Epithelial Cell Model Research Articles

Calycosin prevents NLRP3-induced gut fibrosis by regulating IL-33/ST2 axis

Intestinal interstitial fibrosis is a core event of inflammatory bowel disease (IBD) development. Calycosin has been recognized to carry various therapeutic bioactivities. However, the role of calycosin in intestinal interstitial fibrosis remains to be illustrated. This aim of this study was to explore the effects of calycosin on intestinal interstitial fibrosis in IBD and the underlying mechanisms. The in vitro and in vivo models were established by using TNBS-induced mouse IBD model and co-culture of intestinal epithelial cells and intestinal interstitial cells; moreover, lentivirus-mediated knockdown of NLRP3 expression was applied. The results showed that calycosin significantly improved the intestinal interstitial fibrosis of TNBS-induced IBD. Mechanistically, calycosin downregulated NLRP3 expression and inhibited the activation of IL-33/ST2 signaling in intestinal epithelial cells, which subsequently impedes intestinal interstitial cell migration and activation by regulating the secretion of IL-33/ST2 signaling-induced fibrosis mediators. Notably, combination of calycosin and NLRP3 signaling blockade improved the intestinal interstitial fibrosis extent. Altogether, this study suggests calycosin can improve intestinal interstitial fibrosis by downregulating NLRP3-IL-33/ST2 signaling, reducing inflammation and decreasing pro-fibrotic factors’ secretion, which provides a new perspective for therapeutic options of IBD.

Conditional reprogrammed human limbal epithelial cell model for anti-SARS-CoV-2 drug screening

To minimize the global pandemic COVID-19 spread, understanding the possible transmission routes of SARS-CoV-2 and discovery of novel antiviral drugs are necessary. We describe here that the virus can infect ocular surface limbal epithelial, but not other regions. Limbal supports wild type and mutant SARS-CoV-2 entry and replication depending on ACE2, TMPRSS2 and possibly other receptors, resulting in slight CPE and arising IL-6 secretion, which symbolizes conjunctivitis in clinical symptoms. With this limbal model, we have screened two natural product libraries and discovered several unreported drugs. Our data reveal important commonalities between COVID-19 and ocular infection with SARS-CoV-2, and establish an ideal cell model for drug screening and mechanism research.

Das Zöliakie‐Superantigen Oligomerisiert und Erhöht die Permeabilität in einem Enterozyten‐Zellmodell

AbstractZöliakie (CeD) ist eine Autoimmunerkrankung, die durch Glutenproteine ausgelöst wird und von der etwa 1 % der Weltbevölkerung betroffen ist. Das 33‐mer deamidierte Gliadinpeptid (DGP) ist ein metabolisch modifiziertes Weizengluten‐Superantigen für CeD. Hier zeigen wir, dass das 33‐mer DGP spontan zu Oligomeren mit einem Durchmesser von etwa 24 nm aggregiert. Die 33‐mer‐DGP‐Oligomere weisen zwei sekundäre Hauptstrukturmotive auf: Eine große Polyprolin‐II‐Helix und eine kleine β‐Faltblattstruktur. Wichtig ist, dass in Anwesenheit von 33‐mer‐DGP‐Oligomeren eine statistisch signifikante Erhöhung der Permeabilität im Caco‐2 Darmepithelzellmodell auftritt, begleitet von einer Umverteilung von Zonula occludens‐1, einem Haupt‐Tight‐Junction‐Protein. Diese Ergebnisse bieten neue molekulare und supramolekulare Einblicke in die Auswirkungen von 33‐mer DGP bei CeD und unterstreichen die Bedeutung der Gliadin‐Peptid‐Oligomerisierung.

The Celiac-Disease Superantigen Oligomerizes and Increases Permeability in an Enterocyte Cell Model.

Celiac disease (CeD) is an autoimmune disorder triggered by gluten proteins, affecting approximately 1 % of the global population. The 33-mer deamidated gliadin peptide (DGP) is a metabolically modified wheat-gluten superantigen for CeD. Here, we demonstrate that the 33-mer DGP spontaneously assembles into oligomers with a diameter of approximately 24 nm. The 33-mer DGP oligomers present two main secondary structural motifs-a major polyproline II helix and a minor β-sheet structure. Importantly, in the presence of 33-mer DGP oligomers, there is a statistically significant increase in the permeability in the gut epithelial cell model Caco-2, accompanied by the redistribution of zonula occludens-1, a master tight junction protein. These findings provide novel molecular and supramolecular insights into the impact of 33-mer DGP in CeD and highlight the relevance of gliadin peptide oligomerization.

