Female BALB/c mice, aged 6–8 weeks old and weighing ∼20–25 g, were obtained from the Chinese Academy of Science, Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). A total of six mice were used in each group. Mice were housed in a pathogen-free barrier facility in the Experimental Animal Center of Nanjing Medical University (Nanjing, China) and all experiment protocols were approved by The Animal Care and Use Committee of Nanjing Medical University.

Purified ginsenoside Rg1 was purchased from Sigma (St. Louis, MO, USA) and the ginsenoside Rg1 solution was prepared as follows: 3 mg ginsenoside Rg1 was dissolved in 100 μl acetone (3.0 mg, 6.5 μmol ginsenoside Rg1 in 100 μl acetone/3 cm² mouse skin). Polyclonal antibodies to mouse p53 (sc-6243) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

UVB (280–320 nm) radiation was used, according to the experiment. The UVB source (Spectronics Corp., Lincoln, NE, USA) emitted an average irradiation of 1,243 mW/cm². BALB/c mice were randomly divided into groups as follows: (i) sham-irradiated group, mice subjected to a sham UVB irradiation procedure; (ii) UVB irradiation only; (iii) ginsenoside Rg1 pretreatment plus UVB exposure; and (iv) acetone pretreatment plus UVB exposure, as the control. Prior to UVB exposure, all mice were shaved with electric clippers and were treated topically with ginsenoside Rg1 solution, acetone or nothing for 30 min, and then certain dosages of UVB (30, 60 and 120 mJ/cm²) were delivered at each exposure, respectively, for 30 consecutive days.

At the end of experiment, the treated skin of the ear and dorsal areas were obtained. For histopathological examination and immunohistochemical analysis, the ear biopsies were placed in 10% phosphate-buffered formalin, then dehydrated in ascending concentrations of ethanol, cleared in xylene and embedded in paraffin. Following conventional treatment, 4 mm-thick tissue sections were prepared for regular hematoxylin and eosin staining or immunohisto-chemical staining. For reverse transcription polymerase chain reaction (RT-PCR) detection, the dorsal tissue was preserved in freezing conditions (−70°C). Total RNA was isolated using a total RNA extraction kit (TRIzol^® reagent; Molecular Research Centre, Inc., Cincinnati, OH, USA). The quality of the RNA was confirmed by measuring the optical density (OD) 260/280 ratio.

The sections were treated with 0.01 M sodium citrate buffer, prior to incubation with primary antibodies against p53 (Santa Cruz Biotechnology, Inc.) and incubation in a moist chamber overnight at 4°C. After being washed in phosphate-buffered saline (PBS), the sections were incubated in secondary antibody, immunoglobulin (Ig)G, for 20 min and then were incubated with the streptavidin-biotin-peroxidase complex (SABC) and 3,3′-diaminobenzidine (DAB) substrate solution, respectively. Subsequently, the slides were counterstained with hematoxylin and observed under a light microscope. A positive result was shown as a light to dark brown staining/precipitate in the nuclei of the cells. The p53⁺ nuclei in the epidermis among each 200 basal cells were counted in 10 randomly selected visual fields at ×400 magnification.

The mRNA expression levels of the IFN-γ, IL-10 and TNF-α genes were detected by RT-PCR. In brief, cDNAs were synthesized from 1 μl total RNA using avian myeloblastosis virus (AMV) reverse transcriptase (Takara Co., Ltd., Shiga, Japan) and random oligo (dT) primers. PCR amplification was performed with a thermal cycler (Geneamp^® PCR System 2400, Perkin-Elmer, Norwalk, CT, USA). Thirty-five PCR cycles were run under the following conditions: DNA denaturation at 94°C for 1 min; primer annealing at 60°C for IFN-γ and 56°C for IL-10 for 1 min and at 57°C for TNF-α for 40 sec; and DNA extension at 72°C for 1 min. The housekeeping gene β-actin was amplified as an internal control from the same cDNA product in a separate reaction. The primer sequences (Shanghai BioAsia Co. Ltd, Shanghai, China) specific for the previously mentioned cytokines (IFN-γ, IL-10, TNF-α and β-actin) were as follows: IFN-γ gene (426 bp), 5′-GGCTGTTTCTGGCTG TTACTGC-3′ (upstream) and 5′-GACTCCTTTTCCGCT TCCTGA-3′ (downstream); IL-10 gene (394 bp), 5′-CAA TAACTCACCCACTTCC-3′ (upstream) and 5′-CAT GGCCTTGTAGACACCTT-3′ (downstream); TNF-α gene (212 bp), 5′-TCTCATCAGTTCTATGGCCC-3′ (upstream) and 5′-GGGAGTAGACAAGGTACAAC-3′ (downstream); and β-actin gene (222 bp), 5′-TGACCGGCTTGTATGCTATC-3′ (upstream) and 5′-CAGTGTGAGCCAGGATATAG-3′ (downstream).

All the PCR products were subjected to electrophoresis in a 2.0% agarose gel containing 0.5 mmol/l ethidium bromide and photographic images were subsequently captured. The density of each band was analyzed using a gel imaging system densitometer (Bio-Rad, Hercules, CA, USA).

The results are expressed as the mean ± standard deviation. The statistical significance of the difference between two independent groups was determined using a t-test. P<0.05 was considered to indicate a statistically significant difference.

