| BackgroundSolar UVR consists of UVA (320-400 nm), UVB (280-320 nm), andUVC (200-280 nm).Exposure of human skin to solar UV radiation is amajor environmental factor that has serious adverse effects on thestructure and function of the skin. UVB irradiation may cause sunburn,immunosuppression, oxidative stress, DNA damage, increased rate ofgene mutation and the secretion of cytokines, even skin non-melanomaand melanoma cancers as well as premature aging of the skin so-calledphotoaging.Apoptosis is an active cytocidal process induced by many differentfactors and regulated by various of apoptosis genes, and it is a way of celldeath. Cell apoptosis has its own morphological and biochemical featuresto distinguish from cell necrosis. Apoptosis is a programmed processincluding the expression of many associated proteins and transcriptionfactors, activation of nuclease and fragmentation of DNA, which at lastleads to cell death. Apoptosis intimately relates to embryo formation,cleaning of aged and damaged cells, tumor development andtransformation and other pathological and physiological procedures.Tumor necrosis factor(TNF-α)is an important inflammatory cytokinesecreted by mononuclear macrophage, T lymphocyte and B lymphocyte.It is not only a powerful biologically active factor to kill tumor cells, but an important causative agent involved in pathological and physiologicalprocedures of many diseases. Combination with cell membrane receptorsof TNFR P55 and P75, TNF-αinduces apoptosis, activation of proteinkinase and NF-κB, which leads to cell death and inflammation. TNF-αplays a central action in inflammatory reaction and intimately relates tothe inflammatory reaction induced by UVB irradiation. Meanwhile, it isan inducing factor of apoptosis involved in the mechanism ofkeratinocyte apoptosis induced by UVB. And UVB can increase thesusceptibility of cells to TNF-αfactor in dose dependence.p38 MAPK is involved in numerous cellular processes, such as cellgrowth and survival, differentiation, development, cell cycle regulation,and cell death. Several DNA damage-induced signaling cascades,including ATR/Chk1, jun N-terminal kinase and p38 kinase pathways areactivated following UV radiation, leading to the activation of NER andrecovery mechanisms via transcription factors such as p53, NF-kB andAP-1. These events lead to a complex transcriptional response of the cellsresulting in regulation of DNA damage repair, cell cycle progression andapoptosis. Synthesis of many cytokines, such as tumor necrosis factor-α(TNF-α), IL-1, IL-6, IL-10, and IL-8, is induced by UVB in keratinocytesand these drive cutaneous inflammatory responses. TNF-αhas beendemonstrated to cause apoptosis in a variety of cell types and a direct,albeit minor, role in UVB-induced apoptosis of skin cells. Green tea is consumed worldwide as a beverage. In recent years,many studies have suggested that green tea possesses anti-inflammatoryand anticarcinogenic effects. We and others have demonstrated thattreatment with its major constituent, (-)-epigallocatechin-3-gallate,topically to dorsal skin or through drinking water to mice before UVBirradiation prevents UVB-induced immune suppression andphotocarcinogenesis.A lot of cytological and molecular biological experiments haveconfirmed that EGCG showed nice photo-protection effect. Nowadays,most of the sunscreen product is external preparation, and solution andcream are usually used. Liposome is a new formation. In this study, wetried to investigate not only the effect of EGCG on the apoptosis andexpression of TNF-αand p38 MAPK of skin of BALB/c mice irradiatedby acute and chronic UVB, but also the difference among these differentformations.ObjectiveThe aim of our study is to investigate the effects of UVB irradiationand the potency of (-)-epigallocatechin-3-gallate (EGCG) in differentformation on the apoptosis related TNF-αexpression and activation ofp38 MAPK signal pathway in BALB/c mouse skin.Methods:1. Animals: Female BALB/c mice of 6-8 weeks old were shaved back one day before the experiment and randomly divided into groups asfollows:Ⅰ:acute injury group (180 mJ/cm~2/single):control group, UVBgroup, UVB+EGCG acetone solution group, UVB+EGCG cream group,UVB+EGCG liposome group, UVB+acetone solution group, UVB+cream base group, UVB+liposome base group.