Shortening In Mice Research Articles

Staphylea bumalda Alleviates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice by Regulating Inflammatory Cytokines, Oxidative Stress, and Maintaining Gut Homeostasis

Staphylea bumalda is a rare medicine and edible shrub native to the temperate regions of Asia, possessing significant medicinal potential. In this study, the components of S. bumalda tender leaves and buds extract (SBE) were analyzed and identified by HPLC and LC/MS method, and the safety of SBE was evaluated through mouse acute toxicity models. The protective effects of SBE on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice were investigated in terms of inflammatory factor levels, oxidative stress, and gut microorganisms. Results showed that hyperoside, kaempferol-3-O-rutinoside, isorhoifolin, and rutin were the main components of the extract, and SBE demonstrated good safety in experimental mice. SBE could alleviate weight losing, disease activity index (DAI) raising, and colon shortening in mice. Pathological section results showed that the inflammatory cell infiltration decreased significantly, and the number of goblet cells increased significantly in the SBE group. After SBE treatment, interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) levels in serum were significantly decreased, and the levels of myeloperoxidase (MPO) and nitric oxide (NO) in colon tissues were significantly decreased. SBE inhibited gut inflammation by increasing Lactobacillus. In summary, SBE played a therapeutic role in UC mice by relieving colon injury, reducing inflammatory factor levels, and maintaining gut flora homeostasis. SBE is expected to become an auxiliary means to participate in the prevention and treatment of UC.

Exploring the anti-inflammatory effects of postbiotic proteins from Lactobacillus delbrueckii CIDCA 133 on inflammatory bowel disease model

Lactobacillus delbrueckii CIDCA 133 is a promising health-promoting bacterium shown to alleviate intestinal inflammation. However, the specific bacterial components responsible for these effects remain largely unknown. Here, we demonstrated that consuming extractable proteins from the CIDCA 133 strain effectively relieved acute ulcerative colitis in mice. This postbiotic protein fraction reduced the disease activity index and prevented colon shortening in mice. Furthermore, histological analysis revealed colitis prevention with reduced inflammatory cell infiltration into the colon mucosa. Postbiotic consumption also induced an immunomodulatory profile in colitic mice, as evidenced by both mRNA transcript levels (Tlr2, Nfkb1, Nlpr3, Tnf, and Il6) and cytokines concentration (IL1β, TGFβ, and IL10). Additionally, it enhanced the levels of secretory IgA, upregulated the transcript levels of tight junction proteins (Hp and F11r), and improved paracellular intestinal permeability. More interestingly, the consumption of postbiotic proteins modulated the gut microbiota (Bacteroides, Arkkemansia, Dorea, and Oscillospira). Pearson correlation analysis indicated that IL10 and IL1β levels were positively associated with Bacteroides and Arkkemansia_Lactobacillus abundance. Our study reveals that CIDCA 133-derived proteins possess anti-inflammatory properties in colonic inflammation.

Probiotic properties and the ameliorative effect on DSS-induced colitis of human milk-derived Lactobacillus gasseri SHMB 0001.

Human milk contains a variety of microorganisms that exert benefit for human health. In the current study, we isolated a novel Lactobacillus gasseri strain named Lactobacillus gasseri (L. gasseri) SHMB 0001 from human milk and aimed to evaluate the probiotic characteristics and protective effects on murine colitis of the strain. The results showed that L. gasseri SHMB 0001 possessed promising potential probiotic characteristics, including good tolerance against artificial gastric and intestinal fluids, adhesion to Caco-2 cells, susceptibility to antibiotic, no hemolytic activity, and without signs of toxicity or infection in mice. Administration of L. gasseri SHMB 0001 (1×108CFU per gram of mouse weight per day) reduced weight loss, the disease activity index, and colon shortening in mice during murine colitis conditions. Histopathological analysis revealed that L. gasseri SHMB 0001 treatment attenuated epithelial damage and inflammatory infiltration in the colon. L. gasseri SHMB 0001 treatment increased the expression of colonic occludin and claudin-1 while decreasing the expression of pro-inflammatory cytokine genes. L. gasseri SHMB 0001 modified the composition and structure of the gut microbiota community and partially recovered the Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways altered by dextran sulfate sodium (DSS). Overall, our results indicated that the human breast milk-derived L. gasseri SHMB 0001 exhibited promising probiotic properties and ameliorative effect on DSS-induced colitis in mice. L. gasseri SHMB 0001 may be applied as a promising probiotic against intestinal inflammation in the future. PRACTICAL APPLICATION: L. gasseri SHMB 0001 isolated from human breast milk showed good tolerance to gastrointestinal environment, safety, and protective effect against DSS-induced mice colitis via enforcing gut barrier, downregulating pro-inflammatory cytokines, and modulating gut microbiota. L. gasseri SHMB 0001 may be a promising probiotic candidate for the treatment of intestinal inflammation.

