The intestines are a vital part of animal health and productivity, contributing to the digestion and transport of nutrients, as well as playing a protective role against microbes and harmful substances. However, investigating intestinal functions using traditional models still faces significant challenges, especially in broiler chickens. This article highlights a new model known as “enteroids,” which is considered a promising alternative to traditional models. The research reviews how this model can provide a more accurate laboratory environment for understanding the intestinal response to diseases and stressors, such as oxidative stress and inflammation, by analyzing transport functions and interaction with the immune system. Through this study, we present new prospects in the field of nutrition and veterinary medicine research, enhancing our understanding of gut microbiota and its ability to influence chicken health.
Conventional 2D Model and Its Role in Studying Intestinal Functions
Conventional 2D intestinal epithelial cells are a common model used in research related to intestinal functions. Despite the widespread use of these models, they have numerous limitations when it comes to simulating the true intestinal physiology in birds, especially chickens. The intestines are not just an organ for nutrient transport; they play a complex role in maintaining overall health and growth efficiency through multiple processes from nutrient transport to immune barriers. From this perspective, there was an urgent need to develop more representative models. One of these advancements is the “enteroid” model, which provides greater accuracy and reliability in studying the intestinal role.
The “Enteroid” Model and Its Role in Intestinal Research
The “enteroid” model represents a significant shift in how intestinal functions are studied. This model is based on the formation of intestinal villi units isolated from the intestines of “Cobb 500” chickens to recreate the intestinal environment using intestinal cells in a three-dimensional form. These constructs provide the physiological properties of the intestines, making them important for studying cellular interactions and responses to environmental stresses. Additionally, the results indicate that these models are capable of simulating nutrient flow and response to oxidative stress and inflammation, enhancing their credibility as alternative models.
Nutrient Transport and Barrier Functions in the Enteroid Model
In current research, the basic transport functions of nutrients and barrier functions in this new model have been evaluated. Study results indicated stable cellular activity from day two to day six after the model was established, providing an ideal window for studying intestinal responses. Results showed that permeability to permeable substances increased after exposure to EDTA, reflecting changes in intestinal barrier functions. The study also observed a decrease in glucose absorption rates in the presence of transport inhibitors, enhancing the understanding of the complex mechanisms of nutrient transport in the intestines.
Enteroid Response to Inflammatory Challenges and Oxidative Stress
Inflammatory and oxidative stress triggers came in the form of exposing the enteroids to LPS and “minadience.” Results showed an increase in gene expression of inflammation-related markers, such as IL-1β and IL-6, indicating the response of enteroids to external challenges. Additionally, the study showed an increase in “free radical” production as an indication of oxidative stress levels, reflecting the interaction of intestinal cells with stressful situations. These results highlight the importance of the enteroid model in providing a clearer picture of how the intestines interact with various stresses.
The Future Importance of the Enteroid Model in Intestinal Research
Understanding the complex intestinal physiology requires the development of models that accurately represent the internal environment of the intestines. The enteroid model is an important step in this direction, providing suitable mechanisms to glean information related to nutrient transport and barrier functions. The ability to study and assess the intestinal response to various stressors, such as inflammation and oxidative stress, makes this model an effective and reliable tool. Future research opens the door for using this model in developing new treatments and methods to improve poultry health, contributing to achieving sustainable food production on a global scale.
Challenges
The Future Prospects in Developing the Gut-on-a-Chip Model
Despite the significant success achieved in research using the gut-on-a-chip model, there are challenges related to its further development. This includes working to improve the cultural conditions to make this model more accurate in representing the diverse gut environment. Future studies also need to explore how to leverage this model to study the negative impacts of antibiotics and various environmental factors on gut health. As advanced research in this field continues, it is possible to move towards developing innovative solutions that enhance the effectiveness of gut-on-a-chip models and expand their research capabilities. To this end, researchers should explore the use of new technologies such as genetic engineering and functional cell modification to make these models more closely related to reality.
Measuring Mitochondrial Activity in Gut Organoids Culture
The primary activity of mitochondria is their ability to produce energy through cellular respiration. In this study, the PrestoBlue assay was used to measure mitochondrial activity in gut organoids, monitoring activity from day one (d1) to day seven (d7). Data were expressed as a percentage relative to day one, with cellular activity calculated using the appropriate formula. Monitoring mitochondrial activity is a vital step in understanding the health of gut organoids and the potential use of these models to study gut diseases.
Gut organoids were observed daily under a microscope, which helped to assess their morphology and integrity. By using magnifications of ×40 and ×100, researchers were able to distinguish healthy organoids from damaged ones, which significantly affected the evaluation of the success of experiments in various aspects such as glucose transport and permeability testing. Enhanced mitochondrial activity indicates the organoids’ ability to survive and grow, making them a practical model for studying the clinical response to intestinal diseases.
