目的 观察D-半乳糖致衰老大鼠脑内促红细胞生成素表达的变化,以及熟地黄对这种变化的逆转效果。方法 50、150和250 mg·kg-1 D-半乳糖连续皮下注射8周,复制不同程度的氧化应激致衰老大鼠模型。第8周水迷宫检测大鼠的空间学习记忆能力,按照试剂盒方法检测皮层海马区域的氧化应激损伤程度以及脑内β-半乳糖苷酶的含量。免疫荧光、免疫印迹法检测皮层海马区EPO、EPOR和缺氧诱导因子-2α(HIF-2α)的变化。再取150 mg·kg-1 D-半乳糖组为衰老模型组,另设空白对照组和熟地黄组。熟地黄组在造模的同时,从第5周起灌胃熟地黄水提物4 g·kg-1,每天1次,连续4周,检测指标同上。结果 与空白组相比,150和250 mg·kg-1 D-半乳糖致衰老大鼠水迷宫逃避潜伏期显著延长,穿越原平台位置次数显著减少,脑内β-半乳糖苷酶的含量显著增多,EPO、EPOR、HIF-2α表达显著减少(均P<0.05),且脑内丙二醛(MDA)含量显著增高,超氧化物歧化酶(SOD)活性显著降低(均P<0.01),脑内MDA的含量与EPO(r=-0.897,P<0.01)、EPOR(r=-0.923,P<0.01)表达成负相关性。与150 mg·kg-1D-半乳糖衰老大鼠模型相比,熟地黄水提物4 g·kg-1组大鼠的水迷宫逃避潜伏期显著缩短,穿越原平台位置次数显著增多,脑内β-半乳糖苷酶、MDA含量显著减少,SOD活性显著增强,EPO、EPOR的表达显著增多(均P<0.05)。结论 D-半乳糖致衰老大鼠大脑海马区EPO的表达下降,与脑部氧化应激损伤的增多有关,并受HIF-2α调控,熟地黄水提物可增加脑内EPO的表达并改善衰老大鼠的学习记忆能力。
Abstract
OBJECTIVE To observe the change of EPO in brain of aging rat induced by D-galactose(D-gal) and the EPO-based antiaging of the water extract of Radix Rehmanniae Preparata(WERRP). METHODS D-gal-treated groups were received subcutaneous injection of D-gal at dose of 50, 150 and 250 mg·kg-1 daily for 8 weeks to imitate an aging model that was induced by oxidative stress. After the detection of EPO in hippocampus, the 150 mg·kg-1 D-gal group was chose as the aging model. In addition the WERRP-treated group and vehicle group was set. The WERRP-treated group was given WERRP oral gavage at a dose of 4 g·kg-1 daily starting from the 5th week. Morris water maze (MWM) test was used to assess the spatial learning and memory. SOD, MDA and β-galactosidase in brain were examined by Assay Kits. Finally, EPO, EPOR, and HIF-2α in hippocampus were determined by immunohistochemistry and Western blot. RESULTS D-gal-treated group showed significant longer latency to platform and less times of cross the platform(P<0.05) in MWM. After treated with D-gal, SOD was drastically decreased and MDA and β-galactosidase were remarkably increased in brains compared with vehicle (0.9% saline)-treated rats (P<0.01). In addition, the expression of EPO,EPOR and HIF-2α were significantly decreased in the brains of D-gal-treated rats compared with vehicle-treated rats. Meanwhile, there was a negative correlation between EPOR and MDA in content. Interestingly, WERRP-treated rats showed significant improvement of spatial learning and memory, decrease of oxidative stress and enhancement of EPO/EPOR in the brain compared with 150 mg·kg-1 D-gal-treated rat(P<0.05). CONCLUSION The aging rats induced by D-gal show a significant decline of EPO in brain which indicate the decrease of brain EPO in aging is related to the increase of oxidative stress. That WERRP reverses the decline of the EPO expression in aging model may be the underlying mechanism of the role of anti-aging of WERRP.
关键词
促红细胞生成素 /
D-半乳糖 /
衰老 /
脑 /
氧化应激 /
熟地黄
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Key words
erythropoietin /
D-galactose /
aging /
brain /
oxidative stress /
Radix Rehmanniae Preparata
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然科学基金面上项目(81473549);国家青年科学基金资助项目(31402237);中国博士后基金(2014M562272)
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