目的 运用网络药理学预测酒蒸黄连-石菖蒲治疗糖尿病认知功能障碍的作用机制，并进行相关实验验证。方法 通过TCMSP数据库、SwissTargetPrediction在线平台、GeneCards数据库筛选出酒蒸黄连和石菖蒲的主要活性成分、作用靶点和疾病靶点，借助DAVID 6.8数据库进行京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析，利用STRING数据库和Cystoscope 3.8.1软件分别构建蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络和“药物-关键成分-潜在靶点-核心通路”网络，预测药物防治糖尿病认知功能障碍的作用机制。建立大鼠糖尿病认知功能障碍复合模型，给予酒蒸黄连-石菖蒲进行干预，检测各组大鼠空腹血糖、血清胰岛素、稳态模型胰岛素抵抗指数（homeostasis model of insulin resistance，HOMA-IR）和胰岛素敏感指数（insulin sensitivity index，IAI）；检测各组大鼠海马组织中乙酰胆碱（acetylcholine，Ach）含量以及乙酰胆碱酯酶（acetyl cholinesterase，AchE）和乙酰胆碱转移酶（choline acetyl transferase，ChAT）活性；采用免疫组化法测定各组大鼠海马组织中β-淀粉样蛋白42（β-amyloid protein 42，Aβ₄₂）、AchE和ChAT蛋白表达；采用Western blotting法测定各组大鼠海马组织中与糖代谢相关的胰岛素受体底物（insulin receptor substrate，IRS）、磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）、蛋白激酶B（protein kinase B，Akt）、糖原合成酶激酶3β（glycogen synthase kinase 3β，GSK3β）的蛋白表达及磷酸化水平，以及与炎性反应相关的白细胞介素-1β（interleukin-1，IL-1β）、IL-6、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的蛋白表达，系统观察并验证酒蒸黄连-石菖蒲治疗糖尿病认知功能障碍的作用及机制。结果 筛选出酒蒸黄连-石菖蒲有效活性成分16个，与糖尿病认知功能障碍相关的候选靶点84个；KEGG通路分析发现，酒蒸黄连-石菖蒲可能通过作用于PI3K-Akt信号通路、胰岛素信号通路、胆碱能通路、糖基化终末产物（advanced gycation end products，AGEs）-AGE受体（receptor of AGE，RAGE）信号通路及炎症相关途径治疗糖尿病认知功能障碍。验证性实验结果显示，酒蒸黄连-石菖蒲显著降低糖尿病认知功能障碍大鼠空腹血糖和HOMA-IR（P<0.05、0.01），提高IAI（P<0.01）；抑制海马组织中Aβ₄₂蛋白表达（P<0.05），减少Aβ生成；增加海马组织中Ach含量（P<0.05、0.01），增加ChAT活性及蛋白表达（P<0.05、0.01），降低AchE活性及蛋白表达（P<0.05、0.01）；上调海马组织中IRS、PI3K、Akt和GSK3β的磷酸化水平（P<0.05、0.01），降低IL-1β、IL-6及TNF-α蛋白表达水平（P<0.05、0.01）。结论 网络药理学预测结果和动物实验结果相呼应，均表明酒蒸黄连-石菖蒲组分可能通过调节糖代谢、改善胰岛素抵抗、抑制炎性因子表达、抗神经细胞凋亡以及调控PI3K-Akt、AGE-RAGE、胰岛素、胆碱能等相关通路，发挥治疗糖尿病及其并发的认知功能障碍作用。

Objective To predict the mechanism of wine steamed Huanglian (Coptidis Rhizoma)-Shichangpu (Acori Tatarinowii Rhizoma) by network pharmacology in the treatment of diabetic cognitive impairment, and to verify it by experiments. Methods TCMSP database, Swiss Target Prediction database and GeneCards database were used to screen the main active components, action targets of wine steamed Coptidis Rhizoma-Acori Tatarinowii Rhizoma and disease targets; DAVID 6.8 database was used to conducted Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis; STRING database and Cystoscope 3.8.1 software were used to build protein-protein interaction (PPI) network and "drugs-key components-potential targets-core pathways" network, predict the mechanism of drug prevention and treatment of diabetic cognitive impairment. Diabetic cognitive impairment model in rats was established, wine steamed Coptidis Rhizoma-Acori Tatarinowii Rhizoma were given for intervention, fasting blood glucose, serum insulin, homeostasis model of insulin resistance (HOMA-IR) and insulin sensitivity index (IAI) of rats in each group were detected; Acetylcholine (Ach) content, acetyl cholinesterase (AchE) and acetylcholine acetyl transferase (ChAT) activities in hippocampus of rats in each group were detected; Immunohistochemical method was used to determine the expressions of β-amyloid protein 42 (Aβ₄₂), AchE and ChAT in hippocampal of rats in each group; Western blotting was used to determine the protein expressions and phosphorylation level of insulin receptor substrate (IRS), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), glycogen synthase kinase 3β (GSK3β) related to glucose metabolism in hippocampus of rats in each group, protein expressions of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) related to inflammation, in order to observe and verify the effect and mechanism of wine steamed Coptidis Rhizoma-Acori Tatarinowii Rhizoma in the treatment of diabetic cognitive dysfunction. Results A total of 16 effective active ingredients of wine steamed Coptidis Rhizoma-Acori Tatarinowii Rhizoma, and 84 candidate targets related to diabetic cognitive dysfunction were screened out. KEGG pathway analysis found that wine steamed Coptidis Rhizoma-Acori Tatarinowii Rhizoma treated diabetic cognitive dysfunction through PI3K-Akt signaling pathway, insulin signaling pathway, cholinergic pathway, advanced gycation end products (AGE)-receptor of AGE (RAGE) signaling pathway and inflammation-related pathways. The confirmatory experiment results showed that wine steamed Coptidis Rhizoma-Acori Tatarinowii Rhizoma significantly reduced fasting blood glucose and HOMA-IR (P < 0.05, 0.01) in rats with diabetic cognitive dysfunction, and increased IAI (P < 0.01); Aβ₄₂ protein expression in hippocampus was inhibited (P < 0.05), Aβ production was reduced; Ach content in hippocampus was increased (P < 0.05, 0.01), ChAT activity and protein expression were increased (P < 0.05, 0.01), AchE activity and protein expression were decreased (P < 0.05, 0.01)); The phosphorylation levels of IRS, PI3K, Akt and GSK3β in hippocampus were up-regulated (P < 0.05, 0.01), the protein expressions of IL-1β, IL-6 and TNF-α were reduced (P < 0.05, 0.01). Conclusion The prediction results of network pharmacology correspond to the results of animal experiments, and both indicate that the components of wine steamed Coptidis Rhizoma-Acori Tatarinowii Rhizoma may regulate glucose metabolism, improve insulin resistance, inhibit the expressions of inflammatory factors, resist neuronal apoptosis, regulate PI3K-Akt, AGE-RAGE, insulin, cholinergic and other related pathways, then play the role of treating diabetes and its concurrent cognitive dysfunction.

R285.5

国家自然科学基金资助项目（81302912）；西南民族大学研究生创新型科研项目（CX2020SZ82）