Analysis of Glucose Absorption in Gut Organoids
Researchers tested the capability of gut organoids to absorb glucose using the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2NBDG). Glucose absorption was measured on different days (d2, d4, d6) with the addition of a specific glucose transporter inhibitor, phlorizin (PZ), which affects glucose delivery across the organoid membrane. The goal of this examination was to elucidate the pathways through which glucose is absorbed, which is crucial for understanding nutritional balance and cellular interactions in the gut.
The results indicated significant differences in glucose absorption activity based on the availability of the inhibitor and the age of the organoids, reflecting the importance of time variables and the nature of the challenges imposed. These studies illustrate how various challenges impact the gut cells’ ability to absorb nutrients and their response to changing environmental conditions, which is foundational for understanding gut health and improving nutritional therapies.
Testing the Epithelial Permeability of Gut Organoids
Researchers assessed the epithelial permeability of gut organoids using fluorescent dextran (FD4), a large molecule that can be used to evaluate the integrity of the epithelial barrier. Treatment groups were introduced to induce damage to the gut barrier and respond to agents such as EDTA and LPS. These tests are essential for determining the ability of gut organoids to maintain their epithelial barrier integrity under various conditions, which can reveal the clinical implications of these models in understanding diseases associated with intestinal inflammation.
Factors affecting intestinal permeability, such as inflammation due to pathogen exposure, were considered. Through these tests, researchers concluded the relationship between barrier integrity deterioration and immune response activation. The sensitivity of the epithelial coating of gut organoids is an important topic that should be taken into account in developing clinical strategies for managing gastrointestinal disorders.
Response of Gut Organoids to Inflammatory and Oxidative Challenges
The study evaluated the response of gut organoids to oxidative and inflammatory challenges using chemicals such as LPS and menadione. LPS was used to create an inflammatory state, while menadione was used to assess the cells’ response to oxidative stress. These experiments are an essential part of analyzing protective mechanisms and cellular responses to gut diseases.
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The results indicate that enteroids interact effectively with these challenges, reflecting their ability to respond to environmental stress. RNA analysis and molecular techniques such as qRT-PCR demonstrate the importance of this tool for understanding changes in gene expression during various stages of culture and exposure.
Genetic Isolation and Gene Expression Analysis
The process of genetic isolation in enteroids is a critical step for understanding their response to environmental conditions. Special importance was given to the analysis of gene expression during different days in culture, where researchers used RNA isolation techniques and qRT-PCR to analyze the response of enteroids to inflammatory and oxidative challenges. The main objectives of these procedures were to understand how environmental stress affects gene expression and the functions of enteroids.
The results obtained from these analyses serve as a reference for further studies on intestinal models, as the findings contribute to identifying genetic pathways that influence immune response. This information could aid in the development of strategies to improve gut health and address future gastrointestinal disorders.
Stages of Enteroid Culture
The stages of enteroid culture are a fundamental element for understanding how intestinal cells grow and develop, using a combination of cells isolated from healthy enteroids and individual cells separated from unhealthy enteroids. In this process, cellular activity and mitochondrial metabolism are measured using the PrestoBlue kit. During the first 48 hours of culture, researchers observed a significant increase in cellular activity, peaking on the third day, before gradually declining until the seventh day. With each cultural day, there were notable changes in the number of healthy enteroids, which decreased by 25% by the fourth day and by 55% by the seventh day compared to the first day. These data suggest that the overall structure of the enteroids deteriorated over time, while the activities of individual cells remained relatively stable.
Characterization of Genetic Markers in Chicken Enteroids
The gene expression of epithelial cell markers was studied during the days when the enteroids were cultured. The expression of the stem cell marker, called Lgr-5, showed a significant decrease on the fourth and sixth days compared to the second day. In contrast, the expression of epithelial cell markers such as Paneth cells (LYZ), enterocytes (ALPi), and goblet cells (MUC-2) significantly increased on the fourth and sixth days. These changes in expression led to the conclusion regarding the enteroids’ ability to differentiate and develop over the course of the culture days. Major changes were also observed in the expression of tight junction proteins such as ZO-1, OCCL, and CLDN-2, which increased on the fourth day, indicating a maturation process of the cells.
