Identification of in vitro-in vivo components of Caoguo using accelerated solvent extraction combined with gas chromatography-mass spectrometry integrated with network pharmacology on indigestion.

BACKGROUND: Caoguo (Tsaoko Fructus), a traditional Chinese medicine, is widely used as medicine and dietary spices. Volatile components are among its important bioactive constituents used to treatment of abdominal distension and pain, but the mechanism is not clear up to now. The purpose of this study was to develop a simple, sensitive, and accurate method to analyze and identify components of Caoguo in vitro and in vivo, and further investigate the therapeutic mechanism of Caoguo on indigestion using network pharmacology. METHODS: Caoguo were extracted by accelerated solvent extraction (ASE) and n-hexane:ethyl acetate (1:1, v/v) was selected as the extraction solvent. Gas chromatography-mass spectrometry (GC-MS) was adopted to analyze and identify the volatile components in vitro and in vivo. Network pharmacology including protein-protein network construction, Gene Ontology (GO) enrichment and pathway enrichment analysis and component-target-pathway network construction was applied. RESULTS: By comparing the retention times and mass spectrometry data, as well as retrieving the reference literature, a total of 169 components were tentatively identified in Caoguo extract and 43 components were identified in rats plasma samples for the first time. The results of network pharmacology analysis indicated that the potential mechanism was mainly associated with regulation of lipolysis in adipocytes and serotonergic synapse signaling pathway, which might be responsible for the effect of indigestion. CONCLUSIONS: Caoguo was first extracted by ASE and the volatile chemical components in vivo were first identified by GC-MS. Coupled with network pharmacology analysis, a network of component-target-pathway was constructed to reveal the possible mechanism of Caoguo in treatment of indigestion. This study provided a new reference method for the extraction and analysis of Caoguo, laid a chemical basis for in-depth studies on pharmacodynamics and pharmacology, and revealed an updated understanding of the therapeutic effects of Caoguo on indigestion.

Identification and Validation of a Four-Long Non-coding RNA Signature Associated With Immune Infiltration and Prognosis in Colon Cancer.

The emerging evidence has demonstrated the critical roles of long non-coding RNAs (lncRNAs) as regulators in the tumor immune microenvironment (TIME). However, the tumor immune infiltration-associated lncRNAs and their clinical significance in colon cancer have not yet been thoroughly investigated. This study performed an integrative analysis of lncRNA expression profiles and immune cell infiltration profiles and identified 258 immune infiltration-associated lncRNAs. Of them, four lncRNAs (AC008494.3, LINC00926, AC022034.1, and SNHG26) were significantly and independently associated with the patient's overall survival. Finally, we developed a tumor immune infiltration-associated lncRNA signature (TIILncSig) comprising of these four lncRNAs, which can divide colon cancer patients of The Cancer Genome Atlas (TCGA) into high-risk and low-risk groups with a significantly different outcome [Hazard ratio (HR) = 2.718, 95% CI = 1.955-3.779, p < 0.001]. Prognostic performance of the TIILncSig was further validated in another independent colon cancer cohort (HR = 1.832, 95% CI = 1.045-3.21, p = 0.034). Results of multivariate Cox regression and stratification analysis demonstrated that the TIILncSig is an independent predictive factor from other clinical features (HR = 2.687, 95% CI = 1.912-3.776, p < 0.001 for TCGA cohort and HR = 1.837, 95% CI = 1.047-3.223, p = 0.034 for GSE17538 cohort). Literature analysis provided experimental evidence supporting roles of the TIILncSig in cancer carcinogenesis and progression and immune regulation. Summary, our study will help to understand the mechanisms of lncRNAs in immune regulation in the tumor microenvironment and provide novel biomarkers or targets for prognosis prediction and therapy decision-making for patients with colon cancer.

Silencing of circ_0078607 prevents development of gastric cancer and inactivates the ERK1/2/AKT pathway through the miR-188-3p/RAP1B axis.

