Curative Effect and Mechanisms of Radix Arnebiae Oil on Burn Wound Healing in Rats

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Materials and reagents
• HPLC analysis of chemical components of RAO
• Animal studies
• Measurement of wound healing rate
• HE staining
• Determination of MDA and SOD
• ELISA analysis
• Western blotting
• Gene expression analysis by RT-qPCR
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

Radix arnebiae oil (RAO) is a clinically useful traditional Chinese medical formula with outstanding curative effects on burns. However, the mechanism of the effect of RAO on wound healing remains unclear. The present study investigates the molecular mechanisms of the potential curative effect of RAO on wound healing. The concentrations of the main constituents, shikonin, imperatorin, and ferulic acid in RAO detected by HPLC were 24.57, 3.15, and 0.13 mg/mL, respectively. A rat burn model was established, and macroscopic and histopathological studies were performed. RAO significantly accelerated wound closure and repair scarring, increased superoxide dismutase activities, and reduced malondialdehyde. RAO also downregulated interleukin (IL)-6, IL-1β and tumor necrosis factor-α in wound tissues and increased secretion of vascular endothelial growth factor, epidermal growth factor, and transforming growth factor (TGF)-β1. RAO increased the gene expression of TGF-β1, type I and III collagen, and increased the protein expression of TGF-β1 and phosphorylation of PI3K and Akt. In conclusion, RAO likely promotes wound healing via antioxidant and anti-inflammatory activities and increases re-epithelization. Activation of the TGF-β1/PI3K/Akt pathway may play an important role in the healing efficacy of RAO. These findings suggest that RAO could be a promising alternative local treatment for burn wound healing.

Introduction

Burns are injuries to the skin or other organic tissues [1], with high rates of morbidity and mortality, and they can cause long-term disability and have major psychological and economic impacts on patients [2], [3]. WHO estimates that, annually, there are 180 000 deaths worldwide caused by burns and the majority occur in low- and middle-income countries [1]. The final goal of burn management is wound repair and epithelization as soon as possible in order to prevent infection and abnormal scarring [4]. Burn therapy has always been a challenging medical problem, and various strategies have been used in the treatment of burn injuries, such as skin grafting, wound dressings, and antimicrobial and analgesic drugs [5]. Silver sulfadiazine and antibiotics are commonly used drugs for burns, but they have limitations of poor eschar penetration and cytotoxicity of silver [6], [7] and drug resistance, respectively [8].

In spite of the availability of synthetic drugs and innovative techniques to treat burns, none has achieved outstanding wound closure without adverse effects. However, traditional Chinese medicine has attracted growing scientific interest because of its low cost and safety [9]. It is a promising alternative treatment for burn wounds and has good efficacy and multi-pathway mechanisms of action [10]. Radix arnebiae oil (RAO) is a traditional Chinese formula with an outstanding curative effect in wound care [11]. RAO comprises Arnebia euchroma, Angelica dahurica, Sanguisorba officinalis L., Angelica sinensis, Phellodendron chinense Schneid., borneol, and sesame oil. RAO has antimicrobial and wound-healing activities [12]. A previous study of second-degree burns showed that RAO significantly reduced wound area, accelerated scab shedding, and increased re-epithelialization after 14 days, and scar-free wound healing was achieved after 21 days [11]. However, the scientific rationale and mechanism of action of RAO have not been fully clarified [13].

Physiological wound-healing includes three sequential and overlapping steps: inflammation, proliferation, and tissue remodeling, involving different skin cells, cytokines, growth factors, and regulatory molecules [14], [15]. A prolonged inflammatory and proliferative phase can delay wound healing and cause excessive scarring and skin contraction. Anti-inflammatory strategies by inhibiting the expression of IL-1β, TNF-α, and IL-6 could accelerate the wound-healing process [16]. Moreover, antioxidants can promote wound healing and protect tissues from oxidative damage. Also, the increasing expression of VEGF, EGF, and TGF can lead to the stimulation of re-epithelialization, angiogenesis, formation of granulation tissue, and collagen fiber deposition in the proliferation phase [17]. Phosphatidylinositol 3-kinase (PI3K)/Akt is a key signaling pathway for cell growth and proliferation [18] and has gained growing recognition in wound healing. Also, it has crosstalk and interactions with TGF-β [19]. In this study, an experimental burn wound rat model was used to evaluate the effects of RAO on wound healing. The underlying molecular mechanisms of action of RAO in wound healing were investigated.