Glucose Absorption in Chicken Enteroids
One of the main functions of the small intestine is the absorption of nutrients through the epithelial surface. Researchers used a fluorescent glucose analog called 2NBDG to assess glucose transport function in enteroids. After a period of fixation, fluorescent signals were observed within the enteroids. Conducting experiments with a specific SGLT1 inhibitor showed a significant decrease in fluorescent density, indicating the role of SGLT1 in glucose absorption, with absorption peaking on the fourth day. This data is vital for understanding how epithelial technology contributes to feeding and metabolic processes in living organisms.
Epithelium Barrier Function in Enteroids
The barrier function in the intestine is essential for separating luminal from basal components in the gut. The FD4 molecule was used to evaluate the permeability of the epithelial barrier, and results showed significant leakage of the molecule through tight junctions with certain treatments like EDTA and LPS that increased the permeability of the molecule. The results highlight the importance of structural stability and functional integrity of enteroids in maintaining an effective barrier, reflecting potential challenges from inflammatory effects and may aid in exploring approaches for treating conditions related to gut functions.
Response
Enteroids for Inflammatory Challenges
Upon adding LPS to the enteroids, a notable response was observed, characterized by a decrease in the expression of tight junction proteins and an increase in the expression of inflammatory markers. Several inflammatory cytokines were measured, significantly increasing as a result of the treatment, indicating that inflammatory stimulation affects the cellular composition of the enteroids. These results suggest that inflammatory effects can lead to a reduction in epithelial barrier function, thereby increasing susceptibility to intestinal infections and supporting the theory linking inflammation to impaired gut functions.
Enteroids’ Response to Oxidative Challenges
The use of menadione to highlight the effects of oxidative stress on enteroids provided interesting data regarding the production of ROS molecules and how cells respond to them. A significant increase in ROS production was revealed with increasing concentrations of menadione, but this led to a decline in viability in culture. The results concluded that enteroids can generate ROS and utilize antioxidant enzymes in response to oxidative challenges, reflecting the need for reliable cell systems capable of adapting to physical stressors.
Villus Units and Their Role in Gut Study
The significance of studying villus units lies in their isolation and cultivation methods, which provide more accurate models to understand the biological properties of the gut. Collagenase hydrolysis is used to randomly dissociate the intestinal mucosa, producing villus units of varying lengths. By filtering the cells, the size of these units was standardized to produce relatively homogenous morphologic patterns of enteroids in culture. It was noted that the enteroids contained cells from the crypt layer surrounded by a closed epithelial layer, which prevents their expansion in culture. The activity of enteroid cells was evaluated over days, with an observed increase in cellular mitochondrial activity, indicating stable cellular growth up to the sixth day. However, a discrepancy between individual cell activity and the healthy enteroid suggests the possibility of breaking down the enteroid into individual cells without losing functionality. These results reflect the necessity to understand the expression of key genes during the culture period for accurate measurement of enteroid functionality.
Glucose Absorption and Gut Cell Growth
Glucose absorption in the intestinal epithelium greatly depends on the SGLT1 transporter as well as peripheral diffusion. A fluorescent glucose analogue was used to assess glucose absorption in the enteroids. Prior to conducting the absorption test, enteroids were treated with a substance that inhibits SGLT1 to confirm its role in glucose transport. It was shown that the molecular transport of glucose increased peaking on the fourth day, with glucose absorption significantly reduced upon SGLT1 inhibition. While peripheral diffusion was evident on the second day, it decreased with barrier maturation by the sixth day. These results provide important insights into the lifecycle of gut cells and how they respond to growth and development under certain culture conditions.
Enteroid Response to Inflammatory Challenges
The response of enteroids to inflammatory challenges, particularly the effects of LPS on barrier integrity and gene expression, was studied. Barrier integrity was evaluated using FD4 permeability, revealing increased barrier permeability upon exposure to LPS and EDTA concentrations. Permeability increased significantly, indicating disruption of the intestinal barrier, and these results were corroborated by gene expression data showing upregulation of inflammatory genes. Studies suggest that the poultry’s response strength to LPS may require careful observations due to their resilient nature. This underscores the importance of developing laboratory models like enteroids for comprehensively studying inflammatory effects.
Enteroid Response to Oxidative Stress
The response to oxidative stress is a vital part of understanding how cells interact with environmental stressors. Hydrogen peroxide was used as an oxidizing agent, but it was noted that menadione was more efficient in producing stronger oxidative effects at lower concentrations. The optimal concentration was determined for use in analyzing the response, where a decrease in gene expression of antioxidant enzymes was observed under oxidative stress conditions. These results are significant as enteroid models provide a means to explore the interaction between oxidative stress and gut health, paving the way for future studies on how gut cells cope with various challenges.
