Anti-aging Effects Of Piper Cambodianum Cistanche Extract On Normal Human Dermal Fibroblast Cells And A Wound-healing Model in Mice Ⅱ

Cistanche extract protected NHDF cells from the effects of UV irradiation 

Skin cells respond quickly when exposed to UV or cytokines such as interleukin (IL)-4 and IL-13.19 Upon UV exposure, various factors are stimulated, including EGFR,20,21 TNFR,22 mitogen-activated protein (MAP) kinases, ERK, and c-Jun amino-terminal kinase (JNK).23 Both JNK and p38 phosphorylate and activate the transcription factor c-Jun, which, in turn, elevates c-Fos and increases levels of transcription factor activator protein-1,24 which is required for the transcription of MMPs. These receptors are activated by the phosphorylation of MAP kinase and other downstream proteins.25 MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues.24,26 UV exposure leads to high expression of c-Jun and c-Fos heterodimers, numerous cytokines, and growth factors, which are phosphorylated by JNK and p38.27,28 Similarly, p53 protein levels were elevated, starting from 2.5 hours to 24 hours following UV-exposure.29 We demonstrated that cells exposed to UV irradiation for 24 hours expressed TNFR1 and EGFR protein levels, which decreased in a dose-dependent manner when cells were treated with Cistanche. We examined the effect of UV radiation on both the phosphorylation and activity of p38 (Thr180/Tyr182). Protein levels of c-Jun and p53 were downregulated dramatically in Cistanche-treated cells (Figures 4A and B).

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Cistanche extract protected UV-irradiated NHDF cells from ROS 

UVB light stimulates the production of ROS,30,31 and UV exposure on the skin produces ROS such as O2 ⋅−, H2 O2, and 1 O2. 32 Untreated and Cistanche-treated cells were subjected to UV irradiation for 24 hours, and the generation of ROS was measured by fluorescence-activated cell sorting (FACS). 

Figure 3 Regulation of ECM proteins in Cistanche-treated cells. Notes: (A) NHDF cells were treated with Cistanche for 24 hours. Cell lysates were analyzed by immunoblotting using anti-collagen, anti-elastin, anti-MMP-3, and anti-ERK-2 antibodies. Results are representative of three independent experiments. (B) Actin, collagen, elastin, MMP-3, and ERK-2 in the immunoprecipitates were quantified by Western blotting. Bar heights are the respective mean ± SDs of three independent experiments. *P,0.05; **P,0.01. Abbreviations: Conc, concentration; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; MMP, matrix metalloproteinase; NHDF, normal human dermal fibroblast; Cistanche, Piper cambodianum P. Fourn.; SDs, standard deviations.

Figure 4 Cistanche extract inhibition of ROS production in UV-irradiated cells. Notes: (A) NHDF cells were exposed to UV irradiation at 40 J for 180 seconds and treated with Cistanche for 24 hours. Cell lysates were analyzed by immunoblotting using anti-TNFR6, anti-c-Jun, anti-c-Fos, anti-pp38, or anti-p-JNK antibodies. Results are representative of three independent experiments. (B) Total and tyrosine-phosphorylated p38, JUK, actin, and TNFR6 in the immunoprecipitates were quantified by Western analyses. Bar heights are the mean ± SD of three independent experiments. *P,0.05; **P,0.01. Abbreviations: Conc, concentration; EGFR, epidermal growth factor receptor; JNK, c-Jun amino-terminal kinase; JNK, c-Jun N-terminal kinase; NHDF, normal human dermal fibroblast; Cistanche, Piper cambodianum P. Fourn.; ROS, reactive oxygen species; SD, standard deviation; TNFR, tumor necrosis factor receptor; UV, ultraviolet.

UV irradiation increased ROS production, while Cistanche extract decreased ROS production in NHDF cells. The ROS levels of H2 O2 were measured using the cell permeants 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2 DCFDA) (Figure 5A). UV irradiation increased the ROS levels of H2 O2 , whereas ROS levels were reduced by Cistanche extract in a dose-dependent manner (Figure 5B). These results indicated that Cistanche extract protects dermal fibroblasts from UV irradiation by reducing ROS generation.

Macroscopic observation of wound healing in mice 

Wounds were created on the backs of mice and then treated with Cistanche extract (0, 1, 10, and 50 µg/mL); the wound areas were measured each day for 4 days. On day 4, the areas surrounding the wounds in Cistanche-treated mice showed decreased redness in a dose-dependent manner compared to untreated wound areas (Figure 5). The extract-treated wound areas were smaller than the untreated wound area, showing a dose-dependent pattern. On day 4, the wound areas treated with 50 µg/mL of Cistanche were completely covered by new epithelium. No wound areas were infected (Figure 6A and B). The ratios of wound areas on days 0 and 4 were calculated (Figure 6C).