Human health risk assessment and toxic effect of heavy metal(loid)s in Bamboo shoot products

Bamboo shoots and their products are an important part of the human diet. However, the accumulation and accurate human health risk of heavy metal(loid)s in bamboo shoots are largely unknown. In this study, we determined the total and bioaccessible concentrations of As, Cd, Pb, and Cr in natural and processed bamboo shoot products from markets in southwest China. The health risk assessment and enterotoxicity after consumption of heavy metal(loid)s contaminated samples were further studied using a human colonic epithelial cell (Caco-2 cell) model. The results showed that the average concentrations of As, Pb, Cd, and Cr in the collected samples were 0.036, 0.056, 0.008, and 0.049 mg/kg, while Pb contents in 21 collected fresh bamboo shoot samples exceeded the allowable limit value of 0.1 mg/kg by 1.00–4.60 folds. Besides, As in 2% fresh bamboo shoots exceeded the limit value of 0.50 mg/kg. The average bioaccessibility of heavy metal(loid)s in fresh bamboo shoots was as follows: Cr (47.20%) > Pb (39.56%) > Cd (15.64%)> As (13.29%). Estimated daily intake (EDI) of heavy metal(loid)s after intake of fresh bamboo shoots was higher in children than that in adults, the THQ of As is > 1 based on the total heavy metal(loid)s, indicating a potential non-carcinogenic risk for children. However, health risks assessment based on bioaccessibility were significantly decreased. Furthermore, intestinal digesta of high-risk samples did not trigger detrimental effects on Caco-2 cells, suggesting existing health risk assessment model may overestimate their health risks and should be adjusted by bioaccessibility and cellular toxicity validation to accurately reflect their health risk. Our data may provide a novel and scientific insight into the health risk assessment of bamboo shoots as well as their products and propose a more scientific approach to accurate health risk assessment for heavy metals polluted foods.

Single-cell calcium monitoring of Caco-2 cell co-cultured with intestinal microbiome through carbon fiber based potentiometric microelectrode

The Caco-2 cells were used as intestinal epithelial cell model to illustrate the hyperuricemia (HUA) mechanism under the co-culture of the imbalanced intestinal microbiome in this work. The uric acid (UA) concentration in the HUA process was monitored, and could be up to 425 μmol/L at 8 h co-cultured with the imbalanced intestinal microbiome. Single-cell potentiometry based on ion-selective microelectrode was used to study extracellular calcium change, which is hypothesized to play an important role in the UA excretion. The potential signal of the calcium in the extremely limited microenvironment around single Caco-2 cell was recorded through the single-cell analysis platform. The potential signal of sharp decrease and slow increase followed within a few seconds indicates the sudden uptake and gradually excretion process of calcium through the cell membrane. Moreover, the value of the potential decrease increases with the increase of the time co-cultured with the imbalanced intestinal microbiome ranging from 0 to 8 h. The Ca2+ concentration around the cell membrane could decrease from 1.3 mM to 0.4 mM according to the potential decrease of 27.0 mV at the co-culture time of 8 h. The apoptosis ratio of the Caco-2 cells also exhibits time dependent with the co-culture of the imbalanced intestinal microbiome, and was 39.1 ± 3.6 % at the co-culture time of 8 h, which is much higher than the Caco-2 cells without any treatment (3.9 ± 2.9 %). These results firstly provide the links between the UA excretion with the apoptosis of the intestinal epithelial cell under the interaction of the imbalanced intestinal microbiome. Moreover, the apoptosis could be triggered by the calcium signaling.

