雌激素对大鼠皮肤老化的影响及相关分子机制探讨

Effects of estrogen on skin aging of rats model and its related molecular mechanism

目的探讨雌激素对大鼠皮肤老化的影响及其分子机制,为临床预防和治疗皮肤老化提供依据。方法 2011年8至12月,选择40只清洁级、体重为200~250g、10周龄的雌性SD大鼠为研究对象。采用随机数字表法,将40只SD大鼠随机分为4组,分别纳入去势17β-雌二醇组、去势戊酸雌二醇组,去势对照组、空白对照组,每组SD大鼠各为10只。4组SD大鼠建模成功后,采集4组SD大鼠的血液标本,进行血清雌二醇水平测定。采集4组SD大鼠背部除毛处皮肤标本,用于皮肤厚度测量,以及采用实时荧光定量PCR技术进行皮肤Ⅰ、Ⅲ型胶原及转化生长因子(TGF)-βmRNA水平测定。SD大鼠血清雌二醇水平、皮肤厚度,Ⅰ、Ⅲ型胶原与TGF-βmRNA,以及Ⅲ型与Ⅰ型胶原mRNA相对表达水平比值(Ⅲ/Ⅰ型胶原mRNA相对表达水平)2组间比较,采用成组t检验。上述指标的相关性分析,采用Spearman相关性分析或Pearson相关性分析。本研究实验过程中对动物的处置符合动物伦理学要求。4组SD大鼠的体重、年龄等基本资料比较,差异均无统计学意义(P>0.05),并且均于相同避光条件下饲养。结果 (1)去势17β-雌二醇组SD大鼠血清雌二醇水平较去势戊酸雌二醇组、去势对照组、空白对照组显著增高,差异均有统计意义(t=45.310、303.445、43.563,P<0.001)。去势戊酸雌二醇组SD大鼠血清雌二醇水平较去势对照组显著增高,较空白对照组显著降低,差异均有统计意义(t=303.445、-14.036,P<0.001)。(2)去势17β-雌二醇组SD大鼠皮肤厚度,Ⅰ、Ⅲ型胶原及TGF-βmRNA与Ⅲ/Ⅰ型胶原mRNA相对表达水平,均较去势戊酸雌二醇组、去势对照组显著增加,并且差异均有统计意义(与去势戊酸雌二醇组比较:t=17.213、43.308、165.194、94.328、26.399,P<0.001;与去势对照组比较:t=17.459、46.482、208.517、198.591、40.541,P<0.001)。去势戊酸雌二醇组SD大鼠上述5个指标均较去势对照组显著增加,均较空白对照组显著降低,并且差异均有统计意义(与去势对照组比较:t=5.688、12.067、39.877、66.706、11.180,P<0.001;与空白对照组比较:t=-54.769、-74.981、-172.077、-113.888、-31.136,P<0.001)。去势17β-雌二醇组SD大鼠上述5个指标较空白对照组有所下降,但是差异均无统计学意义(P>0.05)。(3)采用Spearman相关性分析的结果显示,4组SD大鼠的皮肤厚度,Ⅰ、Ⅲ型胶原及TGF-βmRNA与Ⅲ/Ⅰ型胶原mRNA相对表达水平,均与血清雌二醇水平呈正相关关系(r_s=0.649、0.728、0.783、0.754、0.723,P<0.001)。进一步分析显示,SD大鼠血清雌二醇水平≤70pg/mL时,上述指标均与血清雌二醇水平呈线性正相关关系(r=0.740、0.717、0.738、0.757、0.754,P<0.001);但是当血清雌二醇水平>70pg/mL时,上述指标不再随血清雌二醇水平增高而增高。(4)TGF-βmRNA与Ⅰ、Ⅲ型胶原mRNA相对表达水平均呈线性正相关关系(r=0.951、0.981,P<0.001)。结论局部或系统应用雌激素,均可增加皮肤Ⅰ、Ⅲ型胶原mRNA的表达,并且可逆转Ⅲ/Ⅰ型胶原下降,增加皮肤厚度,部分逆转皮肤老化,并且局部用药较系统用药效果更明显。TGF-β是胶原合成的重要调节因子,雌激素通过增加TGF-βmRNA的表达,间接调节Ⅰ、Ⅲ型胶原mRNA的表达。

