七層塔水萃物保護紫外光線誘發人類皮膚細胞死亡功效的研究

The study of the protection effects of Ocimum Gratissimum Linn herb aqueous extract on the ultraviolet rays-induced the cell death in human skin cells.

王冠偉(Guan-Wei Wang)

指導教授 ： 蕭明文

中山醫學大學/健康管理學院/應用化學系碩士班/碩士(2016年)

摘要

Introduction:

紫外線會造成曬傷的影響，也會對皮膚造成傷害而遠紫外線（UVC）為短波紫外光，波長介於100~280奈米，波長更短、更危險，在許多研究顯示UV光(包含A、B及C)照射在皮膚上會產生活性氧(ROS)，使得皮膚發炎，色素沉著，紅斑，過度增生和腫瘤發生，ROS會藉由活化蛋白激酶（MAPK）和核因子-kB（NF-kB）信號路徑使其引起病理及生理作用，因此，如果能抑制活性氧產生的抗氧化劑或試劑就可以減少紫外線引起的組織損傷和細胞凋亡。

Motivation:
中草藥七層塔(Ocimum gratissimum)裡有許多酚成份 (如類黃酮、咖啡酸)，具有抗氧化能力功用，我們猜測七層塔可能有抑制UV光照射產生的氧化性傷害的能力。在本研究裡，利用HaCaT人類表皮皮膚細胞株，來測試七層塔水萃物對於紫外光線影響人類皮膚細胞生長與移轉的保護功效。

Methods and Results:
1. 首先以不同能量UVC去照射皮膚細胞 (HaCaT)，然後加入不同劑量七層塔，隔天測MTT assay，看細胞存活率。結果顯示隨UVC能量越高 (0，40，60及80 J/m2) 細胞存活率分別為100%,32.25%,18.49%,11.49%(控制組)。在UV能量40 J/m2 時我們發現隨著七層塔劑量越高(0,50,75,100,125,150μg/mL)細胞存活率恢復增加愈多分別為32.25% , 46.77% ,57.90% ,56.85% , 54.84 , 68.00，而在UV能量60 J/m2時分別為18.49% , 28.50% , 41.25% , 41.21% , 48.21% , 56.47%，UV能量80 J/m2 時分別為11.49% , 19.07% , 29.60% , 40.00% , 40.14% , 43.04%。

2. 再以流式細胞儀去測試細胞週期，結果顯示UVC的能量(0，40，60，80 J/m2)越高時加入七層塔能使細胞恢復S期及恢復G0/G1的趨勢，而在沒加入七層塔的圖就會明顯的減少各其分佈，所以我們推估這有可能是因為細胞在照射UV後受到了傷害而走向凋亡趨勢而七層塔可能具有保護作用。

3. 在本研究中也做了wound healing傷口癒合測試，結果顯示不論是在未照射UVC的情況下或是有照射UVC的情況下皆會隨著七層塔的劑量上升而加速傷口的癒合。

Conclusion:
綜合以上的結果，我們認為在高劑量的UVC燈照射下，七層塔可能具有保護的作用。我們將繼續利用西方點墨法來探討其機制，而進一步證實七層塔水萃物的功效。

關鍵字

紫外線 ； 皮膚 ； 細胞週期 ； 細胞凋亡

並列摘要

Introduction:
UV radiation can cause damages in the form of sunburns, primarily from wavelengths of 100~280nm, with shorter wavelengths being more harmful. In many previous studies, UV light (including type A, B, and C) creates reactive oxygen species in skin cells, which lead to inflammation, discoloration and tumorigenesis. ROS affect MAPK and NF-kB signal pathways which lead to pathological changes. Therefore, reducing the amount of ROS can reduce the damage caused by UV light.

Objectives:
Ocimum gratissimum contains plant polyphenones such as isoflavones and caffeic acid, which have antioxidant effects. We hypothesize that Ocimum gratissimum can inhibit the oxidative damage on skin cells. In this study, HaCaT skin cells are used to test the protective effects of Ocimum gratissimum on cell proliferation and growth after exposure to UV radiation.

Methods and Results:
After treated with different dose (0，40，60及80 J/m2) of UVC in HaCaT cells, the results showed that the survival rate was 100%,32.25%,18.49%, and 11.49% respectively. When we pre-treated with different dose (0,50,75,100,125,150μg/mL) of 007 extracts, the decrease of survival rate by the 40 J/m2 UVC was restored at 32.25%, 46.77%,57.90%,56.85%, 54.84, and 68.00% respectively, by 60 J/m2 at 18.49%, 28.50%, 41.25%, 41.21%, 48.21%, and 56.47% respectively, and by 80 J/m2 at 11.49%, 19.07%, 29.60%, 40.00%, 40.14%, and 43.04% respectively. The flow cytometry analysis revealed that UVC increased the number of cells arrested at the S phase in a UVC dose dependent manner. When we pre-treated with 007 extracts, the changes were reversed. Moreover, the wound healing also showed that the 007 extracts can restore the inhibition of cell migration by UVC.

From flow cytometry analysis, we found that with increasing UV energy density, adding OGE shows a recovery of cell counts in S phase and G0/G1 phase, whereas the absence of OGE will reduce the cell counts in these phases. This suggests that OGE may have protected the cells against apoptosis after exposure to UV light.

Wound healing was also tested in this study, and the results show that regardless of exposure to UV light, OGE accelerated wound healing.

Conclusion:
Based on the above results, we conclude that at higher amount of UV exposure, OGE has a protective effect on skin cells. We will further confirm our hypothesis using western blot to probe possible mechanisms of OGE.

並列關鍵字

Ultraviolet ； Skin ； cell cycle ； Apoptosis

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