Integrated UPLC/Q-TOF-MS/MS Analysis and Network Pharmacology to Reveal the Neuroprotective Mechanisms and Potential Pharmacological Ingredients of Aurantii Fructus Immaturus and Aurantii Fructus.

Aurantii Fructus (AF) and Aurantii Fructus Immaturus (AFI) have been used for thousands of years as traditional Chinese medicine (TCM) with sedative effects. Modern studies have shown that Citrus plants also have protective effects on the nervous system. However, the effective substances and mechanisms of action in Citrus TCMs still remain unclear. In order to explore the pharmacodynamic profiles of identified substances and the action mechanism of these herbs, a comprehensive approach combining ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS) analysis and network pharmacology was employed. Firstly, UNIFI 2.1.1 software was used to identify the chemical characteristics of AF and AFI. Secondly, the SwissTargetPrediction database was used to predict the targets of chemical components in AF and AFI. Targets for neuroprotection were also collected from GeneCards: The Human Gene Database (GeneCards-Human Genes|Gene Database|Gene Search). The networks between targets and compounds or diseases were then constructed using Cytoscape 3.9.1. Finally, the Annotation, Visualization and Integrated Discovery Database (DAVID) (DAVID Functional Annotation Bioinformatics Microarray Analysis) was used for GO and pathway enrichment analysis. The results showed that 50 of 188 compounds in AF and AFI may have neuroprotective biological activities. These activities are associated with the regulatory effects of related components on 146 important signaling pathways, derived from the KEGG (KEGG: Kyoto Encyclopedia of Genes and Genomes), such as neurodegeneration (hsa05022), the Alzheimer's disease pathway (hsa05010), the NF-kappa B signaling pathway (hsa04064), the hypoxia-inducible factor (HIF)-1 signaling pathway (hsa04066), apoptosis (hsa04210), the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance signaling pathway (hsa01521), and others, by targeting 108 proteins, including xanthine dehydrogenase (XDH), glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B), and glucose-6-phosphate dehydrogenase (G6PD), among others. These targets are thought to be related to inflammation, neural function and cell growth.

Modeling the precise interaction of glioblastoma with human brain region-specific organoids.

Glioblastoma is a highly aggressive malignant tumor of the central nervous system, but the interaction between glioblastoma and different types of neurons remains unclear. Here, we established a co-culture model in vitro using 3D printed molds with microchannels, in which glioblastoma organoids (GB), dorsal forebrain organoids (DO, mainly composed of excitatory neurons), and ventral forebrain organoids (VO, mainly composed of inhibitory neurons) were assembled. Our results indicate that DO has a greater impact on altered gene expression profiles of GB, resulting in increased invasive potential. GB cells preferentially invaded DO along axons, whereas this phenomenon was not observed in VO. Furthermore, GB cells selectively inhibited neurite outgrowth in DOs and reduced the expression of the vesicular GABA transporter (VGAT), leading to neuronal hyperexcitability. By revealing how glioblastoma interacts with brain cells, our study provides a more comprehensive understanding of this disease.

A Scientometric Study to a Critical Review on Promising Anticancer and Neuroprotective Compounds: Citrus Flavonoids.

Flavonoids derived from citrus plants are favored by phytomedicinal researchers due to their wide range of biological activities, and relevant studies have been sustained for 67 years (since the first paper published in 1955). In terms of a scientometric and critical review, the scientometrics of related papers, chemical structures, and pharmacological action of citrus flavonoids were comprehensively summarized. The modern pharmacological effects of citrus flavonoids are primarily focused on their anticancer activities (such as breast cancer, gastric cancer, lung cancer, and liver cancer), neuroprotective effects (such as anti-Alzheimer's disease, Parkinson's disease), and metabolic diseases. Furthermore, the therapeutic mechanism of cancers (including inducing apoptosis, inhibiting cell proliferation, and inhibiting cancer metastasis), neuroprotective effects (including antioxidant and anti-inflammatory), and metabolic diseases (such as non-alcoholic fatty liver disease, type 2 diabetes mellitus) were summarized and discussed. We anticipate that this review could provide an essential reference for anti-cancer and neuroprotective research of citrus flavonoids and provide researchers with a comprehensive understanding of citrus flavonoids.

