Quantitative measurements showed the same. Thus, similar to the results of the migration, this result suggested that arnebin-1 and VEGF had a synergistic effect on the tube formation of HUVECs. 3.5. In vivo diabetic wound healing study 3.5.1. Diabetic induction The mean of FBG levels and weight has been illustrated in Table 1. The body weight and FBG levels of the diabetic rats were measured before and 3 days after injection of alloxan. The rats showed 3–4 fold increase of FBG level compared to their normal level after alloxan injection and a decrease in body weight during this period, indicating that DM was successfully induced in the



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Fig. 4. The ECMatrixTM in vitro angiogenesis assay was used to examine the effects of arnebin-1 on HUVECs capillary tube formation in the absence (A and B) or presence (C and D) of VEGF (1 ng/ml). HUVECs (2 104 cells) were cultured in 96-well plates previously coated with Matrigel and incubated for 12 h at 37 1C in the absence or presence of different concentrations of arnebin-1 (10 3, 10 2, 10 1 μM) with or without VEGF. The total length was counted from 3 randomly selected images. Percentage (%) of control is the total length of tubules expressed as a proportion of that in the control (0.1% DMSO) group. Arnebin-1 significantly enhanced HUVECs tubule formation. Bars represent mean7 SEM. nP o 0.05 vs control; #Po 0.05 vs VEGF (n¼ 4).

Table 1 Effect of arnebin-1 on body weight and blood glucose. Normal rats (n¼ 12)

Body weight (g) 288.3 7 2.3 Blood glucose 6.2 7 0.2 (mmol/L)

Diabetic rats 3 day after injection (n¼ 12)

Diabetic rats 4 day after surgery (n¼ 6)

Diabetic rats 7 day after surgery (n¼6)

nn

nn

nn

260.4 7 3.6 24.37 0.9nn

248.9 7 6.9 24.97 1.8nn

240.07 6.6 26.17 1.6nn

Values are presented as mean 7 SEM. nn

Po 0.01 vs normal rats.

rats. On the 4th and 7th days post surgery, the FBG level remained higher than 16.7 mM. Topical treatment given to the rats wound did not exhibit any effect on the FBG level over the process of study.

3.5.2. Rate of wound closure To estimate the wound repair ability of treatments in diabetic rats, the rate of wound closure was calculated as the percentage of wound reduction from the original wound on the 2nd, 4th and 7th days (Fig. 5). On day 2, there was no significant difference among all groups. However, on the 4th and 7th days post injury, the rate of wound closure was significantly increased in drug ointment

group compared with control and vehicle base ointment groups. There was no significant difference between control group and vehicle base ointment group.

3.5.3. Histopathology analysis Multiple cross-sections of HE stained sections of arnebin-1 treated, basement treated and untreated biopsy punch tissues of rats were examined for infiltration of cells, density of newly formed vessels, epithelial regeneration and the organization of the granulation tissue. Arnebin-1 treatment enhanced wound healing in diabetic wounds (Figs. 6 and 7). On the 4th day post-wounding (Fig. 6), there was inflamed scab that retarded epithelization in the control wounds and the underlying dermis revealed ill-formed granulation tissue with a few newly formed vessels and thin collagen. Granulation tissue of vehicle treated wounds showed a more aggregation of macrophages with few collagen fibers and new vessels than the arnebin1 treated groups. Arnebin-1-treated wounds confirmed more number of congested vessels and collagen deposition in the granulation tissue with fewer macrophages and more fibroblasts. A significant enhancement in collagen content was observed during the process of wound healing in the treated group due to increased migration of fibroblasts and epithelial cells to the wound location. The reduced collagen content in the control group might be due to prolonged inflammatory phase where the degradation of collagen will be more than the synthesis collagen. Furthermore,



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Fig. 5. Arnebin-1 ointment promoted wound healing in diabetic wounds. (A) Photos of diabetic wounds (on the 2nd, 4th and 7th day). Apparently, the arnebin-1 ointment group showed better in wound healing. (B) Healing rate of diabetic wounds. On the 2nd day, there were no significant differences among all groups (P 40.05); after 4 days, arnebin-1 group had the highest rate of healing. Bars represent mean 7SEM. nnPo 0.01 vs control and vehicle groups (n¼6).

collagen is an important protein of the extracellular matrix and is the component that eventually contributes to wound contraction. On the 7th day post-wounding (Fig. 7), organization of the granulation tissue was more advanced with thick bundles of collagen fibers in arnebin-1 treated wounds. The epidermal regeneration was also enhanced by arnebin-1. Arnebin-1 treatment leaded to the formation of dense granulation tissue with neovascularization, whereas many inflammatory cells and a few congested vessels and collagen fibers were seen in the granulation tissue of control and vehicle treated wounds. There was no apparent difference in the wound healing between either of control or vehicle treated wounds, suggesting that vehicle did not affect the wound healing. Wounds on the 7th day were much healthier in arnebin-1-treated wounds.