Results and Discussion

HPLC was used to analyze the potential active constituents of RAO. Three main peaks were obtained indicating shikonin, imperatorin, and ferulic acid ([Fig. 1]), with respective concentrations of 24.57, 3.15, and 0.13 mg/mL. Shikonin is one of the main active components in Arnebia euchroma, which has antibacterial, anti-inflammatory, and antioxidant properties and can promote the wound-healing process [17]. Imperatorin has relatively high abundance in Angelica dahurica, and Angelica dahurica possesses antioxidant and anti-inflammatory activities and can accelerate wound healing by inducing angiogenesis [20]. Ferulic acid is an important active component in Angelica sinensis, which has antioxidant and anti-inflammatory activities, and can be used for wound healing [21], [22].

We assessed the impact of RAO on burn wound healing in rats after wounding on days 3, 6, and 12. Jingwanhong ointment (JO), which is a commercially available traditional Chinese medicine with an excellent curative effect for the treatment of burns and scalds, was used as a positive control [23]. The results showed that all burns had similar appearances on day 3 ([Fig. 2 a]). On day 6 post-burn induction, the wounds in the treated groups showed formation of brown scars that were shed gradually over the next few days. At 12 days after the burn, wound gaps in the drug-treated groups were significantly smaller than in the control group, and granulation tissue formed at the edge of the wound. RAO-treated burn wounds recovered more quickly, as seen on day 12, than those in the control group.

Wound area was measured on days 3, 6, 9, and 12 after wounding to assess wound contraction ([Fig. 2 b]). On days 3 and 6, there was no significant difference between the groups. The RAO-treated group showed marked improvement in wound healing on days 9 and 12 compared with untreated burn wounds. RAO treatment resulted in 48.81% wound closure on day 12. In contrast, only a 36.13% wound-healing rate was obtained for control group at this stage. RAO markedly reduced the wound area and promoted maturation and regeneration of burn wounds, including early scar shedding.

Rat wound skin was stained with hematoxylin and eosin (HE) for histological analysis on days 6 and 12 ([Fig. 3 a]). The normal skin tissue exhibited ordered cell structure and no infiltration of inflammatory cells, while the burn wound skin in the control group showed severe inflammatory cell infiltration compared with normal skin. On day 6 post-burn, drug-treated groups showed fewer inflammatory cells in the wound skin compared with the control group, as well as new blood vessels that were forming. On day 12 post-burn, drug-treated groups exhibited ordered cells and accelerated re-epithelialization. Compared with the control group, wounds around the new granulation exhibited infiltration with fewer inflammatory cells and more new blood vessels in the treated group, suggesting that RAO accelerated burn wound healing.

Delayed wound healing is partly due to oxidative stress, caused by an imbalance between reactive oxygen species and antioxidants [24]. Currently, oxidative stress biomarkers such as malondialdehyde (MDA) and antioxidants such as superoxide dismutase (SOD) are regarded as the most important for evaluation of oxidative stress [25]. The levels of MDA and SOD in wounds were determined ([Fig. 3 b, c]). The MDA content of burn wound skin was notably higher than in normal skin and began to show a downward trend after treatment and was stabilized for normal skin repair over time. As soon as 12 days after the burn, the drug-treated wounds showed significantly less MDA than the control group (p < 0.001) and reached normal levels. Compared with the normal group, SOD activity was significantly reduced in the control group (p < 0.001). RAO or JO promoted SOD activity and reduced MDA in wound tissue on day 12 after the burn, resulting in the inhibition of oxidative stress. The present findings are consistent with a previous study suggesting that reduced oxidative stress promoted wound healing [26].

As wounds heal, a variety of growth factors and cytokines play important roles in modulating and regulating wound closure. It is crucial to inhibit excessive and prolonged inflammation to avoid unexpected complications in skin wound repair. The proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 play an important role in regulating inflammation. IL-1β is mainly responsible for triggering local inflammation during injury and promoting release of other proinflammatory cytokines, such as TNF-α and IL-6 [27]. Antagonism of TNF-α can blunt leukocyte recruitment, enhance extracellular matrix synthesis, and accelerate wound healing [28]. IL-6 is a major regulator of the acute inflammatory response and it indirectly promotes secretion of the proliferative cytokines TGF-β and vascular endothelial growth factor (VEGF), which are crucial for collagen deposition and vascularization, respectively [29].

