ABSTRACT
Vasoplegia observed post cardiopulmonary bypass (CPB) is associated with substantial morbidity, multiple organ failure and mortality. Circulating counts of hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPC) are potential markers of neo-vascularization and vascular repair. However, the significance of changes in the circulating levels of these progenitors in perioperative CPB, and their association with post-CPB vasoplegia, are currently unexplored. We enumerated HSC and EPC counts, via flow cytometry, at different time-points during CPB in 19 individuals who underwent elective cardiac surgery. These 19 individuals were categorized into two groups based on severity of post-operative vasoplegia, a clinically insignificant vasoplegic Group 1 (G1) and a clinically significant vasoplegic Group 2 (G2). Differential changes in progenitor cell counts during different stages of surgery were compared across these two groups. Machine-learning classifiers (logistic regression and gradient boosting) were employed to determine if differential changes in progenitor counts could aid the classification of individuals into these groups. Enumerating progenitor cells revealed an early and significant increase in the circulating counts of CD34+ and CD34+CD133+ hematopoietic stem cells (HSC) in G1 individuals, while these counts were attenuated in G2 individuals. Additionally, EPCs (CD34+VEGFR2+) were lower in G2 individuals compared to G1. Gradient boosting outperformed logistic regression in assessing the vasoplegia grouping based on the fold change in circulating CD 34+ levels. Our findings indicate that a lack of early response of CD34+ cells and CD34+CD133+ HSCs might serve as an early marker for development of clinically significant vasoplegia after CPB.
Subject(s)
Blood Cell Count , Cardiopulmonary Bypass/adverse effects , Endothelial Progenitor Cells , Hematopoietic Stem Cells , Vasoplegia/blood , Adrenergic beta-Antagonists/therapeutic use , Adult , Aged , Angiotensin II Type 1 Receptor Blockers/therapeutic use , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Anthropometry , Comorbidity , Elective Surgical Procedures , Female , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Intraoperative Period , Kinetics , Machine Learning , Male , Middle Aged , Pilot Projects , Postoperative Period , Severity of Illness Index , Vasoplegia/physiopathologyABSTRACT
INTRODUCTION: AMP-activated protein kinase (AMPK) is a drug target for treatment of metabolic and cardiovascular complications. Extracts of Gentianaceace plants exhibit anti-diabetic and anti-atherosclerotic effects, however, whether their phyto-constitutents activate AMPK remains to be determined. METHODS: Molecular docking of Gentiana lutea constituents was performed with crystal structure of human α2ß1γ1 trimeric AMPK (PDB ID: 4CFE). Binding of Amarogentin (AG) to α2 subunit was confirmed through isothermal titration calorimetry (ITC) and in vitro kinase assays were performed. L6 myotube, HUH7 and endothelial cell cultures were employed to validate in silico and in vitro observations. Lipid lowering and anti-atherosclerotic effects were confirmed in streptozotocin induced diabetic mice via biochemical measurements and through heamatoxylin and eosin, Masson's trichrome and Oil Red O staining. RESULTS: AG interacts with the α2 subunit of AMPK and activates the trimeric kinase with an EC50 value of 277 pM. In cell culture experiments, AG induced phosphorylation of AMPK as well as its downstream targets, acetyl-coA-carboxylase (ACC) and endothelial nitric oxide synthase (eNOS). Additionally, it enhanced glucose uptake in myotubes and blocked TNF-α induced endothelial inflammation. Oral supplementation of AG significantly attenuated diabetes-mediated neointimal thickening, and collagen and lipid deposition in the aorta. It also improved circulating levels of lipids and liver function in diabetic mice. CONCLUSION: In conclusion, AG exerts beneficial vasculo-metabolic effects by activating AMPK. GENERAL SIGNIFICANCE: Amarogentin, a naturally occurring secoiridoid glycoside, is a promising lead for design and synthesis of novel drugs for treatment and management of dyslipidemia and cardiovascular diseases.