Ⅱ: chronic injury group(30 mJ/cm~2): groups were divided just the same. There were 6 mice ineach group.2. Preparation of EGCG in different formation: EGCG was made intoacetone solution, cream and liposome at the concentration of 10mg/ml.EGCG liposome was made by Department of Chemistry and ChemicalEngineering of Southeast University.3. UVB irradiation of animals and collection of skin biopsies: Micewere pretreated with topical application of EGCG for 30 min, thenirradiated with UVB in the dosage of 180 mJ/cm~2 once and 30mJ/cm~2once a day for consecutive 30 days. The skin samples were obtained 24hours after UVB irradiation and were conserved in frozen liquid nitrogen.4. Detection of the apoptotic cells: In Situ Cell Apoptosis DetectionKit (TUNEL) was used according to the manufacturer's protocol.5. Detection of the phosphorylation of p38 MAPK: Total proteinswere extracted and p38 and pp38 proteins were measured byWestern-blotting. The protein level expressed by WB was scanned anddetermined by gel imaging system after correcting by GAPDH. 6. Detection of TNF-αcontent: The skin tissue was homogenated with0.1mol/L PBS. TNF-αin supernatant was measured by the Quantitativedetermination for mouse kit and by Enzyme Immunoassay (ELISA).Results1. Acute injury group(180mJ/cm~2/single)(1)UVB irradiation increased the number of epidermal and dermisTUNEL-positive cells by 127 fold compared to the control group;compared to UVB group, the Apoptotic Index (AI) showed no statisticaldifference in UVB+EGCG cream group, UVB+EGCG liposome group,UVB+acetone solution group, UVB+cream base group and UVB+liposome base group(P＞0.05). But in UVB+EGCG acetone solutiongroup, AI was much lower than UVB group(P＜0.05).(2)UVB exposures of mouse skin induced 1.99 fold of TNF-αexpression than control group(P＜0.05). Compared to UVB group, theexpression of TNF-αshowed no statistical difference in UVB+EGCGcream group, UVB+EGCG liposome group, UVB+acetone solutiongroup, UVB+cream base group and UVB+liposome basegroup(P＞0.05), but in UVB+EGCG acetone solution group, theexpression of TNF-αwas 2.54 fold higher than UVB group(P＜0.05).(3)There was no difference in the total amount of p38 proteinexpression(P＞0.05).UVB exposures of mouse skin induced 2.06 foldphosphorylation of p38 than control group(P＜0.05).Compared to UVB group, the expression of pp38 protein showed no statistical difference inUVB+EGCG cream group, UVB+EGCG liposome group, UVB+acetone solution group, UVB+cream base group and UVB+liposomebase group(P＞0.05), but in UVB+EGCG acetone solution group, theexpression of pp38 protein was 2.51 fold higher than UVBgroup(P＜0.05).2. Chronic injury group(30mJ/cm~2/d)(1)Compared to control group, AI was higher in other groups.Compared to UVB group, AI in UVB+EGCG acetone solution group,UVB+EGCG cream group and UVB+EGCG liposome group wasincreased 1.4, 5.60 and 5.77 fold(P＜0.05).(2)The expression of TNF-αwas higher in other groups compared tothe control group. Compared to single UVB group, the expression ofTNF-αin UVB+EGCG acetone solution group, UVB+EGCG creamgroup, UVB+EGCG liposome group was increased by 27.64%, 65.87%and 60.21%(P＜0.05).(3)There was no difference in the total amount of p38 proteinexpression(P＞0.05).The expression of pp38 protein was higher in othergroups compared to the control group. Compared to UVB group, theexpression of pp38 protein in UVB+EGCG acetone solution group,UVB+EGCG cream group and UVB+EGCG liposome groupincreased 1.04, 3.43 and 3.32 fold(P＜0.05). ConclusionsUVB irradiation can increase the rate of apoptotic cells, theexpression of TNF-αand the phosphorylation of p38 MAPK of BABL/cmouse skin. EGCG showed different potency on apoptosis and p38MAPK pathway, which indicated an inhibiting effect in acute injurygroup and the activation in chronic injury group. As to the formation,EGCG solution showed the best effect in acute injury group, and EGCGcream and liposome groups were better than solution group in chronicinjury group. |
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