IL-1β, IL-6, and TNF-α concentrations in wounds showed a significant increase compared with the normal group ([Fig. 4 a]). RAO or JO treatment reduced levels of the three cytokines compared with the control group on day 12 after the burns. The levels of these cytokines in the RAO-treated group on day 12 were markedly lower than those on day 6, indicating that RAO inhibited secretion of proinflammatory cytokines during burn wound healing. Inflammatory cytokine infiltration and IL-1β, IL-6, and TNF-α levels in wounds were markedly reduced after RAO treatment. RAO inhibited inflammation and triggered re-epithelialization to accelerate wound healing.

Another essential phase of wound healing is proliferation, which includes re-epithelialization and angiogenesis, involving several growth factors [30]. Epidermal growth factor (EGF) is closely associated with skin regeneration and has a biological effect on different stages of wound healing through promoting angiogenesis and facilitating injury repair. VEGF is a highly specific and active angiogenic factor, contributing to endothelial cell proliferation and new vessel formation [31]. TGF-β1 plays a crucial role in inflammation, angiogenesis, and granulation tissue generation via the recruitment of neutrophils and activation of epithelial cells and fibroblasts [27].

The levels of these three predominant growth regulators were significantly increased after burn-wound induction compared with normal group on day 6 ([Fig. 4 b]). The concentration of EGF in the RAO-treated group increased on day 12, which showed that RAO significantly increased its production. The release of VEGF after RAO treatment notably increased on days 6 and 12 compared with the control group and showed a downward trend in 12 days. There was also an increased release of TGF-β1 after 12 days following treatment with RAO. The increased expression of the TGF-β1 gene and protein in the RAO-treated group on day 12 corresponded to the proliferation and regeneration phase ([Fig. 6]). RAO significantly enhanced the release of EGF, VEGF, and TGF-β1, contributing to wound healing by re-epithelialization and angiogenesis.

Collagen is an extracellular fibrin, and types I and III are the main extracellular matrix components responsible for wound skin healing [32]. Collagen III is first synthesized in the early phase of wound repair and is later replaced by type I collagen [33]. Collagen I is further reorganized into paralleled fibrils to form a low cellularity scar [14]. In this study, RT-qPCR was used to assess collagen gene expression in wound skin ([Fig. 5]). Gene expression of collagen was markedly lower in wounds compared with that in normal skin, and treatment with RAO or JO promoted the expression of type I and III collagen genes on day 12 after wound induction, contributing to the increase in the tensile strength. The RAO-treated group had significantly higher gene expression of collagen I than the JO-treated group had on day 12, indicating that RAO achieved better healing and scar improvement.

To assess the molecular mechanisms behind the regulatory activity of RAO, we studied the TGF-β1/PI3K/Akt pathway. According to previous studies, TGF-β1 activated the PI3K/Akt pathway, contributing to fibroblast accumulation, differentiation of myofibroblasts, and collagen synthesis, thereby forming new tissue and accelerating burn wound healing [34], [35]. Western blotting revealed an increase in the protein expression of TGF-β1 and phosphorylation of PI3K and Akt on days 6 and 12 compared with the control group ([Fig. 6 a–d]). Compared with the untreated group, the RAO-treated group showed significantly increased P-PI3K/PI3K and P-Akt/Akt, resulting in the activation of the PI3K/Akt pathway, which was likely induced by the high protein expression of TGF-β1. These findings indicated that RAO may accelerate burn repair through activation of the TGF-β1/PI3K/Akt signaling pathway.

In summary, the present study revealed that RAO was a promising therapeutic traditional Chinese formula for treatment of burn wounds. It promoted skin wound healing via anti-inflammatory activity with reduced proinflammatory cytokine release, inhibition of oxidative stress, and promotion of re-epithelialization and angiogenesis with increased secretion of growth factors, as well as upregulation of collagen gene expression. We also showed that the mechanism of action of RAO may be associated with activation of the TGF-β1/PI3K/Akt signaling pathway ([Fig. 7]).

Materials and Methods

Materials and reagents

Radix arnebiae oil (batch number: 210 114) was supplied by the Department of Pharmaceutical Preparation, General Hospital of Ningxia Medical University (Yinchuan, China). Jingwanhong ointment (JO) was supplied by Darentang–Jingwanhong Pharmaceutical Co. Ltd. ELISA kits were purchased from Elabscience Technology Co. Ltd. The murine monoclonal antibodies against PI3K, p-PI3K, Akt, p-Akt, and horseradish-peroxidase-labeled secondary antibody were obtained from Cell Signaling Technology. Anti-TGF-β1 was purchased from Abcam.