Effect of SHP-1 knockout in airway epithelial cells on emphysema phenotype in chronic obstructive pulmonary disease in mice

Objective: To construct and characterize conditional Src homology region 2 protein tyrosine phosphatase 1 (SHP-1) knockout mice in airway epithelial cells and to observe the effect of defective SHP-1 expression in airway epithelial cells on the emphysema phenotype in chronic obstructive pulmonary disease (COPD). Methods: To detect the expression of SHP-1 in the airway epithelium of COPD patients. CRISPR/Cas9 technology was used to construct SHP-1flox/flox transgenic mice, which were mated with airway epithelial Clara protein 10-cyclase recombinase and estrogen receptor fusion transgenic mice (CC10-CreER+/+), and after intraperitoneal injection of tamoxifen, airway epithelial SHP-1 knockout mice were obtained (SHP-1flox/floxCC10-CreER+/-, SHP-1Δ/Δ). Mouse tail and lung tissue DNA was extracted and PCR amplified to discriminate the genotype of the mice; the knockout effect of SHP-1 gene in airway epithelial cells was verified by qRT-PCR, Western blotting, and immunofluorescence. In addition, an emphysema mouse model was constructed using elastase to assess the severity of emphysema in each group of mice. Results: Airway epithelial SHP-1 was significantly downregulated in COPD patients. Genotyping confirmed that SHP-1Δ/Δ mice expressed CC10-CreER and SHP-1-flox. After tamoxifen induction, we demonstrated the absence of SHP-1 protein expression in airway epithelial cells of SHP-1Δ/Δ mice at the DNA, RNA, and protein levels, indicating that airway epithelial cell-specific SHP-1 knockout mice had been successfully constructed. In the emphysema animal model, SHP-1Δ/Δ mice had a more severe emphysema phenotype compared with the control group, which was manifested by disorganization of alveolar structure in lung tissue and rupture and fusion of alveolar walls to form pulmonary alveoli. Conclusions: The present study successfully established and characterized the SHP-1 knockout mouse model of airway epithelial cells, which provides a new experimental tool for the in-depth elucidation of the role of SHP-1 in the emphysema process of COPD and its mechanism.

Protection of Qingfei Xieding prescription from idiopathic pulmonary fibrosis by regulating renin-angiotensin and ferroptosis in MLE-12 cells.

Idiopathic pulmonary fibrosis (IPF) is a lifelong lung disease, but there is no specific drug for treatment. Qingfei Xieding prescription (QF) is active in the treatment of lung diseases. More comprehensive mechanisms over how QF exhibits anti-pulmonary fibrosis need to be elucidated. TGF-β was used to construct a pulmonary fibrosis cell model in vitro. Bleomycin was applied to induce a lung tissue fibrosis model in mice in vivo. Flow cytometry was used to detect cellular ROS and lipid oxidation levels. Cell substructure was observed by Transmission Electron Microscopy. ELISA was used to determine the levels of inflammatory factors. HE staining, Masson staining and immunohistochemistry were performed to evaluate the degree of fibrosis. Western Blot assay was used to determine the protein expressions of different molecules. In TGF-β-exposed lung epithelial MLE-12 cell model, α-SMA and Collagen I were significantly elevated and cell viability was reduced. QF treatment restored the cell viability decreased by exogenous TGF-β. Ferroptosis inducer Erastin administration could reverse the beneficial effects such as lipid oxidation and ROS reduction caused by QF treatment. QF was proven to inhibit ferroptosis and alleviated the process of IPF by activating ACE2 signal axis. In bleomycin induced IPF mice model, QF altered lung coefficient, body weight and the expression of inflammatory factors, which were prevented by ferroptosis activator Erastin. QF was demonstrated to affect the ACE2-ERK signaling axis in vivo. QF alleviated idiopathic pulmonary fibrosis by regulating renin-angiotensin through blocking ferroptosis. This research offers evidence for the potentiality of QF in clinical application for IPF therapy.