Model
Enteroids and Their Effectiveness Compared to Other Models
Traditionally, intestinal studies rely on cellular models such as Caco-2, but they have limitations in simulating the properties of live intestines. More advanced models like organoids offer greater complexity by incorporating multiple cell types, making the chicken enteroid model more efficient for studying intestinal responses to different loads. Current research reflects the effectiveness of the enteroid model in studying nutrient transport and barrier integrity under stress conditions such as inflammation and oxidation. These models provide a strong foundation for understanding how dietary factors can interact with gut health, supporting innovations in the field.
The Relationship Between Gut Physiology and Overall Health
Gut physiology is one of the fundamental factors that determine the overall health of living organisms, including humans. The gut plays a vital role in digesting food and absorbing nutrients, while at the same time forming a protective barrier against harmful microorganisms. Dysfunction of the intestinal barrier can lead to a range of health problems, including inflammatory bowel disease, irritable bowel syndrome, and diabetes. For example, the consumption of unhealthy foods, such as those rich in fats and sugars, has a negative impact on gut performance, leading to increased intestinal permeability. This condition, also known as leaky gut syndrome, can leak harmful components into the bloodstream, causing inflammation and multiple issues in the body.
Additionally, studies have shown that the presence of beneficial bacteria in the gut, such as probiotics, can improve gut health and enhance immune function. Probiotics prevent the growth of harmful bacteria and help generate anti-inflammatory substances, maintaining gut health and contributing to improved digestion. An example of this is the use of probiotic supplements in treating diarrhea caused by infections or in restoring the balance of gut bacteria after antibiotic use.
The Importance of Laboratory Research in Studying Gut Diseases
Laboratory research plays a crucial role in understanding gut-related diseases and how to treat them. Through intestinal cell models, such as Caco-2 cells, researchers can study the effects of drugs and dietary compounds on gut health. These cellular models provide an ideal environment for studying intestinal permeability and testing how new drugs interact with intestinal cells.
For example, the effectiveness of certain natural compounds as treatments for intestinal inflammation has been studied using these models. It has been found that certain substances like ellagic acid can improve the health of intestinal cells when exposed to inflammatory agents. This type of research allows scientists to examine the responses of different cells and track how some treatments can affect chemical interaction pathways within the gut.
Laboratory research also enables the examination of the relationship between pathogens and diseases. By using laboratory intestinal models, scientists can study the effects of different pathogens on gut integrity and how they may play a role in exacerbating conditions such as inflammatory bowel disease or irritable bowel syndrome.
Techniques for Assessing and Maintaining Gut Health
There are various techniques used to assess gut health, aiding in the development of effective strategies to maintain proper intestinal permeability. Researchers use several methods, including measuring transepithelial electrical resistance (TEER), which is a technique used to evaluate the integrity and effectiveness of the intestinal barrier. The higher the resistance, the more organized and robust the cells are in achieving an effective barrier. These measurements are key indicators in studying gut health, as a decrease in these resistances typically signals issues with the cellular barrier.
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the intestinal response and the description of the inflammatory response is also an important aspect in this field. This is done by taking tissue samples and analyzing them under a microscope to check for any changes or damage in the intestinal cells. These analyses are of great importance in the development of drugs aimed at improving gut health.
Future Challenges and Research Opportunities in Gut Health
There are still significant challenges that require more research to understand how to effectively improve gut health. Among these challenges is understanding how environmental factors and dietary patterns affect gut health. With increasing urbanization and modern lifestyles, it has become essential to study how genetic factors, diet, and lifestyle collectively influence gut health.
In addition, research should be enhanced on the relationship between the gut microbiome and systemic diseases, including metabolic and immune diseases. For example, studies suggest that the balance of microorganisms can affect glucose metabolism, which may have direct implications for diabetes. These discoveries have opened up new possibilities for treatment and innovative medical applications.
Gut Development in Poultry
The gut plays a vital role in the health and productivity of animals by managing essential functions including nutrient sensing and transport, ensuring effective growth of the animals. Proper gut development depends on several factors, including the formation of functional epithelium and its interaction with immune cells in the mucosal layer. This interaction is not only essential for basic gut functions but also for protecting animals from microbes and harmful substances that may enter the body through the digestive system.
The gut in poultry, such as chickens, requires a deep understanding of its mechanical and chemical mechanisms due to the complexity of its structure. The gut represents the appropriate environment for the interaction of several types of cells, including immune cells, which helps enhance the immune system’s capacity. For example, chickens can tolerate high levels of bacteria thanks to these advanced immune functions.
With the advancement of technology, alternative laboratory models provide effective ways to study the gut. However, there is still a lack of effective laboratory models for poultry, necessitating the development of more representative models that allow for precise study of metabolic and inflammatory processes.
