Ultraviolet B (UVB) irradiation has been known to cause skin damage, which is associated with oxidative stress, DNA damage, and apoptosis. Echinacoside is a phenylethanoid glycoside isolated from
As the outmost layer of the human body, the skin could be seriously damaged when constantly exposed to chemical pollutants and environmental ultraviolet (UV) radiation. UVB radiations (280–320 nm), which are one of the most damaging of the solar UV emissions, could affect various skin structures, cause edema, erythema, hyperplasia, wrinkling, roughness, and premature aging, and even lead to diagnosed skin malignancy [
Antioxidant supplementation is an effective strategy to counteract the deleterious effects of ROS generated by superoxide radical, hydroxyl radical, and singlet oxygen after UVB irradiation and reduce the harmful effect of DNA damage by excessive exposure to UVB [
Herba Cistanches [
Chemical structure of echinacoside.
Therefore, this study was undertaken to investigate the protective effect and potential mechanism of echinacoside in UVB-mediated response using mice dorsal epidermal model
The present study was performed using an o/w cream formulation containing octadecanoic acid, liquid paraffin, and glycerol monostearate as the oil phase and triethanolamine as the main water phase. Echinacoside (Cat number A1019, 99.5%, purity; Mansite Bio-Tech. Co., Ltd., Chengdu, Sichuan, China) was solubilized in distilled water and incorporated into this system. The drug loading capacities of echinacoside in the creams were 0.5%, 1%, and 5%, respectively.
Male BALB/c mice (6–8 weeks old) were purchased from the Experimental Animal Research Center, the Fourth Military Medical University (Xi’an, China). The animals were allowed to acclimatize for 1 week and maintained in standard conditions (12 h light/dark cycle, 20.3–23.1°C and 40–50% humidity) during the experimental cycle and fed with standard laboratory food and water ad libitum. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University (protocol number 2014-0415-R).
After shaving, clean the dorsal skins with a hand razor in the tail-to-head direction without damaging the skin. The animals were divided into six groups (
UVB source used in the experiment was Philips TL 40 W/12 RS (Holland) emitting a continuous spectrum between 270 nm and 400 nm, with a peak emission at 313 nm. Vehicle cream or echinacoside cream was topically applied to the back of each mice. The dose rates of echinacoside were 0.05 mg/cm2, 0.1 mg/cm2, and 0.5 mg/cm2, respectively. After 30 min, mice were exposed to UVB in a special designed cage, and the irradiation intensity was 0.326 mW/cm2 as measured by a Sentry ST 513 UV light meter (Taiwan, China). Irradiation dose was calculated using the formula: dose (mJ/cm2) = exposure time (sec.) × intensity (mW/cm2).
Mice were exposed to UVB every other day ten times with a total energy dose of 1956 mJ/cm2 and were sacrificed after 24 h of the last UVB exposure. Histopathology examination and immunohistochemical analysis were performed by placing a part of the dorsal skin in 10% phosphate-buffered formalin. The remainder of the skin tissues was stored in liquid nitrogen.
The effect of echinacoside on UVB-induced skin edema was measured as an increase of the dorsal skin weight. After the dorsal skin was excised from the mice, a constant area (5 mm diameter) was delimited using a mold and then weighed.
Five-micrometre-thick sections were obtained from paraffin-embedded tissues for H&E staining. After deparaffinization with xylene and hydration with an alcohol series, the sections were stained with hematoxylin solution (Cat number H6927, Sigma, St. Louis, MO, USA) for 30 s, washed, stained with eosin solution (Cat number 230251, Sigma, St. Louis, MO, USA) for 1 min, washed again, dehydrated with an alcohol series, and cleared with xylene. After mounting, the tissues were observed by Olympus CXX41SF inverted light microscopy (Tokyo, Japan).
The paraffin-embedded skin sections were deparaffinized, rehydrated, and washed in phosphate-buffered saline (PBS) for CPDs and 8-OHdG detection
After the removal of the subcutaneous tissue, the skin tissue fragment was weighed accurately and homogenized with cold Tris-HCl (5 mmol/L, containing 2 mmol/L EDTA, pH 7.4) to prepare 10% (
HaCaT cells (ATCC, Rockville, MD, USA) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100
The cells were irradiated in culture plates placed under a Philips TL 40W/12 RS UVB lamp (Holland) emitting a continuous spectrum between 270 nm and 400 nm, with a peak emission at 313 nm. The emitted radiation dose was measured by a Sentry ST 513 UV light meter (Taiwan, China).