Machine learning-based 3D segmentation of mitochondria in polarized epithelial cells

Mitochondria are dynamic organelles that alter their morphological characteristics in response to functional needs. Therefore, mitochondrial morphology is an important indicator of mitochondrial function and cellular health. Reliable segmentation of mitochondrial networks in microscopy images is a crucial initial step for further quantitative evaluation of their morphology. However, 3D mitochondrial segmentation, especially in cells with complex network morphology, such as in highly polarized cells, remains challenging. To improve the quality of 3D segmentation of mitochondria in super-resolution microscopy images, we took a machine learning approach, using 3D Trainable Weka, an ImageJ plugin. We demonstrated that, compared with other commonly used methods, our approach segmented mitochondrial networks effectively, with improved accuracy in different polarized epithelial cell models, including differentiated human retinal pigment epithelial (RPE) cells. Furthermore, using several tools for quantitative analysis following segmentation, we revealed mitochondrial fragmentation in bafilomycin-treated RPE cells.

Effect of the dietary supplement PERMEAPROTECT+ TOLERANCE© on gut permeability in a human co-culture epithelial and immune cells model

Background and objectiveThe leaky gut syndrome is characterized by an intestinal hyperpermeability observed in multiple chronic disorders. Alterations of the gut barrier are associated with translocation of bacterial components increasing inflammation, oxidative stress and eventually dysfunctions of cellular interactions at the origin pathologies. Therapeutic and/or preventive approaches have to focus on the identification of novel targets to improve gut homeostasis. In this context, this study aims to identify the role of PERMEAPROTECT + TOLERANE©, known as PERMEA, a food complement composed of a combination of factors (including l-Glutamine) known to improve gut physiology. MethodsWe tested the effects of PERMEA or l-Glutamine alone (as reference) on gut permeability (FITC dextran method, expression of tight junctions) and its inflammatory/oxidative consequences (cytokines and redox assays, RT-qPCR) in a co-culture of human cells (peripheral blood mononuclear cells and intestinal epithelial cells) challenged with TNFα. ResultsPERMEA prevented intestinal hyperpermeability induced by inflammation. This was linked with its antioxidant and immunomodulatory properties showing a better efficacity than l-Glutamine alone on several parameters including permeability, global antioxidant charge and production of cytokines. ConclusionPERMEA is more efficient to restore intestinal physiology, reinforcing the concept that combination of food constituents could be used to prevent the development of numerous diseases.

IL-6 mediates olfactory dysfunction in a mouse model of allergic rhinitis

BackgroundImmune-inflammatory response is a key element in the occurrence and development of olfactory dysfunction (OD) in patients with allergic rhinitis (AR). As one of the core factors in immune-inflammatory responses, interleukin (IL)-6 is closely related to the pathogenesis of allergic diseases. It may also play an important role in OD induced by diseases, such as Sjögren’s syndrome and coronavirus disease 2019. However, there is no study has reported its role in OD in AR. Thus, this study aimed to investigate the role of IL-6 in AR-related OD, in an attempt to discover a new target for the prevention and treatment of OD in patients with AR. MethodsDifferential expression analysis was performed using the public datasets GSE52804 and GSE140454 for AR, and differentially expressed genes (DEGs) were obtained by obtaining the intersection points between these two datasets. IL-6, a common differential factor, was obtained by intersecting the DEGs with the General Olfactory Sensitivity Database (GOSdb) again. A model of AR mice with OD was developed by sensitizing with ovalbumin (OVA) to verify the reliability of IL-6 as a key factor of OD in AR and explore the potential mechanisms. Furthermore, a supernatant and microglia co-culture model of nasal mucosa epithelial cells stimulated by the allergen house dust mite extract Derp1 was established to identify the cellular and molecular mechanisms of IL-6-mediated OD in AR. ResultsThe level of IL-6 in the nasal mucosa and olfactory bulb of AR mice with OD significantly increased and showed a positive correlation with the expression of olfactory bulb microglia marker Iba-1 and the severity of OD. In-vitro experiments showed that the level of IL-6 significantly increased in the supernatant after the nasal mucosa epithelial cells were stimulated by Derp1, along with significantly decreased barrier function of the nasal mucosa. The expression levels of neuroinflammatory markers IL-1β and INOS increased after a conditioned culture of microglia with the supernatant including IL-6. Then knockdown (KD) of IL-6R by small interfering RNA (siRNA), the expression of IL-1β and INOS significantly diminished. ConclusionIL-6 plays a key role in the occurrence and development of OD in AR, which may be related to its effect on olfactory bulb microglia-mediated neuroinflammation.

Characterization of the intestinal transport mechanism of polystyrene microplastics (MPs) and the potential inhibitory effect of green tea extracts on MPs intestinal absorption

The aims of the current study included characterizing the intestinal transport mechanism of polystyrene microplastics (MPs) with different charges and sizes in the intestinal epithelial cell model and determining the inhibitory effect of green tea extracts (GTEs) on the intestinal absorption of MPs in Caco-2 cells. The smaller sizes, which included diameters of 0.2 μm, of amine-modified MPs compared to either larger size (1 μm diameter, or carboxylate-MPs (0.2 and 1 μm diameter) significantly lowered the cell viability of caco-2 cells that were measured by MTT assay (p < 0.05). The transported amount (particles/mL of the cell media) of amine-modified MPs by the Caco-2 cell, was not dependent according to the concentrations, energy, or temperature, but it was higher than the carboxylate-modified MPs. The co-treatment of GTEs with the amine-modified MPs inhibited Caco-2 cell cytotoxicity as well as reduced the production of intracellular reactive oxygen species (ROS) in HepG2 generated by the exposure of amine-modified MPs. The GTEs co-treatment also increased trans-epithelial electrical resistances (TEER) and reduced the transportation of Lucifer Yellow via the Caco-2 monolayer compared to only the amine-modified MPs exposure. The GTEs treatment led to a decrease in the number of amine-modified MPs transported to the basal side of the Caco-2 monolayer. The results from our study suggest that the consumption of GTEs could enhance the intestinal barrier function by recovering intestinal epithelial cell damage induced by MPs, which resulted in a decrease of the intestinal absorption of MPs.

Circ_0070934 promotes MGAT3 expression and inhibits epithelial-mesenchymal transition in bronchial epithelial cells by sponging miR-199a-5p

BackgroundCircular RNA (circRNA) has the potential to serve as a crucial regulator in the progression of bronchial asthma. The objective of this investigation was to elucidate the functional dynamics of the circ_0070934/miR-199a-5p/Mannoside acetylglucosaminyltransferase 3 (MGAT3) axis in the development of asthma.MethodsCirc_0070934, miR-199a-5p and MGAT3 in peripheral venous blood of 38 asthmatic patients and 43 healthy controls were detected by qRT-PCR, and the expression of MGAT3 protein was examined by ELISA. The GSE148000 dataset was analyzed for differences in MGAT3. The BEAS-2B cells were transfected with circ_0070934 plasmid and small interfering RNA, miR-199a-5p mimics and inhibitors. The apoptosis level was detected by flow cytometry and MGAT3 was detected by qRT-PCR and Western blot. The expression of E-cadherin, N-cadherin, Vimentin was examined by Western blot. Interleukin-4 (IL-4) and IL-13 were used to co-stimulate BEAS-2B cells as an asthmatic airway epithelial cell model. BEAS-2B cells exposed to type 2 cytokines (IL-4 and IL-13) were treated with circ_0070934 plasmid, and the expression of E-cadherin, N-cadherin, and Vimentin was detected by Western blot. The binding relationships were verified using dual-luciferase reporter assay and miRNA pull-down assay.ResultsThe expression of circ_0070934 and MGAT3 in peripheral venous blood of asthmatic patients was down-regulated, and the expression of miR-199a-5p was up-regulated. And the expression of MGAT3 was reduced in sputum of asthma patients. Down-regulating the expression of circ_0070934 could promote apoptosis of BEAS-2B cells and increase epithelial-mesenchymal transition (EMT), and this effect can be partially reversed by down-regulating miR-199a-5p. Circ_0070934 could inhibit the process of epithelial mesenchymal transition induced by IL-4 and IL-13 in BEAS-2B cells. In addition, miR-199a-5p could respectively bind to circ_0070934 and MGAT3.ConclusionThe findings of this study indicate that circ_0070934 may function as a competitive endogenous RNA (ceRNA) of miR-199a-5p, thereby modulating the expression of MGAT3 and impacting the process of EMT in bronchial epithelial cells. These results contribute to the establishment of a theoretical framework for advancing the prevention and treatment strategies for asthma.

Abstract 899: Development of a deep learning model for cell type mapping in colorectal cancer using H&amp;E images leveraging image-based spatial transcriptomics data

Abstract Purpose The tumor microenvironment (TME) is crucial in colorectal cancer as it influences disease progression, treatment response, and patient outcomes, providing valuable insights for personalized therapies and prognostic assessments. Here, we have developed a deep learning model by integrating hematoxylin and eosin (H&amp;E) stained images of colorectal cancer and image-based spatial transcriptomics (Xenium) to infer spatial mapping of cell types in TME only using H&amp;E images. Methods A total of 30 H&amp;E images of colorectal cancer obtained by tissue microarray were registered with image-based spatial transcriptomics data (Xenium). Utilizing a Variational Autoencoder (VAE) based model and leveraging reference single-cell data enables the acquisition of cell types for individual cells in image-based spatial transcriptomics. A convolutional neural network (CNN) model was developed using H&amp;E image as inputs to predict cell types in H&amp;E-stained tissue image patches of colorectal cancer collected from various patients. The model also estimated the cell types from H&amp;E-stained whole slide tissue image of colorectal cancer of The Cancer Genome Atlas (TCGA-COAD). Results The accuracy of the model's predictions for cell types using H&amp;E image patches was notably high and exhibited a significant concordance with the results obtained through the validation. The Intersection over Union (IoU) metric for image segmentation indicated a value of 0.66 for epithelial cells and 0.44 for TNK cells. The output of deep learning model for epithelial cells and T/NK cells from TCGA-COAD tissue images showed a correlation with human-labeled regions of cancer epithelium and immune cells. Conclusions Leveraging image-based spatial transcriptomics, we developed a deep learning model capable of discerning various cell types within the tumor microenvironment solely from H&amp;E images. This clinically translatable approach is valuable for investigating tumor microenvironment to develop biomarkers associated with various cancer therapeutics particularly immuno-oncology drugs. This approach can yield objective deep learning-based models without human labels for characterizing the tumor microenvironment in single-cell resolution, particularly regarding spatial immune distribution. Citation Format: Seungho Cook, Dongjoo Lee, Myunghyun Lim, Jae Eun Lee, Daeseung Lee, Hyung-Jun Im, Jung-Soo Pyo, Kwon Joong Na, Hongyoon Choi. Development of a deep learning model for cell type mapping in colorectal cancer using H&amp;E images leveraging image-based spatial transcriptomics data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 899.

Abstract 1528: Flowcytometric based approach to investigate prostate tumor microenvironment

Abstract Prostate cancer (PC) and its tumor microenvironment (TME) are heterogenous and evolving along with the treatment patients are exposed to. Modelling of the TME represents an attractive approach to understand how PC evolves and spreads. Flow cytometry is a technique for quantitative measurement of single cell profiling based on the cell surface as well as intracellular markers. We designed a flowcytometric based approach to investigate the interplay between the PC cells and immune cells in a direct coculture. Cells are fluorescently tagged with a cell tracer and the analysis is performed separately on the adherent and non-adherent cells. To optimize the readout method, we investigate the interplay between Jurkat cells and PC cells (LNCaP) as well as immortalized prostate epithelial cell models modified to mimic early events in prostate carcinogenesis (RWPE-1 cells overexpressing androgen receptor, RWPE-1-AR, and having PTEN deletion, RWPE-1-AR-PTENdel).Jurkat (suspension culture) cocultured with LNCaP (adherent culture) exhibited 40% decrease in viability but only 30% of LNCaP dying cell in the floating population compared to LNcaP only which is 60%. When Jurkat were activated using Phorbol 12-myristate 13-acetate (PMA), we observed an increase in LNCaP cell death. Jurkat viability was not impacted in presence of RWPE-1 neither when exposed to RWPE1-AR-PTENdel. In summary our Flowcytometric-based approach will help to understand the interplay between immune cells and prostate cancer cells. Citation Format: karishma sajnani, Sini Eerola, Konsta Kukkonen, Hanna Rauhala, Tapio Visakorpi, Alfonso Urbanucci. Flowcytometric based approach to investigate prostate tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1528.

Methionyl-Methionine Dipeptide Enhances Mammogenesis and Lactogenesis by Suppressing the Expression of a Novel Long Noncoding RNA MGPNCR to Inhibit eIF4B Dephosphorylation.

