基于基因网络调控的黄芩素改善衰老性学习记忆障碍作用机制研究

Aged learning and memory impairment seriously affects the physical and mental health, and life quality of older people. Previous research found that baicalein could significantly improve the learning and memory ability in D-galactose-induced aging rat model, however, the molecular mechanisms causing this effect are still unclear. Based on previous work, this project intends to: 1) further clarify the effects and characteristics of baicalein on improving learning and memory impairment by intervention of baicalein in senescence-accelerated mouse prone/8 (SAMP8) model; 2) high throughput sequencing technology will be used to analyze the genes significantly influenced by baicalein for improving learning and memory, and to construct gene networks; 3) multiple node ranking approaches and network module enrichment analysis were applied to predict the key genes, biological processes and pathways regulated by baicalein; 4) real-time PCR and RNA interference were used to validate the top 10 genes obtained by different ranking algorithms; western blot and in vitro luciferase reporter gene assay were used to validate one significant biological process or pathway regulated by baicalein. Our results will provide some insights on understanding the mechanisms of learning and memory loss and the pharmacological mechanisms of baicalein. It will also suggest a network-based approach for the future drug mechanism research, which will be of great academic values.

衰老性学习记忆障碍严重影响着老年人的身心健康和生活质量。前期研究发现黄芩素能显著改善D-半乳糖致大鼠衰老模型学习记忆障碍，然而，其作用机制尚不清楚。本项目拟在前期研究基础上，1）采用黄芩素干预快速老化小鼠SAMP8模型，进一步明确黄芩素改善学习记忆障碍的作用和特点；2）采用转录组测序技术，研究并分析黄芩素改善学习记忆显著影响的基因，构建基因网络；3）应用多种节点排序方法和网络模块分析，预测黄芩素发挥活性所调节的关键基因、关键生物过程和通路；4）采用Real-time PCR、RNA干扰等技术对不同排序方法所得前10位基因进行验证；并用Western blot、体外荧光素酶报告基因实验对黄芩素影响较大的1个关键生物过程或通路进行验证。研究结果不仅为深入理解学习记忆衰退及黄芩素的药理机制奠定基础，而且将提供一条基于网络的药物作用机制研究新途径，具有重要的学术价值。

衰老性学习记忆障碍严重影响着老年人的身心健康和生活质量。前期研究发现黄芩素能显著改善D-半乳糖致大鼠衰老模型学习记忆障碍，但是其作用机制尚不清楚。本项目采用快速老化小鼠SAMP8模型，通过衰老评分明确了黄芩素能够延缓SAMP8小鼠衰老；通过Morris水迷宫、新型物体识别、嗅觉记忆等实验，明确黄芩素能够改善SAMP8小鼠的衰老性认知功能障碍。采用转录组测序技术，发现黄芩素能够回调SAMP8小鼠的214个差异表达基因，使基因表达与正常老化小鼠SAMR1更接近。对上述差异表达基因进行网络药理学分析，采用平均最短路径长度（ASPL）和介数（BC）这两个参数进行网络分析和节点排序，得到黄芩素能够显著回调的50个差异表达基因。通过对这些基因进行通路富集，得到8条信号通路，主要包括细胞因子-细胞因子受体相互作用、Jak-STAT信号通路、PI3K-Akt信号通路、神经活性配体-受体相互作用、趋化因子信号通路等。采用qRT-PCR和Western blot对Jak-STAT信号通路中关键基因及相关蛋白进行验证，结果表明黄芩素给药后显著降低了SAMP8小鼠皮层Il7、Il-20rb、Prlr和Socs1的mRNA表达水平，与转录组结果一致；同时显著抑制Jak2和STAT1的磷酸化，这些结果提示黄芩素能够抑制SAMP8小鼠皮层Jak2-STAT1信号通路的活化。对相关蛋白进行了后续验证，最终结果提示黄芩素显著改善SAMP8小鼠的学习记忆障碍可能与抑制Aβ和RAGE/Jak2/STAT1信号通路密切相关。研究结果不仅为深入理解学习记忆衰退及黄芩素的药理机制奠定基础，而且提供了一条基于网络的药物作用机制研究新途径。