Experimental Study Of Photo-protection Of Hydroxychloroqine And TCMs On Human Skin Damaged From Ultraviolet-B Irradiation

BackgroundUltraviolet irradiation (UVA and UVB) of sunlight is the most effective wavelength in causing sunburn, photoaging and skin tumor. The photo-damage effect of UVB is 800~ 1000 times stronger than that of UVA under the same dosage irradiation. UVB radiation can cause the generation of free radicals and relevant reactive oxygen species (ROS), the secretion of cytokines, the immunosuppression of topical and general immune systems, DNA damage and mutation, and contribute to photo-aging and photo-carcinogenesis. IL-1, IL-6 and TNF-a, etc, are mainly produced and secreted from keratinocytes, which play some important parts in cellular responses to UVB. It has been proved clinically that hydroxychloroqine has powerful efficacy on dermatoses related to photodamage. And traditional Chinese medicines (TCMs) have a potent antioxidant responses in mice liver and in human lymphocytes in vitro study. EGCG is the major and most effective component extracted from green tea, which has been proved to be able to improve microcirculation, antioxidation, anti-inflammation and anti-carcinogenesis. The results to investigate photodamage of ultraviolet-B irradiation on human skin cells and to evaluate photo-protective efficiency of hydroxychloroqine (HCQ) and TCMs (EGCG , szechwan lovge rhizome and baikal skullcap root) will contribute to the development and application of natural sunscreens.ObjectiveTo investigate and compare damage effects of UVB on human primary keratinocytes(KC), eternal keratinocyte -HaCaT cells and fibroblasts; and toevaluate photo-protective efficiency of hydroxychloroqine and traditional Chinese medicines (EGCG, szechwan lovge rhizome and baikal skullcap root) on above cells damaged from UVB irradiation and their mechanisms. Materials and methods1. Cell culture: primary keratinocytes(KC) and fibroblasts(FB) were isolated from human skin samples and cultured in K-SFM and DMEM with 10% fetal bovine serum separately; HaCaT cells were cultured in RMPI-1640 media with 10% fetal bovine serum; Three kinds of above cells were plated in 3.5 cm dishs and 96 wells with an equal number (104~106).2. Ultraviolet irradiation: Subconfluent KC, fibroblasts and HaCaT cells were shammed or irradiated with different dosages of UVB irradiation (30^ 60 and 90 mJ/ cm2) and treated with above TCM agents and (or) hydroxychloroqine.3. Cell viability assay: The dose- and time- dependent photodamage was observed by light microscopy; cell count and MTT assay were used to record cell proliferation and cellular activity.4. ELISA analysis: The conditioned medium from HaCaT cultures was collected and stored at -70 C; the secretion levels of IL-6 and TNF-a were detected with Human Cytokine Sandwich ELISA Kits.5. DNA flow cytometric analysis: HaCaT cells were harvested by trypsin-ethylene diamine tetraacetic acid (EDTA) release and fixed in 70% ethanol. The cell cycle and apoptotic rate were determined with a DNA flow cytometer.6. Reverse transcription-polymerase chain reaction (RT-PCR):Total RNA of HaCaT cells was extracted by trizol kits. cDNA was synthesized by reverse-transcriptas reaction. The gene fragments of p53, p21, c-fos and GADPH were amplified by PCR. The mRNA expression level of above genes was evaluated by the scanned intensity of each gene band to the correspondingband of GAPDH PCR product and normalized to the control sample.7. Statistical analysis: The experimental data were analyzed with SPSS. P values less than 0.05 were considered statistically significant. Results1. The irradiation damage of skin cells (KC, HaCaT cells, fibroblasts) was dependent on the irradiated dosages (0, 30, 60, 90mJ/cm ). The intervention of the tested drugs could protect the cellular activity partly.2. As to cytokine secretion, EGCG could decline the secretion amount of IL-6 and TNF-a apparently; hydroxychloroqine and baikal skullcap root could reduce the secretion of IL-6; szechwan lovge rhizome only reduce the secretion of IL-6 at 90mJ/cm2 level; the secretion of TNF-a...