Laboratory Models and Their Relevance to Poultry
Laboratory models are considered essential tools for clinical studies, especially when internal studies on animals are costly and time-consuming. Laboratory models contribute to providing a less expensive and more efficient environment to study gut health. Among the types of these models, the intestinal epithelial cell line stands out, as it forms monolayers in culture that mimic the functions of intestinal cells. Although they are effective in cultivating animal models such as humans and pigs, a corresponding model for chickens has not been developed yet.
Recent studies highlight the need for more representative models that allow for accurate understanding of intestinal characteristics. Poultry tissue models are a promising model, as they have three-dimensional characteristics that reflect the natural environment of the gut, allowing for the study of nutrient transport interactions and disease responses. A good example of this is using poultry intestinal tissues to evaluate the response to inflammation and oxidative stress, enabling specialists to study the health conditions affecting chicken growth.
Tissue Cell Isolation Techniques
Effective methods have been developed to isolate tissue cells from chickens, through a complex process involving the use of enzyme extracts and filtration, allowing for the collection of intestinal villus units. These units represent important cell aggregates, which can be used in laboratory models to study many vital processes. In these processes, collagenase is used as a means of digesting intestinal tissue and isolating the cells, which allows for the construction of a cell reservoir that can be expanded and developed later.
The studied processes serve as a solution to pave the way for in-depth studies, where isolated cells can be cultured and stimulated to extend over time periods. As a result, researchers can monitor behavioral and biochemical changes in cells during different stages. For example, cellular activity was monitored and quantities were estimated through multiple measurement screens and tools.
Intestinal Responses to Oxidative Stress and Inflammation
The response to diseases and oxidative stress poses a significant challenge for poultry, as intestinal inflammation is one of the main causes of poor poultry health, leading to high economic costs in the livestock production sector. The technique of using avian tissues allows for an understanding of the mechanisms of stress response, as it can address the environmental and nutritional effects at the microscopic level. These analyses shed light on how changes in foods and bioactives affect intestinal health.
Scientific applications can help develop new strategies to improve the quality of chicken nutrition and enhance its ability to resist diseases. Additionally, these models can be used in efficacy tests of new treatments designed specifically to boost immunity and improve stress response. Thus, the use of avian tissues represents an important step towards improving the overall health of poultry production.
Future Applications of Avian Tissue Models
Ongoing research into avian tissue culture techniques and disease responses offers new hopes for understanding the depths of digestive and productive health in poultry. The introduction of these models may provide unprecedented insights into how to manage intestinal health and achieve higher levels of productivity efficiently. The future opens doors for the development of new strategies in nutrition and disease treatment using avian tissues as a study model.
Furthermore, collaboration between researchers and farmers may contribute to the application of technology and the delivery of increasing understanding to farmers, enabling them to choose the most effective feed and nutrition types in terms of productive health. Consequently, these achievements in scientific research represent a significant step towards achieving a healthier and more prosperous future in the poultry industry.
Cell Activity and Measuring the Effect of Light Intensity
The activity of enterocyte cells was measured based on the intensity of PrestoBlue dye on the first day of the experiment. Enterocytes were monitored daily from day one to day seven using an inverted microscope. The goal was to assess the morphological status and integrity of the enterocytes, using magnifications of ×40 and ×100, and the healthy enterocytes with a clear epithelial layer were classified. As part of this process, 8 wells from a 96-well plate were used, and images were documented under the same conditions. Data were expressed as a percentage compared to day one, providing a standardized metric for monitoring the condition over the time period.
This method demonstrates how changes in cell activity in enterocytes can reflect environmental or therapeutic conditions. For instance, if a specific substance is added that may affect cellular activity, measuring PrestoBlue intensity gives an indication of effective changes occurring and can indicate negative or positive effects. Relying on enterocytes as a basis in studies enhances the chances of obtaining accurate information regarding cell responses to various factors.
Testing Glucose Absorption and the Effect of Inhibitors
The glucose absorption capacity of enterocytes was evaluated using the fluorescent glucose molecule 2-NBDG. On days two, four, and six, the enterocytes were treated with 2-NBDG at a concentration of 300 micromolar for 30 minutes to monitor how enterocytes absorbed glucose. The unique inhibitor of the glucose receptor SGLT1, named phlorizin, was added before adding 2-NBDG. This approach enables researchers to understand the mechanism used by cells to take in glucose, which has important implications for research in diabetes and malnutrition.
Unabsorbed glucose was removed from the medium by washing with cold PBS solution, and fluorescence intensity was measured using a fluorescence plate reader. These data reflect the capacity of enterocytes to absorb glucose and provide insight into the role of SGLT1 in this process. Focusing on glucose absorption in the enterocyte cells is crucial for understanding the metabolic processes stored in living tissues.
