Research on the Material Basis and Mechanism of Kudzu Root in Preventing and Treating Cerebral Ischemia based on Network Pharmacology.

BACKGROUND: It has been shown that Kudzu root has significant pharmacological effects such as improving microcirculation, dilating coronary arteries, and increasing cerebral and coronary blood flow, but its material basis and mechanism of action are not clear. OBJECTIVE: The aim of this study was to investigate the mechanism of action of Kudzu root in the prevention and treatment of cerebral ischemia (CI) through network pharmacology combined with animal experiments. METHODS: The components of kudzu root were screened by using the Chemistry Database, Chinese Academy of Science. Linpinski's five rules were used to perform pharmacophore-like analysis to obtain the active ingredients of Kudzu root. The Swiss Target Prediction Service database was used to predict the potential protein targets of kudzu root components associated with CI. An active ingredient-target network was constructed by using Cytoscape 3.6.0. A rat model of middle cerebral artery occlusion (MCAO) was established, then the main targets and signaling pathways predicted were verified by observing the area of cerebral infarction and Western blot experiments. RESULTS: In total, 84 major active compounds and 34 targets included gerberoside, belonging to the isoflavone class, gallic acid, amino acid class, 4-Methylphenol, phenolic class, and quercetin, and flavonoid class (Flavonoids). The targets covered were proteins related to excitatory amino acids and calcium overload, including Excitatory amino acid transporter 2 (SLC1A2), Glutamate receptor ionotropic, kainate 1 (GRIK1), Glutamate receptor ionotropic, NMDA 1 (GRIN1), Glutamate receptor 2(GRIA2), Calcium/calmodulin-dependent protein kinase II (CaMKII), Neuronal nitric oxide synthase(nNOS). Glutamatergic energy is prominent, and calcium transport across the membrane is central to the network and occupies an important position. CONCLUSION: Kudzu root can significantly reduce neurological damage in rats with CI, and also significantly reduce the rate of cerebral infarction. It is worth noting that Kudzu root can prevent and treat CI by reducing excitatory amino acid toxicity and improving calcium overload.

[Different processed products of Polygonati Rhizoma treat Alzheimer's disease in rats: urine metabolomics based on UPLC-Q/TOF-MS].

The study investigated the effects of different processed products of Polygonati Rhizoma(black bean-processed Polygonati Rhizoma, BBPR; stewed Polygonati Rhizoma, SPR) on the urinary metabolites in a rat model of Alzheimer's disease(AD). Sixty SPF-grade male SD rats were randomized into a control group, a model group, a donepezil group, a BBPR group, and a SPR group, with twelve rats in each group. Other groups except the control group were administrated with D-galactose injection(100 mg·kg~(-1)) once a day for seven weeks. The control group was administrated with an equal volume of normal saline once a day for seven consecutive weeks. After three weeks of D-galactose injection, bilateral hippocampal Aß_(25-35) injections were performed for modeling. The rats were administrated with corresponding drugs(10 mL·kg~(-1)) by gavage since week 2, and the rats in the model and control group with an equal volume of double distilled water once a day for 35 continuous days. The memory behaviour and pathological changes in the hippocampal tissue were observed. The untargeted metabolites in the urine were detected by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q/TOF-MS). Principal component analysis(PCA) and orthogonal partial least square-discriminant analysis(OPLS-DA) were employed to characterize and screen differential metabolites and potential biomarkers, for which the metabolic pathway enrichment analysis was conducted. The results indicated that BBPR and SPR increased the new object recognition index, shortened the escape latency, and increased the times of crossing the platform of AD rats in the Morris water maze test. The results of hematoxylin-eosin(HE) staining showed that the cells in the hippocampal tissue of the drug administration groups were closely arranged. Moreover, the drugs reduced the content of interleukin-6(IL-6, P<0.01) and tumor necrosis factor-α(TNF-α) in the hippocampal tissue, which were more obvious in the BBPR group(P<0.05). After screening, 15 potential biomarkers were identified, involving two metabolic pathways: dicoumarol pathway and piroxicam pathway. BBPR and SPR may alleviate AD by regulating the metabolism of dicoumarol and piroxicam.

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

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 Aß and ER_ß 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 Aß-positive cells(P<0.05), declining expression of ER_ß-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 Aß-positive cells, and elevated expression of ER_ß-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.

Evidence-based complementary and alternative medicine bioinformatics approach through network pharmacology and molecular docking to determine the molecular mechanisms of Erjing pill in Alzheimer's disease.

