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. 2014 Jun 20;9(6):e99703.
doi: 10.1371/journal.pone.0099703. eCollection 2014.

The protective effect of baicalin against UVB irradiation induced photoaging: an in vitro and in vivo study

Jia-an Zhang  1 , Zhi Yin  1 , Li-wen Ma  1 , Zhi-qiang Yin  1 , Yan-yan Hu  1 , Yang Xu  1 , Di Wu  1 , Felicia Permatasari  1 , Dan Luo  1 , Bing-rong Zhou  1
Affiliations

The protective effect of baicalin against UVB irradiation induced photoaging: an in vitro and in vivo study

Jia-an Zhang et al. PLoS One. .

Abstract

Objective: This study was aimed to evaluate the anti-photoaging effects of baicalin on Ultraviolet B (UVB)-induced photoaging in the dorsal skin of hairless mice and premature senescence in human dermal fibroblasts.

Methods: We established in vivo and in vitro photoaging models by repeated exposures to UVB irradiation. By HE staining, masson staining, immunohistostaing and real-time RT-PCR, we analyzed epidermal thickness, collagen expression and the mRNA and protein levels of type I collagen, type III collagen, interstitial collagenase (MMP-1 and MMP-3) in UVB exposed dorsal mice skin. The aging condition in human dermal fibroblasts was determined by senescence-associated β-galactosidase (SA-β-gal) staining. Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The G1 phase cell growth arrest was analyzed by flow cytometry. The senescence-related protein levels of p16INK-4a, p21WAF-1, and p53 and protein levels of phosphorylated histone H2AX were estimated by Western blotting.

Results: Topically application of baicalin treatment reduced UVB-induced epidermal thickening of mouse skin and also result in an increase in the production of collagen I and III, and a decrease in the expression of MMP-1 and MMP-3. Compared with the UVB-irradiated group, we found that the irradiated fibroblasts additionally treated with baicalin demonstrated a decrease in the expression of SA-β-gal, a increase in the cell viability, a decrease in the G1 phase cell proportion, a downregulation in the level of senescence-associated and γ-H2AX proteins. However, Baicalin had no difference in the normal fibroblasts without UVB irradiation and long-term Baicalin incubation of UVB-SIPS fibroblasts gave no effects on the cell proliferation.