The optimum level of UVB irradiation intensity was determined by incubating the cells at a density of 1 × 105 cells/mL in 96-well plates. At a confluence of 80–90%, the cells were exposed to UVB in the range of 25–500 mJ/cm2 in 200
Cell proliferation was assessed by MTT assay. Cells were grown in 96-well plates at a density of 1 × 105 cells/mL. At a confluence of 80–90%, cells were pretreated with various concentrations (25, 50, and 100
Cell injury was measured by quantifying the amount of LDH, a cytosolic enzyme released in the supernatant of cultures by damaged cells. The conditioned media of the UVB-exposed cells were collected for LDH measurement following the supplier’s instructions (Cat number A020, Nanjing Jiancheng Bioengineering Institute, China). The absorbance was determined at a wavelength of 450 nm immediately using a Model 680 Microplate Reader (Bio-Rad, Hercules, CA, USA).
After treatment, the cells were washed twice with cold PBS and lysed with RIPA buffer. The protein concentration was determined using the BCA protein assay kit (Beyotime Institute of Biotechnology, China). The activities of SOD (Cat number A001), CAT (Cat number A007) and GSH-Px (Cat number A005), and the cellular content of MDA (Cat number A003-1) were determined following kit’s specifications (Nanjing Jiancheng Bioengineering Institute, China).
The level of intracellular ROS generation was detected using 2′, 7′-dichlorofluorescein diacetate (DCFH-DA). HaCaT cells at a concentration of 1 × 105 cells/mL were seeded in 6-well plates. After treatment, DCFH-DA (10
UV-induced CPDs were quantitated using the OxiSelect UV-induced DNA damage enzyme-linked immunosorbent assay (ELISA) kit (Cat number SAT-326, Cell Biolabs, Inc., San Diego, CA) according to the manufacturer’s instruction.
HaCaT cells were cultured in sterilized cover slips. At the end, the cells were treated with lysis buffer, and the lysates were centrifuged at 10,000 rpm for 3 min. The supernatants were collected to determine 8-OHdG levels. A standard ELISA procedure was conducted according to the manufacturer’s manual (Cat number QS440011, Beijing Gersion Bio-Technology Co., Ltd., China).
DNA fragmentation was quantitatively assayed by cytoplasmic histone-associated DNA fragmentation ELISA kit (Cat number 11585045001, Roche, Indianapolis, IN, USA) and agarose gel electrophoresis. HaCaT cells were seeded in 24-well plates and cultured as described above. The cells were collected, and cytoplasmic histone-associated DNA fragmentation was measured according to the manufacturer’s instructions.
The pattern of DNA cleavage was analyzed by agarose gel electrophoresis. DNA was extracted by DNA purification kit (Cat number C0007, Beyotime Institute of Biotechnology, Haimen, China) as described by the supplier’s instructions. Then the DNA samples mixed with loading buffer [glycerol (40%,
Apoptotic cells were measured using the Cell Death Detection kit (Cat number 11684817910, Roche, Indianapolis, IN, USA). The cells were washed twice with PBS, fixed in 2% paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 30 min. They were then incubated with TUNEL reaction buffer at 37°C for 1 h in the dark, rinsed twice with PBS, and incubated with Hoechst 33342 at 37°C for 5 min. The stained cells were visualized under a Nikon TE2000-E inverted fluorescence microscope (Nikon Instruments Inc., Lewisville, TX, USA).
Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining was used to measure percentile of apoptosis in HaCaT cells. Cells at a concentration of 1 × 105 cells/mL were seeded in 6-well plates. Finally, the cells were resuspended in 500
HaCaT cells were seeded in 90 mm dishes. After treatment, cells were washed twice with cold PBS and lysed with RIPA buffer. The lysates were centrifuged at 12,000 rpm for 5 min, and supernatants were collected for gel electrophoresis. Protein concentration of each sample was determined using a BCA protein assay kit (Cat number P0012, Beyotime Institute of Biotechnology, China). Isolated proteins were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF)/nitrocellulose membranes. The blocked membranes (in 5% nonfat dry milk resolved in PBS containing 0.01% Tween 20, PBST) were then incubated with the primary antibodies against ataxia telangiectasia- and Rad3-related protein kinase (ATR) (Cat number sc-28901), phospho-ATR(Cat number sc-109912), p53(Cat number sc-47698), phospho-p53 (Ser15) (Cat number sc-135772), protein inhibitor of activated signal transducer and activator of transcription 3 (PIAS3) (Cat number sc-46682), heterogeneous nuclear ribonucleoprotein K (hnRNP K) (Cat number sc-53620), small ubiquitin-related modifier (SUMO-1) (Cat number sc-5308), cleaved poly ADP-ribose polymerase (PARP) (Cat number sc-56197), xeroderma pigmentosum group A (XPA) (Cat number sc-28353) (1 : 500, Santa Cruz Biotechnology Inc., Heidelberg, Germany), and 8-OHdG (Cat number MOG-100P, 1 : 50, clone N45.1, JaICA, Tokyo, Japan) overnight at 4°C and reacted with HRP-conjugated secondary antibodies (Cat number sc-2789, 1 : 3000, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h. The immunoreactive bands were detected using the Bio-Rad ChemiDoc XRS imaging system and quantity One software (Bio-Rad, Hercules, CA).
Quantitative values were determined in at least three independent experiments and expressed as the means ± standard deviation (SD). A statistical comparison of different treatment groups was determined by one-way analysis of variance (ANOVA) using GraphPad Prism 5.01 (GraphPad Software, Le Jolla, CA). A
Compared to unexposed mice, UVB irradiation induced an obvious increase in skin weight (
Echinacoside improved UVB-induced skin damage in BALB/c mice. (a) Vehicle or echinacoside cream was administered topically to the back of each mouse. After 30 min, mice were exposed to UVB and exposures were made 10 consecutive days. Animals were sacrificed at 24 h after the last UVB exposure. A constant area (5 mm diameter) was weighed to measure the skin edema. (b) Dorsal skin was photographed. And five-micrometre-thick sections were obtained from paraffin-embedded tissues for H&E staining. The red arrow shows the epidermis thickness of BALB/c mice. (c) Epidermal thicknesses in each group of mice were measured and analyzed. Data are presented as means ± SD (
We employed MTT and LDH leakage assays to evaluate whether echinacoside prevented UVB-induced cytotoxicity. In MTT assay, UVB radiation at all doses, in the range of 25–500 mJ/cm2, both reduced the viability of HaCaT cells, and 300 mJ/cm2 UVB irradiation probably induced a 50% decrease in cell viability compared with that in the control group without UVB irradiation (
Echinacoside prevented UVB-induced cytotoxicity. (a) Cells were exposed to different doses of UVB (0, 25, 50, 100, 300, and 500 mJ/cm2). (b) Cells were pretreated with echinacoside (25, 50, and 100
Data in Figures
Echinacoside improved the activities of antioxidant enzymes and inhibited MDA production
Echinacoside improved the activities of antioxidant enzymes and inhibited MDA production
As an indicator of ROS production, DCFH-DA fluorescence intensity was measured by flow cytometry. Apparently, although the progressive increments in ROS levels were observed in the UVB irradiation group, a significant decrease occurred in HaCaT cells treated with echinacoside (Figure
Echinacoside suppressed intracellular ROS generation. (a) Cells were pretreated with echinacoside (25, 50, and 100
Our result showed strong and intensive staining for CPDs in mouse skin after UVB irradiation. Whereas, comparatively lower intensity of CPDs was easily detectable in the epidermis and dermis following topical treatment of echinacoside (Figure
Echinacoside decreased CPD levels in BALB/c mice and HaCaT cells exposed to UVB. (a) Paraffin-embedded sections of the mouse skin were used to immunohistological staining with CPDs. The red arrow shows the epidermis thickness of BALB/c mice, and the black arrow shows CPD-positive cell. (b) Cells were pretreated with echinacoside (25, 50, and 100
Immunohistochemical staining revealed that in the UVB group, a representative 8-OHdG was localized mainly between the epidermis and dermis. In comparison, a significant inhibition in 8-OHdG induction was observed following topical application of echinacoside (Figure
Echinacoside reduced the level of 8-OHdG in BALB/c mice and HaCaT cells exposed to UVB. (a) Paraffin-embedded sections of the mouse skin were used to immunohistological staining with 8-OHdG. The red arrow shows the epidermis thickness of BALB/c mice, and the black arrow shows 8-OHdG-positive cell. (b) Cells were pretreated with echinacoside (25, 50, and 100
