Expression data from the hearts of aged Norway Brown rats (18-22 months-old)

Despite an abundance of evidence to the contrary from animal studies, large clinical trials on humans have shown that estrogen administered to post-menopausal women increases the risk of cardiovascular disease. However, timing may be everything, as estrogen is often administered immediately after ovariectomy (ovx) in animal studies, while estrogen administration in human studies occurred many years post-menopause. This study investigates the discrepancy by administering 17ß-estradiol (E2) in a slow-release capsule to Norway Brown rats both immediately following ovx and 9 weeks post-ovx (Late), and studying differences in gene expression between these 2 groups as compared to age-matched ovx and sham operated animals. Two different types of microarray were used to analyze the left ventricles from these groups: an Affymetrix array (2 samples/group, each sample contained total RNAs pooled from 3 rats) and an Inflammatory Cytokines and Receptors PCR array (N=4 /group). Key genes were analyzed by western blotting. Ovx without replacement led to an increase in caspase 3, caspase 9, calpain 2, MMP9, and TNFa. Caspase 6, STAT3, and CD11b increased in the Late group, while TIMP2, MMP14, and collagen I a1 were decreased. MADD and fibronectin were increased in both Ovx and Late. TNFa protein levels increased with Late replacement. Many of these changes were prevented by early E2 replacement. These findings suggest that increased TNFa may be involved in some of the deleterious effects of delayed E2 administration seen in human studies.尽管动物研究已积累大量相反证据，但针对人类的大型临床试验显示，向绝经后女性施用雌激素会增加心血管疾病风险。然而，时机或许是关键所在：动物研究中通常会在卵巢切除术（ovariectomy, ovx）后立即施用雌激素，而人类研究中的雌激素给药则是在绝经多年后进行的。本研究通过以下方式探究这一差异：向棕色挪威大鼠分别在卵巢切除术后即刻以及术后9周（晚期给药组，Late）投喂17β-雌二醇（17ß-estradiol, E2）缓释胶囊，并对比这两组与年龄匹配的卵巢切除组及假手术组动物的基因表达差异。本研究采用两种不同类型的微阵列（microarray）对各组大鼠的左心室组织进行分析：其一为Affymetrix基因芯片（每组2个样本，每个样本包含3只大鼠的混合总RNA）；其二为炎症细胞因子与受体聚合酶链式反应（PCR）芯片（每组N=4）。核心基因则通过蛋白质印迹（Western blotting）进行分析。未进行雌激素替代的卵巢切除组大鼠，其胱天蛋白酶3（caspase 3）、胱天蛋白酶9（caspase 9）、钙蛋白酶2（calpain 2）、基质金属蛋白酶9（MMP9）以及肿瘤坏死因子α（TNF-α）的表达水平均有所升高。晚期给药组中，胱天蛋白酶6（caspase 6）、信号转导与转录激活因子3（STAT3）以及分化簇11b（CD11b）的表达水平升高，而金属蛋白酶组织抑制剂2（TIMP2）、基质金属蛋白酶14（MMP14）以及Ⅰ型胶原蛋白α1链（collagen I α1）的表达水平则有所下降。卵巢切除组与晚期给药组中，丝裂原活化蛋白激酶激活的死亡结构域蛋白（MADD）与纤连蛋白（fibronectin）的表达均有所升高。晚期给药组的肿瘤坏死因子α（TNF-α）蛋白水平有所升高。早期雌激素替代治疗可阻断其中多数变化。本研究结果表明，肿瘤坏死因子α（TNF-α）水平升高，可能参与了人类研究中观察到的延迟施用雌激素所带来的部分有害效应。