fecal deposit analysis of drosophila for the assessment of antidiarrheal drugs and plant extracts

초파리 약물 발견을 위한 분변 침전물 분석

The gastrointestinal (GI) tract, also called the digestive tract, is responsible for the digestion and absorption of nutrients and excretion of undigested products1. The GI tract is vulnerable to a range of disorders that can cause discomfort, pain, and disruption to daily life. Gastrointestinal disorders include abdominal pain and discomfort, bloating, heartburn, indigestion or dyspepsia, nausea, vomiting, diarrhea, and constipation2. Diarrhea is the most common symptom of GI disorder3, and it is defined as a disease with at least three loose and watery stool during a 24 h period4. Diarrhea is caused by a wide range of pathogens, including bacteria, viruses, parasites, fungi, and can also be caused by drugs5,6. Worldwide, diarrhea continues to be the second leading cause of mortality among children under 5 years7. Although diarrhea can resolve itself, it can also indicate a more severe underlying condition if it lasts for more than a few days.
To study the intestinal tract, researchers turn to animal models such as mice, rats, and pigs8,9. However, the use of these animals can be expensive and time-consuming because they require specialized facilities and ethical considerations. Recent studies have shown that D. melanogaster can be used as a model to study the GI tract and investigate some mechanisms such as the maintenance of regenerative homeostasis, the development of immune senescence, the loss of epithelial barrier function, and the decline in metabolic homeostasis10,11. D. melanogaster, known as the fruit fly, shares a high degree of genetic homology with humans; approximately 75% of human disease genes are believed to have a functional homolog in fly12. They also have a simple digestive system consisting of a foregut, a midgut, and a hindgut13. D. melanogaster is easy to culture in the laboratory and can be genetically modified in different ways14. Therefore, using D. melanogaster for in vivo testing is a powerful tool that allows researchers to study complex biological processes in a controlled setting.
According to the World Health Organization (WHO), about 80% of people living in developing countries use traditional medicine for their primary health needs15. The high use of medicinal plants can be explained by the fact that they are easily available, inexpensive, and have few side effects16. The main plant parts used in herbal therapy include leaves, bark, roots, seeds17 while the main methods of preparation are infusion, decoction, and maceration18. These herbal remedies contain phytochemical substances such as alkaloids, terpenoids, flavonoids, steroids, tannins and carbohydrates19, which have therapeutic effects on the human body. People use a variety of medicinal plants to treat GI disorders such as diarrhea, stomachache, and dysentery20. For example, Psidium guajava is one of the most commonly used plants to treat diarrhea in the world. Various pharmacological and clinical tests have already showed its safety, which make it a good antidiarrheal candidate to study21,22. However, the major limitations of herbal medicines are the lack of efficiency and safety assessment, as well as a lack of definite and complete information about the composition of plant extracts used23. To validate the efficiency and the safety of herbal medicines, a systematic approach involving experimental and clinical validation is required and the approach should be supported by enough data from in vivo and in vitro studies.
To evaluate traditional remedies for their efficacy in the treatment of diarrhea, the use of mice and rats have been predominant in recent decades24,25. Due to the main advantages mentioned previously, i.e., ease of use, affordable, replicable, conserved absorptive and digestive functions between flies and mammals, we propose to use D. melanogaster as a model to evaluate the antidiarrheal activity of plants. The diarrheic phenotype in D. melanogaster can be characterized by several features, including increased abundance of fecal deposits, larger deposit sizes, a lighter coloration (less concentrated), and higher fecal material26. This phenotype can be quantified using various parameters: number of fecal deposits, total area of deposits, mean lightness, and total integrated optical density (IOD). Total IOD is defined as the total dye content of the deposit, meaning the total fecal material excreted27. Previously, an assay has been developed to analyze fecal deposits of D. melanogaster27,28. In this assay, the ultimate reader of dung (T.U.R.D.) was used as a fecal analysis tool, which allows to check for the number, size and lightness of fecal deposits and thus to monitor the intestinal physiology of the fruit flies. However, this method was never applied to evaluate the diarrheic phenotype in flies. The Ion Transport Peptide (ITP) gene is an important endocrine regulator of thirst and excretion and combines water homeostasis with feeding in D. melanogaster. In a recent study, it was shown that the speed of food transit throughout the GI tract and the frequency of defecation events were decreased by ITP over-expression and increased by ITP knockdown. The latter phenotype was described as diarrheic by the authors of this study29.
In this protocol, a modified version of the fecal deposit assay is employed to assess the effect of an antidiarrheal agent (i.e., guava leaf extract) on the gastrointestinal tract of D. melanogaster by using the ITPi strain as a diarrheic model. The overall goal of this method is: 1) to provide an easy and reliable method to evaluate the antidiarrheal effect of drugs and plant extracts, and 2) to allow the discovery of bioactive compounds responsible for the antidiarrheal effect in plant extracts by applying a bioactivity-guided approach.

