The Effects Of Licoflavone On IRE1 Signaling Pathway In HaCaT Cells ERS Induced By UVB

As a serious environmental problem, the ultraviolet radiation has attracted more and more people’s attention. Long term exposure to ultraviolet light can induce or aggravate many skin diseases, such as photosensitive skin diseases, photo aging, skin cancer, systemic lupus erythematosus, etc. In which, UVB radiation is the main cause of skin damage. The research results of our research team found that the licoflavone had a better defensive effect on UVB radiation. However, the protective effect of licoflavone on ERS induced by UVB is still not clear. For this, our study use the human immortalized keratinocytes(Ha Ca T cells) as the study object, which biological characteristics is similar to normal human keratinocytes, with GRP78, CHOP, XBP1 as the detection index, to study the effects of licoflavone on IRE1 signaling pathway in keratinocytes cells ERS induced by UVB, and to lay the foundation for further research and development of external anti ultraviolet preparation with licoflavone as main ingredient.Objective:1. To determine the effective dose of ultraviolet B on the immortalized human keratinocyte cell line(Ha Ca T cells).2. To determine the safe and effective concentrations of licoflavone, that effects on Ha Ca T cells.3. To study the regulatory effect of licoflavone on the expression of endoplasmic reticulum stress marker molecules GRP78/Bip, CHOP and XBP1 in Ha Ca T cells induced by UVB, and the effects of licoflavone on IRE1 signaling pathway in Ha Ca T cells ERS induced by UVB.Methods:1. Screened the safe and effective concentrations of licoflavone on Ha Ca T cells by MTT method. Recovery of Ha Ca T cells from cryopreservation of liquid nitrogen and cultured at 37℃ in a humidified atmosphere containing 5%CO2 with the medium containing 10% fetal calf serum. When the cell density grew to about 80%, then selected the well grown cells to seed in 96 well plate. The experiment was divided into blank control group(only cell culture, no special treatment) and licoflavone experimental group(applied with the corresponding concentration of licoflavone solution in cultured cells), while set blank wells(only added medium). After the cells adherent growth, the experimental groups were treated with different concentrations of licoflavone, the normal control group and the blank wells were added to 1640 medium containing 10% fetal bovine serum. After 24 hours of cultivation, then screened the safe and effective concentrations of licoflavone on Ha Ca T cells by MTT method.2. The safe and effective dose of UVB was screened by MTT method. The cells on the 96 well plate were divided into two groups: control group(cells were not irradiated with UVB) and UVB irradiation group(cells were not irradiated with corresponding dose of UVB), while set blank wells, which had no cells but medium. After the cells adherent growth, the UVB irradiation group received different doses of UVB irradiation(10、20、30、40、50、60m J/cm2), the control group and blank wells were without special treatment. After irradiation, the medium was added to each hole and continued cultivation for 24 hours, then used MTT test to screened the safe and effective dose of UVB irradiation on Ha Ca T cells.3.The effects of licoflavone on the expression of GRP78/Bip, CHOP and XBP1 in Ha Ca T cells induced by UVB. The well grown Ha Ca T cells were selected to seed in the 6 well plate with a density of 2×105 /m L. The experiment was divided into blank control group(without any special treatment), UVB control group(only irradiated with UVB), matrix control group(irradiated by UVB, and added with the medium containing DMSO) and the licoflavone experiment group(cells were treated with5μg/m L licoflavone solution and irradiated by UVB). After the cells adherent growth, the licoflavone experiment group was added with 5μg/m L licoflavone solution, the blank control group and UVB control group were applied with fresh 1640 