P141 Engineered probiotics treat inflammatory bowel disease by releasing all trans retinoic acid in the intestine

Abstract Background All trans retinoic acid (ATRA) is a potential candidate drug for the treatment of inflammatory bowel disease (IBD). ATRA can affect multiple pathways involved in the pathogenesis of IBD by binding to retinoic acid receptors. However, the shortcomings of poor compliance and significant side effects in patients who directly use ATRA limit its clinical application. Methods Here, we have developed a live biotherapeutic products (ELBPRA) based on synthetic biology methods for controllable delivery of ATRA on the gut. We first expressed four enzyme genes required for the synthesis of β-carotene, a precursor of retinoic acid, in Escherichia coli Nissle 1917 (EcN). In addition, we introduced the exogenous synthesis pathway of isopentane pyrophosphate into EcN to increase the yield of β-carotene. Then, β-carotene oxygenase and retinal dehydrogenase, were expressed in EcN to convert β-carotene into retinoic acid. However, considering the poor solubility of retinoic acid, we knocked out some genes related to vesicle formation in the EcN genome to increase extracellular production of retinoic acid. Finally, we investigated the therapeutic effect of ELBPRA using a colitis model induced by sodium dextran sulfate (DSS). Results ELBPRA continuously produces retinoic acid and delivers it in the form of vesicles. ELBPRA reduced weight loss and disease activity index in DSS-induced colitis. Similarly, the spleen index also demonstrates the effectiveness of ELBPRA in treating colitis. The ELBPRA intervention group reduced the degree of colon shortening in mice. In addition, ELBPRA improved crypt loss and disorder of crypt structure. Finally, we also evaluated the inflammatory cytokines in the serum. ELBPRA significantly increased the concentration of anti-inflammatory cytokine IL-10 and decreased the concentration of pro-inflammatory cytokine IL-1β and TNF-α. Conclusion Engineered probiotics ELBPRA can deliver retinoic acid in the form of vesicles to alleviate DSS-induced colitis in mice, which provides a safe and effective treatment strategy for ulcerative colitis.

Butyrate ameliorated ferroptosis in ulcerative colitis through modulating Nrf2/GPX4 signal pathway and improving intestinal barrier

Oxidative stress and intestinal inflammation are main pathological features of ulcerative colitis (UC). Ferroptosis, characterized by iron accumulation and lipid peroxidation, is closely related to the pathologic process of UC. 16S rRNA sequencing for intestinal microbiota analysis and gas chromatography–mass spectrometry (GC–MS) for short-chain fatty acid (SCFA) contents clearly demonstrated lower amounts of butyrate-producing bacteria and butyrate in colitis mice. However, the precise mechanisms of sodium butyrate (NaB) in treating UC remain largely unclear. We found that ferroptosis occurred in colitis models, as evidenced by the inflammatory response, intracellular iron level, mitochondria ultrastructural observations and associated protein expression. NaB inhibited ferroptosis in colitis, significantly rescued weight loss and colon shortening in mice and reduced inflammatory lesions and mitochondrial damage. Furthermore, NaB improved intestinal barrier integrity and markedly suppressed the expression of pro-ferroptosis proteins. Conversely, the protein expression of anti-ferroptosis markers including nuclear factor erythroid-related Factor 2 (Nrf2) and glutathione peroxidase 4 (GPX4), was significantly upregulated with NaB treatment. Moreover, the knockdown of Nrf2 reversed the anti-colitis effect of NaB. Taken together, NaB exhibited a protective effect by ameliorating ferroptosis in experimental colitis through Nrf2/GPX4 signaling and improving intestinal barrier integrity, which provides a novel mechanism for NaB prevention of UC.

Limosilactobacillus fermentum HF06-derived paraprobiotic and postbiotic alleviate intestinal barrier damage and gut microbiota disruption in mice with ulcerative colitis.

