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Resveratrol and Its Natural Analogs Mitigate Immune Dysregulation and Oxidative Imbalance in the Endometriosis Niche Simulated in a Co-Culture System of Endometriotic Cells and Macrophages
Background: Inflammation and immune cell dysfunction are critical facilitators of endometriosis pathophysiology. Macrophages are renowned for stimulating lesion growth, vascularization, innervation, and pain generation. By combining macrophages and endometriotic cells, we determined if resveratrol and its natural analogs can target the immune dysregulation and oxidative imbalance in endometriosis. Methods: After treatment with compounds (5, 10, 25 µM), we evaluated the expression of key inflammatory and oxidative stress markers, cytokines release, and ROS production by applying q-PCR, ELISA, Cytometric Beads Array, and multiplexed fluorogenic staining and flow cytometry analysis with bioimaging. Results: The results showed that endometriosis-related macrophages treated with stilbenes have impaired expression of pro-inflammatory markers (IL6, IL8, IL1B, TNF, CCL2, CXCL10, PTGS2). The effect of resveratrol, pterostilbene, and piceatannol was observed, especially in reducing IL1B, CCL2, and CXCL10 genes up to 3.5-, 5-, and 7.7-fold at 25 µM, respectively. Also, with piceatannol or polydatin exposure, the IL-6 decrease was noticeable. This study reported an antioxidant effect by reducing ROS-positive cells from 96% to 48% by pterostilbene. Results from flow cytometry correlated with the transcript activation of detoxification enzymes (SOD, GPX). Conclusions: Prospects for potential therapy based on regulating the immune microenvironment and reducing the accumulation of free radicals with stilbenes application were described in the article.
Thermo-oxidative stability of asymmetric distigmasterol-modified acylglycerols as novel derivatives of plant sterols
Anti-proliferative potential and oxidative reactivity of thermo-oxidative degradation products of stigmasterol and stigmasteryl esters for human intestinal cells
AbstractStigmasterol in free and esterified form is incorporated in LDL cholesterol-lowering food products, intended for direct consumption and cooking, baking, and frying. Under thermal treatment, stigmasterol compounds may constitute a source of thermo-oxidative degradation products and oxyderivatives with potentially adverse health effects. This study aimed to analyze the anti-proliferative potential and genotoxicity of thermo-oxidatively treated stigmasterol (ST), stigmasteryl linoleate (ST-LA), and oleate (ST-OA). The effects on cell viability and proliferation, cell cycle progression, intracellular reactive oxygen species (ROS) generation, and DNA damage were analyzed in normal human intestinal cells. The mutagenic potential was assessed in a bacterial reverse mutation test using Salmonella enterica serovar Typhimurium strains involving metabolic activation. Stigmasteryl esters showed a significantly lower potential to affect intestinal cell viability and proliferation than non-esterified ST, regardless of heating. Thermo-oxidatively treated ST suppressed intestinal cell proliferation by arresting the cell cycle in the G2/M phase and DNA synthesis inhibition. The enhanced intracellular ROS generation and caspase 3/7 activity suggest targeting intestinal cells to the apoptosis pathway. Also, heated ST-LA intensified ROS production and elicited pro-apoptotic effects. Thermo-oxidative derivatives of ST and ST-LA may evoke harmful gastrointestinal effects due to their high oxidative reactivity towards intestinal cells.
High-capacity adsorption and pH-responsive release of an antihypertensive drug via urea- and cetyltrimethylammonium bromide-functionalized mesoporous carbons
Modulating effects of steviol and steviol glycosides on adipogenesis, lipogenesis, glucose uptake and insulin resistance in 3T3-L1 adipocyte model
Heating and storage of structured acylglycerols with succinyl-linked stigmasterol residue does not cause negative chemical or biological changes
AbstractFour structured acylglycerols with stigmasterol bonded by a succinyl linker were investigated and their stability were analyzed. Samples were heated to 60 °C and kept at that temperature to simulate storage, and to 180 °C to simulate frying conditions. The degradation of the synthesized compounds and formed derivatives was determined, and their cytotoxicity and genotoxicity on normal human cells from the digestive system was determined. Holding at 180 °C resulted in greater degradation of the compounds than holding at 60 °C. The most stable compound in each sample proved to be one with oleic acid in its structure—1,3-dioleoyl-2-stigmasterylsuccinoyl-sn-glycerol (DO2SSt) at 60 °C and 1,2-dioleoyl-3-stigmasterylsuccinoyl-sn-glycerol (DO3SSt) at 180 °C. These results indicate that the type of fatty acid in the molecule is more important than its position in the glycerol structure. None of the diacylmonostigmasterylsuccinoyl-sn-glycerols (DASStGs) before or after heating exhibited cytotoxic or genotoxic potential to small intestine and colon mucosa cells.