The aim of this study is to explore the expression and mechanism of circ_0078607 on proliferation and apoptosis of gastric cancer. Real time PCR (RT-PCR) was performed to detect the expression of circ_0078607 in gastric cancer tumor tissues, plasma and cell lines. Cell viability was detected by cell counting Kit-8. Cell proliferation ability was assessed by cell cycle assay. The samples were analyzed by flow cytometry for the detection of apoptosis. Luciferase assay and RNA immunoprecipitation (RIP) were carried out to verify the relationship between circ_0078607 and miR-188-3p, miR-188-3p, and RAP1B. Western blot was employed to detect the protein level of RAP1B, ERK1/2 and AKT. In vivo, the effect of circ_0078607 on gastric cancer tumor growth was detected by lentivirus vector injection. Here, we found the increased level of circ_0078607 in gastric cancer tissues, gastric cancer patients plasma and cell lines. Knockdown of circ_0078607 could prevent proliferation and induce cell apoptosis in MKN-28 cells. Then we verified that circ_0078607 could interact with miR-188-3p by performed luciferase assay and RIP. Furthermore, we observed that RAP1B was a potential target of miR-188-3p. Next, we found that miR-188-3p inhibitor or overexpression of RAP1B could prevent the anti-tumor function of sh-circ_0078607. Silencing of circ_0078607 inhibited ERK1/2/AKT signal pathways via regulating miR-188-3p/RAP1B. In vivo, knockdown of circ_0078607 inhibited tumor growth. Knockdown of circ_0078607 inhibits the proliferation and induces apoptosis of gastric cancer via miR-188-3p/RAP1B signal pathway.

Toll-Like Receptors Recognize Intestinal Microbes in Liver Cirrhosis.

Liver cirrhosis is one major cause of mortality in the clinic, and treatment of this disease is an arduous task. The scenario will be even getting worse with increasing alcohol consumption and obesity in the current lifestyle. To date, we have no medicines to cure cirrhosis. Although many etiologies are associated with cirrhosis, abnormal intestinal microbe flora (termed dysbiosis) is a common feature in cirrhosis regardless of the causes. Toll-like receptors (TLRs), one evolutional conserved family of pattern recognition receptors in the innate immune systems, play a central role in maintaining the homeostasis of intestinal microbiota and inducing immune responses by recognizing both commensal and pathogenic microbes. Remarkably, recent studies found that correction of intestinal flora imbalance could change the progress of liver cirrhosis. Therefore, correction of intestinal dysbiosis and targeting TLRs can provide novel and promising strategies in the treatment of liver cirrhosis. Here we summarize the recent advances in the related topics. Investigating the relationship among innate immunity TLRs, intestinal flora disorders, and liver cirrhosis and exploring the underlying regulatory mechanisms will assuredly have a bright future for both basic and clinical research.

Identification of a TLR-Induced Four-lncRNA Signature as a Novel Prognostic Biomarker in Esophageal Carcinoma.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of Toll-like receptor (TLR) signaling to control innate immunity, and this regulatory mechanism has recently been implicated in esophageal carcinoma (ESCA). However, a comprehensive analysis of TLR-induced lncRNAs and their roles in diagnosis and prognosis in ESCA is still lacking. In this study, we first investigated the precise relationship between lncRNA perturbations and alteration of TLR signaling by constructing the lncRNA-TLRs co-expression network involved in ESCA, and identified 357 TLR-related lncRNAs. Of them, four TLR-related lncRNAs (AP000696.1, LINC00689, LINC00900, and AP000487.1) are significantly associated with the overall survival (OS) of ESCA patients, and utilizing this four-lncRNA signature is capable of stratifying patients into high-risk and low-risk groups with significantly different OS in the discovery set. Further analysis in different independent patient sets also confirmed the robustness of the prognostic value of the four-TLR-lncRNA signature in predicting the OS of ESCA patients. Moreover, the results of multivariate analysis in different patient sets indicated that the four-TLR-lncRNA signature is an independent factor after adjusted by other clinical factors. Thus, we have identified a TLR-induced four-lncRNA signature, which represents a promising prognosis biomarker for ESCA, and our study might provide new candidate targets for therapeutic intervention via targeting TLR-induced lncRNAs in ESCA patients.

Characterization of lncRNA-Perturbed TLR-Signaling Network Identifies Novel lncRNA Prognostic Biomarkers in Colorectal Cancer.

