不同浓度17β-雌二醇对雌性大鼠晶状体上皮细胞凋亡的影响

Effects of 17β-estradiol with different concentrations on lens epithelial cells apoptosis in female rats

目的观察不同浓度17β-雌二醇对H2O2诱导的雌性大鼠晶状体上皮细胞凋亡的影响及其对凋亡相关蛋白Bcl-2和Bax表达的调控,探讨不同浓度雌激素对晶状体上皮细胞凋亡的作用及其机制,为合理应用雌激素防治白内障提供实验依据。方法摘取健康成年雌性SD大鼠的透明晶状体48枚,随机分为6组培养:17β-雌二醇+H2O2组1、17β-雌二醇+H2O2组2、17β-雌二醇+H2O2组3、17β-雌二醇+H2O2组4、H2O2组以及空白对照组。除空白对照组外,其他组以终浓度为300μmol.L-1过氧化氢制作晶状体上皮细胞凋亡模型,并分别加入不同终浓度17β-雌二醇干预,17β-雌二醇+H2O2组1至4的17β-雌二醇干预浓度分别为1×10-8mol.L-1、1×10-7mol.L-1、1×10-6mol.L-1、1×10-5mol.L-1。于细胞恒温培养箱中孵育24h后,撕取各组晶状体前囊及赤道部囊膜铺片,采用TUNEL法检测晶状体上皮细胞凋亡率,免疫组织化学SP法检测凋亡相关蛋白Bcl-2和Bax的阳性表达率。结果 TUNEL法检测晶状体上皮细胞凋亡率结果显示:17β-雌二醇+H2O2各组晶状体上皮细胞凋亡率(0.4508±0.0203、0.4245±0.0196、0.4077±0.0163、0.3770±0.0205)均显著低于H2O2组(0.9048±0.0186),差异均具有显著统计学意义(均为P=0.000),并且与17β-雌二醇的浓度呈负相关。免疫组织化学SP法检测Bcl-2及Bax阳性表达率结果显示:在空白对照组晶状体上皮细胞中,Bcl-2和Bax均有表达,且Bcl-2的表达(0.8907±0.0190)远较Bax表达(0.0777±0.0234)强;H2O2可明显降低Bcl-2表达(0.0930±0.0110),提高Bax表达(0.9248±0.0238);与H2O2组比较,17β-雌二醇可明显提高Bcl-2表达(0.5662±0.0564、0.5872±0.0441、0.6212±0.0548、0.6682±0.0568),降低Bax表达(0.4677±0.0352、0.4472±0.0451、0.4077±0.0154、0.3587±0.0310),差异均有显著统计学意义(均为P=0.000)。结论 17β-雌二醇对H2O2诱导的离体培养雌性大鼠晶状体上皮细胞的凋亡有抑制作用,此抑制作用在一定范围内与17β-雌二醇的浓度呈正相关;对凋亡相关蛋白Bcl-2和Bax表达的调控可能是17β-雌二醇抑制晶状体上皮细胞凋亡的分子机制之一。

Objective To observe the effects of 17β-estradiol with different concentrations on lens epithelial cells（LEC） apoptosis induced by H2O2 in vitro in female rats,investigate its regulation on expression of Bcl-2 and Bax protein,approach the action and mechanism of different concentration of 17β-estradiol on LEC apoptosis and provide the experimental evidence of estrogen prevention and treatment of cataract with estradiol.Methods Forty-eight diaphanous lens of healthy adult female SD rats were extracted and randomly divided into 6 groups for culture：17β-estradiol ＋H2O2 group 1,17β-estradiol ＋H2O2 group 2,17β-estradiol ＋H2O2 group 3,17β-estradiol ＋H2O2 group 4,H2O2 group and control group.Except for control group,the apoptosis model of LEC was established by H2O2 with the final concentration of 300 μmol·L-1,then 17β-estradiol with different final concentrations（1×10-8 mol·L-1,1×10-7 mol·L-1,1×10-6 mol·L-1,1×10-5 mol·L-1） were added into 17β-estradiol＋ H2O2 group 1-4 for intervention at the same time,respectively.After the lens were cultured in incubator for 24 hours,the anterior lens capsule and the ambitus peplos were obtained and stretched,then the apoptosis rate was detected by TUNEL method,the expression of Bcl-2 and Bax protein were detected by immunohistochemical method.Results The apoptosis rates of LEC in 17β-estradiol groups（0.450 8±0.020 3,0.424 5±0.019 6,0.407 7±0.016 3,0.377 0±0.020 5）were obviously lower than that in H2O2 group（0.904 8±0.018 6）,there were significant differences（all P=0.000）,furthermore,the apoptosis rate was negative related with the concentration of 17β-estradiol.The expression of Bcl-2 and Bax protein in control group were 0.890 7±0.019 0 and 0.077 7±0.023 4,the former was higher than the latter;H2O2 could up-regulate the expression of Bax protein（0.924 8±0.023 8）and down-regulate the expression of Bcl-2 protein（0.093 0±0.011 0）;Compared with H2O2,17β-estradiol could significantly up-regulate the expression of Bcl-2 protein（0.566 2±0.056 4,0.587 2±0.044 1,0.621 2±0.054 8,0.668 2±0.056 8） and down-regulate the expression of Bax protein（0.467 7±0.035 2,0.447 2±0.045 1,0.407 7±0.015 4,0.358 7±0.031 0）,there were significant differences（all P=0.000）,furthermore,the expression of Bcl-2 and Bax protein were positive related with the concentration of 17β-estradiol.Conclusion 17β-estradiol can inhibit the LEC apoptosis induced by H2O2 in vitro,the effect was positive related with the concentration of 17β-estradiol;The regulation on expression of Bcl-2 and Bax protein may be the molecular mechanism of inhibiting the LEC apoptosis by 17β-estradiol.