Assessment
Permeability of the enterocyte layer and safety testing
FD4 permeability testing was conducted to evaluate the safety of the enterocyte epithelial layer after structural treatment using EDTA or LPS as positive controls. This experiment indicates how enterocytes respond to safety changes, where the lowest level of ethylene diamine tetraacetic acid at varying concentrations was used to monitor the effects on the structure. Regarding LPS monitoring, different doses were applied to determine the initial effects on enterocytes.
The results show that enterocytes are very sensitive to changes in the chemical composition around them, which is very important for understanding how cells respond to inflammatory agents, preservative substances in the structure, and some irritants. The FD4 permeability analysis aims to confirm the defensive capacities of enterocytes against harmful environmental factors. This study highlights the importance of cellular defense mechanisms and opens new avenues for research on interactions based on various environmental challenges.
Response of enterocytes to inflammatory and oxidative challenges
Studying the response of enterocytes to inflammatory challenges is one of the important research aspects. Enterocytes were treated with LPS and Menadione to provide contexts for inflammation and oxidation. After the experiment, RNA was isolated for gene expression analysis. qRT-PCR techniques were used to determine how various substances can alter gene expression levels, thus affecting cellular activity.
The cellular response to oxidation is a pivotal point in multiple diseases such as diabetes and heart disease. The research shows how enterocytes can combat toxic substances and thus adapt to disturbed environments. A deep understanding of such responses contributes to the development of new therapeutic strategies that may be effective in managing complex health conditions and also provides evidence on how to enhance the functional performance of various organs.
Generation of reactive oxygen species (ROS) and their response to challenges
The generation of reactive oxygen species is recognized as an important marker of cellular damage. Using the CellRox commercial kit, it is possible to measure how enterocytes are affected by high levels of reactive oxygen. Experimental procedures that involve diverse platforms to study the extent of the cells’ ability to confront these challenges indicate their defensive and adaptive skills.
Experimental results indicate direct outcomes in ROS production upon exposure to chemicals such as menadione. Experimental data may reveal new pathways to understand the cellular processes occurring under oxidative stress, and also provide indicators of when and how to develop appropriate treatments to mitigate the effects of oxidative stress. These hypotheses include enhancing cellular responses or augmenting natural defenses against cellular damage.
RNA isolation and gene expression analysis
RNA was isolated from enterocytes at different culture stages to evaluate basic cellular activity. The aim of these procedures is to determine how cells may be affected by various factors and analyze gene expression as a standard tool. In this approach, an RNA isolator device and a qRT-PCR system were used to analyze changes in gene expression, contributing to determining whether there is a difference due to exposure to irritant substances.
Gene expression studies are a vital part of understanding biological functions and the potential for internal damage or morphological changes that might adversely affect overall performance. This study can help unveil genetic patterns associated with increased risk and heightened sensitivity to harmful factors, potentially contributing to the development of suitable therapeutic intervention strategies.
Statistical analysis and the impact of different experiments
The statistical analysis required accurate measurement of the results obtained during the experiments, where means and standard deviations of cellular activity measurements were calculated. Various analytical methods, including ANOVA, were used for statistical evaluation and the impact of different treatments. The study design ensured transparency and accuracy in data collection, thereby increasing the reliability of the results.
These results are not only significant for scientific research but also shed light on how scientific findings translate from the lab to healthcare practices. Providing reliable analytical tools is crucial for gene research and cellular response. This also aids in offering effective insights for developing new treatments or improving existing therapies, contributing to evidence-based solutions for numerous health issues.
Analysis
Activity of Enterocytes in Culture
The study results showed a significant enrichment in the activity of enterocyte cells during the first 48 hours of culture, peaking on the third day before starting to decline until the seventh day. The healthy enterocyte cells concentrated to approximately 150-200 cells per well on the first day, and over time, the total numbers of healthy enterocytes began to gradually decrease. After 24 hours of culture, we observed a notable decrease of about 25% by the fourth day and 55% by the seventh day compared to the first day. These data suggest that the overall structure of the enterocytes began to deteriorate during the culture process, although individual cellular activity remained relatively stable. For example, the research team used the PrestoBlue assay to measure cell activity as a means to determine their viability impairments as culture progressed.
Description of Gene Markers in Chicken Enterocytes
The expressions of epithelial cell genes changed significantly over the days of culture. For the first time, researchers noted a substantial decrease in the expression of the stem cell marker, G-protein-coupled receptors (Lgr-5), on the fourth and sixth days compared to the second day, indicating a reduction in stem cells. In contrast, there was a notable increase in the expression of genes associated with differentiated epithelial cells such as Paneth cells, intestinal cells, and goblet cells. These results indicate that there is a disparity between cellular activities and gene expression, as the decline in the expression of hormonal cell markers continued on the fourth and sixth days.