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Endothelium, Vascular/drug effects , Iridoids/pharmacology , Animals , Atherosclerosis/prevention & control , Calorimetry , Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Experimental/metabolism , Endothelium, Vascular/metabolism , Enzyme Activation , Glucose/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Lipids/blood , Male , Mice , Mice, Inbred C57BL , Molecular Docking Simulation , Rats , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Viola odorata L. (Violaceae), an Indian medicinal plant, contains a plethora of cyclotides, which are a class of cyclic peptides derived from plants, possessing several applications. Somatic embryo culture of V. odorata was developed, via indirect somatic embryogenesis, to serve as an alternative to natural plant biomass for sustainable and continuous production of its bioactive ingredients, such as cyclotides. Among the various combinations of phytohormones tested, Murashige and Skoog medium supplemented with 1â¯mg/l thidiazuron gave rise to the maximum frequency of induction (86.7%) and a high number of somatic embryos (3) from an embryogenic callus. Identification and characterization of cyclotides in the somatic embryos were carried out using a Fourier transform mass spectrometer coupled with liquid chromatography (LC-FTMS). Among the cyclotides identified in the study, few were found to be exclusively present in the somatic embryo culture. Furthermore, the relative abundance of the cyclotides was higher in somatic embryo extract than in the natural plant extract. The biological activities (cytotoxic, haemolytic and antimicrobial) of the somatic embryos and the parent plant were compared. Unlike the natural plants, the somatic embryo extracts demonstrated specificity i.e. they were found to be potent against cancerous cells but not against non-cancerous cell line or red blood cells. In contrast to the plant extract, the somatic embryos extracts were found to be potent against Escherichia coli and Staphylococcus aureus. These results suggest that somatic embryos of V. odorata (rich in cyclotides) can be used as an alternative to plant biomass for its therapeutic applications and germplasm conservation.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cyclotides/pharmacology , Plant Extracts/pharmacology , Viola/metabolism , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/chemistry , Antineoplastic Agents, Phytogenic/biosynthesis , Antineoplastic Agents, Phytogenic/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclotides/biosynthesis , Cyclotides/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Escherichia coli/drug effects , HEK293 Cells , Humans , Microbial Sensitivity Tests , Plant Extracts/biosynthesis , Plant Extracts/chemistry , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects , Structure-Activity Relationship , Viola/chemistry , Viola/embryologyABSTRACT
Volumetric productivity of camptothecin from the suspension culture of the endophyte Fusarium solani was enhanced up to â¼152 fold (from 0.19µgl(-1)d(-1) to 28.9µgl(-1)d(-1)) under optimized fermentation conditions including initial pH (6.0), temperature (32°C) and agitation speed (80rpm) with (5% (v/v)) ethanol as medium component. Among various elicitors and precursors studied, tryptamine (0.5mM) as precursor and bovine serum albumin (BSA) (0.075mM) as an elicitor added on day 6 of the cultivation period resulted in maximum enhancement of camptothecin concentration (up to 4.5 and 3.4-fold, respectively). These leads provide immense scope for further enhancement in camptothecin productivity at bioreactor level. The cytotoxicity analysis of the crude camptothecin extract from the fungal biomass revealed its high effectiveness against colon and mammary gland cancer cell lines.
ABSTRACT
Volumetric productivity of camptothecin from the suspension culture of the endophyte Fusarium solani was enhanced up to â¼152 fold (from 0.19 µg l(-1) d(-1) to 28.9 µg l(-1) d(-1)) under optimized fermentation conditions including initial pH (6.0), temperature (32 °C) and agitation speed (80 rpm) with (5% (v/v)) ethanol as medium component. Among various elicitors and precursors studied, tryptamine (0.5 mM) as precursor and bovine serum albumin (BSA) (0.075 mM) as an elicitor added on day 6 of the cultivation period resulted in maximum enhancement of camptothecin concentration (up to 4.5 and 3.4-fold, respectively). These leads provide immense scope for further enhancement in camptothecin productivity at bioreactor level. The cytotoxicity analysis of the crude camptothecin extract from the fungal biomass revealed its high effectiveness against colon and mammary gland cancer cell lines.
Subject(s)
Camptothecin/biosynthesis , Fermentation , Fusarium/metabolism , Antineoplastic Agents, Phytogenic/biosynthesis , Antineoplastic Agents, Phytogenic/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Camptothecin/pharmacology , Cell Line, Tumor/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Endophytes/metabolism , Ethanol/metabolism , Female , Humans , Hydrogen-Ion Concentration , Industrial Microbiology/methods , TemperatureABSTRACT
Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis.