Intervention Study Of Photoprotective Medicines In Different Pharmaceutical Preparations On UVB Photodamage And Their Effects On Relevant Regulative Molecules

BackgroundUltraviolet irradiation of sunlight including UVA and UVB is the most powerful wavelength which causes sunburn, photoaging and skin tumors. The photo-damage effect of UVB is 800~1000 times stronger than that of UVA under the same dosage irradiation. UVB irradiation can cause the generation of free radicals and relevant reactive oxygen species (ROS), the cytokine production and secretion, the local and systemic immune suppression, DNA damage and mutation, and contribute to photo-aging and photo-carcinogenesis. Keratinocytes of the skin are the important target cells of UV irradiation and the epidermis is also the first protective line to UV irradiation. UVB can cause DNA damage to form 6-4 PPs and CPDs of skin cells and then the photodamaged cells proceed removing the photoproducts which are performed mainly by the pathway of nucleotide excision repair (NER). More than 30 kinds of genes and proteins are involved in the procedures of DNA damage and repair. Some NER related proteins such as p53, PCNA,RPA play some important parts in NER.It has been clinically proved that vitamin E has a efficacy on dermatoses related to photodamage and traditional Chinese medicines(TCMs) also have potential antioxidant properties in mouse liver and in human lymphocytes in vitro study. Szechwan lovge rhizome, baikal skullcap root and EGCG(the major and most effective component of TP extracted from green tea) have been proved to be able to improve antioxidation, anti-inflammation and anti-carcinogenesis. Nano techniques are introduced into the medicine vehicle and transfer system because of its features of high efficiency of envelopment and loading, stable release and nontoxicity, etc. The experimental results and data to investigate penetrating capacity of medicines in nano-preparation, to evaluate photo-protective efficiency of vitamin E submicron emulsion(vitamin E SME) and TCMs (szechwan lovge rhizome, baikal skullcap root, EGCG and EGCG nano-preparation), and to explore the mechamisms of such photoprotection by NER related regulation proteins will contribute to the development and application of the natural sunscreens.ObjectiveTo investigate the penetrating capacity of tested medicines in nano-preparation in vitro; to observe releasing efficacy and toxicity of VitE SME in cell culture system; to study the photodamage of UVB on eternal keratinocyte -HaCaT cells and photoprotection of VitE SME; to study the production and removal of CPDs in HaCaT cells after UVB irradiation and the intervention of photoprotective medicines; to observe traditional Chinese medicines (szechwan lovge rhizome solution, baikal skullcap rootsolution, EGCG solution and EGCG nano-preparation) on HaCaT cells damaged by UVB irradiation and to clarify the probable regulation mechanisms of NER related proteins. Materials and methods1 Preparation of VitE SME and TP SLN VitE SME was prepared by high pressure homogenization. TP SLN was prepared by microemulsion technique.2 Experiment of transdermal capacity The penetrating capacity test of medicines in nano-preparation were conducted on the rabbit skin with the experimental device.3 Time-dependent releasing efficacy of VitE SME The containing amount of VitE SME in HaCaT culture system was measured by HPLC method.4 Cells culture HaCaT cells were cultured in RMPI-1640 medium with 10% fetal bovine serum and cells were plated in 3.5 cm dishs and 96-well plate with an equal number of cells (104~ 106 cells).5 Ultraviolet irradiation The time and dosage of UVB irradiation were conducted according to experiment design. Tested medicines were added into the medium before or after UVB irradiation.6 Cell viability assay The dose- and time- dependent photodamage was observed by light microscopy; MTT assay was used to record cell proliferation and cellular activity.7 Photoproducts removal assay The production and removal of CPDs in HaCaT cells after UVB irradiation were dectected by immunohistochemical method. Positive stainded cells are calculated by microscope.8 Reverse transcription-polymerase chain r...