Glucose Absorption Function in Chicken Enterocytes
The function of the small intestine in absorbing nutrients through the apical membrane is central to studies related to enterocytes. Researchers used a fluorescent glucose probe (2NBDG) to assess glucose absorption function. After 30 minutes of exposure to 2NBDG, fluorescent signaling was detected inside each enterocyte. The interaction with the SGLT1 inhibitor showed a significant decrease in fluorescence intensity, confirming that this absorption occurs via the SGLT1 transporter.
Epithelial Barrier Function in Enterocytes
The intestinal barrier function is crucial for separating different components within the intestine. Results showed that the FD4 molecule leaked through the epithelial tight junctions, resulting in a significant increase in FD4 permeability due to safety treatments such as EDTA and LPS. The study highlighted that both EDTA and LPS can cause significant changes in the function of this barrier, indicating the need for a deeper understanding of how these substances interact with enterocytes.
Enterocyte Response to Inflammatory Challenge
Upon adding LPS to the enterocytes, a significant decrease in the expression of tight junction proteins was observed, alongside a substantial increase in the expression of inflammatory markers. The resulting inflammatory response was similar to that observed in intestinal tissues, suggesting that enterocytes could be considered a good model for studying the effects of inflammation in the gut.
Enterocyte Response to Oxidative Challenge
The study used melatonin to enhance oxidative challenges within the enterocytes. A notable increase in the production of reactive oxygen species (ROS) was recorded at all the concentrations used. Specific doses were chosen to induce oxidative stress, where a reduction in the expression of genes responsible for combating free radical oxidation was observed. These results indicate the capability of enterocytes to generate ROS and utilize antioxidant enzyme defenses when exposed to oxidative challenges.
Entrocyte Models of Bacteria and Their Use in Research
Entrocyte models are among the advanced biological models used to study intestinal physiology, especially in poultry. These models are derived from intestinal tissues that are miniaturized in vitro, allowing researchers to study multiple cell interactions. These models enable the study of live intestinal properties, such as nutrient absorption and intestinal barrier integrity. Entrocyte models represent the cellular diversity found in actual intestines, making them a powerful tool for analyzing nutritional mechanisms and host-microbe communication.
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Recent research indicates that these models can be used to study the intestinal response to various challenges it faces. For example, the enteroids were used to study the effect of sugars on nutrient absorption. The absorption of sugars in the intestine requires the transport protein SGLT1, which has proven effective in transporting glucose into intestinal cells. Through various experiments, it has been documented that glucose absorption significantly increases during the early days of enteroid culture, reflecting cell maturation and a decrease in glucose absorption by day 6.
Enteroid Responses to Inflammatory Challenges
The intestinal response to inflammatory crises is an important subject addressed in research. Various compounds such as LPS were used to assess their impact on the integrity of the intestinal barrier and cellular responses. In studies, it was observed how exposure to LPS leads to increased permeability of the intestinal barrier, allowing hypothetical molecules to flow through. Gene expression was also observed to be significantly affected, with increased expression of inflammatory genes measured.
The importance of understanding how inflammation affects living organisms, especially in poultry, increases. The rapid reactions to increased inflammation on immune cell stimulation and tissue response highlight the importance of these studies. An assay was also used to study the effects of toxic substances such as EDTA, which weaken the intercellular connections and contribute to understanding how the front barrier of the intestine collapses. This understanding provides deeper insights for researchers in the fields of biology and veterinary medicine.
Studying the Effects of Oxidative Stress on Enteroids
Oxidative stress is one of the critical factors affecting intestinal health. In the context of research, the compound H₂O₂ was used as a stimulus to assess responses to oxidative stress. Studies have shown that substances such as menadione can be used for a better response in evaluating the negative effects of oxidative stress on intestinal cells.
In the experiments, different levels of menadione were administered to monitor their response, resulting in enhanced production of reactive oxygen species in enteroids. Finally, some changes in gene expression were observed, where the study showed a decrease in the expression of antioxidant enzyme genes, indicating that enteroids can be an important tool for exploring the effects of oxidative stress and how to interact with different factors and their impact on the overall health of living tissues.
Nutritional Transport Analysis in Enteroid Models
Nutritional transport is a vital component of any study related to the intestines. Enteroid models were used to study how glucose, minerals, and other nutrients are absorbed. Through experiments, the peak of glucose absorption was identified on day 4 of enteroid culture, indicating functional maturation of intestinal cells. The results suggest that the SGLT1 protein is the key element responsible for glucose transport, highlighting its importance in nutritional processes overall.