medium containing serum; while the matrix control group was applied with medium containing 0.02% DMSO(the concentration of DMSO was same with 5μg/m L licoflavone group). After 24 hours of cultivation, except the blank control group, the other groups were irradiated with 30 m J/cm2 UVB. After irradiation, the corresponding medium or 5μg/m L licoflavone solution was added to each group, and continued cultivation for 24 hours, then collected the cells for experiment. The expression level of GRP78/Bipm RNA, CHOPm RNA and XBP1 m RNA in Ha Ca T cells were detected by real-time quantitative PCR(q RT-PCR); while the protein expression level of GRP78/Bip, CHOP and XBP1 in Ha Ca T cells were tested by immunohistochemistry(ICC) and Western blotting(WB).Results:1. Compared with the blank control group, the cell survival rate of 5μg/m L licoflavone group was increased, and the difference was significant(P<0.05), the 1.25, 2.5μg/m L licoflavone had no significant effect on cell survival rate(P>0.05), but the cell survival rate of 10, 20, 40μg/m L licoflavone groups were significantly decreased(P<0.05). It was suggested that 1.25, 2.5μg/m L licoflavone was a relative safe concentration of licoflavone effectd on Ha Ca T cells, but had no obvious proliferation, and the 10, 20, 40μg/m L licoflavone solution showed different levels of cytotoxicity, but the 5μg/m L licoflavone had a promoting effect on cell proliferation, so we choosed the 5μg/m L licoflavone solution in this study;2. Compared with the blank control group, after the 30 m J/cm2 UVB irradiation, the cell survival rate decreased slightly, but higher than the 40, 50, 60 m J/cm2 dose group(P<0.05), there was no significant difference of the cell survival rate between10, 20 m J/cm2 dose group and the control group(P>0.05), and the cell survival rate of 40, 50, 60 m J/cm2 dose group was significantly decreased(P<0.05). It showed that 10, 20 m J/cm2 dose of UVB irradiation had no obvious photodamage effects on Ha Ca T cells, after irradiation the Ha Ca T cells with 40, 50, 60 m J/cm2 dose of UVB, the cells were severely injured and died more, so we choosed the 30 m J/cm2 UVB to irradiate the cells and to establish photodamaged model;3. The result of q RT-PCR showed that, the expression levels of GRP78/Bipm RNA(1±0.00 vs 1.314±0.077), CHOPm RNA(1±0.00 vs 1.698±0.155) and XBP1 m RNA(1±0.00 vs 1.568±0.037) in the UVB control group were upregulated after UVB irradiation for 24 hours, compared with the blank control group, and there were significant differences between the two groups(P<0.05); Compared with the UVB control group, the expression levels of GRP78/Bipm RNA(1.314±0.077 vs 0.755±0.102), CHOPm RNA(1.698±0.155 vs 1.098±0.108) and XBP1 m RNA(1.568±0.037 vs 1.425±0.017) were downregulated in the licoflavone experimental group, and the difference was significant(P<0.05); it was suggested that UVB irradiation could induce Ha Ca T cells to ERS, and the gene expression level of ERS molecular makers GRP78/Bip, CHOP and XBP1 were up-regulated, but licoflavone had a certain protective effect on UVB induced Ha Ca T cell ERS, and downregulated the gene expression level of the above molecular markers.The results of ICC and WB showed that, the expression of GRP78/Bip(256.80±19.29 vs 112.27±15.28), CHOP(206.79±24.23 vs 102.74±15.90) and XBP1(229.86±8.16 vs 171.47±12.49) proteins were significantly higher than the control group(P<0.05); compared with the UVB control group, the expression of GRP78/Bip(146.31±7.26 vs 256.80±19.29), CHOP(121.01±13.64 vs 206.79±24.23)and XBP1(197.88±11.26 vs 229.86±8.16)protein in 5μg/m L licoflavone experimental group was significantly decreased(P<0.05).Conclusion:The 5μg/m L licoflavone had obvious protective effect on ERS induced by UVB irradiation in Ha Ca T cells. The possible mechanism may be that, licoflavone could inhibit the expression of signal molecules GRP78/Bip, CHOP and XBP1, which were related to the ERS signal pathway, then alleviate the ERS, and promote cell survival.