Paraprobiotics and postbiotics have shown potential in the treatment of ulcerative colitis (UC). However, their in vivo application is still in its infancy and their mechanisms of action are not well understood. Here, we investigated the mitigation effects of Limosilactobacillus fermentum HF06-derived paraprobiotic (6-PA) and postbiotic (6-PS) on dextran sulfate sodium induced UC and the potential mechanisms. Results indicated that the administration of 6-PA and 6-PS resulted in the inhibition of weight loss and colon shortening in mice with UC. Furthermore, they led to a significant reduction in both fecal moisture content and the levels of proinflammatory cytokines and oxidative stress in the intestine of the mice. 6-PA and 6-PS treatment strengthened the intestinal mucosal barrier by dramatically upregulating the levels of zonula occludens-1 and occludin proteins. In addition, 6-PA and 6-PS restored intestinal dysbiosis by regulating abundances of certain bacteria, such as Bifidobacterium, Faecalibaculum, Muribaculaceae, Corynebacterium, Escherichia-Shigella and Clostridium_sensu_stricto_1, and regulated the level of short-chain fatty acids. These findings illustrated for the first time that L. fermentum HF06-derived paraprobiotic and postbiotic enhanced the intestinal barrier function, and restored gut microbiota alterations. © 2023 Society of Chemical Industry.

Bergamot polysaccharides relieve DSS-induced ulcerative colitis via regulating the gut microbiota and metabolites

This study aimed to explore the efficacy of polysaccharides from bergamot (BP) in alleviating DSS-induced colitis. Results showed that BP was primarily composed of two components, BP-1 and BP-2, with similar monosaccharide compositions to BP (mainly glucose and xylose) and molecular weights (Mw) of 4.50 × 105 and 2.35 × 105 Da. This study found BP relieved disease symptoms such as weight loss and colon shortening in mice with colitis. Gut microbiota and metabolomics analysis revealed that the BP could also promote the proliferation of beneficial bacteria such as Bifidobacteria, Butyrivibrio, and Blautia, resulting in increased levels of SCFAs and L-phenylalanine, which were associated with phenylalanine, tyrosine, and tryptophan metabolism pathways. Further analysis validated the inflammatory activity of L-phenylalanine. Hence, BP may relieve colitis symptoms by regulating the gut microbiota and metabolism, which reduced inflammation and enhanced the expression of tight junctional proteins (TJ proteins) and mucin in the intestine.

Effect of ethyl gallate and propyl gallate on dextran sulfate sodium (DSS)-induced ulcerative colitis in C57BL/6J mice: preventive and protective.

Inflammatory bowel disease (IBD) is an idiopathic inflammatory condition of the digestive system marked by oxidative stress, leukocyte infiltration, and elevation of inflammatory mediators. In this study, we demonstrate the protective effect of ethyl gallate (EG), a phytochemical, and propyl gallate (PG), an anti-oxidant, given through normal drinking water (DW) and copper water (CW) in various combinations, which had a positive effect on the amelioration of DSS-induced ulcerative colitis in C57BL/6J mice. We successfully determined the levels of proinflammatory cytokines and anti-oxidant enzymes by ELISA, tracked oxidative/nitrosative stress (RO/NS) by in vivo imaging (IVIS) using L-012 chemiluminescent probe, disease activity index (DAI), and histopathological and morphometric analysis of colon in DSS-induced colitis in a model. The results revealed that oral administration of ethyl gallate and propyl gallate at a dose of 50mg/kg considerably reduced the severity of colitis and improved both macroscopic and microscopic clinical symptoms. The level of proinflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ) in colonic tissue was considerably reduced in the DSS + EG-treated and DSS + PG-treated groups, compared to the DSS alone-treated group. IVIS imaging of animals from the DSS + EG and DSS + PG-treated groups showed a highly significant decrease in RO/NS species relative to the DSS control group, with the exception of the DSS + PG/CW and DSS + EG + PG/CW-treated groups. We also observed lower levels of myeloperoxidase (MPO), nitric oxide (NO), and lipid peroxidation (LPO), and restored levels of GST and superoxide dismutase (SOD) in DSS + EG-DW/CW, DSS + PG/DW, and DSS + EG + PG/DW groups compared to DSS alone-treated group. In addition, we showed that the EG, PG, and EG + PG treatment significantly reduced the DAI score, and counteracted the body weight loss and colon shortening in mice compared to DSS alone-treated group. In this 21-day study, mice were treated daily with test substances and were challenged to DSS from day-8 to 14. Our study highlights the protective effect of ethyl gallate and propyl gallate in various combinations which, in pre-clinical animals, serve as an anti-inflammatory drug against the severe form of colitis, indicating its potential for the treatment of IBD in humans. In addition, propyl gallate was investigated for the first time in this study for its anti-colitogenic effect with normal drinking water and reduced effect with copper water.