Low-Density sEMG-Based Pattern Recognition of Unrelated Movements Rejection for Wrist Joint Rehabilitation.

sEMG-based pattern recognition commonly assumes a limited number of target categories, and the classifiers often predict each target category depending on probability. In wrist rehabilitation training, the patients may make movements that do not belong to the target category unconsciously. However, most pattern recognition methods can only identify limited patterns and are prone to be disturbed by abnormal movement, especially for wrist joint movements. To address the above the problem, a sEMG-based rejection method for unrelated movements is proposed to identify wrist joint unrelated movements using center loss. In this paper, the sEMG signal collected by the Myo armband is used as the input of the sEMG control method. First, the sEMG signal is processed by sliding signal window and image coding. Then, the CNN with center loss and softmax loss is used to describe the spatial information from the sEMG image to extract discriminative features and target movement recognition. Finally, the deep spatial information is used to train the AE to reject unrelated movements based on the reconstruction loss. The results show that the proposed method can realize the target movements recognition and reject unrelated movements with an F-score of 93.4% and a rejection accuracy of 95% when the recall is 0.9, which reveals the effectiveness of the proposed method.

Study on Active Components of Cuscuta chinensis Promoting Neural Stem Cells Proliferation: Bioassay-Guided Fractionation.

Neural stem cells (NSCs) exist in the central nervous system of adult animals and capable of self-replication. NSCs have two basic functions, namely the proliferation ability and the potential for multi-directional differentiation. In this study, based on the bioassay-guided fractionation, we aim to screen active components in Cuscuta chinensis to promote the proliferation of NSCs. CCK-8 assays were used as an active detection method to track the active components. On the basis of isolating active fraction and monomer compounds, the structures of these were identified by LC-MS and (1H, 13C) NMR. Moreover, active components were verified by pharmacodynamics and network pharmacology. The system solvent extraction method combined with the traditional isolation method were used to ensure that the fraction TSZE-EA-G6 of Cuscuta chinensis exhibited the highest activity. Seven chemical components were identified from the TSZE-EA-G6 fraction by UPLC-QE-Orbitrap-MS technology, which were 4-O-p-coumarinic acid, chlorogenic acid, 5-O-p-coumarinic acid, hyperoside, astragalin, isochlorogenic acid C, and quercetin-3-O-galactose-7-O-glucoside. Using different chromatographic techniques, five compounds were isolated in TSZE-EA-G6 and identified as kaempferol, kaempferol-3-O-glucoside (astragalin), quercetin-3-O-galactoside (hyperoside), chlorogenic acid, and sucrose. The activity study of these five compounds showed that the proliferation rate of kaempferol had the highest effects; at a certain concentration (25 µg/mL, 3.12 µg/mL), the proliferation rate could reach 87.44% and 59.59%, respectively. Furthermore, research results using network pharmacology techniques verified that kaempferol had an activity of promoting NSCs proliferation and the activity of flavonoid aglycones might be greater than that of flavonoid glycosides. In conclusion, this research shows that kaempferol is the active component in Cuscuta chinensis to promote the proliferation of NSCs.

Zebularine elevates STING expression and enhances cGAMP cancer immunotherapy in mice.

DNA methylation abnormality is closely related to tumor occurrence and development. Chemical inhibitors targeting DNA methyltransferase (DNMTis) have been used in treating cancer. However, the impact of DNMTis on antitumor immunity has not been well elucidated. In this study, we show that zebularine (a demethylating agent) treatment of cancer cells led to increased levels of interferon response in a cyclic guanosine monophosphate-AMP (cGAMP) synthase (cGAS)- and stimulator of interferon genes (STING)-dependent manner. This treatment also specifically sensitized the cGAS-STING pathway in response to DNA stimulation. Incorporation of zebularine into genomic DNA caused demethylation and elevated expression of a group of genes, including STING. Without causing DNA damage, zebularine led to accumulation of DNA species in the cytoplasm of treated cells. In syngeneic tumor models, administration of zebularine alone reduced tumor burden and extended mice survival. This effect synergized with cGAMP and immune checkpoint blockade therapy. The efficacy of zebularine was abolished in nude mice and in cGAS-/- or STING-/- mice, indicating its dependency on host immunity. Analysis of tumor cells indicates upregulation of interferon-stimulated genes (ISGs) following zebularine administration. Zebularine promoted infiltration of CD8 T cells and natural killer (NK) cells into tumor and therefore suppressed tumor growth. This study unveils the role of zebularine in sensitizing the cGAS-STING pathway to promote anti-tumor immunity and provides the foundation for further therapeutic development.