저자 스포트라이트: Advancing Antidiarrheal Research with the Drosophila Model (초파리 모델을 통한 지사제 연구의 발전) 

Drosophila melanogaster에 대한 지사제 및 식물 추출물의 효과 평가

Our research focuses on the scientific validation of traditional remedies, including medicinal plants. We are trying to better understand the pharmacological action, especially antidiarrheal and antibacterial effects of these remedies. To test the antidiarrheal effects of medicinal plants, mice models are usually employed, but the process is time consuming and expensive, and it requires a large quantity of plant material to be tested.By using Drosophila melanogaster as a model, we aim to improve the discovery process of new antidiarrhea agents by reducing the experimental time and also the quantity of plant material used. We also aim to provide a scientific rationale for the use of antidiarrhea plants in traditional medicine by using a simple and effective model. Our findings will help scientists to perform bio-guided fractionation for plant extracts and thus allow the discovery of compounds that are responsible for the antidiarrhea activity.

Watch this Scientific Journal Video about Fecal Deposit Analysis of Drosophila for the Assessment of Antidiarrheal Drugs and Plant Extracts at JoVE.com

Fecal Deposit Analysis of Drosophila for the Assessment of Antidiarrheal Drugs and Plant Extracts  

Fecal Deposit Analysis of Drosophila for the Assessment of Antidiarrheal Drugs and Plant Extracts

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327 조회수.  UMR 152 PharmaDev, Université Paul Sabatier, Institut de Recherche pour le Développement. Fecal Deposit Analysis of Drosophila for the Assessment of Antidiarrheal Drugs and Plant Extracts

비디오: Fecal Deposit Analysis of Drosophila for the Assessment of Antidiarrheal Drugs and Plant Extracts  

Assessment of The Effect of Antidiarrheal Drugs and Plant Extracts on Drosophila melanogaster

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a model to study intestinal function, only a few reports have focused on Drosophila melanogaster (D. melanogaster). Compared to mice, fruit flies present many advantages, such as a short life cycle, cost-effective and simple maintenance, and no ethical issues. Furthermore, the mammalian gastrointestinal physiology, anatomy, and signaling pathways are highly conserved in D. melanogaster. Plant extracts have been used traditionally to treat diarrhea and constipation. For example, Psidium guajava (P. guajava) is one of the most known antidiarrheal agents in the tropics. However, no studies have evaluated the effect of antidiarrheal and laxative drugs and plant extracts in D. melanogaster, and it remains unknown if similar effects (e.g., smaller, more concentrated, and less abundant fecal deposits in the case of antidiarrheal drugs) can occur in the fruit flies compared to mammals. In this study, an antidiarrheal effect induced by P. guajava is demonstrated in a D. melanogaster strain that presents a diarrheic phenotype. Fecal sampling produced by flies is monitored using a dye-supplemented food. This protocol outlines the method used for preparing food with drugs, evaluating the fecal deposits of flies fed on these food preparations, and interpreting the data obtained.

Here, a method is described to feed Drosophila melanogaster with drugs and plant extracts and assess their effect on the gastrointestinal tract by analyzing the fruit flies fecal deposits. The drug-treated flies can be used as a model for further research.

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a ...