| Background:The environmental medium is the material environmental condition which is closely related to humans,the environmental factor contained in the environmental medium is the complexity of composition and the diversity of species.Appropriate content contained in the environmental factors is the material basis for the survival of human and maintenance of health.Harmful substances polluted environment are seriously damaged the health of the organisms and destroyed the ecological environment.With the evolution of the natural environment and the development of human society,global environmental problems such as “global warming”,“hollow ozone layer”,“acid rain settlement” and “brown atmosphere cloud” have become increasingly prominent.Studies show that ozone can absorb almost all of the short-wave ultraviolet light and absorb a small part of the long-wavelength ultraviolet light from the sun.Ozone-depleting substances such as N2 O,CFSC,and CH4 can deplete ozone and cause the formation of ozone holes by interacted with ozone.The depletion of ozone can weaken the absorption of UV.According to studies,the weakened region of ozone layer where the absorption of ultraviolet radiation is the region contained in the 310 to 315 nm wavelength,the region is within the wavelength range of UVB.The rapid increase of UVB reaching the surface of the earth is mainly caused by the destruction of ozone layer.Excessive exposure to UVB can increase the incidence of skin erythema,edema,photoaging and skin cancer.With the increased incidence of skin cancer,the prevention and treatment of skin cancer has caused widespread concern.The 90% of non-melanoma skin cancer patients mainly caused by ultraviolet radiation.The statistic data shows that the 1% reduction of atmospheric ozone can increase the 2% to 3% incidence of skin cancer.A large amount of ultraviolet radiation to the surface of the earth caused by the continuous expansion of the ozone layer can increase by 120,000 people per year of the global incidence of skin cancer.In the United States,there will be more than 3 million patients diagnosed as skin cancer each year.Therefore,it is very important for us to explore the mechanism of UVB damage.NF-?B signaling pathway plays an important role in both the cell proliferation and differentiation.ROS as a "second messenger" can activate NF-?B,the positive feedback regulation of NF-?B can increase cell damage and stimulate cells to produce excessive ROS and inflammatory cytokines.Keratinocytes in the human body among the layers of the epidermis are more susceptible to the influence of the endogenous and exogenous factors.Keratinocytes as the first line of defense for human skin are first damaged to UVB exposure.Studies have shown that improving the oxidative stress state of the body can improve the degree of skin cell damage mainly caused by oxidative stress.PDTC is a specific inhibitor of NF-?B and Ginkgo biloba has a strong antioxidant effect.This study investigated the role of PDTC in the UVB-induced HaCaT cell damage by inhibiting NF-?B.The combine PDTC with GBE to study the protective effect of PDTC and GBE on UVB-induced HaCaT cell injury and its mechanism.Objective:To investigate the adverse effects of UVB on HaCaT cells and relevant mechanisms underlying these effects.Methods:1.The determinination of the radiation dose and the concentration of PDTC and GBE: The determinination of the radiation dose and the concentration of PDTC and GBE were used by MTT assay detected cell survival rate.2.Cell grouping: The cells were divided into 7 groups: normal group,UVB group,UVB+PDTC group,UVB+GBE group,UVB+PDTC+GBE group,PDTC group,and GBE group;Control: No treatment;UVB: Only UVB irradiation treatment;UVB + PDTC: PDTC pretreatment was given 2 hours,then given UVB irradiation 5 minutes;UVB + GBE: Pretreated with GBE for 2 hours,then given UVB irradiation for 5 minutes;UVB+PDTC+GBE: Pretreatment with GBE and PDTC was performed for 2 hours,followed by UVB irradiation for 5 minutes.PDTC: Only PDTC treatment;GBE: Only give GBE treatment;3.Cell morphology: HE staining was used to observe the morphological changes of cells;4.Cell scratch test was uesd to detect