红景天通过EGFR/PI3K/AKT通路抑制神经炎症改善糖尿病认知功能障碍的机制研究

Rhodiola rosea Alleviates Diabetic Cognitive Dysfunction by Inhibiting Neuroinflammation through the EGFR/PI3K/AKT Signaling Pathway

关键词:  红景天  糖尿病认知功能障碍  网络药理学  EGFR/PI3K/AKT通路  神经炎症

Key words:  Rhodiola rosea  diabetic cognitive dysfunction  network pharmacology  EGFR/PI3K/AKT pathway  neuroinflammation

摘要:

目的 通过网络药理学和分子对接探讨红景天改善糖尿病认知功能障碍(diabetic cognitive dysfunction,DCD)的作用机制,并建立糖尿病小鼠模型进行验证。方法：通过TCMSP和HERB数据库寻找红景天(Rhodiola rosea)的活性成分,结合Swiss Target Prediction和 SEA数据库寻找相关靶点。通过GeneCards、TTD、OMIM数据库,搜索DCD相关靶点。利用微生信网站将红景天有效成分靶点和DCD相关靶点通过Venn图取交集获得共同靶点,通过String平台进行蛋白质互作网络构建和分析,通过Cytoscape软件可视化后筛选核心靶点,最终构建PPI网络模型。同时将交集靶点导入Metacape网站进行GO功能富集和KEGG通路富集分析,通过Auto dock软件对红景天苷与核心靶点进行分子对接。雄性C57BL/6J小鼠50只,实验分5组,分别为正常组,模型组,治疗组,溶剂对照组,抑制剂组。高脂饮食4周后腹腔注射链脲佐菌素(streptozotocin,STZ,剂量150mg/kg)建立Ⅱ型糖尿病小鼠模型。通过Western blot检测核心靶点蛋白的表达水平,Elisa检测脑海马组织炎症相关因子的表达水平,通过水迷宫试验检测小鼠空间记忆和学习功能。结果：网络药理学共筛选出16种单体化合物,其中3种具备成为药物的潜力,经过筛选最中确定红景天治疗DCD的主要活性成分为红景天苷(salidroside,SAL),共获取80个潜在靶点,DCD相关靶点8968个,交集靶点42个,其中核心靶点10个。GO和KEGG富集分析表明红景天治疗DCD主要是通过EGFR/PI3K/AKT信号通路调控神经炎症反应过程。分子对接结果显示SAL与核心靶点IL-6、IL-2、FGF2对接效果更高。与模型组相比,红景天苷治疗后明显下调EGFR、p-PI3K/PI3K、p-AKT/AKT蛋白表达水平(P<0.05),mRNA水平和蛋白水平上显著抑制IL-6 、IL-2和FGF2因子的表达(P<0.05)。水迷宫试验结果显示红景天苷改善糖尿病小鼠学习记忆能力(P<0.05)。与治疗组相比,抑制剂组显著逆转相关指标,使SAL对DCD的保护作用降低。结论 红景天改善糖尿病认知功能障碍可能的作用机制是红景天苷通过调控EGFR/PI3K/AKT信号通路调节IL-6、IL-2、FGF2的表达进而降低神经炎症反应。

Abstract:

Objective The mechanism of Rhodiola rosea improving diabetic cognitive dysfunction (DCD) was explored by network pharmacology and molecular docking, and a diabetic mouse model was established for validation. Methods The active ingredients of Rhodiola rosea were obtained from the TCMSP and HERB databases, and the relevant targets were obtained by Swiss Target Prediction and SEA database. GeneCards, TTD, and OMIM databases were used to search for targets related to DCD. Using the “Wei Sheng Xin” website, the common targets of active ingredients of Rhodiola rosea and the related targets of DCD were obtained by the Venn diagram. The protein interaction network was constructed and analyzed using the String platform. The core targets were screened after visualization with Cytoscape software, and the PPI network model was constructed. Then the intersecting targets were imported into the Metacape website for Gene Ontology (GO) functional enrichment and KEGG pathway enrichment analysis. Molecular docking was performed using Autodock software to analyze the interaction between salidroside and the core targets. 50 male C57BL/6J mice were randomly divided into 5 groups: normal group, model group, treatment group, solvent control group, and inhibitor group. After 4 weeks of a high-fat diet, streptozotocin (STZ, dose of 150mg/kg) was intraperitoneally injected to establish a type II diabetes model in mice. The expression levels of core target proteins were detected by western blot, the expression levels of inflammation-related factors in the hippocampus were detected by Elisa, and the spatial memory and learning functions of mice were assessed using the water maze test. Results Network pharmacology screening identified 16 monomeric compounds, three of which showed potential as drugs. After screening, the main active ingredient of Rhodiola rosea for treating DCD was determined to be salidroside (SAL). A total of 80 potential targets of SALwere obtained, with 8,968 DCD-related targets and 42 intersecting targets, including 10 core targets. GO and KEGG enrichment analysis indicated that Rhodiola rosea treatment of DCD mainly regulates the inflammatory response process through the EGFR/PI3K/AKT signaling pathway. Molecular docking results showed that SAL had a higher docking effect with the core targets IL-6, IL-2, and FGF2. Compared with the model group, treatment with SAL significantly downregulated the expression levels of EGFR, p-PI3K/PI3K, and p-AKT/AKT proteins (P<0.05) and obviously inhibited the expression of IL-6, IL-2, and FGF2 factors at both mRNA and protein levels (P<0.05). The results of the water maze test showed that SAL remarkably improved the learning and memory abilities of diabetic mice (P<0.05). Compared with the treatment group, the inhibitor group significantly reversed the related indicators, reducing the protective effect of SAL on DCD. Conclusion The possible mechanism by which Rhodiola rosea improves diabetic cognitive dysfunction is that SAL regulates the expression of IL-6, IL-2, and FGF2 by modulating the EGFR/PI3K/AKT signaling pathway, thereby reducing neuroinflammatory response.