Discussion

The use of herbal materials and different plant parts has been increasing with concomitant advances in phytotherapy. Herbal materials have been used in various forms such as mono- or polyherbal drugs, dietary supplements, and dietary ingredients, and many have become well-known and safe commercial commodities.33 Various plants, including Cistanche, have been investigated for their potential pharmacological properties, but no comparative study was performed previously with Cistanche extract. This study was performed to determine the effects of Cistanche extract on wound-healing activities using incision wound models in mice. Wound healing involves the re-establishment of tissue integrity through processes associated with inflammation, proliferation, and remodeling stages.34 The skin remodeling stage is characterized by the reformulation and reformation of collagen fiber components that improve tensile strength.35 The healing process is regulated mostly by the biosynthesis and deposition of new collagen and subsequent maturation.36 In our wound study, Cistanche extract showed increased wound-healing effects compared with the control (Figures 1C and 6). Such healing might be due to the increased collagen concentration and stabilization of the fibers. In the tissue repair process, inflammatory cells enhance the migration and proliferation of endothelial cells, leading to the neovascularization of connective tissue cells that synthesize extracellular matrices, including collagen and keratinocytes, resulting in the reepithelialization of wounded tissue.37 The faster wound contraction rate may increase the gap junctional intracellular communication in fibroblasts and enhance the faster maturation of granulation tissue.38 In the incision wound model, mice treated with Cistanche extract showed faster wound healing compared with controls (Figure 6). Numerous studies have revealed that downregulation of the p38 kinase pathway protects various wild-type p53-expressing cell lines from genotoxic stress.39,40 Our data showed that p38 expression in UV-irradiated cells was downregulated by Cistanche treatment. Therefore, Cistanche extract obtained from leaves and stems demonstrated effective treatment in wound healing. Cistanche extract decreased ROS production from UV irradiation by scavenging ROS in NHDF cells (Figures 5A and B). Moreover, Cistanche extract accelerated wound healing after skin irradiation ((Figure 6A–C). These results suggested that Cistanche extract protects skin from UV irradiation damage, and might be useful as a novel raw material in cosmetic applications.

Figure 5 Cistanche-mediated inhibition of ROS production in UV-irradiated cells. Notes: (A) Cells were treated with UVB irradiation (40 J) for the indicated time and then incubated with 2′,7′-dichlorodihydrofluorescein diacetate for 30 minutes. The generation of ROS was measured by FACS. (B) Cells were exposed to UVB (40 J) for 180 seconds and treated with the indicated concentration of Cistanche for 24 hours. ROS production were estimated with the staining of 2′,7′-dichlorodihydrofluorescein diacetate and analyzed by FACS. Abbreviations: FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; NHDF, normal human dermal fibroblast; Cistanche, Piper cambodianum P. Fourn.; ROS, reactive oxygen species; UV, ultraviolet; UVB, ultraviolet B.

Figure 6 Wound healing effects on impaired mouse skin. Notes: (A) Wounds of 5-mm diameter were created on the dorsal skin of 7-week-old male C57Bl/6 mice (n=3). The recovery of wounds was evaluated with Cistanche treatment in the different concentrations (negative control and post wound treatment with Cistanche at 1, 10, and 50 µg/mL). (B) The wound area was determined by measuring the diameter of the open wounds. (C) The wound area was calculated using the dimension of the open wounds; *P,0.05; **P,0.01. Abbreviations: Cistanche, Piper cambodianum P. Fourn; Conc, concentration.

Conclusion 

This study investigated the potential anti-aging and wound-healing effects of Cistanche stem and leaf extract in NHDF cells and a mouse model of wound healing. The treatment of NHDF cells with Cistanche extracts promotes the recovery of the scratched area in a dose-dependent manner as well as the enhancement of ECM gene expression. In addition, Cistanche extracts were able to protect the cell from UV exposure by reducing ROS production. Finally, these observations are further evaluated and confirmed in the model mice for in-vivo wound healing. Therefore, Cistanche extract represents a promising new therapeutic agent for anti-aging and wound-healing treatment.

Acknowledgments: This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2014R1A1A3050752). The authors would like to thank the Korea Research Institute of Bioscience & Biotechnology (KRIBB) for providing to Cistanche extracts.

Disclosure The authors report no conflicts of interest in this work.

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