the migration ability of cells;5.Reactive oxygen species(ROS): DCFH-DA probe was used to detect the production of ROS in HacaT cells;6.Apoptosis-associated protein: The Bax,Bcl2,Caspase8,and FADD were detected by Western blot;7.Oxidative Stress Signal Pathway-associated Proteins: Western blot was used to detect NF-?B,TLR2/4,P38 MAPK and PI3K;8.Endoplasmic reticulum stress-related protein:Western blot was used to detect Bip,CHOP,PERK,IRE1 and eIF2 a.Results:The determinination of the radiation dose and the concentration of PDTC and GBE:1.The determinination of the radiation dose: UVB dose determination: With the prolongation of UVB irradiation time,the survival rate of HaCaT cells gradually decreased.The survival rate of HaCaT cells with a UVB irradiation time of 1 min was not statistically different from that of the normal group(P>0.05).The survival rate of HaCaT cells was significantly lower in the UVB irradiation groups at 3 minutes,5 minutes,7 minutes,9 minutes,11 minutes,13 minutes,and 15 minutes than in the normal group(P<0.05).The survival rate of the cells in the 1min group was 96%,the survival rate in the 3min group was approximately 80%,and the survival rate in the 5min group was only approximately 70%.In order to ensure that the number of cells during the experiment is not too small,therefore,choose 5min as the best irradiation time.(1)Determination of PDTC concentration:The survival rate of UVA-irradiated HaCaT cells in PDTC group with different concentrations was lower than that in normal group(P<0.05).Except for normal group,the survival rate of UVA-irradiated HaCaT cells in PDTC 75?M group was higher than that in normal group.In other concentrations,the difference was statistically significant(P<0.05).The PDTC 75 ?M concentration group had a survival rate of 74.40% of the HaCaT cells irradiated with UVB.Therefore,the study determined that 75 ?M PDTC was the concentration of this experiment.(2)Determination of GBE concentration:With the increase of GBE concentration,the survival rate of HaCaT cells increased gradually.The survival rate of HaCaT cells reached a peak at 200 ?g/ml,and then the survival rate of HaCaT cells decreased gradually with the increase of GBE concentration.The survival rate of GBE 200?g/ml concentration group was higher than that of other groups,and the difference was statistically significant(P<0.05).Therefore,the concentration of 200?g/ml GBE was determined in this study.2.Changes in cell morphology: Normal cell cytoplasm nucleus was stained uniformly,cell membrane and nucleus were intact,cell outline was clear,and cell density was uniform;UVB group cells had a rupture phenomenon,cytoplasm was red stained,and few cells were visible.The phenomenon of nuclear blue dyeing,cell membrane rupture,unclear cell outlines,cytoplasmic outflow,and the density of living cells decreased;the UVB+PDTC group,UVB+GBE group,and UVB+PDTC+GBE group had clearer cell outlines and had a more cytoplasmic red staining.Evenly,some cells appeared broken and uneven staining.3.Changes in cell migration: Compared with UVB irradiation at 0,the width of cell scratches at 12 hours and 24 hours gradually decreased in all group cell scratches(P<0.05),except for UVB group.The 24-hour relative width of each group was significantly less than the 12-hour group,and the difference was statistically significant(P<0.05).In terms of the migration speed,the 12-hour period,the cell migration speed of the first 12 hours of each group of cells was significantly faster.There was a statistically significant difference in cell migration rate at the end of 12 hours(P<0.05);for the degree of healing of the scratches in each group,UVB group and normal group were compared at 0 and 12 hours at 12 and 24 hours.In comparison,the degree of healing of the cells was significantly decreased,and the difference was statistically significant(P<0.05).The degree of wound healing in the UVB+PDTC and UVB+GBE and UVB+PDTC+GBE groups was significantly higher than that in the UVB group.There was a statistically significant difference(P<0.05),and the healing process of UVB+PDTC+GBE group compared with UVB+PDTC group and UVB+GBE group was better,and the difference was