The histological changes in the irradiated and non-irradiated control mouse skin samples were examined 30 days subsequent to the UVB-irradiation at different doses. Fig. 1 demonstrates the pathological results of the hematoxylin and eosin-stained specimens observed in the different groups of BALB/c mice. The UVB-exposed mouse skin appeared thicker compared with that of the mice in the control and ginsenoside Rg1-pretreated groups, with hyperkeratosis, acanthosis, sponge-like edematization and sunburn occurring in the epidermis. In addition, edema in the papillary layer of the dermis, telangiectasis, an intense diffuse inflammatory leukocyte infiltration (predominantly monocytes/macrophages and neutrophils) and tissue necrosis were observed in the UVB-irradiated mice. Following chronic UVB irradiation, infiltrating cells were present in the dermis in higher numbers, compared with those in the non-UVB-exposed skin of the control mice. The damage was more marked in the mice exposed to a higher dosage of UVB irradiation.

The application of ginsenoside Rg1 protected against UVB-induced damage and maintained the normal structure of the epidermis and dermis. A significant alleviation of skin swelling, slight epidermal injury and a reduced lymphocyte and polymorphonuclear cell infiltration with minimal dermal tissue destruction were observed in the groups pretreated with ginsenoside Rg1 compared with that in the UVB-irradiated mice that did not receive pretreatment. Therefore, morphological differences between the irradiation-only mouse skin samples and samples pretreated with ginsenoside Rg1 were able to be distinguished in the histopathological images.

In addition, to examine whether ginsenoside Rg1 exerted a toxic effect on the skin of BALB/c mice, one group was treated with ginsenoside Rg1 without UVB irradiation. The result demonstrated that the morphology of sham-irradiated skin treated with ginsenoside Rg1 for 1 month appeared similar to the skin of the normal control mice. Thus, it was concluded that 30 mg/ml ginsenoside Rg1 did not affect the histopathology of the mouse skin. For the group treated with acetone plus UVB irradiation, the pathological changes appeared to be similar to those in the UVB-irradiated mouse skin, which suggested that the acetone solvent possessed no marked photoprotective capacity on the UVB-irradiated mouse skin.

To examine the effects of different doses of UVB irradiation and ginsenoside Rg1 on local p53 expression in BALB/c mice, skin biopsies were performed following 30 days of consecutive specific treatments and immunohistochemical analysis was used to determine the number of p53⁺ stained cells (which were stained brown). Subsequent to UVB exposure, the majority of the p53⁺ cells appeared in the basal layer of the epidermis, while some were present around the hair follicles in the skin of irradiated mice (Fig. 2). The number of p53⁺ cells was markedly increased in comparison with that in the non-UVB-exposed skin, with a noticeable upregulation at 30 mJ/cm² and marginal increases at 60 and 120 mJ/cm². Abundant p53⁺ cells were observed in the skin of the UVB-irradiated groups; however, only a few p53⁺ cells remained in the group pretreated with ginsenoside Rg1. Significant differences between the ginsenoside Rg1-pretreated and the UVB irradiation-only groups were observed at each parallel control under 30, 60 and 120 mJ/cm² UVB irradiation (Fig. 3). With regard to the p53 protein expression induced by UVB irradiation, pretreatment with ginsenoside Rg1 resulted in marked reductions of 69.50, 23.53 and 12.93% at 30, 60 and 120 mJ/cm², respectively. However, in the acetone-pretreated mice, the number of p53⁺ nuclei was 78% of the number in the UVB irradiation group (data not shown), which further indicated that acetone did not have any photoprotective capacity under such circumstances.

To examine the effects of different doses of multiple UVB irradiation on the local expression of the IFN-γ, IL-10 and TNF-α genes, skin samples were collected following 30 consecutive days of irradiation and processed for mRNA detection by RT-PCR. There were statistically significant differences in the mRNA expression of cytokines between mice irradiated with different doses of UVB and non-irradiated control mice (Fig. 4). Compared with the sham-irradiated group, the mRNA expression of the IFN-γ cytokine was downregulated by 19.6, 36.3 and 39.6% following 30, 60 and 120 mJ/cm² UVB irradiation, respectively, and the difference was statistically significant. In addition, the levels of IL-10 mRNA in the irradiated groups demonstrated a dose-dependent induction and increased by 40.1, 71.0 and 89.4% following 30, 60 and 120 mJ/cm² UVB irradiation, respectively, and the upregulation was statistically significant at each dose of UVB. Furthermore, the expression of TNF-α mRNA in each UVB-irradiated group was upregulated by 36.4, 18.4 and 8.6% versus the sham-irradiated group, respectively. The expression level of TNF-α mRNA peaked significantly at 30 mJ/cm² and fell marginally at 60 and 120 mJ/cm².

While UVB exposure reduced IFN-γ mRNA expression and induced IL-10 and TNF-α mRNA expression in the skin of BALB/c mice (Fig. 4), pretreatment with ginsenoside Rg1 prior to UVB exposure resulted in an attenuation of the effects on the expression of the cytokines caused by 30 mJ/cm² UVB irradiation (Fig. 5). The conditioned skin samples were collected and the RT-PCR results revealed that ginsenoside Rg1 increased the UVB-reduced IFN-γ mRNA level by 19.7% and led to significant reductions in the mRNA expression levels of IL-10 and TNF-α of 25.7% and 20%, respectively. Unlike ginsenoside Rg1, acetone had no such effect on the mRNA expression of the three cytokines.