Previous research has demonstrated that in pregnant mice deficient in l-methionine (Met), the mixture of the dipeptide l-methionyl-l-methionine (Met-Met) with Met was more effective than Met alone in promoting mammogenesis and lactogenesis. This study aimed to investigate the role of a novel long noncoding RNA (lncRNA), named mammary gland proliferation-associated lncRNA (MGPNCR), in these processes. Transcriptomic analysis of mammary tissues from Met-deficient mice, supplemented either with a Met-Met/Met mixture or with Met alone, revealed significantly higher MGPNCR expression in the Met group compared to the mixture group, a finding recapitulated in a mammary epithelial cell model. Our findings suggested that MGPNCR hindered mammogenesis and milk protein synthesis by binding to eukaryotic initiation factor 4B (eIF4B). This interaction promoted the dephosphorylation of eIF4B at serine-422 by enhancing its association with protein phosphatase 2A (PP2A). Our study sheds light on the regulatory mechanisms of lncRNA-mediated dipeptide effects on mammary cell proliferation and milk protein synthesis. These insights underscore the potential benefits of utilizing dipeptides to improve milk protein in animals and potentially in humans.

Kynurenine 3-monooxygenase limits de novo NAD+ synthesis through dietary tryptophan in renal proximal tubule epithelial cell models.

Nicotinamide adenine dinucleotide (NAD+) is a pivotal coenzyme, essential for cellular reactions, metabolism, and mitochondrial function. Depletion of kidney NAD+ levels and reduced de novo NAD+ synthesis through the tryptophan-kynurenine pathway are linked to acute kidney injury (AKI), whereas augmenting NAD+ shows promise in reducing AKI. We investigated de novo NAD+ biosynthesis using in vitro, ex vivo, and in vivo models to understand its role in AKI. Two-dimensional (2-D) cultures of human primary renal proximal tubule epithelial cells (RPTECs) and HK-2 cells showed limited de novo NAD+ synthesis, likely due to low pathway enzyme gene expression. Using three-dimensional (3-D) spheroid culture model improved the expression of tubular-specific markers and enzymes involved in de novo NAD+ synthesis. However, de novo NAD+ synthesis remained elusive in the 3-D spheroid culture, regardless of injury conditions. Further investigation revealed that 3-D cultured cells could not metabolize tryptophan (Trp) beyond kynurenine (KYN). Intriguingly, supplementation of 3-hydroxyanthranilic acid into RPTEC spheroids was readily incorporated into NAD+. In a human precision-cut kidney slice (PCKS) ex vivo model, de novo NAD+ synthesis was limited due to substantially downregulated kynurenine 3-monooxygenase (KMO), which is responsible for KYN to 3-hydroxykynurenine conversion. KMO overexpression in RPTEC 3-D spheroids successfully reinstated de novo NAD+ synthesis from Trp. In addition, in vivo study demonstrated that de novo NAD+ synthesis is intact in the kidney of the healthy adult mice. Our findings highlight disrupted tryptophan-kynurenine NAD+ synthesis in in vitro cellular models and an ex vivo kidney model, primarily attributed to KMO downregulation.NEW & NOTEWORTHY Nicotinamide adenine dinucleotide (NAD+) is essential in regulating mitochondrial function. Reduced NAD+ synthesis through the de novo pathway is associated with acute kidney injury (AKI). Our study reveals a disruption in de novo NAD+ synthesis in proximal tubular models, but not in vivo, attributed to downregulation of enzyme kynurenine 3-monooxygenase (KMO). These findings highlight a crucial role of KMO in governing de novo NAD+ biosynthesis within the kidney, shedding light on potential AKI interventions.

