基于蛋白质组学探讨九蒸九晒地黄延缓去卵巢小鼠脑衰老的药效与机制

Mechanism analysis of repeatedly steamed and sundried Rehmanniae Radix Praeparata in delaying brain aging in ovariectomized mice based on proteomics

探究九蒸九晒地黄延缓去卵巢小鼠脑衰老的药效与机制。小鼠去卵巢之后随机分成模型组、戊酸雌二醇组(0.3 mg·kg^(-1))以及九蒸九晒地黄低、中、高剂量组(1.0、2.0、4.0 g·kg^(-1)),另设假手术组,每组15只。术后1周,开始灌胃给药连续15周。跳台实验和水迷宫实验检测小鼠行为学变化。HE染色和尼式染色观察小鼠脑组织形态学变化。免疫组化检测小鼠脑组织Aβ和ER_(β)的表达。试剂盒检测小鼠血清雌激素水平和脑组织胆碱酯酶及胆碱酯酶转移酶水平。提取海马蛋白,Nano-ESI-LC-MS联用系统检测蛋白,Protein Discovery软件鉴定蛋白,SIEVE软件进行蛋白相对定量定性分析,PANTHER Classification System数据库对差异蛋白进行GO分析和KEGG数据库信号通路富集。与假手术组比较,模型组学习记忆能力下降,跳台潜伏期显著缩短(P<0.05),Morris水迷宫实验中,逃避潜伏期显著延长(P<0.05),穿越平台次数和目标象限停留时间缩短;海马区神经细胞排列散乱,细胞间隙变大,Aβ阳性细胞表达显著增多(P<0.05),ER_(β)阳性细胞表达显著减少(P<0.05),雌激素水平明显下降(P<0.05),胆碱功能减退(P<0.05)。与模型组相比,九蒸九晒地黄组学习记忆能力改善,跳台潜伏期显著延长,雌激素水平明显升高(P<0.05),海马区神经细胞排列整齐,形态完整,Aβ阳性细胞数量减少,ER_(β)阳性细胞表达增多。蛋白质组学筛选差异蛋白得到146个,KEGG信号通路富集分析得到75条信号通路,其中参与dopaminergic synapse信号通路的蛋白最多,有13个蛋白参与。综上,九蒸九晒地黄可延缓去卵巢小鼠脑衰老进程,其机制可能为提高雌激素水平,介导dopaminergic synapse信号通路,提高胆碱功能。

The present study explored the effect and mechanism of repeatedly steamed and sundried Rehmanniae Radix Praeparata(RRP)in delaying brain aging in ovariectomized mice.After ovariectomy,the mice were randomly divided into a model group,an estradiol valerate group(0.3 mg·kg^(-1)),and low-(1.0 g·kg^(-1)),medium-(2.0 g·kg^(-1)),and high-dose(4.0 g·kg^(-1))RRP groups,and a sham operation group was also set up,with 15 mice in each group.One week after the operation,intragastric administration was carried out for 15 consecutive weeks.The step-down test and Morris water maze test were used to detect the behavioral changes of mice.HE staining and Nissl staining were used to observe the morphological changes of mouse brain tissues.Immunohistochemistry was used to detect the expression of Aβand ER_(β)in mouse brain tissues.The serum estrogen levels and cholinesterase and cholinesterase transferase levels in brain tissues of mice were detected by assay kits.The extracted hippocampal protein was detected by the Nano-ESI-LC-MS system,identified by the Protein Discovery,and analyzed quantitatively and qualitatively by the SIEVE.The PANTHER Classification System was used for GO analysis and KEGG pathway enrichment analysis of the differential proteins.Compared with the sham operation group,the model group showed decreased learning and memory ability,shortened step-down latency(P<0.05),prolonged escape latency(P<0.05),reduced platform crossings and residence time in the target quadrant,scattered nerve cells in the hippocampus with enlarged intercellular space,increased expression of Aβ-positive cells(P<0.05),declining expression of ER_(β)-positive cells and estrogen level(P<0.05),and weakened cholinergic function(P<0.05).Compared with the model group,the RRP groups showed improved learning and memory ability,prolonged step-down latency(P<0.05),increased estrogen level(P<0.05),neatly arranged nerve cells in the hippocampus with complete morphology,declining Aβ-positive cells,and elevated expression of ER_(β)-positive cells.A total of 146 differential proteins were screened out by proteomics,and KEGG pathway enrichment yielded 75 signaling pathways.The number of proteins involved in the dopaminergic synapse signaling pathway was the largest,with 13 proteins involved.In summary,RRP can delay brain aging presumedly by increasing the level of estrogen,mediating the dopaminergic synapse signaling pathway,and improving cholinergic function.