本研究旨在观察六味地黄(Liu Wei Dihuang,LWDH)对D-半乳糖(D-galactose,D-gal)致衰老大鼠的学习记忆能力及中枢胆碱能神经系统的保护作用。采用成年Sprague-Dawley(SD)大鼠64只,雌雄各半。雄性、雌性大鼠均随机分为4组(n=8):空白对照组即生理盐水+生理盐水(N+N)组;对照组即生理盐水+LWDH(N+L)组;模型组即D-gal+生理盐水(D+N)组和治疗组即D-gal+LWDH(D+L)组。D+N组和D+L组连续每天项部皮下注射D-gal(100mg/kg)6周,N+N和N+L组大鼠注射相同体积生理盐水。第三周起给D+L和N+L组大鼠连续每天LWDH汤剂灌胃6周,同时给N+N组和D+N组连续每天相同体积生理盐水灌胃6周。D-gal衰老大鼠造模结束后,采用Morris水迷宫测定大鼠学习、记忆能力,用HE染色法观察海马和视皮质的形态学变化,检测视皮质中乙酰胆碱(acetylcholine,ACh)含量,以及胆碱乙酰转移酶(choline acetyltransferase,ChAT)及乙酰胆碱酯酶(acetylcholinesterase,AChE)的活性,用免疫组织化学方法观察视皮质中胆碱能神经元ChAT、AChE的表达。结果显示:与N+N组相比,D+N组表现出明显的学习记忆能力下降,提示D-gal衰老模型建立成功;D+L组逃避潜伏期显著缩短,游泳速度明显增加,同时其对靶象限搜寻时间百分比和平台穿越次数均显著增加,提示LWDH显著改善了D-gal诱导的学习记忆障碍。LWDH显著地增加了D+L组的视皮质匀浆ACh含量和ChAT活性,而降低了AChE活性。免疫组织化学结果显示:D+L组视皮质神经元AChE和ChAT均明显高于D+N组。HE染色结果显示:与N+N组相比,D+N组海马和视皮质神经元凋亡增加,细胞密度明显下降,而LWDH显著地增加了D+L组神经元密度。LWDH对衰老雌、雄大鼠的神经保护作用存在差异,D+N组雄性大鼠平台穿越次数的改善情况优于雌性大鼠,而雌性大鼠在ChAT表达水平和视皮质神经元?
This study was aimed to investigate the protective effect of Liu Wei Dihuang(LWDH) against D-galactose(D-gal)-induced brain injury in rats and the existence of sex-dependent differences in LWDH protection.Sixty-four rats evenly composed of males and females were randomly assigned into 4 groups(n = 8):normal saline(NS) + NS(N + N),NS + LWDH(N + L),D-gal + NS(D + N) and D-gal + LWDH(D + L) groups.Rats in D + N and D + L groups received daily injection of D-gal(100 mg/kg,s.c.) for six weeks to establish the aging model,while rats in N + N and N + L groups were injected with the same volume of NS.From the third week,rats in N + L and D + L groups were orally administered with a decoction of LWDH for subsequent six weeks.Rats in N + N and D + N groups were orally administered just with the same volume of NS simultaneously.Morris water maze test was employed to evaluate the ability of learning and memory of the rats in all the groups.Acetylcholine(ACh) content,activities of choline acetyltransferase(ChAT) and acetylcholinesterase(AChE) in visual cortex were assayed.Hematoxylin and eosin(HE) staining were used to observe the morphologic injury in hippocampus and visual cortex,and immunohistochemistry was performed to evaluate ChAT and AChE expression levels in the visual cortex.The results showed that the rats in D + N groups exhibited a longer escape latency to platform,lower swimming speed,less percent of target quadrant search time and platform crossings,compared with N + N groups,suggesting the establishment of aging model,while LWDH improved these indexes in D-gal-treated rats.Compared with D + N groups,LWDH increased ACh content and ChAT activity,and decreased AChE activity in visual cortex.Remarkable loss of neurons was found in hippocampus and visual cortex of aging rats,and the injury was significantly attenuated by LWDH.Immunohistochemistry showed D-gal-induced decreases of ChAT and AChE expressions were restored by LWD