3.5.4. Angiogenesis study At days 4 and 7 post surgery, the area of neovascularization increased in the arnebin-1 treated group compared to the control and vehicle groups (Fig. 8A and B). VEGF effectively improves the proliferation and migration of endothelial cells in vitro and triggers a series of events that induce new blood vessel growth in diabetic wounds (Yancopoulos et al., 2000), thereby improve the blood perfusion and metabolism in wound tissues, and then promote wound healing (Yan et al., 2010). From our results, topical administration of the arnebin-1 cream

would have the possibility to accelerate angiogenesis by upregulation of VEGF expression in diabetic rats (Fig. 8C and D). Moreover, PCNA is a protein of proliferation marker and is seen to be strongly associated in the nuclear region where DNA synthesis is occurring (Sidhu et al., 1999). PCNA expression showed an increase in the proliferating cells in the granulation tissue in arnebin-1-treated wounds compared with the control and vehicle groups during diabetic wound healing (Fig. 8C and E), suggesting that arnebin-1 facilitated the infiltration of proliferating endothelial cells into the wound bed.

4. Discussion This study demonstrates the potential effect of arnebin-1 in treating diabetic wound healing. We aimed to find whether arnebin-1 could significantly promote angiogenesis of HUVECs and accelerates wound closure in type І diabetic rats. There is an intricate cascade of events in skin repair, but angiogenesis is considered a pivotal process during wound healing (Bitto et al., 2013). Chronic wound healing is a common complication of diabetes, and diabetes has been shown to hinder the angiogenesis (Bek et al., 2002; Da Costa Pinto and Malucelli, 2002). On the other hand, a generalized microangiopathy could decrease nutrient provision to the ulcer tissues and result in impaired diabetic foot ulcer healing. Thus angiogenesis is a crucial


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Fig. 6. Histopathology (HE staining) of rat wounds treated with arnebin-1 ointment for 4 days. A, D, G Control wound. Ill-formed granulation tissue with a few newly formed vessels and thin collagen. B, E, H Vehicle treated wound. Granulation tissue of vehicle treated wounds showed a more aggregation of macrophages with few collagen fibers and new vessels. C, F, I Arnebin-1 ointment treated wound. Granulation tissue is dense with more number of congested vessels, fibroblasts and fewer macrophages as compared to the control and vehicle treated wound.

Fig. 7. Histopathology (HE staining) of rat wounds treated with arnebin-1 ointment for 7 days. A, D, G Control wound and B, E, H Vehicle treated wound. Many inflammatory cells and a few congested vessels and collagen fibers were seen in the granulation tissue. C, F, I Arnebin-1 ointment treated wound. The epidermal regeneration was enhanced. Organization of the granulation tissue was more advanced with thick bundles of well-aligned collagen fibers and moderate neovascularization.



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Fig. 8. Effect of arnebin-1 on angiogenesis of wound healing in diabetic rats. (A and B) The area of neovascularization was measured at day 4 (A) and 7 (B) post surgery. The numbers of pixels were intended as amount of neovascularization. (C) Western blot analysis of protein expressions of VEGF and PCNA in skin tissue of wound healing at day 4 post surgery. The expression of α-tubulin was used as internal control. (D and E) The bar chart represented the relative fold change of protein expressions of VEGF (D) and PCNA (E). Data were presented as a mean 7 SEM. nnP o0.01 vs control and vehicle groups (n¼6).