HPLC analysis of chemical components of RAO

First, 10 mL RAO was extracted with methanol (40 mL) and the methanol layer was separated and then concentrated to 2 mL in a water bath. Second, the oil part was extracted twice with 30 mL NaOH solution (0.25 mmol/L) and the combined extracted liquid was adjusted to pH 2.5 – 3.5 with hydrochloric acid, followed by extraction with trichloromethane (20 mL) three times. Then, 2 mL of methanol was used to dissolve the residue after the combined organic phases were evaporated to dryness. Finally, the two parts of the methanol solution were blended and analyzed with HPLC to quantify the content of shikonin, imperatorin, and ferulic acid. HPLC analysis was carried out on a C18 column at 35 °C and 10 µL samples were detected at 316 nm with a flow rate of 1.0 mL/min; mobile phase: methanol (A) and 0.1% phosphoric acid (B). The gradient program was set as follows: 0 – 5 min, 10 – 20% A; 5 – 8 min, 20 – 30% A; 8 – 12 min, 30 – 55% A; 12 – 15 min, 55 – 65% A; 15 – 30 min, 65 – 85% A; 30 – 35 min, 85 – 90% A.

Animal studies

A total of 64 male Sprague-Dawley rats (6 – 8-weeks-old and 180 – 220 g body weight) were supplied by the Laboratory Animal Center of Ningxia Medical University. The animals were used in accordance with the “Laboratory Animal Administration Rules”. All animal research was approved by the Laboratory Animal Ethical and Welfare Committee for Animal Experimentation on December 14, 2021 (No. IACUC-NYLAC-2020 – 151). After being raised in cages under standard conditions for 1 week, the dorsal surfaces of the rats were shaved. The rats were injected with 3% sodium pentobarbital (40 mg/kg) under abdominal anesthesia and a circular burn wound was created using a 2 cm diameter aluminum bar. The aluminum bar was applied perpendicularly to the back surface for 20 s after preheating in boiling water for 2 min (day 0), except for the normal group (n = 16). The rats were divided into three main groups (n = 16) as follows: untreated group (control), the wounds received sesame oil mixed with 10% beeswax; positive control, the wounds were treated with JO; and the RAO group, the burn wounds received topical treatment with RAO containing 10% beeswax. Each main group was divided into two subgroups (n = 8 animals per group), according to the time of death. Wound treatment started with an equal amount (0.3 g) of topical formulation twice daily on day 1. The rats were killed with lethal doses of intraperitoneal sodium pentobarbital (120 mg/kg) on days 6 and 12, which was the recommended method of killing. The wound was removed and rinsed. Some skin was fixed with 4% paraformaldehyde for HE staining and the rest was stored at − 80 °C for further analysis.

Measurement of wound healing rate

Wound closure was evaluated by taking photographs on days 1, 3, 6, and 12 after wounding. The wound area was traced on transparent tracing paper and calculated using Image J. According to the wound area at different times, the wound healing rate was calculated as follows: wound healing rate (%) = (wound area on day 0 – wound area on the indicated day)/wound area on day 0 × 100%.

HE staining

Wound tissues were collected, followed by fixation and dehydration. The tissues were embedded in paraffin. Tissue sections (5 µm thick) were stained with HE and then scanned on a Leica Aperio AT2 scanner.

Determination of MDA and SOD

The wound tissue (100 mg) was added to 0.9 mL PBS (pH 7.4) and homogenized for 10 min. After centrifugation, the MDA content of 0.2 mL tissue homogenate was measured at 532 nm using an assay kit from Nanjing Jiancheng Corp, and 30 µL tissue homogenate (1%) was detected at 550 nm using a SOD assay kit from Nanjing Jiancheng.

ELISA analysis

The burn wound tissues of each group were collected, and ELISA kits were used to measure the levels of IL-6, IL-1β, TNF-α, EGF, VEGF-α, and TGF-β1. The optical density value was detected at 450 nm by an enzyme marker.

Western blotting

Tissue total protein concentration was measured using a BCA kit after homogenizing the wound tissue. Protein samples (30 µg) were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were blocked with 5% fat-free milk in Tris-buffered saline with Tween-20 for 1.5 h. The membranes were incubated with the corresponding primary antibody and horseradish-peroxidase-labeled secondary antibody. ECL chemiluminescence was used to visualize the signals. Finally, protein band densities were detected by an Amersham ImageQuant 800 Western blot imaging station (Cytiva).