Quercetin improves diabetic kidney disease by inhibiting ferroptosis and regulating the Nrf2 in streptozotocin-induced diabetic rats

Diabetic kidney disease (DKD) is a leading factor in end-stage renal disease. The complexity of its pathogenesis, combined with the limited treatment efficacy, necessitates deeper insights into potential causes. Studies suggest that ferroptosis-driven renal tubular damage contributes to DKD's progression, making its counteraction a potential therapeutic strategy. Quercetin, a flavonoid found in numerous fruits and vegetables, has demonstrated DKD mitigation in mouse models, though its protective mechanism remains ambiguous. In this study, we delved into quercetin’s potential anti-ferroptotic properties, employing a DKD rat model and high glucose (HG)-treated renal tubular epithelial cell models. Our findings revealed that HG prompted unusual ferroptosis activation in renal tubular epithelial cells. However, quercetin counteracted this by inhibiting ferroptosis and activating NFE2-related factor 2 (Nrf2) expression in both DKD rats and HG-treated HK-2 cells, indicating its renal protective role. Further experiments, both in vivo and in vitro, validated that quercetin stimulates Nrf2. Thus, our research underscores quercetin’s potential in DKD treatment by modulating the ferroptosis process via activating Nrf2 in a distinct DKD rat model, offering a fresh perspective on quercetin’s protective mechanisms.

Selective Replacement of Cholesterol with Cationic Amphiphilic Drugs Enables the Design of Lipid Nanoparticles with Improved RNA Delivery.

The delivery of RNA across biological barriers can be achieved by encapsulation in lipid nanoparticles (LNPs). Cationic amphiphilic drugs (CADs) are pharmacologically diverse compounds with ionizable lipid-like features. In this work, we applied CADs as a fifth component of state-of-the-art LNPs via microfluidic mixing. Improved cytosolic delivery of both siRNA and mRNA was achieved by partly replacing the cholesterol fraction of LNPs with CADs. The LNPs could cross the mucus layer in a mucus-producing air-liquid interface model of human primary bronchial epithelial cells following nebulization. Moreover, CAD-LNPs demonstrated improved epithelial and endothelial targeting following intranasal administration in mice, without a marked pro-inflammatory signature. Importantly, quantification of the CAD-LNP molar composition, as demonstrated for nortriptyline, revealed a gradual leakage of the CAD from the formulation during LNP dialysis. Altogether, these data suggest that the addition of a CAD prior to the rapid mixing process might have an impact on the composition, structure, and performance of LNPs.

An inulin-type fructan CP-A from Codonopsis pilosula attenuates experimental colitis in mice by promoting autophagy-mediated inactivation of NLRP3 inflammasome

Inulin-type fructan CP-A, a predominant polysaccharide in Codonopsis pilosula, demonstrates regulatory effects on immune activity and anti-inflammation. The efficacy of CP-A in treating ulcerative colitis (UC) is, however, not well-established. This study employed an in vitro lipopolysaccharide (LPS)-induced colonic epithelial cell model (NCM460) and an in vivo dextran sulfate sodium (DSS)-induced colitis mouse model to explore CP-A's protective effects against experimental colitis and its underlying mechanisms. We monitored the clinical symptoms in mice using various parameters: body weight, disease activity index (DAI), colon length, spleen weight, and histopathological scores. Additionally, molecular markers were assessed through enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting assays. Results showed that CP-A significantly reduced reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), and interleukins (IL-6, IL-1β, IL-18) in LPS-induced cells while increasing IL-4 and IL-10 levels and enhancing the expression of Claudin-1, ZO-1, and occludin proteins in NCM460 cells. Correspondingly, in vivo findings revealed that CP-A administration markedly improved DAI, reduced colon shortening, and decreased the production of myeloperoxidase (MPO), malondialdehyde (MDA), ROS, IL-1β, IL-18, and NOD-like receptor protein 3 (NLRP3) inflammasome-associated genes/proteins in UC mice. CP-A treatment also elevated glutathione (GSH) and superoxide dismutase (SOD) levels, stimulated autophagy (LC3B, P62, Beclin-1, and ATG5), and reinforced Claudin-1 and ZO-1 expression, thereby aiding in intestinal epithelial barrier repair in colitis mice. Notably, the inhibition of autophagy via chloroquine (CQ) diminished CP-A's protective impact against colitis in vivo. These findings elucidate that CP-A's therapeutic effect on experimental colitis possibly involves mitigating intestinal inflammation through autophagy-mediated NLRP3 inflammasome inactivation. Consequently, inulin-type fructan CP-A emerges as a promising drug candidate for UC treatment.