Increasing evidence has suggested that long non-coding RNAs (lncRNAs) are critical regulators in the Toll-like receptors (TLR)-signaling network to modulate colorectal cancer (CRC) development and progression. However, the mechanism and clinical significance for lncRNAs regulating TLR signaling pathways in CRC remained largely unknown. In this study, we performed an integrative network analysis of transcriptomics by focusing on a lncRNA-perturbed TLR-signaling network, identifying 280 lncRNAs and 122 mRNAs. We found a profound phenomenon that abnormal expression of some lncRNAs can perturb the TLR-signaling network to contribute to CRC development and progression. Furthermore, we identified a novel TLR-related prognostic gene signature (TLRLncSig) composed of three lncRNAs (MCHR2, AC011472.4, and AC063944.1), and one mRNA (CDKN2B). Utilizing TLRLncSig could classify CRC patients of training set into two groups with significantly different overall survival. The prognostic value of the TLRLncSig was further validated in the other two independent CRC datasets with different platforms. Results of multivariate and stratification analysis indicated that the TLRLncSig is an independent prognostic factor, and our study underscores the clinical significance of TLR-related lncRNAs in CRC development and progression.

Pharmacokinetic-pharmacodynamic modeling to study the anti-dysmenorrhea effect of Guizhi Fuling capsule on primary dysmenorrhea rats.

BACKGROUND: Primary dysmenorrhea (PDM) is one of the most common gynaecological disorders among women, which seriously affects women's life quality due to its high incidence rate. Guizhi Fuling capsule (GZFLC), a well-known traditional Chinese medical prescription, has been widely used to treat gynecological blood stasis syndromes such as PDM. However, its mechanisms of action and combination were still unknown. PURPOSE: The aim of this study was to develop a pharmacokinetic-pharmacodynamic (PK-PD) model to assess time-concentration-effect relationships for anti-dysmenorrhea effect of GZFLC and provide better understanding for mechanisms of action and combination of GZFLC. STUDY DESIGN AND METHODS: The PDM rats model was induced by oxytocin exposure following estradiol benzoate pretreatment. Gallic acid (GA), amygdalin (AMY), albiflorin (ALB), prunasin (PA) and cinnamic acid (CA) were evaluated as bioactive ingredients for investigating PK processes. GA, AMY, ALB and PA exhibited appropriate PK parameters and were selected as the PK markers to map the anti-dysmenorrhea effect of GZFLC. A PK-PD model was established on the basis of GA, AMY, ALB and PA plasma concentrations vs. the values of two ratios (PGE2/PGF2α and 6-Keto-PGF1α/TXB2), by a two-compartment PK model with a simple Emax model to explain the time delay between the drug plasma concentrations of PK markers and the anti-dysmenorrhea effect. RESULTS: The PDM rat model has been successfully established. Compared with the normal treated group, the bioactive ingredients in PDM treated group exhibited significant changing trends of PK behaviors, such as better absorption and distribution, slower elimination and delays in reaching the maximum concentration (Tmax). The analysis of PK-PD parameters indicated that the active metabolites and prototypes of bioactive ingredients in GZFLC were inclined to regulate the activity of prostacyclin synthetase and thromboxane synthetase to control the production of TXA2 and PGI2 so as to treat PDM. As the main effective medicinal materials for the treatment of PDM in GZFLC prescription Persicae Semen, Moutan Cortex and Paeonia lactiflora Pall, Persicae Semen played the most important role, while the role of Paeonia lactiflora Pall was the weakest. CONCLUSION: The PK-PD model results provided scientific basis for clarifying compatibility mechanisms of GZFLC prescription and a better understanding for biosynthetic mechanisms of four prostaglandins (PGE2, PGF2α, 6-Keto-PGF1α and TXB2) in the treatment of PDM by GZFLC. Investigations on the relationship between the effects and the bioactive ingredients are of benefit to explore the mechanisms of action and combination for traditional Chinese medical prescriptions (TCP) and facilitate the development of future clinical applications of TCP.

Investigation of potential toxic components based on the identification of Genkwa Flos chemical constituents and their metabolites by high-performance liquid chromatography coupled with a Q Exactive high-resolution benchtop quadrupole Orbitrap mass spectrometer.

Genkwa Flos, a famous traditional Chinese medicine has been reported to have significant hepatotoxicity. A high-throughput and reliable method was established to explore potential toxic components by high-performance liquid chromatography coupled with a Q Exactive high-performance benchtop quadrupole-Orbitrap mass spectrometer. A total of 68 compounds including 22 chemical components and 46 metabolites were tentatively identified based on the accurately measured mass value, retention time, and fragmentation pattern. Besides, the metabolic pathways of main components in Genkwa Flos were also illustrated. The results indicated that hydroxylation, demethylation, methylation, glucuronidation, sulfation, cysteine conjugation, and glutathione conjugation participated in the metabolic reactions of Genkwa Flos. Moreover, 12 Genkwa Flos chemical components and 26 metabolites were detected in cell lysate, which were considered as the bound components to HL-7702 cells. In view of cell affinity theory, these compounds were preliminarily deduced to be potential toxic ingredients for the hepatotoxicity induced by Genkwa Flos. The results demonstrated that the developed method was a very feasible and efficient approach for the components identification even in the complex matrix. In conclusion, this study will provide a deep insight into the toxic substances of Genkwa Flos and lay a chemical basis for in-depth toxic studies on Genkwa Flos hepatotoxicity.