The Effects of Cellular Membrane Damage on the Long-Term Storage and Adhesion of Probiotic Bacteria in Caco-2 Cell Line
Adhesion is one of the main factors responsible for the probiotic properties of bacteria in the human gut. Membrane proteins affected by cellular damage are one of the key aspects determining adhesion. Fluid-bed-dried preparations containing probiotic bacteria were analyzed in terms of their stability (temperature of glass transition) and shelf life in different conditions (modified atmosphere, refrigeration). Imaging flow cytometry was utilized to determine four subpopulations of cells based on their physiological and morphological properties. Lastly, adhesion was measured in bacteria cultured in optimal conditions and treated with heat shock. The results show that the subpopulations with no or low levels of cell membrane damage exhibit the ability to adhere to Caco-2 cells. The temperature of protein denaturation in bacteria was recorded as being between 65 °C and 70 °C. The highest glass transition temperature (Tg) value for hydroxypropyl methylcellulose (used as a coating substance) was measured at 152.6 °C. Drying and coating can be utilized as a sufficient treatment, allowing a long shelf-life (up to 12 months). It is, however, worth noting that technological processing, especially with high temperatures, may decrease the probiotic value of the preparation by damaging the bacterial cells.
In vitro modeling of endometriosis and endometriotic microenvironment – Challenges and recent advances
Effect of gastrointestinal digestion on the stability and cytotoxicity of conventional and pegylated liposomes encapsulated with stigmasterol and its esters
Permeability of the Perindopril Arginine under In Vitro Conditions across Caco-2 Monolayer and Biomimetic Phospholipid Membrane
Perindopril arginine (PA) as an angiotensin-converting enzyme (ACE) inhibitor is widely used in cardiovascular diseases, especially in systemic hypertension and heart failure. Although the pharmacokinetics of PA are well documented, there is no available detailed data on its permeation in in vitro conditions. The present study aimed to assess the transport of PA across both biological membranes and artificial biomimetic ones. For the determination of PA transport, the Caco-2 cell line was selected as a reliable in vitro model of gastrointestinal biological barriers. Additionally, a novel 96-well plate with phospholipid membrane PermeaPad was used to evaluate the transport of PA by passive diffusion. We confirmed that PA is relatively poorly permeable across the Caco-2 monolayer. The permeability results obtained from the non-cell-based model demonstrated higher transport of PA as compared to that of Caco-2. Thus, PA transport across the biological membranes might be suggested to be regulated by the membrane transporters.
Bioactive Substances of Potato Juice Reveal Synergy in Cytotoxic Activity against Cancer Cells of Digestive System Studied In Vitro
More and more literature data indicate the health-promoting effect of potato juice (PJ). However, to date, it has not been precisely explained which of the many compounds present in PJ exhibit biological activity. The work aimed to establish the antiproliferative effect of gastrointestinal digested PJ and the products of its processing. Fresh PJs derived from three edible potato varieties, industrial side stream resulting from starch production, partially deproteinized PJ derived from feed protein production line, and three different potato protein preparations subjected to digestion in the artificial gastrointestinal tract were used in this study. The cytotoxic potential of glycoalkaloids (GAs), phenolic acids, digested PJ, and products of PJ processing was determined in human normal and cancer cells derived from the digestive system. The results showed that GAs exhibit concentration-dependent cytotoxicity against all analyzed cell lines. In contrast, phenolic acids (caffeic, ferulic, and chlorogenic acid) do not show cytotoxicity in the applied cell lines. A correlation between cytotoxic potency and GAs content was found in all PJ products studied. The most potent effects were observed under treatment with deproteinized PJ, a product of industrial processing of PJ, distinguished by the highest effective activity among the fresh juice products studied. Moreover, this preparation revealed a favorable cytotoxicity ratio towards cancer cells compared to normal cells. Statistical analysis of the obtained results showed the synergistic effect of other bioactive substances contained in PJ and its products, which may be crucial in further research on the possibility of using PJ as a source of compounds of therapeutic importance.
Economic Analysis of the Production Process of Probiotics Based on the Biological and Physiological Parameters of the Cells
Probiotic bacteria confer a range of health benefits and are a focus of a growing number of studies. One of the main issues is their stability during drying and storage, which is why techniques, such as fluid bed drying and coating or treatment with stress factors during culturing, are utilized. The methods of the evaluation of probiotic viability and quality are, however, lacking and we need a way of distinguishing between different subpopulations of probiotic bacteria. To address this issue, imaging flow cytometry (IFC) has been utilized to assess cells after simulated in vitro digestion of dried and coated preparations treated with pH stress and heat shock. Samples were analyzed fresh and after 12 months of storage using RedoxSensor green and propidium iodide dyes to assess metabolic activity and cell membrane integrity of the cells. The results were then used to design a drying process on an industrial scale and evaluate the economic factors in the SuperPro Designer v13 software. Based on the number of biologically active and beneficial cells obtained utilizing tested methods, the coating process and treatment with heat shock and pH stress have been the most effective and up to 10 times cheaper to produce than only by drying. Additionally, samples after 12 months of storage have shown an increase in the proportion of cells with intermediate metabolic activity and small amounts of cell membrane damage, which are still viable in probiotic products. This subpopulation of bacteria can still be considered live in probiotic products but is not necessarily effectively detected by pour plate counts.
Immunomodulatory Effect of Raspberry (Rubus idaeus L.) Fruit Extracts on Activated Macrophages and Dysfunctional Vascular Endothelial Cells
Background: Growing evidence highlights the beneficial effects of flavonoids, including anthocyanins, as key components in reducing cardiovascular risk, and emphasizes that incorporating anthocyanin-rich fruits into the daily diet significantly impacts public health. Methods: The effect of bioactive polyphenols from raspberry fruit (RBF) on molecular pathways in inflammation was studied in activated RAW 264.7 macrophages and their protective potential against endothelial dysfunction was analyzed using TNF-α-induced human umbilical vein endothelial cells (HUVECs). Results: The results have shown that RBF extract, along with its anthocyanin and polyphenol fractions, has a significant anti-inflammatory effect in macrophage cell culture by inhibiting the LPS-induced expression of pro-inflammatory genes, including IL-6, IL-1β, TNF-α, and NF-κB. Moreover, RBF and both fractions have demonstrated a protective effect on endothelial function by decreasing the expression of several inflammation-related genes and adhesion molecules, such as IL-6, IL-1β, VCAM-1, ICAM-1, and SELE, in TNF-α-induced HUVECs. Conclusions: The consumption of RBF and/or polyphenol-rich extracts may help prevent the onset of early atherosclerosis. This is attributed to their ability to improve inflammation status and enhance vascular endothelial function. Given the strong anti-inflammatory properties of RBF, incorporating them into a daily diet could significantly reduce the risk of non-communicable diseases related to inflammation.
Co-interaction of nitrofuran antibiotics and the saponin-rich extract on gram-negative bacteria and colon epithelial cells
AbstractLarge-scale use of nitrofurans is associated with a number of risks related to a growing resistance to these compounds and the toxic effects following from their increasing presence in wastewater and the environment. The aim of the study was to investigate an impact of natural surfactant, saponins from Sapindus mukorossi, on antimicrobial properties of nitrofuran antibiotics. Measurements of bacterial metabolic activity indicated a synergistic bactericidal effect in samples with nitrofurantoin or furazolidone, to which saponins were added. Their addition led to more than 50% greater reduction in viable cells than in the samples without saponins. On the other hand, no toxic effect against human colon epithelial cell was observed. It was found that exposure to antibiotics and surfactants caused the cell membranes to be dominated by branched fatty acids. Moreover, the presence of saponins reduced the hydrophobicity of the cell surface making them almost completely hydrophilic. The results have confirmed a high affinity of saponins to the cells of Pseudomonas strains. Their beneficial synergistic effect on the action of antibiotics from the nitrofuran group was also demonstrated. This result opens promising prospects for the use of saponins from S. mukorossi as an adjuvant to reduce the emission of antibiotics into the environment.
Rosehip Extract Decreases Reactive Oxygen Species Production and Lipid Accumulation in Hypertrophic 3T3-L1 Adipocytes with the Modulation of Inflammatory State
Background: Rosa canina L. (rosehip) is used worldwide in traditional medicine as a plant with medicinal properties. However, its anti-obesity effects are not fully explained on a transcriptional level. Methods: In the present work, the 3T3-L preadipocytes were utilized to explore the impact of R. canina fruit extract (RCE) on the cellular and molecular pathways involved in adipocyte hypertrophy. Results: Obtained results showed the ability of RCE to reduce lipid overloads in hypertrophic adipocytes associated with the down-regulation of mRNA expressions of adipogenic transcription factors such as PPARγ, C/EBPα, and SREBP-1c as well as genes involved in lipid biosyntheses such as FAS, LPL, and aP2. Moreover, obesity-associated oxidative stress (antioxidant enzyme activities and ROS generation) and inflammation were ameliorated in RCE-treated hypertrophic adipocytes. The mRNA and protein levels of adipokines such as leptin, resistin, and adiponectin were restored to more favorable levels. Conclusions: Rosa canina fruit might be a valuable source of phytochemicals in preventing obesity and obesity-related metabolic complications.