Objective To explore the effects of estrogen on skin aging of rats and its related molecular mechanism, to provide references for prevention and treatment of skin aging.Methods From August to December 2011,a total of 40 clean-grade,200-250 g,10-week-old and female SD rats were selected as study subjects.They were divided into ovariectomized 17β-estradiol group,ovariectomized estradiol valerate group,ovariectomized control group and blank control group by random digits table method,and 10 SD rats in each group.After the success of the modeling,blood samples of 4 groups were collected for serum estradiol level detection.The tissue samples were acquired from the dorsal skin of SD rats for morphometric determination of skin thickness and mRNA levels of collagen Ⅰ,Ⅲ and transforming growth factor（TGF）-βby real time-PCR.Serum estradiol levels,skin thickness,relative expression levels of collagen Ⅰ,Ⅲ and TGF-βmRNA,and ratios of relative expression levels of collagenⅢand collagenⅠ mRNA between two groups were compared by independent-samples t test.Correlation analyses of the above indicators were performed by Spearman correlation analysis or Pearson correlation analysis.The treatment of animals in the experimental process of this study met the animal ethical requirements.There were no significant differences in the body weight,age,and other basic data among 4 groups（P〈0.05）,and they were all raised under the same dark condition.Results（1）The serum estradiol level of ovariectomized 17β-estradiol group was significantly higher than that of ovariectomized estradiol valerate group,ovariectomized control group and blank group respectively,and all the differences were statistically significant（t=45.310,303.445,43.563;P〈0.001）.The serum estradiol level of ovariectomized estradiol valerate group was significantly higher than that of ovariectomized control group,while significantly lower than that of blank control group,and both the differences were statistically significant（t=303.445,-14.036;P〈0.001）.（2）The skin thickness,relative expression levels of collagen Ⅰ,Ⅲ and TGF-βmRNA,and ratios of relative expression levels of collagen Ⅲ and collagen Ⅰ mRNA of SD rats in ovariectomized 17β-estradiol group all were significantly higher than those of ovariectomized estradiol valerate group and ovariectomized control group,and all the differences were statistically significant（compared with ovariectomized estradiol valerate group：t=17.213,43.308,165.194,94.328,26.399,P〈0.001;compared with ovariectomized control group：t=17.459,46.482,208.517,198.591,40.541,P〈0.001）.The above-mentioned five indexes of SD rats in ovariectomized estradiol valerate group were significantly higher than those of ovariectomized control group,while significantly lower than those of blank control group,and all the differences were statistically significant（compared with ovariectomized control group：t=5.688,12.067,39.877,66.706,11.180,P〈0.001;compared blank control group：t=-54.769,-74.981,-172.077,-113.888,-31.136,P〈0.001）.The above-mentioned five indexes of SD rats in ovariectomized estradiol valerate group were relatively lower than those of blank control group,but all the difference were not statistically significant（P〈0.05）.（3）Spearman correlation analysis showed that the skin thickness,relative expression levels of collagenⅠ,Ⅲand TGF-βmRNA,and ratios of relative expression levels of collagen Ⅲ and collagen Ⅰ mRNA of SD rats were positively correlated with serum estradiol level（r_s=0.649,0.728,0.783,0.754,0.723;P〈0.001）.Further analysis showed that when serum estradiol levels of SD rats were ≤70 pg/mL,there were a linear positive correlations between the above-mentioned indicators and serum estradiol levels（r=0.740,0.717,0.738,0.757,0.754;P〈0.001）.However,when serum estradiol levels were 70 pg/mL,the above-mentioned indicators would no longer increase with serum estradiol levels.（4）The relative expression level of TGF-βmRNA was positively correlated with the relative expression levels of collagenⅠ andⅢ mRNA,respectively（r=0.951,0.981;P〈0.001）.Conclusions Local or systemic application of estrogen can increase the expression of collagen Ⅰ and Ⅲ mRNA,and can reverse the decrease of ratio of relative expression levels of collagen Ⅲ and collagen Ⅰ mRNA,increase the thickness of skin and partially reverse the aging of skin.The effectiveness of local administration is better than that of systemic administration.TGF-βis an important regulatory factor of collagen synthesis,estrogen can indirectly regulate the expression of collagen Ⅰ and Ⅲ mRNA through increasing the expression of TGF-βmRNA.