凋亡和自噬交互调控元件Bax、Bcl-2、Beclin-1在慢性紫外线损伤小鼠模型皮肤角质形成细胞中的表达

Expressions of three regulatory elements involved in the cross-talk between apoptosis and autophagy, including Bax, Bcl-2, and Beclin-1, in keratinocytes of mouse skin exposed to chronic ultraviolet radiation

目的观察慢性紫外线损伤对小鼠皮肤角质形成细胞凋亡和自噬交互调控元件Bax、Bcl-2、Beclin-1及半胱氨酸天冬氨酸蛋白酶3（caspase3）的调控效应。方法健康昆明小鼠30只，雌雄各半，鼠龄6—8周，按性别随机分成慢性紫外线损伤组（20只）和对照组（10只），每组雌雄各半，根据性别将小鼠分笼饲养。采用模拟日光紫外线（UVA＋UVB）光源对实验小鼠进行照射，从最小红斑量（UVB0．07J／cm2，UVA0．7J／cmO开始，每周增加0．5个最小红斑量，每日1次，每周照射5d，共9周，UVB总剂量达9．45J／cm2，UVA总剂量达94．5J／cm2。通过组织学观察、特殊化学染色和表皮厚度测量确定皮肤损伤，应用免疫组化法分别对照射前（w0）和实验9周末（w9）Bax、Bcl-2、Beclin-1和caspase3的表达进行检测，表达强度以免疫反应强度分布指数（IRIDI）表示，并与对照组进行比对分析。分别采用配对t检验和Wilcoxon符号秩和检验对各参数进行比较。结果慢性紫外线照射后，小鼠表皮厚度明显增加。肉眼观察、组织学观察和胶原纤维、弹性纤维特殊染色均显示损伤组小鼠皮肤临床表现和组织学改变均与慢性紫外线光损伤的人类皮肤较为吻合。在损伤组，照光前Beclin-1、caspase3、Bax、Bcl-2的表达计分均值分别为0．30、0．25、0．35、0．25，照光后分别为2．70、3．30、3．35、0．25。Beclin-1、caspase3、Bax蛋白表达显著升高，且差异有统计学意义（z值分别为4．034、4．001、3．970，均P〈0．05），Bcl-2蛋白表达变化不明显（z=0，P〉0．05）。对照组小鼠Beclin-1、caspase3、Bax、Bcl-2的均值在wo时和W9时表达差异均无统计学意义（z值分别为0、0．577、0、0．577，均P〉0．05）。结论慢性紫外线损伤对小鼠皮肤角质形成细胞中凋亡和自噬的交互调控元件Beclin-1和Bax具有上调表达的作用，而对Bcl-2表达调控效应不显著。这一系列的调控效应可能参与了凋亡和自噬在慢性紫外线皮肤损伤中的交互调控。

Objective To study the regulatory effect of chronic ultraviolet （UV） radiation on the expressions of three regulatory elements involved in the cross-talk between apoptosis and autophagy （including Bax, Bcl-2 and Beclin-l）, as well as cysteine-containing aspartate-specific protease 3 （caspase 3） in keratinocytes of mouse skin. Methods Thirty healthy Kunming mice at 6-8 weeks of age were included in this study, and randomly divided into two groups with the ratio of female to male mice being 1 ： 1 in each group： chronic UV damage group （n = 20） receiving sunlight simulator UV radiation, and control group （n = 10） receiving rio radiation. The irradiation was performed on the back of mice once a day for 5 consecutive days per week, and lasted for 9 weeks. The dose of irradiation began at one minimal erythema dose （UVB： 0.07 J/cm2, UVA： 0.7 J/cm2）, and increased by 0.5 minimal erythema dose per week, with the total dose of UVB and UVA being 9.45 J/cm2 and 94.5 J/cm2 respectively. Skin samples were resected from the back of these mice before and at 9 weeks after the beginning of the radiation. Skin damage was evaluated by histological observation, specificchemical staining and epidermal thickness measurement. Immunohistochemistry was conducted to detect the expressions of Bax, Bcl-2, caspase 3 and Beclin-1 in epidermal keratinocytes. Paired t test and Wilcoxon signed rank test were carried out for statistical analysis. Results After UV irradiation, the mice showed an obvious increase in epidermal thickness. Histological examination and specific staining for collagen and elastic fibers both revealed a histological change characteristic of chronic UV damage in the mice receiving UV radiation. In the chronic UV damage group, the average immunoreactivity intensity distribution index （IRIDI） was statistically increased for Beclin-1, caspase 3 and Bax in the skin sample （2.70 vs. 0.30, 3.30 vs. 0.25, 3.35 vs. 0.35, Z = 4.034, 4.001, 3.970, respectively, all P 〈 0.05）, but remained unchanged for Bcl-2 （0.25 vs. 0.25, Z = 0, P 〉 0.05） at 9 weeks after the radiation compared with that before radiation. No significant changes were observed in the control group for the expression intensity of Beclin-1, caspase 3, Bax or Bcl-2 during the 9 weeks （Z = 0, 0.577, 0, 0.577, respectively, all P 〉 0.05）. Conclusions Chronic UV irridiation shows no obvious effect on the expression of Bcl-2, but can up-regulate the expression of Beclin-1 and Bax, both of which may be involved in the cross-talk between apoptosis and autophagy in chronic UV radiation-induced skin damage.