Prophylactic Neuroprotection of Total Glucosides of Paeoniae Radix Alba against Semen Strychni-Induced Neurotoxicity in Rats: Suppressing Oxidative Stress and Reducing the Absorption of Toxic Components.

Strychnos alkaloids (SAs) are the main toxic constituents in Semen Strychni, a traditional Chinese medicine, which is known for its fatal neurotoxicity. Hence, the present study was carried out to evaluate the neurotoxicity induced by SAs and the pre-protective effects of the total glucosides of Paeoniae Radix Alba (TGP). An SA brain damage model was firstly established. The neurotoxicity induced by SAs and the pre-protective effects of TGP were confirmed by physical and behavioral testing, biochemical assay, and histological examination. Then, a liquid chromatography-tandem mass spectrometry method was developed and validated to investigate the time-course change and distribution of strychnine and brucine (two main SAs) in the brain after oral SA administration with or without TGP pretreatment. Biochemical analysis results indicated that TGP could ameliorate the oxidative stress status caused by SAs. Time-course change and distribution studies demonstrated that strychnine and brucine were rapidly absorbed into the brain, peaked early at 0.5 h, and were mainly located in the hippocampus and cerebellum. TGP showed a pre-protective effect against neurotoxicity by reducing the absorption of toxic alkaloids into the brain. These findings could provide beneficial information in facilitating future studies of Semen Strychni neurotoxicity and developing herbal medicines to alleviate neurotoxicity in the clinic.

Investigation of metabolic profile of pimavanserin in rats by ultrahigh-performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry.

RATIONALE: Pimavanserin, a selective serotonin 2A receptor inverse agonist, is a promising candidate for treating Parkinson's disease psychosis. Our previous study revealed that there might be the presence of extensive metabolites of pimavanserin in rats. However, the metabolic fate of pimavanserin in vivo remains unknown. Thus, it is essential to develop an efficient method to investigate the metabolic profile of pimavanserin in rats. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to date has the highest mass measurement accuracy and resolution of any mass spectrometry platform. METHODS: After a single intragastric administration of pimavanserin at a dose of 50 mg kg-1 , plasma, bile, urine and feces were collected from rats. A novel and efficient strategy was developed to analyze the metabolic profile of pimavanserin in vivo based on ultrahigh-performance liquid chromatography (UHPLC) coupled with FT-ICR-MS. RESULTS: A total of 23 metabolites were detected and tentatively identified through comparing their mass spectrometry profiles with those of pimavanserin. These metabolites were found in feces (22), bile (21), rat urine (16) and plasma (15). Results demonstrated that metabolic pathways of pimavanserin in rats included dehydrogenation, demethylation, deethylation, depropylation, debutylation, hydroxylation, dihydroxylation and trihydroxylation. CONCLUSIONS: A total of 22 phase I metabolites of pimavanserin were detected and tentatively identified. This report presents the first study of screening and identification of the metabolites of pimavanserin. The UHPLC/FT-ICR-MS method is a powerful tool for exploring and identifying metabolites in complex biological samples.

Granulocyte colony-stimulating factor enhances the therapeutic efficacy of bone marrow mesenchymal stem cell transplantation in rats with experimental acute pancreatitis.

INTRODUCTION: Acute pancreatitis (AP) is one of the most common diseases involving necrotic inflammation. Bone marrow mesenchymal stem cells (BMMSCs) have the potential of multi-directional differentiation and self-renewal for tissue repair. It remains less clear if granulocyte colony-stimulating factor (G-CSF) can improve the therapeutic effect of BMMSC transplant in AP. Therefore, we explored this issue in a rat model of experimental AP. RESULTS: Transplanted PKH26-positive BMMSCs were present in the injured pancreatic tissue, with some cells co-expressed pancreatic cellular markers, including Pax-4, Ngn3 and Nkx-6. Pathological, biochemical and serological data suggested an improvement in histological and functional recovery in these animals relative to control. Overall, the AP model rats received BMMSCs and G-CSF co-treatment showed better recovery in terms of tissue regeneration and blood biochemical levels relative to other groups. MATERIALS AND METHODS: BMMSCs from donor rats were labeled with the fluorescent dye PKH26 and transfused into recipient rats with AP induced by L-arginine. The animals were divided into a control group, and groups treated with BMMSCs, G-CSF, and BMMSCs together with G-CSF. Therapeutic effects were evaluated histologically with immunohistochemistry and immunofluorescence, together with biochemical measurement of pancreatic markers. CONCLUSION: G-CSF therapy with BMMSC transplantation improves histological and functional outcomes in rats with experimental AP.

Surface modification via strain-promoted click reaction facilitates targeted lentiviral transduction.

As a result of their ability to integrate into the genome of both dividing and non-dividing cells, lentiviruses have emerged as a promising vector for gene delivery. Targeted gene transduction of specific cells and tissues by lentiviral vectors has been a major goal, which has proven difficult to achieve. We report a novel targeting protocol that relies on the chemoselective attachment of cancer specific ligands to unnatural glycans on lentiviral surfaces. This strategy exhibits minimal perturbation on virus physiology and demonstrates remarkable flexibility. It allows for targeting but can be more broadly useful with applications such as vector purification and immunomodulation.

Chemoselective modification of viral surfaces via bioorthogonal click chemistry.

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development. Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction. Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible. The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically or analytically, as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness, (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access. In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only. In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein - strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Magnetic resonance imaging of nonaqueous phase liquid during soil vapor extraction in heterogeneous porous media.

Soil vapor extraction (SVE) is commonly used to remediate nonaqueous phase liquids (NAPLs) from the vadose zone. This paper aims to determine the effect of grain size heterogeneity on the removal of NAPL in porous media during SVE. Magnetic resonance imaging (MRI) was used to observe and quantify the amount and location of NAPL in flow-through columns filled with silica gel grains. MRI is unique because it is nondestructive, allowing three-dimensional images to be taken of the phases as a function of space and time. Columns were packed with silica gel in three ways: coarse grains (250-550 microm) only, fine grains (32-63 microm) only, and a core of fine grains surrounded by a shell of coarse grains. Columns saturated with water were drained under a constant suction head, contaminated with decane, and then drained to different decane saturations. Each column was then continuously purged with water-saturated nitrogen gas and images were taken intermittently. Results showed that at residual saturation, a sharp volatilization front moved through the columns filled with either coarse-grain or fine-grain silica gel. In the heterogeneous columns, the volatilization front in the core lagged just behind the shell because gas flow was greater through the shell and decane in the core diffused outward to the shell. When decane saturation in the core was above residual saturation, decane volatilization occurred near the inlet, the relative decane saturation throughout the core dropped uniformly, and decane in the core flowed in the liquid phase to the shell to replenish volatilized decane. These results indicate that NAPL trapped in low-permeability zones can flow to replenish areas where NAPL is lost due to SVE. However, when residual NAPL saturation is reached, NAPL flow no longer occurs and diffusion limits removal from low-permeability zones.

Effect of soil properties on saturated and unsaturated virus transport through columns.

Viruses from contaminant sources can be transported through porous media to drinking water wells. The objective of this study was to investigate inactivation and sorption of viruses during saturated and unsaturated transport in different soils. Bacteriophages phiX174 and MS-2, and Br- tracer in a phosphate-buffered saline solution were introduced into saturated and unsaturated soil columns as a step function under constant flow rate and hydraulic conditions. Results showed that significantly greater virus removal occurred in the unsaturated columns than in the saturated columns in the two soils containing high metal oxides content. However, the increase in virus retention under unsaturated conditions was not significant in two other soils having high phosphorus and calcium contents and high pH, and in another soil with high organic matter content. The results imply that the extent of water content effect on inactivation and sorption of viruses can range from significant to minimal depending on the properties of the transport medium. We found that the presence of in situ metal oxides was a significant factor responsible for virus sorption and inactivation. Therefore, soils with high metal oxides content may have the potential to be used as hydrological barriers in preventing microbial contamination in the subsurface environments. We also found that the water content effect on virus removal and inactivation strongly depended on solid properties of the testing medium.