基于网络药理学和分子对接探讨“酸枣仁-石菖蒲”药对改善小鼠失眠的机制

Explore the Mechanism of “Ziziphi Spinosae Semen-Acori Tatarinowii Rhizoma” in improving insomnia Based on network pharmacology and molecular docking

关键词:  酸枣仁  石菖蒲  失眠  网络药理学  分子对接

Key words:  Ziziphi Spinosae Semen  Acori Tatarinowii Rhizoma  insomnia  network pharmacology  molecular docking

摘要:

目的 利用网络药理学和分子对接探究酸枣仁-石菖蒲药对改善失眠的物质基础和潜在分子机制，并建立对氯苯丙氨酸(p-chlorophenylalanine，PCPA)诱导的失眠小鼠模型进行体内实验验证。方法 首先通过TCMSP数据库收集酸枣仁和石菖蒲的化学成分并筛选得到潜在活性成分及其靶点；在GeneCards、OMIM和TTD数据库中获得失眠相关的基因，取得酸枣仁-石菖蒲药对和失眠疾病的交集靶点，然后在Cytoscape 3.6.1软件中构建中药-化合物-靶点-疾病网络图；利用STRING数据库建立蛋白相互作用网络图；借助DAVID数据库进行靶点GO功能富集分析和KEGG通路富集分析；接着采用分子对接技术进行初步验证；ICR雄性小鼠60只随机分为正常组，模型组，酸枣仁-石菖蒲高、中、低剂量组(8.0，4.0，2.0 g·kg^-1)，地西泮组(3 mg·kg^-1)；除正常组外，第1天和第2天腹腔注射PCPA(30 mg·mL^-1)建立失眠模型；然后连续给药7 d，正常组和模型组小鼠灌胃等体积生理盐水。测定戊巴比妥钠阈上剂量(55 mg·kg^-1)所致的小鼠睡眠潜伏期和持续期，行为学中旷场实验的垂直得分和水平得分，以及高架十字迷宫的进入开放臂次数百分比(open arm entry，OE%)和开臂活动百分比(open arm time，OT%)；利用HE染色观察脑组织病理学情况；通过Elisa法检测各组小鼠血清中TNF-α和CASP3表达水平；最后利用Western blotting法检测各组小鼠脑组织中AKT1和p-AKT1蛋白的表达情况。结果 筛选获得酸枣仁和石菖蒲的潜在活性成分为9个和5个，与失眠共同靶点为34个；GO富集分析结果共得到160条GO分析条目，KEGG通路分析发现信号通路主要与炎症信号通路有关，其中AKT1、CASP3和TNF为关键靶点；分子对接结果显示筛选得到的化合物与关键靶点具有较高的结合活性。动物实验结果显示酸枣仁-石菖蒲药对可显著缩短失眠模型小鼠睡眠潜伏期，延长睡眠持续期，降低垂直得分和水平得分，提高OE%和OT%，恢复海马组织病理损伤，显著降低TNF-α和CASP3的含量，提高AKT1蛋白在脑组织中的表达水平。结论 酸枣仁-石菖蒲可通过作用于TNF-α、CASP3、AKT1、p-AKT1等靶点调节TNF信号通路发挥治疗失眠的作用。

Abstract:

OBJECTIVE to explore the material basis and potential molecular mechanism of Ziziphi Spinosae Semen- Acori Tatarinowii Rhizoma in the treatment of insomnia using network pharmacology and molecular docking, and establish insomnia mouse model by p-chlorophenylalanine(PCPA) for verification in vivo.METHODS Firstly, the chemical constituents of Ziziphi Spinosae Semen and Acori Tatarinowii Rhizoma were collected through TCMSP database and the potential active constituents were screened. The genes related of insomnia were obtained from GeneCards, OMIM and TTD databases, and the intersection targets of Ziziphi Spinosae Semen-Acori Tatarinowii Rhizoma and insomnia diseases were obtained. Then the network map of Chinese medicine-compound-target-disease was constructed in Cytoscape 3.6.1 software. The protein interaction network diagram was established in the STRING database. Functional enrichment analysis of target GO and KEGG pathways were performed using the DAVID database. Then the molecular docking technology was used for preliminary verification. The 60 male ICR mice were randomly divided into normal group, model group, high, medium and low dose groups(8.0, 4.0, 2.0 g·kg^-1) and diazepam group(3 mg·kg^-1). In addition to the normal group, PCPA(30 mg·mL^-1) was intraperitoneally injected on the first and second days to establish the insomnia model. Then the drug was administered continuously for 7 d, and the normal group and the model group were given the same volume of normal saline. The sleep latency and duration of mice induced by the upper threshold dose of pentobarbital sodium(55 mg·kg^-1), vertical and horizontal scores in the behavioral open-field experiment, open arm entry(OE%) and open arm time(OT%) of elevated cross maze were determined. HE staining was used to observe the hypothalamic histopathological situation. Serum levels of TNF-α and CASP3 were detected by ELISA. Finally, Western blotting was used to detect the expression of AKT1, p-AKT1 protein in the hypothalamus of the mice in each group. RESULTS The potential active components of Ziziphi Spinosae Semen and Acori Tatarinowii Rhizoma were 9 and 5, and the common targets with insomnia were 34. A total of 160 GO items were obtained through GO enrichment analysis. KEGG pathway analysis found that the signaling pathway was mainly related to inflammatory signaling pathway, among which AKT1, CASP3 and TNF were the key targets. The results of molecular docking showed that the selected compounds had high binding activity with the key targets. Animal experiment results showed that the Ziziphi Spinosae Semen-Acori Tatarinowii Rhizoma for insomnia could significantly shorten the model mice sleep latency, prolong sleep duration, reduce the vertical and horizontal score, improve the OE% and OT%, restore the hypothalamus pathological tissue damage, significantly reduce the content of TNF-α and CASP3, raise the level of AKT1 protein expression in the tissue of the hypothalamus. CONCLUSION Ziziphi Spinosae Semen-Acori Tatarinowii Rhizoma can regulate TNF signaling pathway by acting on TNF-α, CASP3, AKT1, p-AKT1 and other targets to treat insomnia.