Chen et al. BMC Complementary and Alternative Medicine (2016) 16:487 DOI 10.1186/s12906-016-1473-6 RESEARCHARTICLE Open Access Anti-inflammatory and burn injury wound healing properties of the shell of Haliotis diversicolor Zhi-Cheng Chen1, Shing-Yi Sean Wu2, Wei-Yang Su1, Yuan-Chuan Lin3, Yi-Hsin Lee1, Wei-Hao Wu1, Chun-Hong Chen4,5 and Zhi-Hong Wen1,4* Abstract Background: The shell of Haliotis diversicolor, or shijueming (SJM), is a type of traditional Chinese medicine. The SJM has appeared in historical records as early as the third and fourth centuries. Historical records have revealed that SJM had mainly been used to treat eye diseases. After the Qing Dynasty (1757), records had emerged, detailing the use of SJM for treating skin injuries, particularly for treating poorly managed ulcers or traumatic wounds. Furthermore, in our anti-inflammation-screening system, SJM significantly inhibited the expression of pro- inflammatory proteins. Previous studies have yet to adopt an animal model to verify the phenomenon and described in the historical records regarding the efficacy of SJM in promoting wound healing. Besides, the mechanism of wound healing effect of SJM is also not clear. Methods: This study applied in vitro and in vivo models, tissue section analysis, and western blotting to evaluate the effect of SJM on wound healing. The RAW 264.7 cells were used in anti-inflammatory activity assay and phagocytic assay. Male Wistar rats were used to evaluate the effect of SJM on burn injury healing. A copper block (2 × 2 cm, 150 g) preheated to 165 °C in a dry bath was used to contact the skin area for 10 s, thus creating a full-thickness burn injury. The results were analyzed by hematoxylin and eosin staining, picrosirius red staining and Western blotting. Results: The results revealed that in the in vitro model, the presence of SJM decreased the inducible nitric oxide synthase (iNOS) expression and enhanced the functions of macrophages. The results of the rat burn injury model revealed that SJM decreased neutrophil infiltration, promoted wound healing, thus increasing the collagen I content and promoting the expression of transforming growth factor-beta 1 (TGF-β1) protein. We speculate that the effect and mechanism of SJM on promoting wound healing is related to macrophage activation. In the inflammation phase, SJM alleviates inflammation by inhibiting iNOS expression and removing neutrophils through phagocytosis. Furthermore, SJM induces the secretion of TGF-β1, converting collagen during the tissue remodeling phase. Conclusions: According to our review of relevant literature, this is the first study that applied an evidence-based method to verify that SJM alleviates inflammation, enhances phagocytosis, and triggers wound healing after burn injury. The study findings reveal that SJM provides a promising therapeutic option for treating burn injury. Keywords: Haliotis diversicolor, Burn injury, Wound healing, Transforming growth factor-beta * Correspondence: [email protected] 1Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan 4Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung, Taiwan Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. BMC Complementary and Alternative Medicine (2016) 16:487 Page 2 of 12 Background caused by the invasions of foreign pathogens. However, Burn injuries, particularly those at the full-thickness prolonging the inflammatory response and delaying granu- level, can develop into acute wounds that are difficult to lation will cause delayed healing. Because excessive produc- manage and heal [1]. Currently, standard treatment for tion of NO also damages normal tissue [20, 21]. Thus, the burn injuries involves using silver-containing antibiotics inhibition of inflammation is essential to wound treatment. to prevent infection [2]. Because silver ion is highly re- In our anti-inflammation-screening system, SJM signifi- active, it can bond with deoxyribonucleic acid, ribo- cantly inhibited the expression of pro-inflammatory pro- nucleic acid, chloride ions, and other negatively charged teins. Moreover, decrease in oxidative stress may lead to proteins [3]. Additionally, medical dressings are used to acceleration of the wound healing [22, 23]. A recent study maintain the hydration environment and air permeability reported that a SJM solution significantly increased the of a burn injury [4]. However, the aforementioned treat- survival rate of human lens epithelial cells that are damaged ment methods cannot be used to alleviate inflammation by hydrogen peroxide-induced oxidative stress [24]. There- or facilitate healing a burn injury. Previous studies have fore, based in the effect of protect oxidative damage and indicated that