Erjing pill, a Traditional Chinese Medicine (TCM) formulation composed of Polygonatum sibiricum and Lycium chinense, has an important role in the treatment of Alzheimer's disease (AD). However, the underlying mechanisms of the action of Erjing pill in AD have remained elusive. In the present study, the key ingredients of Erjing pill were investigated and the active components and their mechanisms of action on AD were analyzed based on networks pharmacology. By using the TCM and TCM Systems Pharmacology and databases, the components of Erjing pill were screened and the data were captured using Discovery Studio. The SwissTarget webserver database was used to predict the potential protein targets of Erjing pill components for pathologies related to AD. The data were further analyzed with the disease targets of AD based on analysis of the Online Mendelian Inheritance in Man, DiGSeE and Therapeutic Target Database. Subsequent analysis of mechanistic pathways of the screened components and protein targets allowed us to construct a network by using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, which revealed potential molecular mechanisms of Erjing pill against AD. Finally, the protective effect of three active components on neurons was verified using an in vitro injury model of PC12 cells induced by Aß25-35. The results indicated that 65 bioactive components of Erjing pill, including lauric acid and zederone, and 6 targets, including acetylcholinesterase, butylcholinesterase and amyloid protein precursor, were closely associated with the prevention and treatment of AD. The molecular components of Erjing pill were indicated to be involved in various biological signaling processes, mainly in synaptic signal transmission, transsynaptic signal transmission and chemical synaptic transmission. Furthermore, related pathways targeted by Erjing pill in AD included the regulation of neuroactive ligand-receptor interactions, the PI3K-Akt signaling pathway, serotoninergic synapses, calcium signaling pathways and dopaminergic synapses. A cell viability assay indicated that the compounds (polygonatine A, polygonatine C and 4',5-dihydroxyflavone) assessed were able to significantly improve the survival rate and increase the Ca2+ level in a PC12 cell model of AD induced by amyloid-ß25-35. The present study revealed that the mechanisms of action of Erjing pill to prevent and treat AD included a multicompound, multitarget and multipathway regulatory network.

Network pharmacology-based strategy to investigate pharmacological mechanisms of Tinospora sinensis for treatment of Alzheimer's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora sinensis (Lour.) Merr. belongs to the family Menispermaceae. It is called LeZhe and is widely used as a kind of folk medicine especially in the Tibetan Plateau of China. T. sinensis has the functions of clearing away heat and detoxification, dispelling wind and dredging collaterals, calming and soothing the nerves. T. sinensis is an effective medicine for the prevention and treatment of aging diseases such as Alzheimer's disease (AD) in the Tibetan Plateau of China, whereas its material basis and underlying mechanisms are not clear. The aim of this study was to investigate the material basis and potential mechanisms of T. sinensis in the treatment of AD by using network pharmacology and molecular docking. MATERIALS AND METHODS: In this study, targets were collected from DrugBank database, Therapeutic Target Database (TTD) and literatures reports for the treatment of AD. Compounds were searched by literatures and systematic separation from T. sinensis. The molecular docking experiment was carried out by using Autodock Vina software to screen the bioactive compounds in T. sinensis and target proteins for AD. Then, the "compound-target network" was constructed by Cytoscape software. The drug-like properties of the active compounds were analyzed by pKCSM performs, and the protein-protein interaction (PPI) network was constructed by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). The Kyoto Encyclopedia of Genes and Genomes (KEGG) target pathway enrichment analysis was carried out by Database for Annotation, Visualization and Integrated Discovery (DAVID). Furthermore, the protective effect of neurons of two active compounds were verified with the injury cell model of PC12 and primary hippocampus neurons induced by Aß25-35. Finally, the key proteins of related pathways were quantitatively analyzed with Western blot method. RESULTS: In total, 105 compounds and 38 targets have been screened. The main active compounds contained berberine, which belongs to alkaloids, Aurantiamide acetate, N-P-coumaroyltyramine, which belongs to amides, Trans-syringin and 3-demethyl-phillyrin, which belongs to phenylpropanoids. The targets covered inflammation-related proteins, including Protein kinase B (AKT), Phosphoinositide 3-kinase (PI3K), Tyrosine-protein kinase JAK1 (JAK1), mammalian target of rapamycin (mTOR), tumor necrosis factor alpha (TNF-α), Neuronal NOS (NOS1), and cholinergic function-related proteins, including α4-Nicotinic acetylcholine receptor (α4 nAChR), Muscarinic acetylcholine receptor M1 (Muscarnic M1). Inflammation and cholinergic dysfunction were the center of the network and occupy a dominant position. And the results of enrichment analysis shown the pathways mainly contained phosphoinositide-3-kinase/Akt (PI3K/Akt) signal pathway, neurotrophic factors (NTFs) signal pathway, Hypoxia-inducible factor 1 (HIF-1) signal pathway, mechanistic Target of Rapamycin (mTOR) signal pathway, Tumor necrosis factor (TNF) signal pathway, insulin resistance (IR). The results of in vitro assays showed that the tested compounds could significantly improve the survival rate and inhibit the apoptosis of PC12 cells and primary hippocampal neurons injured by Aß25-35. Western blot results showed that T. sinensis had a significant effect on the expression of protein PI3K and Akt. CONCLUSION: Our results revealed that T. sinensis could prevent and treat AD through a multi-compound-multi-target-multi-pathway regulatory network. Our work also expected to provide new ideas and theoretical bases for searching for the active compounds in T. sinensis and potential mechanism in the prevention and treatment of AD by the network pharmacology and molecular docking. The results of in vitro assay and in vivo assay supported the results of molecular docking.