Inhibition of cGAS-Mediated Interferon Response Facilitates Transgene Expression.

DNA transfection is often the bottleneck of research and gene therapy practices. To explore the mechanism regulating transgene expression, we investigated the role of the cGAS-STING signaling pathway, which induces type-I interferons in response to DNA. We confirmed that deletion of cGAS enhances transgene expression at the protein level by ~2- to 3-fold. This enhancement is inversely correlated with the expression of interferons and interferon stimulated genes (ISGs), which suppress expression of transfected genes at the mRNA level. Mechanistically, DNA transfection activates the cGAS-STING pathway and induces the expression of the OAS family proteins, leading to the activation of RNaseL and degradation of mRNA derived from transgenes. Administration of chemical inhibitors that block cGAS-mediated signaling cascades improves the expression of transgenes by ~1.5- to 3-fold in multiple cell lines and primary cells, including T cells. These data suggest that targeting the cGAS-STING pathway can improve transgene expression, and this strategy may be applied to gene therapy.

Compound C Reducing Interferon Expression by Inhibiting cGAMP Accumulation.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a major DNA sensor responsible for cytosolic DNA-mediated innate immune response. Inhibition of cGAS may be an effective strategy for treating autoimmune diseases such as Aicardi-Goutieres syndrome and systemic lupus erythematosus. Compound C (also known as Dorsomorphin) has been annotated as a potent and reversible inhibitor for AMPKs as well as ALK protein kinases. Here, we report a new function of Compound C which can suppress dsDNA-dependent type I interferon induction. These effects were not dependent on the activities of AMPK proteins. In vitro assays and liquid chromatograph-mass spectrometry data show that Compound C has the capability of reducing cGAMP accumulation, suggesting that Compound C may function as a modulator involved in the cGAS-STING-mediated DNA sensing pathway. Furthermore, Compound C is able to rescue the autoimmune phenotypes in a mouse model carrying the Trex1 gene deficiency. These data demonstrate a new and inverse correlation between Compound C and type I interferon production in response to dsDNA signaling.

[Study on therapeutic mechanism of Rehmanniae Radix Praeparata- Corni Fructus in sequelae of ischemic stroke based on network pharmacology technology].

In ischemic stroke sequela phase, Rehmanniae Radix Praeparata-Corni Fructus drug pair has the effect in protecting damaged neurons, but its mechanism has not been clear. In this study, network pharmacology was used to predict the mechanism of Rehmanniae Radix Praeparata-Corni Fructus in the treatment of ischemic stroke sequela. Through database search and literature retrie-val, 40 active ingredients of Rehmanniae Radix Praeparata and Corni Fructus were obtained, and their targets were obtained through STITCH and TCMSP databases. The targets of ischemic stroke sequela were obtained through OMIM,GAD,TTD and DrugBank databases. By screening the intersections of active ingredients targets and stroke treatment targets, 21 potential targets were obtained. The DAVID database was used for GO enrichment analysis and KEGG pathway analysis of potential targets. GO enrichment analysis showed that Rehmanniae Radix Praeparata-Corni Fructus were mainly involved in regulation of blood pressure, negative regulation of extrinsic apoptotic signaling and positive regulation of angiogenesis. KEGG pathway analysis showed that Rehmanniae Radix Praeparata-Corni Fructus could inhibit inflammatory response and apoptosis signaling pathway by regulating HIF-VEGFA signaling pathway in neural stem cell proliferation, TNF signaling pathway and NF-kappaB signaling pathway. Molecular docking technique was used to verify that Rehmanniae Radix Praeparata-Corni Fructus component has a good binding activity with potential targets. The results showed that in ischemic stroke sequela phase, Rehmanniae Radix Praeparata-Corni Fructus drug pair could play an important role in recovering neural function, promoting the proliferation of neural stem cells, angiogenesis, preventing neural cells apoptosis and regulating inflammatory factors.

A modified bisulfite conversion method for the detection of DNA methylation.

