Research On Protective Effects Of 17beta-estradiol On HaCaT Cells Exposed To UVB

Background:Skin is the first physical barrier of human for defending outside micro-organisms, chemical and physical injuries, it is the sign of aging. Skin aging is classified into two categories, natural aging and photoaging. Researches showed that ultraviolet radiation was the major cause of skin photoaging. The thinning of the ozone layer rendered more UV radiation onto the ground. UV radiation caused skin aging through damaging skin cell DNA, inducing the production of ROS and cytokines and lipid peroxidization. UV radiation induces early skin aging as well as skin inflammation and immune suppressive diseases even skin cancer. How to reduce, delay skin aging, and prevent damages to skin caused by UV radiation is becoming the hot issue of skin research. Estrogen is a type of steroid hormone, which plays important role in several kinds of physiological functions, such as bone metabolsim, cardiovascular and central nerve system functions etc. In fighting skin aging, estrogen has its unique function, especially in skin nourishing and its function maintaining. As a member of estrogen, estradiol was showed to play a role in antioxidation and antiapoptosis. In the present study, HaCaT cells were first treated by estradiol and then irradiated with UVB to explore the functions of estradiol in the process of skin photoaging by testing oxidative damages and apoptosis in cells, to elucidate the effects of estradiol on HaCaT cells exposed to UVB.Objective:Cell photoaging model was established by irradiating HaCaT cells with UVB. HaCaT cells were treated with estradiol and then irradiated with UVB to explore the protective effects of estradiol on HaCaT cells and roles of estradiol in skin photoaging.Methods:HaCaT cells were used in the present study to explore the function of estradiol in the skin photoaging. HaCaT cells were culitivated in RPMI 1640 supplemented with 10 % fetal bovine serum at the 5 % atmosphere at 37℃. When the cells confluence was 90 %, the cells were randomly classified into three groups, that was, normal cell group, UVB group, and E2+UVBgroup. DMSO was added to the cells in UVB group and17β-E2 was added to the cells in E2+UVBgroup with the final concentration of 1×10-7 mol?L-1 and cultivated for 24 h more. Before irradiation, RPMI 1640 was replaced by PBS, UVB light( 15W, wavelength 306 nm) was 40 cm above the cells and irradiation time was 5 min. The cells in normal group was covered by tin foil during irradiation. After irradiation, the cells was washed by 4℃precooling PBS and cultivated in RPMI 1640, 12 h later, the cells from all groups were collected and analysed.Trypan staining was used to test the viability of cells.The collected cells were washed by PBS three times and resuspected by PBS, Fluo-3/AM Ester(final concentration 5μmol?L-1)to the cells to test the mitochondrial potential by flow cytometry with excitation wavelength 488 nm, emission wavelength 530 nm. Rh123(final concentration 5 mg?L-1)was added to the cells to test intracellular calsium concentration with the same testing condition of mitochondrial potential.1 ml precooling PBS was added to the collected cells and cooled on ice for 5 min, then the cells were lysed by supersound machine and the supernatant were collected to test the level of SOD, MDA, superoxide anion, hydroxyl radical, total protein according to the test kit procedure. The cells in different groups were collected 12 h after being irradiated by UVB and the morphology of cells were observed under electronic microscope.The data was analysed by SPSS statistics software 13.0, the variable was expresssed as x±S, the difference of data among different groups was done by variance analysis.Results:1. Trypan staining test: the results from three times trypan staining showed that the number of viable cells in normal group was significantly higher than that of other groups(P<0.05). The number of viable cells in E2+UVB group was significantly higher than that in UVB group(P<0.05).2. The detection of mitochondrial potential: the cells in normal group had the highest mitochondrial potential, and those in UVB group the lowest. The mitochondrial potential in normal cells was significantly higher than that in UVB and E2+UVBgroup(P<0.05); the mitochondrial potential in E2+UVB group cells was significantly higher than that in UVB group cells(P<0.05).3. The test of intracellular calcium concentration: the intracellular calcium concentration in normal cells was the lowest, significantly lower than that in UVB group and E2+UVBgroup(P<0.05); the intracellular calcium concentration in E2+UVB group cells was significantly than that in UVB group cells(P<0.05).4. The content detection of SOD, MDA, superoxide anion, hydroxyl radical, and total protein: the production of superoxide anion, hydroxyl radical and MDA content in normal cells were significantly lower than those in UVB and E2+UVBgroup cells(P<0.05); the activity of SOD was significantly higher(P<0.05). The production of superoxide anion, hydroxyle radical and MDA content in E2+UVBgroup cells were significantly lower than those in UVB group cells(P<0.05), the activity of SOD was higher(P<0.05).5. Under electronic microscope, the normal HaCaT was oval with big, round nucleus, the cells are oval-shaped, the nuclear was large and circular, had clear nucleolus, the nuclear type was irregular; many microvilli and lappet-shaped structure can be seen on the suface of the cells, the distribution of the nuclear chromatin were more even, many nuclear disintegration, rough endoplasmic reticulum and mitochondria can be seen in the cytoplasm. 6 h after UVB irradiational damadge, the shape of HaCaT cells started to become smaller, nuclear pyknosis showed up, some of the nuclear shape became to be extremely irregular, intranuclear heterochromatin increased and tend to condense in the fringe of the cells, the microvilli on the surface disappeared, showed the modification of initial apoptosis, there were even apoptotic bodies. Rough endoplasmic reticulum and some crack-like structures can be seen in the cell, and secondary lysosomes and mitochondria cavitation phenomenon showed in many times. 12 h after UVB irradiational damadge, the change of the cells was more appearant, cells started to be apoptotic, apoptotic bodies increased. In the17β-E2 protection group, 6 h after UVB irradiational damadge, most HaCaT cells were in good condition, the cells size were large, and nuclears were large, and the distribution of intranuclear heterochromatin was even, many sliver-shaped mitochondria can be seen in the cytoplasm, the cristae of mitochondria is clearly visible. Lysosome and crack-like structures can be seen less. 12 h after UVB irradiational damadge, the majority of cells were in a better condition, the cells size were large and oval-shaped, there were more microvilli on the surface of the cells, nuclears were large, and the distribution of intranuclear heterochromatin was even, there were less lysosome in the cytoplasm.Conclusion:1. UVB radiation can induce the oxidative stress and apoptosis in HaCaT cells.2. UVB radiation can cause the reduction of SOD level in HaCaT cells.3. 17β-E2 can protect HaCaT cells from oxidative damages caused by UVB.