Conclusions: Taken together, these results suggest that baicalin significantly antagonizes photoaging induced by UVB in vivo and in vitro, indicating the potential of baicalin application for anti-photoaging treatment.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Baicalin prevents UVB induction of increased mouse epidermal thickness.
(A) After mice were treated as described for methods, the dorsal skin was excised, sectioned, mounted onto slides, and stained with hematoxylin and eosin for measurement of epidermal thickness. Images are representative of results from 5 tissue samples. Original magnification ×40. (B) Bars represent the mean thickness (µm) of epidermis from 5 animals (40 measurements/section). Results are shown as means±SD (n = 5). The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-irradiated group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and non-treated groups of irradiated mice.
Figure 2
Figure 2. Baicalin protects against UVB irradiation-induced dermal collagen fiber loss.
(A) Mouse skin samples were fixed in 10% formalin and embedded in paraffin. Each section (6 mm) was stained with masson-trichrome for collagen fibers. Images are representative of results from 5 tissue samples. Original magnification ×40. (B) Bars represent the mean density value of the collagen fiber, which was stained in blue color, from 5 animals (10 measurements/section). Results are shown as means±SD (n = 5). The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-irradiated group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and non-treated groups of irradiated mice.
Figure 3
Figure 3. Baicalin protects against UVB irradiation-induced type I collagen fiber loss.
(A) Skin specimens of dorsal trunk were harvested for immuno-staining using primary antibodies of procollagen type I collagen. Images are representative of results from 5 tissue samples. Original magnification ×40. (B) Total RNA was extracted from biopsied skin samples of different groups. Type I procollagen mRNA was determined by real-time RT-PCR analysis. Results are shown as means±SD (n = 5). The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-irradiated group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and non-treated groups of irradiated mice.
Figure 4
Figure 4. Baicalin protects against UVB irradiation-induced type III collagen fiber loss.
(A) Skin specimens of dorsal trunk were harvested for immuno-staining using primary antibodies of procollagen type III collagen. Images are representative of results from 5 tissue samples. Original magnification ×40. (B) Total RNA was extracted from biopsied skin samples of different groups. Type III procollagen mRNA was determined by real-time RT-PCR analysis. Results are shown as means±SD (n = 5). The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-irradiated group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and non-treated groups of irradiated mice.
Figure 5
Figure 5. Baicalin protects against UVB irradiation-induced amplification of MMP-1 expression.
(A) Skin specimens of dorsal trunk were harvested for immuno-staining using primary antibodies of MMP-1. Images are representative of results from 5 tissue samples. Original magnification ×40. (B) Total RNA was extracted from biopsied skin samples of different groups. MMP-1 mRNA was determined by real-time RT-PCR analysis. Results are shown as means±SD (n = 5). The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-irradiated group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and non-treated groups of irradiated mice.
Figure 6
Figure 6. Baicalin protects against UVB irradiation-induced amplification of MMP-3 expression.
(A) Skin specimens of dorsal trunk were harvested for immuno-staining using primary antibodies of MMP-3. Images are representative of results from 5 tissue samples. Original magnification ×40. (B) Total RNA was extracted from biopsied skin samples of different groups. MMP-3 mRNA was determined by real-time RT-PCR analysis. Results are shown as means±SD (n = 5). The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-irradiated group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and non-treated groups of irradiated mice.
Figure 7
Figure 7. Baicalin protects cultured HDFs against UVB-SIPS induced impaired cell viability.
Baicalin exerted a protective effect in a concentration- dependent manner. HDFs were irradiated with UVB at a subcytotoxic dose of 10 mJ/cm2 twice a day for 5 days, and then cultured with 6.25, 12.5 and 25 µg/ml baicalin. 24 hours after last treatment, the cell viability was assayed by using a CCK-8 assay kit. Values are given as mean ± SD (n = 5). The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-SIPS group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and UVB-SIPS cells.
Figure 8
Figure 8. Baicalin protects cultured HDFs against UVB induced premature senescence, while has no protection for the replicative senescence.
Fibroblasts were stained for SA-β-gal and photographed under 100× magnifications (A) (C) The percentage of SA-β-gal positive cells is shown in Figure 8. (B) (D) Statistical analysis was carried out with the Student's t-test. The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-SIPS group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and UVB-SIPS cells.
Figure 9
Figure 9. Baicalin protects cultured HDFs against UVB-SIPS induced G1 arrest.
The percentage of cells in G1 blockage after treatment in each group. Data are expressed as the mean of at least three determinations. Statistical analysis was carried out with Student's t-test. The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-SIPS group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and UVB-SIPS cells.
Figure 10
Figure 10. Baicalin protects cultured HDFs against UVB-SIPS induced expression of senescence-related proteins.
(A) Expression of p-P53, p21WAF-1, and p16INK-4a after UVB stress treated with or no with baicalin were detected by Western blotting. (B) Band-Scan software was used to analyze each band in the gray scale. The ratio of gray scale values represent the ratio of amount of protein of interest/actin, and we calculated the relative ratio of every treatment group/control group. The results represented the mean relative ratio of triplicates. These results were corresponding to the features of senescence, as described above. Statistical analysis was carried out with Student's t-test. The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-SIPS group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and UVB-SIPS cells.
Figure 11
Figure 11. Baicalin protects cultured HDFs against UVB-SIPS induced expression of γ-H2AX proteins.
(A) Expression of γ-H2AX after UVB stress treated or untreated with baicalin were detected by Western blotting. (B) Band-Scan software was used to analyze each band in the gray scale. The ratio of gray scale values represents the ratio of amount of protein of interest/actin, and we calculated the relative ratio of every treatment group/control group. The results represented the mean relative ratio of triplicates. Statistical analysis was carried out with Student's t-test. The symbol (#) indicates a significant difference (p<0.05) between the control group and the UVB-SIPS group. Asterisks (*) indicate significant differences of p<0.05, respectively, between the baicalin-treated and UVB-SIPS cells.
Figure 12
Figure 12. Baicalin does not display any promotion on the long term proliferation of fibroblasts after UVB irradiation.
HDFs were irradiated with UVB at a subcytotoxic dose of 10/cm2 twice a day for 5 days, and then cultured with 6.25, 12.5 and 25 µg/ml baicalin. With the culture over 8 weeks, the cell viability was assayed by using a CCK-8 assay kit. Values are given as mean ± SD (n = 5).
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Grants and funding

This work was supported by grants from the China National Natural Science Foundation (81000700 and 81171518), science project from traditional Chinese medicine Bureau of Jiangsu Province (LZ11084) and Jiangsu National Natural Science Foundation (BK2012877). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.