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Protective effect of EGCG on epidermal keratinocytes after acute UVB radiation protection mechanisms to investigate and mice after chronic UVA radiation skin photoaging

Author: ChenXiaoE
Tutor: LuoDan
School: Nanjing Medical University
Course: Clinical
Keywords: UVB HaCaT cells Apoptosis p38 mitogen-activated protein kinase EGCG TNF-α IL-1β IL-6
CLC: R285
Type: Master's thesis
Year: 2008
Downloads: 175
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Abstract

Part of EGCG on HaCaT cells of acute UVB radiation protection mechanism Background: the eyes of long-wave ultraviolet (UVA) and ultraviolet B (UVB) radiation can cause acute injury such as sunburn and chronic cumulative damage such as photoaging and skin cancer, pose a potential hazard to human health. The wave length of high energy UVB (290-320nm) a major role in forming cells in the epidermis, horny its target cells, induced skin reactions such as erythema, edema, blisters, skin pigmentation, dysplasia, photoaging and non-melanoma skin cancer, while the body through a variety of protective measures, such as apoptosis and inflammation to protect the skin to monitor and fight against the proliferation of potentially carcinogenic cells. Signaling pathways in UV-induced inflammatory response and cell malignant transformation process attracted widespread attention in recent years, which is a research focus that mitogen-activated protein kinase (MAPK) signal transduction pathway, this pathway can be activated by ultraviolet, so crucial in the regulation of UV-induced multiple cell response. MAPK is mainly composed of the following four families: ERK, JNK, p38 and ERK5/BMK1 p38-mediated inflammatory reaction and apoptosis plays an important role in the maintenance of epidermal integrity and against UV-induced tumor effect. Green tea is a popular beverage worldwide, phenols are in green tea catechins, wherein epigallocatechin gallate (EGCG) is the effective free radical scavengers and antioxidants, can prevent chemical carcinogenesis and ultraviolet induced oxidative stress. Our previous studies have confirmed that a single UVB radiation can cause acute light damage including reduced cell activity and proliferation slows down, increased apoptosis, light adduct formation increased. The cumulative UVA / UVB irradiation can even lead to DNA mutations and skin cancer caused by UVB irradiation can reduce the injury changes after adding EGCG intervention, but p38 mitogen-activated protein kinase (p38MAPK) signaling pathway role in apoptosis and inflammation due to its protection mechanism of EGCG in UVB whether this route yet to see the report in the country, so we formed human skin keratinocytes cell line HaCaT object of observation, research p38MAPK pathway in UVB radiation the role induced apoptosis and inflammatory mediators secretory activity and p38 inhibitors positive reference to the intervention of the contrast observed EGCG whether the protective effect of the p38MAPK pathway. Objective: The first part of the study UVB radiation-induced apoptosis of HaCaT cells probability secretion of cytokines TNF-α, IL-1β, IL-6 and p38MAPK and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK) expression explore the links between apoptosis, cytokine secretion p38MAPK determine p38MAPK signal transduction pathways are involved in UVB-induced apoptosis and cytokine secretion. The second part of the study of EGCG on apoptosis after UVB radiation, cytokine secretion and expression of p38MAPK, p-p38MAPK investigate the light protection mechanisms EGCG whether p38MAPK pathway. Method: use 0,20,30,40,60,70 90mJ/cm ~ 2UVB, irradiated HaCaT cells after irradiation Oh, 1h, 2h, 3h, 4h, 6h, 24 and 48h the cells were collected and the supernatant. Flow cytometry cell cycle and apoptosis; ELISA detected in the supernatant of TNF-alpha, IL-1β, IL-6 levels; Western Blot was used to detect the expression of p38MAPK and p-p38MAPK. Results: ① with the blank control group apoptosis rate (0) compared 30mJ/cm ~~ 24h 2UVB after irradiation, HaCaT cells apoptosis rate was 21.25 ± 0.33%, measured after the application of p38 inhibitor apoptosis rate reduced to 10.14 ± 0.27%, compared with a the UVB group of nearly 1/2 lower. ② 30mJ/cm ~ 2UVB irradiated HaCaT cells after irradiation, HaCaT cells secrete TNF-α gradually increased and reached the peak at 24h, after gradually declined p38 inhibitor SB203580 completely inhibited the secretion of TNF-α. UVB radiation, IL-1β, IL-6, with the incubation time, a substantial increase, SB203580 significantly inhibited IL-1β and IL-6 secretion levels. 