event in diabetic foot ulcer healing (Chabbert-Buffet et al., 2003). Several studies have reported that the expression of VEGF or its receptor was decreased in the type I and type II diabetic animals (Zhang et al., 2011; Lima et al., 2012; Zandifar et al., 2012). It has been reported that VEGF can promote angiogenesis and accelerate the blood supply in diabetic wounds, and many reports suggest a beneficial effects of VEGF, but the safety of it in treatment is still an important concern. Low doses of VEGF may issue in tardive angiogenesis but high doses of VEGF may cause the formation of hemangioma, and the diffusion of VEGF may lead to undesirable side effects (Lee et al., 2000). Besides, in practical use, there are still two puzzlers: firstly, the half-life of VEGF is too short, and the metabolic degradation is too rapid to be maintained at the target location for adequate time. Secondly, inflammatory exudates make VEGF metabolic loss easily through its scouring effect, which cannot produce an effective partial concentration of VEGF (Yan et al., 2010). At present, tests extended on cell cultures are one of the most universal and efficient methods to experiment with the sensitivity of chosen group of cells to substances exist in their microenvironment. Endothelial cell cultures have been considered as a method for testing angiogenesis of wound-healing activity in vitro. We firstly developed HUVECs culture technique to assess the arnebin-1. In vitro experiments we adopted MTT test. This cell proliferation measurement was implemented after seeding the endothelial cells for 24 h. We found that arnebin-1 enhanced the proliferation of HUVECs only in the presence of VEGF. Arnebin-1 increased migration and tube formation of HUVECs in the absence of VEGF. Moreover, in the presence of VEGF, the increased migration and tube formation of HUVECs induced by arnebin-1 was enhanced further. These findings suggest that arnebin-1 acts as a proangiogenic agent, and has a synergetic effect with VEGF. The significant concentrations of arnebin-1 in the presence of VEGF is at 10 2 μM and 10 1 μM in proliferation, migration, and tube formation assays, but in the absence of VEGF the concentration of arnebin-1 is at 10 2 μM and 10 3 μM in migration and tube formation assays. This difference is probably due to the difference in the experimental conditions or could not be explained by present knowledge. The findings obtained indicate that arnebin1 can accelerate the wound-healing activity, and its mechanism of

action is to promote angiogenesis by the activation of endothelial cell, which was additionally supported by histopathological studies in vivo. in vivo experiment, alloxan was used to induce diabetes in experimental rats intravenously. Three days after the injection of alloxan, all the rats exhibited increase of FBG level. The FBG remained continually elevated in time of all the experimental duration. The topical application of arnebin-1 ointment showed no effect on the FBG. Rate of wound closure is a helpful measurement to evaluate the development of wound healing. It had been proved that there was a reduction in rate of wound closure in diabetic rats compared with the normal rats (Teoh et al., 2009). In the present study, application of 0.1% arnebin-1 ointment was able to increase the rate of wound closure in diabetic rats compare with the untreated and basement treated groups. Angiogenesis during progress of wound repair serves the dual function of providing the nutrients required by supply essential nutrients and oxygen to the wound site, and accelerating granulation tissue formation (Brem and Tomic-Canic, 2007; Martin et al., 2003; Roy et al., 2006). In the present study, by observing the capillary number, histological evaluation revealed that more blood vessel formation were found in the granulation tissue of arnebin-1 ointment-treated wounds of diabetic rats on day 4 and 7. Besides, arnebin-1 significantly upregulated the expressions of PCNA and VEGF in diabetic wound skins, which suggested that arnebin-1 facilitated the infiltration of proliferating endothelial cells into the wound bed and accelerated angiogenesis by upregulation of PCNA and VEGF expressions in diabetic rats. In this study, we showed the angiogenic activity of arnebin-1, but other biological functions of arnebin-1 are still unclear. The origin of VEGF in the skin is mainly from macrophages, fibroblasts and keratinocytes. Many researches have demonstrated that secretion of VEGF is evidently reduced due to damage of these cells in diabetes (Frank et al., 1995; Lerman et al., 2003). VEGF not only can promote angiogenesis, but also can increase collagen deposition, enhance granulation tissue formation and promote epithelialization (Brem et al., 2009). Besides induction of angiogenesis, our histological studies also showed that a general trend of better granulation tissue as well as re-epithelialization was


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found in the arnebin-1 ointment-treated group after 4 and 7 days of treatment. Thus, Arnebin-1 also may have a positive effect on fibroblast cells and keratinocytes. In diabetic patients, delayed wound healing is a crucial clinical problem resulting in chronic nonhealing ulcers which may necessitate limb amputation. This is a considerable health-care expense, so efficient dressings for these injuries would afford efficacious health benefits (Inpanya et al., 2012). These data provided in the present suggests that our arnebin-1 could indeed fulfill this much needed clinical application.