statistically significant(P<0.05).4.Reactive oxygen detection: Compared with the normal group,the relative level of ROS in the UVB group was significantly increased(P<0.05),and compared with the UVB group,UVB+PDTC group,UVB+GBE group,UVB The relative levels of ROS in the +PDTC+GBE group significantly decreased,and the difference was statistically significant(P<0.05).The ROS levels in the UVB+PDTC+GBE group were also significantly lower than those in the UVB+PDTC group and the UVB+GBE group.5.Expression of apoptotic protein: The relative expression of Bax,Caspase8,FADD and Cyt c protein in UVB group was higher than that in normal group(P<0.05);the relative expression of protein Bcl 2 was lower than that of normal group.In the normal group,the difference was statistically significant(P<0.05);The expression levels of Bax,Caspase 8,FADD,and Cyt c proteins were lower in the UVB+PDTC group,the UVB+GBE group,and the UVB+PDTC+GBE group than in the UVB group.The difference was statistically significant(P<0.05).The expression of Bcl2 protein was higher than that of UVB group(P<0.05).Compared with UVB+PDTC group and UVB+GBE group,UVB+PDTC+GBE group was significantly different.The expression levels of Caspase8,FADD,and Cytc proteins decreased,and the difference was statistically significant(P<0.05).The Bax/Bcl2 ratio in the UVB group was higher than that in the normal group(P<0.05).UVB+PDTC The ratio of Bax/Bcl2 in group,UVB+GBE group and UVB+PDTC+GBE group was lower than that in UVB group(P<0.05),and the ratio of Bax/Bcl2 in UVB+PDTC+GBE group was higher than that in UVB+PDTC group.The group,UVB + GBE group was lower,the difference was statistically significant(P <0.05).6.Oxidative Stress Signal Pathway-associated Protein: Compared with the normal group,the expression of NF-?B and TLR2/4 protein in the UVB group was significantly increased(P<0.05),and compared with the UVB group.The expression of NF-?B and TLR2/4 protein in UVB+PDTC group,UVB+GBE group and UVB+PDTC+GBE group were decreased,and the difference was statistically significant(P<0.05).UVB+PDTC+GBE group The expression of NF-?B and TLR2/4 was significantly lower than that of UVB+PDTC group and UVB+GBE group(P<0.05).Compared with the normal group,the expression of P38-MAPK protein in UVB group was significantly increased.The difference was statistically significant(P<0.05).Compared with the UVB group,the expression of P38-MAPK and protein in the UVB+PDTC group,the UVB+GBE group,and the UVB+PDTC+GBE group were all decreased,and the difference was statistically significant.Significance(P<0.05),and the expression of P38-MAPK protein in UVB+PDTC+GBE group was lower than UVB+PDTC group and UVB+GBE group(P<0.05).Compared with normal group,UVB The expression level of PI3 K was significantly lower in the group(P<0.05).The expression of PI3 K in UVB+PDTC group,UVB+GBE group and UVB+PDTC+GBE group was significantly higher than that in UVB group.The level was significantly higher than the UVB group,the difference was statistically significant(P<0.05).7.Endoplasmic reticulum stress-related signal pathway proteins: Compared with the control group,the UVB group endoplasmic reticulum stress proteins Bip,CHOP,PERK,IRE1,eIF2 a,etc.were significantly higher,the difference was statistically significant(P<0.05).Compared with the UVB group,the relative expression of ER stress protein in the UVB+PDTC group,the UVB+GBE group,and the UVB+PDTC+GBE group decreased,with a statistically significant difference(P<0.05).The expression of eIF2 a in +PDTC+GBE group was significantly lower than that in UVB+PDTC group and UVB+GBE group(P<0.05).Conclusion:1.UVB can lead to oxidative stress of cells,cause cell damage and reduce the ability of cell migration;2.GBE combined with PDTC can significantly reduce intracellular ROS levels,improve cell damage and increase the ability of the cell migration.The combined use of PDTC is superior to single application;3.Combined application of GBE and PDTC can reduce UVB damage to HaCaT cells by inhibiting the activation of NF-?B signaling pathway;4.Combined application of GBE and PDTC can alleviate HaCaT cell ERS induced by UVB. |
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