3 different doses of UVB radiation can activate the HaCaT cells p38MAPK ways, increased levels of p-p38 phosphorylation was statistically significant compared to phototherapy group and non-illuminated group. Highest expression level the irradiation 30mJ/cm ~ 2UVB p-p38, but non-phosphorylated p38 expression levels did not like the obvious changes. The above data show that acute UVB irradiation between chronological order on the regulation of relations, namely the rapid phosphorylation of p38 p38MAPK signaling pathway involved in the regulation of subsequent cytokine the p38MAPK pathway activation, cytokine secretion and increase in apoptotic cells secretory activity and its role in apoptosis mediated by inflammatory mediators. In addition the experimental results show that p38 inhibitors do not inhibit UVB induced p38 phosphorylation. (4) application of EGCG apoptosis rate reduced to 6.63 ± 7.5%, while adding that EGCG can inhibit the expression of p-p38MAPK and TNF-α, IL-1β and of IL-6 secretion. Conclusion: UVB radiation HacaT cell injury; UVB p38MAPK pathway by activating p38 phosphorylation to promote increased secretion of inflammatory cytokines induced apoptosis of HaCaT cells. The treatment inhibits p38MAPK pathway activation and inflammatory cytokine secretion activities plus EGCG intervention, reducing UVB-induced apoptosis, play EGCG to withstand UVB acute injury efficacy. Part II of EGCG on chronic UVA irradiation mice skin photoaging protective effect of background: high UV exposure crowd skin aging risk than low exposure to high population aging occurs in advance. Photoaging of the skin not only detrimental to the beauty, but also with the occurrence of skin diseases with etiology contact. In the the etiological factors Photoageing, UVA due to its long wavelength can penetrate the epidermis to reach the dermis play a role, it has important significance in the process of photoaging. Photoaging clinical characteristic changes include: wrinkles, texture deepened, dry, rough, loose skin, decreased elasticity, blotchy pigmentation and cancer. Histologically, mainly as follows: dermal connective tissue changes, namely the reduction of the collagen component and abnormal elastic fibers deposition. Collagen to make the skin having the strength and elasticity, is the main component of the skin proteins, approximately 90% of the protein in human dermis is composed mainly composed of type I collagen (80%) and a small amount of collagen type III (10%). When the skin from ultraviolet radiation damage, connective tissue reconstruction: clearance and degradation of extracellular matrix synthesis and deposition of new ingredients. Collagen as the most abundant component of the extracellular matrix play an important role in this process. The experimental group has EGCG cream protection UVB BALB / c mice skin due to acute and chronic photodamage been studied and the results are shown: EGCG intervention chronic UVB radiation-induced skin pathological changes, p53 protein expression and inflammatory cytokines change, but also promote apoptosis of damaged cells. But both at home and abroad due to chronic UVA radiation of animal skin photoaging as well as EGCG cream with or without intervention role currently little studied, so the design of the experiment, each UVA irradiation before topical EGCG cream by morphological methods observe the protective effect of EGCG on mouse skin. Objective: To study EGCG on chronic UVA radiation protective effect of BALB / c mice skin photoaging. Methods: The experimental mice were divided into six groups: control group, EGCG group, matrix group, UVA irradiation group, EGCG UVA group and matrix UVA group, EGCG cream and matrix coated 30 minutes before UVA irradiation, UVA irradiation dose Section 1 week 20min for 0.5J/min x, a week after gradually increasing 20min, until the radiation time is to stop the increase at 2h, lasting a total of 12 weeks after treatment the mice from skin common pathological examination and scanning electron microscopy. Results: Mice exposed to UV radiation for 12 weeks the histopathological significant changes in the skin, increase in dermal thickness. Treated with EGCG cream group can reduce UV-induced skin photoaging effects. Scanning electron microscopy UVA group dermis layer thickening, loosely arranged collagen distorted without rules, and the blank control group dermal collagen fibers dense. EGCG UVA the groups and UVA group compared dermal thickness and collagen fibers arranged direction were significantly improved, while the matrix UVA group Microscopically no obvious improvement. Conclusion: Topical application of EGCG cream can prevent tissue damage after UV radiation, to delay the process of photoaging.

Protective effect of EGCG on epidermal keratinocytes after acute UVB radiation protection mechanisms to investigate and mice after chronic UVA radiation skin photoaging

Abstract

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