5. Conclusion From these overall outcomes, we demonstrated for the first time that Arnebin-1 can significantly promote angiogenesis of HUVECs in vitro and the topical application of arnebin-1 ointment has positive effect on wound healing process in type І diabetic rats. The wound healing action of arnebin-1 may probably be a function of either the individual or the additive effects of the VEGF. The exact mechanisms of arnebin-1 on endothelial cells, fibroblast cells and keratinocytes remain to be investigated.

Acknowledgments This study was supported by grants from the Joint Project of National Education Ministry and Guangdong province (Nos. 2007B090400089) and (2007A032702001). References Akkol, E.K., Koca, U., Pesin, I., Yilmazer, D., Toke,r, G., Yesilada, E., 2009. Exploring the wound healing activity of Arnebia densiflora (Nordm.) Ledeb. by in vivo models. Journal of Ethnopharmacology 124, 137–141. Bek, E.L., McMillen, M.A., Scott, P., Angus, L.D., Shaftan, G.W., 2002. The effect of diabetes on endothelin, interleukin-8 and vascular endothelial growth factormediated angiogenesis in rats. Clinical Science 103, 424s–429s. Bitto, A., Irrera, N., Minutoli, L., Calo, M., Lo Cascio, P., Caccia, P., Pizzino, G., Pallio, G., Micali, A., Vaccaro, M., Saitta, A., Squadrito, F., Altavilla, D., 2013. Relaxin improves multiple markers of wound healing and ameliorates the disturbed healing pattern of genetically diabetic mice. Clinical Science 125, 575–585. Brem, H., Kodra, A., Golinko, M.S., Entero, H., Stojadinovic, O., Wang, V.M., Sheahan, C.M., Weinberg, A.D., Woo, S.L.C., Ehrlich, H.P., Tomic-Canic, M., 2009. Mechanism of sustained release of vascular endothelial growth factor in accelerating experimental diabetic healing. Journal of Investigative Dermatology 129, 2275–2287. Brem, H., Tomic-Canic, M., 2007. Cellular and molecular basis of wound healing in diabetes. Journal of Clinical Investigation 117, 1219–1222. Chabbert-Buffet, N., LeDevehat, C., Khodabandhelou, T., Allaire, E., Gaits, P.J., Tribout, L., Abdoucheli-Baudot, N., Vayssairat, M., 2003. Evidence for associated cutaneous microangiopathy in diabetic patients with neuropathic foot ulceration. Diabetes Care 26, 960–961. Chen, X., Yang, L., Oppenheim, J.J., Zack Howard, O.M., 2002. Cellular pharmacology studies of shikonin derivatives. Phytotherapy Research 16, 199–209. Chithra, P., Sajithlal, G.B., Chandrakasan, G., 1998. Influence of Aloe vera on the glycosaminoglycans in the matrix of healing dermal wounds in rats. Journal of Ethnopharmacology 59, 179–186. Da Costa Pinto, F.A., Malucelli, B.E., 2002. Inflammatory infiltrate, VEGF and FGF-2 contents during corneal angiogenesis in STZ-diabetic rats. Angiogenesis 5, 67–74.

