An effective LC-MS method for the simultaneous determination of a potential anti-rheumatoid arthritis drug, carboxyamidotriazole, and its major metabolite in rat plasma.

Carboxyamidotriazole (CAI) was previously recognized as a well-tolerated anticancer drug. It has also demonstrated significant anti-inflammatory effects in various cell and animal model experiments, prompting its investigation as a potential treatment for rheumatoid arthritis. In this study, the potential biotransformation metabolites of CAI were identified both in vitro and in vivo. A sensitive, specific, and accurate LC-MS method was developed for the quantitative analysis of CAI and its major metabolite, CAI-OH, in rat plasma. CAI, CAI-OH, and telmisartan (used as an internal standard) were separated using a Zorbax SB C18 column. The mobile phase consisted of water (phase A, containing 0.1% formic acid) and acetonitrile (phase B, containing 0.1% formic acid) at a flow rate of 0.2 mL/min. The analytes were examined using a high-resolution mass spectrometer, with detected mass-to-charge ratios of m/z 424.01293 for CAI, m/z 440.00785 for CAI-OH, and m/z 515.24415 for telmisartan. Good linearity was observed within the range of 10-5000 ng/mL. Both inter- and intra-batch precision (relative standard deviation, %) were below 6%, and the accuracy ranged from 94.9% to 106.1%. The analytes remained stable throughout the entire experimental period. This method was successfully applied in a pharmacokinetic study of CAI following oral administration in rats.

Establishment of Epithelial Inflammatory Injury Model Using Intestinal Organoid Cultures.

Intestinal epithelial dysfunction is critical in the development of inflammatory bowel disease (IBD). However, most cellular experiments related to epithelial barrier studies in IBD have been based on tumor cell line that lack a variety of intestinal epithelial cell types. Thus, intestinal organoids can present the three-dimensional structure and better simulate the physiological structure and function of the intestinal epithelium in vitro. Here, the crypts were isolated from the small intestine of mice; with the participation of major cytokines (EGF, Noggin, and R-Spondin 1 included), the intestinal organoids were established at a density of 100 crypts per well, containing intestinal stem cells (ISC), Paneth cells, goblet cells, and intestinal endocrine cells. We found that tumor necrosis factor-alpha (TNF-α) could induce the inflammatory response of intestinal organoids, and a dose of 10 ng/mL could maintain stable passaging of organoids for dynamic observation. After stimulation with TNF-α, the intestinal organoid cultures showed lower expression of the cell proliferation-related protein identified by monoclonal antibody Ki 67 (Ki67), the ISC marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), and the intestinal tight junction proteins occludin (Ocln) and claudin-1 (Cldn1) while higher expression of the inflammatory cytokine interleukin- (IL-) 15 and the chemokines C-X-C motif ligand 2 (Cxcl2) and Cxcl10 significantly. In this study, we successfully established an epithelial inflammatory injury model of intestinal organoids, which provides an effective in vitro model for studying the pathogenesis and treatment of IBD.

The Distinguishing Bacterial Features From Active and Remission Stages of Ulcerative Colitis Revealed by Paired Fecal Metagenomes.

Ulcerative colitis (UC) is a serious chronic intestinal inflammatory disease, with an increased incidence in recent years. The intestinal microbiota plays a key role in the pathogenesis of UC. However, there is no unified conclusion on how the intestinal microbiota changes. Most studies focus on the change between UC patients and healthy individuals, rather than the active and remission stage of the same patient. To minimize the influences of genetic differences, environmental and dietary factors, we studied the intestinal microbiota of paired fecal samples from 42 UC patients at the active and remission stages. We identified 175 species of microbes from 11 phyla and found no difference of the alpha and beta diversities between the active and remission stages. Paired t-test analysis revealed differential microbiota at levels of the phyla, class, order, family, genus, and species, including 13 species with differential abundance. For example, CAG-269 sp001916005, Eubacterium F sp003491505, Lachnospira sp000436475, et al. were downregulated in the remission, while the species of Parabacteroides distasonis, Prevotellamassilia sp900540885, CAG-495 sp001917125, et al. were upregulated in the remission. The 13 species can effectively distinguish the active and remission stages. Functional analysis showed that the sporulation and biosynthesis were downregulated, and the hydrogen peroxide catabolic process was upregulated in remission of UC. Our study suggests that the 13 species together may serve as a biomarker panel contributing to identify the active and remission stages of UC, which provides a valuable reference for the treatment of UC patients by FMT or other therapeutic methods.