silver sulfadiazine is toxic to keratinocytes anti-inflammation potential, SJM may has potential to en- and fibroblasts, thus decreasing the healing rate of a hance wound healing. This study applied in vitro and in wound [5–8]. Consequently, scholars have suggested vivo models, tissue section analysis, and western blotting to that the use of silver-containing drugs should be avoided evaluate the effect of SJM on wound healing. when treating a wound [3]. Therefore, developing safe drugs that promote wound healing is necessary. Methods Early studies have verified that macrophages facilitate Haliotis diversicolor shell preparation healing wounds through promoting angiogenesis and colla- The shell of Haliotis diversicolor (Zheng-Der Chinese gen formation [9–12]. Laboratory rats with macrophage herbal apothecary, Taipei, Taiwan) was placed in a metal depletion exhibit reductions in wound healing rate, collagen pot and calcined (300 °C for 15 min) until its surface formation, granulation tissue, angiogenesis, and secretion of gloss was altered (Fig. 1). The shell was then cooled and transforming growth factor-beta (TGF-β1; [13–15]), indi- ground to fine powder (particle diameter < 75 μm). Be- cating that macrophages facilitate healing wounds. cause SJM is slightly soluble, the SJM powder was dis- The shell of Haliotis diversicolor,orshijueming(SJM),is solved in a culture medium, and only the supernatant of a type of traditional Chinese medicine. The exact time the medium was used in the in vitro experiment. Specif- period in which this medicine was first used is debatable, ically, the SJM powder was weighed and then loaded but this medicine has appeared in historical records as early into Dulbecco’s modified Eagle’s medium (DMEM, Invi- as the third and fourth centuries. Historical records have trogen, Grand Island, NY, USA) containing 10% fetal bo- revealed that SJM had mainly been used to treat eye dis- vine serum. The resulting medium was evenly mixed, eases. After the Qing Dynasty (1757), records had emerged, sonicated for 15 min, and centrifuged for 10 min at detailing the use of SJM for treating skin injuries, particu- 180 × g. The supernatant was acquired and loaded into a larly for treating poorly managed ulcers or traumatic new centrifuge tube and then centrifuged for 10 min at wounds. In recent years, multiple scholars have analyzed 400 × g. The resulting supernatant was filtered using the pharmaceutical property of SJM using scientific 0.22 μm membrane to obtain a nonbacterial SJM solu- methods and techniques, thereby providing systematic, tion. The solution was kept in a 4 °C refrigerator and scientized evidence supporting the use of this medicine. used as the culture medium. In this study, when 1 g of Accordingly, the medical functions of SJM can be further SJM powder was added to 10 ml of cell growth medium, explored and verified. the resulting supernatant solution was defined as having Several studies have demonstrated the use of lipopolysac- a concentration of 10 mg/ml. Regarding the animal ex- charide (LPS) challenged RAW 264.7 murine macrophage periment, the SJM powder was added to a mineral oil cells, as a well-established model for in vitro anti- (Macron, Center Valley, PA, USA) to prepare a high- inflammatory screening. Using this screening model, we concentration 600 mg/mL solution (SJM-H) and a low- have successfully illustrate the several marine-derived sub- concentration 200 mg/mL solution (SJM-L). stances can produce efficacious anti-inflammatory activity in in vitro and in vivo models [16–19]. Thus, screening for Cell culture and cell survival assay inhibitors of the pro-inflammatory mediators in marine The RAW 264.7 cells were grown in 1:1 DMEM (Invitro- natural bioactive substances is a promising avenue for drug gen, Grand Island, NY, USA) containing 10% heat- development. Inflammation is the initial phase of wound inactivated fetal bovine serum, penicillin G (100 U/ml), healing which the macrophages mainly take the role. Resi- and streptomycin (100 μg/ml). For the cell survival assay, dent macrophages are activated and generate inflammatory the cells were seeded in 96-well culture dishes (Corning, mediators such as nitric oxide (NO) to prevent infections NY, USA) at an initial density of 3000 cells/well. The SJM Chen et al. BMC Complementary and Alternative Medicine (2016) 16:487 Page 3 of 12 separately loaded into the selected wells. For every 1 ml of culture medium, 100 μl of Latex Beads-Rabbit IgG-FITC solution was loaded. The plate was cultured at 37 °C, and the culture medium was replaced after 8 h. Finally, the cultured cells were placed under a microscope to observe the conditions of the fluorescently-labeled latex beads.