以人類纖維母細胞模式探討紅光LED對於由UVA引起的光傷害之影響

Effects of red LED on UVA induced photodamage in Human fibroblasts

何珮芸(Pei-Yun Ho)

指導教授 ： 余幸司 ； 楊瑞成

高雄醫學大學/醫學院/生理及分子醫學研究所/碩士(2011年)

摘要

許多研究指出，紫外光A（UVA）照射下會刺激纖維母細胞產生活性氧分子（reactive oxygen species，ROS），透過MPAK pathway讓MMP-1的表現量增加，進而去分解膠原蛋白（collagen），而產生光傷害的現象。然而在另一方面，某些特定波長的光卻可以修復光所造成的皮膚傷害。傳統的光療例如雷射或是脈衝光具有一定的療效，但卻會引起患者不適。發光二極體（light-emitting diodes，LEDs）是新一代的光療設備，有效、安全、無痛、不產熱且具有鎖定特定波長的特性。臨床研究指出，波長595 nm或是630 nm的低能量的可見光（LEVL）具回春效果，也有報導指出波長400-700 nm的可見光對於細胞是有益處的。但是對於LEDs確切的生物效應還是不甚清楚。為了解LEDs是如何影響由UVA引起的光傷害效應，我們設計實驗以探討紅光LED照射前處理是否透過調控MAPK pathway來避免光傷害的產生，並進一步觀察與光傷害有關的下游蛋白質：MMP-1的表現。本研究以人類之包皮組織真皮層部分進行體外實驗，以不同波長之紅光LED先行照射細胞，而後再以暴露於UVA下（5焦耳（J）），再進行下列測試： （1）細胞活性測試； （2）細胞ROS（H2O2）生成量測試； （3）測定MMP-1之mRNA表現量；再測定 （4）MAPK pathway中p-JNK、p-P38、STAT-1及其下游MMP-1之蛋白質表現量。研究結果顯示，單獨照射UVA（5焦耳（J））後的細胞活性有顯著的下降；在ROS的生成量則有明顯增加的趨勢；在基因與蛋白質表現量的部分，MMP-1的表現也有顯著上升。而預先處理紅光LED的細胞存活率相較於未做LEDs前處理的組別明顯提高許多；同時經過紅光LED前處理再照射UVA的組別，能反轉由UVA照射所引起的負面效果。另外我們也發現在MAPK pathway部分，LED在調控 JNK kinase較 p38 kinase上有較明顯的效果。而單獨照射紅光LED的組別並不影響上述蛋白質的表現。為更確定UVA是透過降低ROS生成進而抑制JNK這條路徑影響MMP-1蛋白質的表現，我們利用加入ROS及JNK抑制劑的方式並以西方墨點法（western blot）方式偵測細胞內蛋白質之表現，再次證明藉由紅光LED保護由UVA引起的傷害是透過降低ROS生成進而抑制JNK pathway來進行。從實驗結果我們發現，紅光LED在保護由UVA引起的光傷害是透過降低ROS產生進而抑制JNK pathway。

關鍵字

紫外線 ； 發光二極體 ； 人類纖維母細胞

並列摘要

Ultraviolet A (UVA) contains wavelength of 320~400nm that can pass through the epidermis to the dermis, the location of fibroblasts. Fibroblast is the major extracellular matrix producer that secretes collagen to function as the major supporter in skin tissue. Studies have shown that reactive oxygen species (ROS) produced by fibroblasts are increased under UVA irradiation and lead to up-regulation of MMP-1 protein via MAPK pathway, which results in collagen degradation and photo damage. However, light with specific wavelength can also help skin tissue to be recovered from photo damage in skin. Traditional phototherapy uses lasers or pulsed dye laser (PDL) to reverse or repair sun-induced damages, which is effective but painful. Recently, Light-emitting diode (LED) is discovered as a new way for light therapy. Phototherapeutic LED-based systems have been shown to be an effective, safe, well-tolerated and painless treatment. Clinical studies have shown that low-energy visible light (LEVL), such as yellow LED (595nm) and red LED (630nm) have beneficial effects on aging prevention.Several studies also reported that cellular functions could be influenced by visible light (400–700 nm) irradiation. However, the exact beneficial effects of LED irradiation still remain elusive. To better understand how red LED prevent UVA-induced cell damage, we investigated the effects of LED irradiation on UVA-induced cell damage. In this study, we used fibroblasts which were isolated from humen’ foreskins and the primary fibroblasts were sub-cultured for the subsequent experiments. We used MTS to observe cell viability. ROS (H2O2) production was measured by flow cytometry. The protein levels of p-JNK, p-P38, and MMP-1 were each quantified by Western blot.
Our data showed that under UVA irradiation, mitochondria activity was declined accompanied with an increase of ROS in fibroblasts, and MMP-1 mRNA and protein expression level is up-regulated. Cells pretreated with LED and irradiated with UVA had higher cell viability compared to non-LED pretreatment group. We also demonstrated that intracellular ROS production and MMP-1 protein level were altered by UVA irradiations which were reversed by LED pretreatment. Furthermore, we observed that LED pre-treatment groups showed significant decrease in p-JNK level while it was not observed in p-P38 level under the UVA irradiation. We also exploited ROS and JNK inhibitors to show that LED protection against UVA-induced injury was mediated through ROS-induced JNK pathway. These results suggested that LED irradiation protected human dermal fibroblasts cells from UVA damage through regulation of the MAP kinase pathway.

並列關鍵字

UVA ； LED ； human fibroblast

參考文獻

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