AIM: Our purpose is to improve the conventional procedures for bisulfite conversion used to detect 5-methylcytosine in DNA. METHODS: Impacts of different bisulfite salts, bisulfite conversion temperature, antioxidants and denaturants on DNA conversion and degradation were assessed by methylation-sensitive melt curve analysis. The modified method was tested on different genes and the conversion efficiency was analyzed by bisulfite sequencing. RESULTS: We developed a modified bisulfite conversion method that completes this process within 2 h. We demonstrate that high temperature denaturation is the major cause for DNA degradation, and the addition of ethylene glycol dimethyl ether is an effective way to accelerate the bisulfite conversion. The conversion efficiency is comparable to many other commercial kits. CONCLUSION: Our modified bisulfite conversion method is simple, cost efficient and less time consuming and is compatible with different genes and samples, thus has a great potential for the future research and clinical applications.

cGAS is essential for cellular senescence.

Cellular senescence is a natural barrier to tumorigenesis and it contributes to the antitumor effects of several therapies, including radiation and chemotherapeutic drugs. Senescence also plays an important role in aging, fibrosis, and tissue repair. The DNA damage response is a key event leading to senescence, which is characterized by the senescence-associated secretory phenotype (SASP) that includes expression of inflammatory cytokines. Here we show that cGMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor that activates innate immunity, is essential for senescence. Deletion of cGAS accelerated the spontaneous immortalization of mouse embryonic fibroblasts. cGAS deletion also abrogated SASP induced by spontaneous immortalization or DNA damaging agents, including radiation and etoposide. cGAS is localized in the cytoplasm of nondividing cells but enters the nucleus and associates with chromatin DNA during mitosis in proliferating cells. DNA damage leads to accumulation of damaged DNA in cytoplasmic foci that contain cGAS. In human lung adenocarcinoma patients, low expression of cGAS is correlated with poor survival. These results indicate that cGAS mediates cellular senescence and retards immortalization. This is distinct from, and complementary to, the role of cGAS in activating antitumor immunity.

The relationship between DNA methylation and Reprimo gene expression in gastric cancer cells.

Reprimo (RPRM) is a tumor suppressor involved in the development of a number of malignant tumors including gastric cancer which is highly related to its gene hypermethylation. However, the regulation of RPRM gene expression by DNA methylation in gastric cancer is not well understood. We examined the RPRM gene methylation in gastric cancer tissues or plasma samples by bisulfite sequencing, and investigated the relationship between DNA methylation and the RPRM gene expression by quantitative reverse transcription-PCR and Western blotting. We found that the RPRM gene promoter region is hypermethylated in gastric cancer tissues (75%, 45/60), plasma samples (86.3%, 44/51) and various cancer cell lines (75%, 3/4), which is correlated with the decrease of RPRM gene expression. The hypermethylation-induced RPRM reduction can be recovered by treating with zebularine, a demethylating agent, and by inhibition of the DNA methyltransferases via RNA interference and CRISPR/Cas9-mediated gene knockout. In addition, we generated RPRM gene-knockout cells and studied the effects of the RPRM deficiency on tumor formation by inoculating these cells in mice. The data show that the loss of RPRM can promote tumorigenesis. These data suggest that the RPRM expression is inhibited by DNA methyltransferases and the RPRM normal function can be restored by treating with DNA methylation inhibitors. The study provides important information regarding the role of RPRM and its methylation related to gastric cancer development.

Methylation-Sensitive Melt Curve Analysis of the Reprimo Gene Methylation in Gastric Cancer.

Reprimo (RPRM) is a p53-induced tumor suppressor gene. Its aberrant DNA methylation is correlated with carcinogenesis and may be used as a surrogate marker for the early detection of gastric cancer. However, the detail information regarding its DNA methylation has not been revealed. Here, we investigated the RPRM gene methylation in gastric cancer tumor and plasma samples by methylation-sensitive melt curve analysis (MS-MCA) and bisulfite sequencing in depth. We developed a semi-quantitative method based on MS-MCA for detecting DNA methylation and unraveled the RPRM gene methylation pattern in gastric cancer. This study provides a solid foundation for the future application of detecting RPRM gene methylation in human plasma or serum samples to help diagnose gastric cancer or for prognosis evaluation.