Silymarin, a flavonoid from milk thistle (Silybum marianum L.), inhibits UV-induced oxidative stress through targeting infiltrating CD11b+ cells in mouse skin

Abstract

Phytochemicals have shown promise in inhibiting UV-induced oxidative stress, and therefore are considered as potent inhibitors of UV-induced oxidative stress-mediated skin diseases. We have shown previously that topical treatment of silymarin, a flavonoid from milk thistle (Silybum marianum), inhibits UV-induced oxidative stress in mouse skin. However, the cellular targets responsible for the inhibition of UV-induced oxidative stress by silymarin are not clearly defined. To address this issue, C3H/HeN mice were UV irradiated (90 mJ cm(-2)) with or without topical treatment with silymarin (1 mg cm(-2) skin area). Mice were killed 48 h later and skin samples collected. Flow cytometric analysis of viable dermal cells revealed that the number of infiltrating CD11b+ cells were the major source of oxidative stress (31.8%) in UV-irradiated skin compared with non-UV-exposed skin (0.4%). Treatment of silymarin inhibited UV-induced oxidative stress through inhibition of infiltrating CD11b+ cells. The analysis of myeloperoxidase also indicated that silymarin significantly (P < 0.001) decreased UV-induced infiltration of leukocytes, and this effect of silymarin was similar to that of intraperitoneal treatment of mice with monoclonal antibodies to CD11b. The inhibitory effect of silymarin, regardless of whether it is topically treated before or after UV irradiation, was of similar magnitude. Intraperitoneal administration of monoclonal antibodies to CD11b (rat IgG2b) to C3H/HeN mice inhibited UVB-induced oxidative stress generated by both epidermal and dermal cells as is evident by relative fluorescence intensity of oxidized rhodamine. Similar to the effect of anti-CD11b, silymarin also inhibited UV-induced oxidative stress in both epidermal and dermal cells. Further, CD11b+ and CD11b- cell subsets from UV-treated or silymarin+UV-treated mice were separated by immunomagnetic cell isolation technique from total epidermal and dermal single cell suspensions and analyzed for reactive oxygen species (ROS)/H2O2 production. Analytic data revealed that CD11b+ cell population from UV-irradiated skin resulted in significantly higher production of ROS in both epidermis and dermis than CD11b- cell population, and that silymarin inhibited UV-induced oxidative stress through targeting infiltrating the CD11b+ cell type in the skin.

Figures

Figure 1

Silymarin inhibits the percentage of hydrogen peroxide producing CD11b+ (CD11b+ H2O2 +) cells in the UV-exposed mouse skin compared to those mice which were not treated with silymarin but exposed to UVB. Silymarin was dissolved in 200 μl of acetone and topically applied (1mg/cm2 skin area) on the skin 30 min before UV exposure. Mice were sacrificed 48 h later and skin samples were collected. Panel A, Quantitative analysis of CD11b+ H2O2 + cell subset was performed in dermal single cell suspension using flow cytometry. Dermal single cell suspension was prepared from the dorsal skin of each mouse (4cm2/mouse) of each treatment group, and subjected to three-color flow cytometric analysis, as detailed in Materials and Methods, n=5. A representative analytical FACS histogram from three independent experiments is shown. Quadrant B and A represents respectively the cells expressing low and high expression of CD11b. Panel B, Total percent CD11b+ H2O2+ cells are presented as mean± SD from three independent experiments. *Significant difference between UVB-exposed mice versus normal (non-UV-exposed), and UVB versus silymarin +UVB-treated mice, p<0.001.

Figure 2

In vivo intraperitoneal treatment of anti-CD11b or topical treatment of silymarin (1mg/cm2 skin) inhibits UVB-induced myeloperoxidase (MPO) both in the epidermis and dermis. Mice were treated with silymarin either 30 min before UV irradiation or just after UV irradiation, sacrificed 48 h later and skin samples (4 cm2) were collected. Epidermal or dermal cytosolic fractions were prepared and used to analyze MPO activity, as detailed in Materials and Methods, n=5. MPO was determined as a marker of tissue infiltration of leukocytes. Experiments were repeated twice and resultant data are presented as means ± SD. E= epidermis; D= dermis. *Significant inhibition of MPO in anti-CD11b or silymarin treated groups versus UV+CD11b−treated isotype controls, p<0.001.

Figure 3

Topical treatment of silymarin or in vivo intraperitoneal treatment of anti-CD11b mAb inhibits UV-induced intracellular production of ROS in epidermal or dermal cells. Animals were treated as detailed in Figure 2. Quantitative analysis of UVB-induced intracellular production of ROS including H2O2 was performed in single cell suspensions from epidermis and dermis separately collected from the skin samples of five mice in each treatment group. Total ROS level was determined using dihydrorhodamine 123 as a fluorescent dye probe as described in Materials and Methods. Experiments were repeated twice and resultant data are presented as means ± SD in terms of relative fluorescence intensity of oxidized rhodamine. The mice in control group were not UV irradiated. E= epidermis; D= dermis in control group.

Figure 4

Silymarin inhibits UVB-induced ROS production in UV-exposed mouse skin through targeting the inhibition of infiltrating CD11b−positive cells. Mice were treated with silymarin either 30 min before UV irradiation or just after UV irradiation, sacrificed 48 h later and skin samples (4 cm2) were collected. Epidermal and dermal single cell suspensions were subjected to analysis of intracellular ROS production using dihydrorhodamine 123 as described in Materials and Methods. Experiments were performed twice and resultant data are presented as means ± SD in terms of relative fluorescence intensity of oxidized rhodamine, n=5. *Significantly higher amount of ROS is produced by CD11b+ cells versus CD11b− cells, p<0.001 *Significant difference of ROS level between UV and silymarin+UV-treated groups, p