UVB irradiation resulted in a marked and significant increase in histone-associated DNA fragmentation compared with the non-UVB group (
Echinacoside inhibited UVB-induced DNA fragmentation and apoptosis in HaCaT cells. (a) Cells were pretreated with echinacoside (25, 50, and 100
As illustrated, there were a large number of TUNEL-positive cells in the UVB-irradiated group compared with very few apoptotic cells in the control group. However, after pretreating with 25, 50, or 100
As shown in Figure
Echinacoside modulated the expression of ATR, p53, hnRNP K, PIAS3, PARP, and XPA in HaCaT cells. (a) Cells were pretreated with echinacoside (25, 50, and 100
Studies have revealed that the photodamaged skin involves increased epidermal thickness and alterations in connective tissue organization [
Evidences have been shown that UVB radiation produced DNA damage directly and indirectly through oxidative stress in human skin and also the induction of apoptosis as a protective mechanism relevant in limiting the survival of cells with irreparable DNA damage caused by UVB [
Oxidative stress plays a significant role in UVB-induced skin damage [
UVB is strongly absorbed by cellular DNA in the skin and results in several different types of premutagenic lesions, which could alter the structure of DNA and consequently inhibit polymerases and arrest replication. CPDs are common photochemical products with respect to photocarcinogenesis [
Phosphoinositol-3-phosphate kinase-like kinase ATR is critical to the proper function of DNA damage and can be activated in response to a variety of damaging agents, particularly to the UV irradiation damage [
As a component of the hnRNP complex, hnRNP K plays an essential role in RNA and DNA binding [
It has been reported that apoptosis could be an oxidative response which is closely associated with DNA damage, and changes in UV-induced apoptosis may have a profound impact in the induction of skin damage. PARP appears to be involved in DNA repair in response to environmental pressure. Cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis [
Additionally, XPA deficiency is known to decrease antioxidant defense and increase susceptibility to UVB-induced skin cancer [
A proposed working model related to the protective effect of echinacoside against UVB irradiation-mediated skin damage was described in Figure
A proposed working model related to potential signaling that is involved in the protective effect of echinacoside against UVB-induced skin damage.
In conclusion, the present study demonstrated that echinacoside manifested significant protective effects against UVB-induced DNA damage and apoptosis. This effect could be at least partly mediated by its antioxidation as well as suppressing the activation of ATR and the downstream genes. This finding might provide evidence supporting the beneficial effects of echinacoside in the prevention and treatment of UVB-associated skin diseases. However, studies are still needed to discriminate the crosstalk among different signaling involved in the protective effect of echinacoside, especially including the use of ATR knockout mice and specific inhibitors. And also, whether posttreatment of echinacoside would be effective for protecting the skin against UVB radiation as well as the effect of echinacoside to malignant cells or UVA/UVC-related skin injury will be identified in further investigations.
Ultraviolet B
Immortalized human keratinocyte
Hematoxylin and eosin
Cyclobutane pyrimidine dimers
8-Hydroxy-2′-deoxyguanosine
Phosphate-buffered saline
Superoxide dismutase
Catalase
Glutathione peroxidase
Malondialdehyde
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Lactate dehydrogenase
2′, 7′-dichlorofluorescein diacetate
Fluorescent terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-fluorescein nick end-labeling
Ataxia telangiectasia- and Rad3-related protein kinase
Protein inhibitor of activated signal transducer and activator of transcription 3
Heterogeneous nuclear ribonucleoprotein K
Small ubiquitin-related modifier
Poly ADP-ribose polymerase
Xeroderma pigmentosum group A
Annexin V-fluorescein isothiocyanate
Propidium iodide.
The authors declare that they have no competing financial interests.
Di Zhang, Chengtao Lu, and Zhe Yu contributed equally to this work. Di Zhang conceived and designed the experiments, performed the experiments, and wrote the original manuscript. Chengtao Lu performed the experiments and analyzed the data. Zhe Yu analyzed the data, prepared the figures, and crafted the final manuscript. Xiayin Wang and Li Yan prepared the figures. Juanli Zhang helped in the analysis of data. Hua Li collected the data, provided constructive comments, and drafted the manuscript. Jianbo Wang and Aidong Wen conceived and supervised the study. Di Zhang, Chengtao Lu, and Zhe Yu are the co-first authors of the manuscript. All authors have reviewed and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (Program no. 81302695) and Natural Science Basic Research Plan in Shaanxi Province of China (Program no. 2016JQ8035).