Gene expression analysis by RT-qPCR

Total RNA was collected from the frozen wound tissues of each group using TRIzol reagent. The gene primers of internal reference GAPDH are listed in [Table 1]. The RNA content of samples was measured by Nanodrop (Thermo Fisher Scientific). The reverse transcription of total RNA to cDNA was carried out using a cDNA reverse transcription kit (Takara Bio), and RT-qPCR was conducted on a QuantStudio^™ Real-Time PCR System (Applied Biosystems by Thermo Fisher Scientific). The results were based on 2^−ΔΔCT calculations.

Statistical analysis

The results were analyzed for statistical significance in SPSS software (version 26.0; IBM Corp.) by one-way analysis of variance, followed by Tukeyʼs post hoc test, and presented as means ± standard deviation.

Contributorsʼ Statement

Ting Gao and Jianhong Yang designed the study. Yuna Zhao and Yanping He performed HPLC. Ting Gao and Yu Zhao performed the animal experiments and biological tests. Qi Huang carried out data analysis. Ting Gao wrote the paper. Liming Zhang and Jing Chen guided the research and reviewed the paper. All authors have agreed to publish the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

Funding for this study was provided by the Key Research and Development Projects of Ningxia (No. 2022BEG03141), and the Natural Science Foundation of Ningxia (No. 2022AAC03579).

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Correspondence

Publication History

Received: 19 July 2022

Accepted after revision: 09 December 2022

Accepted Manuscript online:
13 December 2022

Article published online:
17 April 2023

© 2023. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 World Health Organization. Burns (06.03.2018). Accessed March 06, 2018 at: https://www.who.int/en/news-room/fact-sheets/detail/Burns
  PubMed
• 2 Nele B, Stan M, Dirk V, Eric H, Stijn B. Severe burn injury in Europe: A systematic review of the incidence, etiology, morbidity, and mortality. Critical Care 2010; 14: R188
  CrossrefPubMedSearch in Google Scholar
• 3 James S, Justin PG, Sazzadul K, Dan C, James MB, Jitender S, Jessica E, Malcolm D, Marni B, Sarvesh L. Outcomes in adult survivors of childhood burn injuries as compared with matched controls. J Burn Care Res 2016; 37: e167
  PubMedSearch in Google Scholar
• 4 Salas Campos L, Fernándes Mansilla M, Martínez de la Chica AM. Topical chemotherapy for the treatment of burns. Rev Enferm 2005; 28: 67-70
  PubMedSearch in Google Scholar
• 5 Oryan A, Alemzadeh E, Moshiri A. Burn wound healing: Present concepts, treatment strategies and future directions. J Wound Care 2017; 26: 5-19
  CrossrefPubMedSearch in Google Scholar
• 6 Aziz Z, Abu SF, Chong NJ. A systematic review of silver-containing dressings and topical silver agents (used with dressings) for burn wounds. Burns 2012; 38: 307-318
  CrossrefPubMedSearch in Google Scholar
• 7 Atiyeh BS, Costagliola M, Hayek SN, Dibo SA. Effect of silver on burn wound infection control and healing: Review of the literature. Burns 2007; 33: 139-148
  CrossrefPubMedSearch in Google Scholar
• 8 Ghavari A, Miller C, McMullin B, Ghahary A. Potential application of gaseous nitric oxide as a topical antimicrobial agent. Nitric Oxide 2006; 14: 21-29
  CrossrefPubMedSearch in Google Scholar
• 9 Calixto JB. Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz J Med Biol Res 2000; 33: 179-189
  CrossrefPubMedSearch in Google Scholar
• 10 Lu Y, Yongping Z, Lihua P. Research progress on role of Chinese medicinal materials and their topical preparations in wound healing by regulating cytokines and growth factors. Chin J Chin Mat Med 2021; 46: 5173-5184
  PubMedSearch in Google Scholar
• 11 Ting G, Yu Z, Liming Z, Jing C. Clinical observation of external application of compound oleum lithospermi oil gauze in the treatment of moderate burns cases. Tradit Chin Med 2021; 10: 581-591
  CrossrefPubMedSearch in Google Scholar
• 12 Shuhui P, Maoyuan X, Lu G, Song Z, Hongxiang C, Dawei L, Yan L, Xinpei L. Plasma activated radix arnebiae oil as innovative antimicrobial and burn wound healing agent. J Phys D Appl Phys 2019; 52: 335201
  CrossrefPubMedSearch in Google Scholar
• 13 Jiangyong S, Qiang M, Zhibin Y, Jingjing G, Yinsheng W. Effects of arnebia root oil on wound healing of rats with full-thickness skin defect and the related mechanism. Chin J Burns 2017; 33: 562-567
  PubMedSearch in Google Scholar
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