Frank, S., Hübner, G., Breier, G., Longaker, M.T., Greenhalgh, D.G., Werner, S., 1995. Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing. Journal of Biological Chemistry 270, 12607–12613. Galeano, M., Bitto, A., Altavilla, D., Minutoli, L., Polito, F., Calo, M., Lo Cascio, P., Stagno d'Alcontres, F., Squadrito, F., 2008. Polydeoxyribonucleotide stimulates angiogenesis and wound healing in the genetically diabetic mouse. Wound Repair and Regeneration 16, 208–217. Greenhalgh, D.G., 2003. Wound healing and diabetes mellitus. Clinics in Plastic Surgery 30, 37–45. Hsiao, C.Y., Tsai, T.H., Chak, K.F., 2012. The molecular basis of wound healing processes induced by lithospermi radix: a proteomics and biochemical analysis. Evidence-based Complementary and Alternative Medicine, 508972. Inpanya, P., Faikrua, A., Ounaroon, A., Sittichokechaiwut, A., Viyoch, J., 2012. Effects of the blended fibroin/aloe gel film on wound healing in streptozotocininduced diabetic rats. Biomedical Materials 7, 035008. Lau, T.W., Lam, F.F., Lau, K.M., Chan, Y.W., Lee, K.M., Sahota, D.S., Ho, Y.Y., Fung, K.P., Leung, P.C., Lau, C.B., 2009. Pharmacological investigation on the wound healing effects of Radix Rehmanniae in an animal model of diabetic foot ulcer. Journal of Ethnopharmacology 123, 155–162. Lee, R.J., Springer, M.L., Blanco-Bose, W.E., Shaw, R., Ursell, P.C., Blau, H.M., 2000. VEGF gene delivery to myocardium: deleterious effects of unregulated expression. Circulation 102, 898–901. Lee, Y.H., Chang, J.J., Chien, C.T., Yang, M.C., Chien, H.F., 2012. Antioxidant sol–gel improves cutaneous wound healing in streptozotocin-induced diabetic rats. Experimental Diabetes Research, 504693. Lerman, O.Z., Galiano, R.D., Armour, M., Levine, J.P., Gurtner, G.C., 2003. Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia. American Journal of Pathology 162, 303–312. Lima, M.H., Caricilli, A.M., de Abreu, L.L., Araújo, E.P., Pelegrinelli, F.F., Thirone, A.C., Tsukumo, D.M., Pessoa, A.F., dos Santos, M.F., de Moraes, M.A., Carvalheira, J.B., Velloso, L.A., Saas, M.J., 2012. Topical insulin accelerates wound healing in diabetes by enhancing the AKT and ERK pathways: a double-blind placebocontrolled clinical trial. PLoS One 7, e36974. Majtan, J., 2011. Methylglyoxal-a potential risk factor of manuka honey in healing of diabetic ulcers. Evidence-Based Complementary and Alternative Medicine, 295494. Martin, A., Komada, M.R., Sane, D.C., 2003. Abnormal angiogenesis in diabetes mellitus. Medicinal Research Reviews 23, 117–145. Papageorgiou, V.P., Assimopoulou, A.N., Ballis, A.C., 2008. Alkannins and shikonins: a new class of wound healing agents. Current Medicinal Chemistry 15, 3248–3267. Roy, H., Bhardwaj, S., Yla-Herttuala, S., 2006. Biology of vascular endothelial growth factors. FEBS Letters 580, 2879–2887. Sidhu, G.S., Singh, A.K., Banaudha, K.K., Gaddipati, J.P., Patnaik, G.K., Maheshwari, R.K., 1999. Arnebin-1 accelerates normal and hydrocortisone-induced impaired wound healing. Journal of Investigative Dermatology 113, 773–781. Stadelmann, W.K., Digenis, A.G., Tobin, G.R., 1998. Impediments to wound healing. American Journal of Surgery. 176, 39–47. Teoh, S.L., Latiff, A.A., Das, S., 2009. The effect of topical extract of momordica charantia (bitter gourd) on wound healing in nondiabetic rats and in rats with diabetes induced by streptozotocin. Clinical and Experimental Dermatology 34, 815–822. Wang, W., Lin, S., Xiao, Y., Huang, Y., Tan, Y., Cai, L., Li, X., 2008. Acceleration of diabetic wound healing with chitosan-crosslinked collagen sponge containing recombinant acidic fibroblast growth factor in healing-impaired STZ diabetic rats. Life Science 82, 190–204. Yan, X., Chen, B., Lin, Y., Li, Y.J., Xiao, Z.F., Hou, X.L., 2010. Accelration of diabetic wound healing by collagen-binding vascular endothelial growth factor in diabetic rat model. Diabetes Research and Clinical Practice 90, 66–72. Yancopoulos, G.D., Davis, S., Gale, N.W., Rudge, J.S., Wiegand, S.J., Holash, J., 2000. Vascular-specific growth factors and blood vessel formation. Nature 407, 242–248. Zandifar, E., Sohrabi Beheshti, S., Zandifar, A., Haghjooy Javanmard, S., 2012. The effect of captopril on impaired wound healing in experimental diabetes. International Journal of Endocrinology, 785247. Zhang, Z.F., Zhao, M., Wang, J.B., Ding, Y., Dai, X.Q., Li, Y., 2011. Oral administration of skin gelatin isolated from Chum Salmon (Oncorhynchus keta) enhances wound healing in diabetic rats. Marine Drugs 9, 696–711.


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