| Background and ObjectivesSolar UV radiation, particularly its UVB component (290–320nm), is the major cause which leads to a series of photodamage including erythema, sunburn, inflammation, hyperplasia, skin aging, hyperpigmentation, actinic keratoses, immunosuppression and skin cancer.Absorption of UVB by the DNA of skin cells causes DNA photolesions, predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4)-photoproducts (6-4PPs). These photoproducts are important inducers of photodamage. One crucial cellular response occurring after formation of UV-caused photolesions is phosphorylation of p53 at various serines that increases its half-life causing its accumulation in nucleus. The activating of p53 protein results in the synthesis of its target genes that helps the cells in repairing photolesions by inducing the activities of nucleotide excision repair (NER) pathway or modulating Bcl-2/Bax levels causing apoptosis to remove seriously damaged cells. If UVB-induced photolesions are not repaired, DNA mutations representing initial events of multi-step carcinogenesis could develop.Baicalin, isolated from baical skullcap root, has been proved to show properties of antioxidation, anti-inflammation and anti-carcinogenesis. Earlier studies conducted by our research group have shown that Baicalin shows its photoprotective effects on cultured skin cells (keratinocytes and fibroblasts) from several aspects in vitro. However, whether application of Baicalin can protect skin cells from UVB-induced DNA damage in vivo and its underlying molecular mechanisms are still not investigated. Consistent with above reports, in the present study, we assessed the effects of Baicalin on UV induced DNA damage in terms of CPDs positive cells, apoptosis as well as the role of p53 molecules in these UV-caused early adaptive changes, in human skin fibroblasts and female Balb/C mouse epidermis.MicroRNAs (miRNAs) are about 22-nucleotides, short, noncoding RNAs that are thought to regulate gene expression through sequence-specific base pairing with the 3’-untranslated region (3’-UTR) of target mRNAs. In recent years, several articles have been published showing a possible link between miRNAs and cancer. Differential expression profiles of microRNAs in NIH 3T3 cells in response to UVB irradiation has also been reported. However, until now, there is no studies of mechanisms of photoprotective drugs from the aspect of miRNAs. Understanding the mechanisms that contribute to photodamge and find effect targets of photoprotective drugs will be of great value for the development of improved anti-UV strategies. In our study, we compare the profiles of miRNA expression in 3 pairs of UVB irradiated mice, baicalin treated irradiated mice and untreated mice, trying to reveal some underlying mechanisms associated with photodamage.MethodsA photo-damage model of UVB irradiation-induced human skin fibroblasts and mice epidermis were established. The immunohistochemical staining, southwestern dot blotting were used for CPDs detection; western blotting was used for P53 detection; terminal deoxynucleotidyl transferase-mediated Dutp nick end labeling (TUNEL) was used to detect apoptotic cells; histochemical staining was used to detect hydrogen peroxide. We next analyzed the miRNA expression profiles in 3 pairs of UVB irradiated mice, baicalin treated irradiated mice and untreated mice, using a mammalian miRNA microarray containing whole mice mature and precursor miRNA sequences. TargetScan and GO-analysis were employed for the prediction the functions of miRNA targets.Results1. Effect of baicalin on production of photoproducts in human skin fibroblasts induced by UVB irradiation: Pre-treatment of fibroblasts with Baicalin dose-dependently reduced the amount of UVB-generated CPDs. Compared with UVB irradiated cells, UVB-induced p53 accumulation was less pronounced in Baicalin treated cells.2. Effect of baicalin on production and removal of photoproducts induced by UVB irradiation in mice skin: Topical application of Baicalin on Balb/C mice skin significantly decreased the amount of epidermal CPDs 1, 24 and 48 hours after 180mJ/cm2 of UVB irradiation as compared to untreated mice. UVB-induced apoptosis was less pronounced in Baicalin treated mice epidermis which was accompanied by less p53 accumulation compared to that of untreated mice.3. Effect of baicalin on UVB-induced photodamage in mice skin: Topical application of baicalin, either as a pre-UVB irradiation or post-UVB irradiation, resulted in a significant decrease in UVB-mediated increases in skin erythema, edema, skin hyperplasia and infiltration of leukocytes. Further, baicalin treatments (pre and post-UVB irradiation) also resulted in a significant decrease in UVB mediated (1) generation of H2O2 and (2) formation of DNA photoproducts: cyclobutane pyrimidine dimers (CPDs).4. Effect of baicalin on UVB-induced microRNA expression changes in mice skin: 3 miRNAs were down-regulated and 3 miRNAs were up-regulated in UVB irradiated mice compared with untreated counterpart. Differentially expressed miRNAs were predicted to have some relationships with photocarcinogenesis, hypomethylation and apoptosis. 3 miRNAs were down-regulated and 1 miRNA was up-regulated in baicalin treated irradiated mice compared with UVB irradiated mice. Differentially expressed miRNAs were predicted to have some relationships with DNA repair signaling.ConclusionsThe results obtained in the present study indicated that pre-treated with Baicalin can prevent DNA photo-damage in cultured human fibroblasts and this effects were accomplished by the less p53 accumulation compared with untreated cells; baicalin treatment to mouse skin significantly inhibits the erythema, edema, hyperplasia, H2O2 and CPDs production as well as infiltration of leukocytes caused by UVB exposure; topically application of Baicalin mitigates DNA photo-damage in mice epidermis which accompanied by blocking the p53 accumulation and apoptosis in mice epidermis and these protective effects may be mediated via its antioxidant, sunscreening and DNA damage removal properties; miRNAs are potential diagnosis biomarkers and probable factors involved in the pathogenesis of photodamage. Our study of miRNAs may lead to provide a novel researching method for photodamage and photocarcinogensis. |
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