Reproductive and Developmental Toxicity Assessment of Human Umbilical Cord Mesenchymal Stem Cells in Rats.

Objective: Human umbilical cord mesenchymal stem cells (hUC-MSCs) have shown very attractive potential in clinical applications for the treatment of various diseases. However, the data about the reproductive and developmental toxicity of hUC-MSCs remains insufficient. Thus, we assessed the potential effects of intravenous injection of hUC-MSCs on reproduction and development in Sprague-Dawley rats. Methods: In the fertility and early embryonic development study, hUC-MSCs were administered at dose levels of 0, 6.0 × 106, 8.5 × 106, and 1.2 × 107/kg to male and female rats during the pre-mating, mating and gestation period. In the embryo-fetal development study, the pregnant female rats received 0, 6.0 × 106, 1.2 × 107, and 2.4 × 107/kg of hUC-MSCs from gestation days (GD) 6-15. Assessments made included mortality, clinical observations, body weight, food consumption, fertility parameters of male and female, litter, and fetus parameters, etc. Results: No hUC-MSCs-related toxicity was observed on the fertility of male and female rats, and no teratogenic effect on fetuses. hUC-MSCs at 1.2 × 107/kg caused a mildly decrease in body weight gain of male rats, transient listlessness, tachypnea, and hematuria symptoms in pregnant female rats. Death was observed in part of the pregnant females at a dose of 2.4 × 107/kg, which could be due to pulmonary embolism. Conclusion: Based on the results of the studies, the no-observed-adverse-effect levels (NOAELs) are 8.5 × 106/kg for fertility and early embryonic development, 1.2 × 107/kg for maternal toxicity and 2.4 × 107/kg for embryo-fetal development in rats intravenous injected with hUC-MSCs, which are equivalent to 8.5-fold, 12-fold, and 24-fold respectively of its clinical dosage in humans. These findings may provide a rational basis for human health risk assessment of hUC-MSCs.

Guanylin ligand protects the intestinal immune barrier by activating the guanylate cyclase-C signaling pathway.

Inflammatory bowel disease (IBD) impacts patient quality of life significantly. The dysfunction of intestinal immune barrier is closely associated with IBD. The guanylate cyclase-C (GC-C) signaling pathway activated by the guanylin (Gn) ligand is involved in the occurrence and development of IBD. However, how it regulates the intestinal immune barrier is still unclear. To investigate the effect of the GC-C pathway on intestinal mucosal immunity and provide experimental basis for seeking new therapeutic strategies for IBD, we focused on Caco-2 cells and intestinal intra-epithelial lymphocytes (IELs), which displayed inflammatory responses induced by lipopolysaccharide (LPS). GC-C activity was modulated by transfection with Gn overexpression or GC-C shRNA plasmid. Levels of Gn, GC-C, and CFTR; transepithelial electrical resistance (TER); paracellula r permeability; and levels of IL-2, IFN-γ, and secretory IgA (sIgA) were examined. The study found that after stimulation with LPS, Gn, GC-C, CFTR, TER, and sIgA levels were all significantly reduced, IL-2 and IFN-γ levels as well as paracellular permeability were significantly increased. These indicators changed inversely and significantly after transfection with the Gn overexpression vector. Compared to the vector controls, GC-C-silenced cells displayed significantly decreased levels of GC-C, CFTR, and TER and increased levels of IL-2, IFN-γ, and paracellular permeability stimulated by LPS. The results show that Gn ligand can protect the intestinal immune barrier by activating the GC-C signaling pathway, which may be helpful for the development of new treatments for IBD. DATA AVAILABILITY STATEMENT: The data used to support the findings of this study are available from the corresponding author upon request.