It was observed how the absorption pattern changes as the days progress, with a set of proteins identified as being used in tight junctions, which indicated the development of the intestinal barrier. This suggests that the models functionally represent the reality of the intestine in various contexts, such as the presence of nutrients and microbes. Understanding the mechanism of nutritional transport and the constraints placed on it aids in veterinary, nutritional studies, and public health research.
The Importance of the Model in Future Studies
Enteroid models represent a significant advancement in biological and food research. Their ability to be used in understanding intestinal responses to different challenges enhances our capacity to conduct clinical and cellular experimental studies. This model showcases the boundary between traditional research tools and modern ones, allowing for the opportunity to expand the scope of experiments with new methods.
Researching the links between nutrition, public health, and microbes within the models embodies a deeper understanding of how they influence intestinal health. With the capability to incorporate external factors such as feed additives or microbes, the importance of researching enteroid models is highlighted within the context of future studies. The results derived from such studies illuminate how biological models can be utilized to address the increasing challenges of public and veterinary health worldwide.
Understanding
Cultural Isolation of the Intestines
The concept of cultural isolation of the intestines is related to enhancing a good understanding of the physiological and biological characteristics of the intestines through cell models and their specific isolates. These isolates allow researchers to study cellular interactions and vital processes in a controlled environment, providing deep insights into the health status of the intestines. The intestines play a significant role in immunity and digestion, so understanding intestinal tissues and cells is vital for developing research related to various diseases and disorders.
For example, intestinal isolates can be used to study how the body responds to infections or certain diseases. Through these models, scientists can clarify the complex mechanisms that govern intestinal health and how external factors such as diet or pollutants can affect this system.
Moreover, cultural isolation not only provides scientists with a means to understand diseases but also enables them to test new drugs or treatments more effectively. These intestinal isolates are used in drug development and quality assessment, facilitating more precise and effective treatments for patients. There is also a need to continue developing these isolates to improve their accuracy and ability to interact with human systems.
Conflicts and Interests of the Authors
Conflict of interest is a sensitive topic in the world of scientific research. Researchers must be transparent about any financial or commercial affiliations that may affect their research. In this context, the authors mentioned were employees of Eastman Chemical Company, which may raise questions about the reliability of the results and research trends aimed at serving the company’s interests. These considerations require attention and deep study from the scientific community and critics.
The potential impacts arising from conflicts of interest indicate the necessity to develop clear systems and processes to ensure the reliability of scientific data. It is important to establish clear standards for transparency and encourage researchers to report any affiliations that may affect the integrity of the research. This not only protects the reputation of researchers but also ensures that science advances in reliable and fair ways.
The pressures that may come from pharmaceutical companies or aging agencies emphasize the need for an objective and unbiased view in scientific research. Certainly, this ethical balance requires raising societal awareness and engaging in open discussions about the role of what is known as “conflict of interest” in scientific research.
Challenges and Innovations in Intestinal Models
Engineered models of intestinal cells have radically changed our understanding of intestinal functions. Such models allow scientists to study the complex interactions between intestinal cells and environmental factors. However, these models are not without challenges. Most research relies on in vitro cellular models, which may lead to differences in how tissues respond when they show interactions in real biological environments.
Three-dimensional intestinal models presented by researchers exemplify how these systems can be improved to accommodate more real-life factors affecting intestinal health. Such models provide valuable information about how the intestines interact with dietary and pharmaceutical factors, enhancing the necessary understanding of the implications of lifestyle choices.
One important example is studying the effect of certain dietary supplements on intestinal healing. Through live models, the effectiveness of these supplements in promoting intestinal health can be assessed, providing vital information for the food and dietary supplement industry.
Awareness and Innovation in Intestinal Research
Research on intestinal health is closely related to overall health and innovation in developing preventive support strategies. Ongoing research in diverse techniques for analyzing and studying the intestines opens a wide field for thinking about new methods to improve digestive health. Learning from previous research contributes to enhancing modern designs of treatments and food products.
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For example, biographic techniques make it easy to measure the various factors that affect the intestinal walls, helping researchers uncover ways that can enhance or weaken gut health based on surrounding factors.
The need for innovation is clearly evident in living models, and studies have continued to further develop these models to keep up with changing needs. This requires scientists to partner with institutions and companies to develop new tools that support their research and make it more accurate and reliable. These partnerships may contribute to expanding knowledge and applying results in broader fields such as diagnosis and treatment.
Source link: https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2024.1470009/full
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