Fermented Sargassum fusiforme Mitigates Ulcerative Colitis in Mice by Regulating the Intestinal Barrier, Oxidative Stress, and the NF-κB Pathway.

In recent years, Sargassum fusiforme has gained increasing attention for its ability to improve human health and reduce the risk of disease. Nevertheless, there have been few reports on the beneficial functions of fermented Sargassum fusiforme. In this study, the role of fermented Sargassum fusiforme in the mitigation of ulcerative colitis was investigated. Both fermented and unfermented Sargassum fusiforme demonstrated significant improvement in weight loss, diarrhea, bloody stools, and colon shortening in mice with acute colitis. Fermented Sargassum fusiforme further protected against goblet cell loss, decreased intestinal epithelium permeability, and enhanced the expression of tight junction proteins. Fermented Sargassum fusiforme reduced oxidative stress, which was demonstrated by a decrease in nitric oxide (NO), myeloperoxidase (MPO), and malondialdehyde (MDA) concentrations in the colon of mice and an increase in total superoxide dismutase (T-SOD) activity in the colon. Meanwhile, catalase (CAT) concentrations in both the colon and serum of mice were significantly increased. Fermented Sargassum fusiforme also attenuated the inflammatory response, which was evidenced by the decreased level of pro-inflammatory cytokines in the colon. Moreover, fermented Sargassum fusiforme inhibited the nuclear factor-κB (NF-κB) signaling pathway and increased the production of short-chain fatty acids in the intestine. These findings indicate that fermented Sargassum fusiforme may have the potential to be developed as an alternative strategy for alleviating colitis.

Therapeutic effect of ursodeoxycholic acid-berberine supramolecular nanoparticles on ulcerative colitis based on supramolecular system induced by weak bond

Ulcerative colitis(UC) is a recurrent, intractable inflammatory bowel disease. Coptidis Rhizoma and Bovis Calculus, serving as heat-clearing and toxin-removing drugs, have long been used in the treatment of UC. Berberine(BBR) and ursodeoxycholic acid(UDCA), the main active components of Coptidis Rhizoma and Bovis Calculus, respectively, were employed to obtain UDCA-BBR supramolecular nanoparticles by stimulated co-decocting process for enhancing the therapeutic effect on UC. As revealed by the characterization of supramolecular nanoparticles by field emission scanning electron microscopy(FE-SEM) and dynamic light scattering(DLS), the supramolecular nanoparticles were tetrahedral nanoparticles with an average particle size of 180 nm. The molecular structure was described by ultraviolet spectroscopy, fluorescence spectroscopy, infrared spectroscopy, high-resolution mass spectrometry, and hydrogen-nuclear magnetic resonance(H-NMR) spectroscopy. The results showed that the formation of the supramolecular nano-particle was attributed to the mutual electrostatic attraction and hydrophobic interaction between BBR and UDCA. Additionally, supramolecular nanoparticles were also characterized by sustained release and pH sensitivity. The acute UC model was induced by dextran sulfate sodium(DSS) in mice. It was found that supramolecular nanoparticles could effectively improve body mass reduction and colon shortening in mice with UC(P<0.001) and decrease disease activity index(DAI)(P<0.01). There were statistically significant differences between the supramolecular nanoparticles group and the mechanical mixture group(P<0.001, P<0.05). Enzyme-linked immunosorbent assay(ELISA) was used to detect the serum levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6), and the results showed that supramolecular nanoparticles could reduce serum TNF-α and IL-6 levels(P<0.001) and exhibited an obvious difference with the mechanical mixture group(P<0.01, P<0.05). Flow cytometry indicated that supramolecular nanoparticles could reduce the recruitment of neutrophils in the lamina propria of the colon(P<0.05), which was significantly different from the mechanical mixture group(P<0.05). These findings suggested that as compared with the mechanical mixture, the supramolecular nanoparticles could effectively improve the symptoms of acute UC in mice. The study provides a new research idea for the poor absorption of small molecules and the unsatisfactory therapeutic effect of traditional Chinese medicine and lays a foundation for the research on the nano-drug delivery system of traditional Chinese medicine.

Ononin alleviates DSS-induced colitis through inhibiting NLRP3 inflammasome via triggering mitophagy.

Ononin, a flavonoid isolated from Astragalus membranaceus root, is the active ingredient of A. membranaceus and has potential anti-inflammatory properties, but its effect on colitis is unclear. This study aimed to explore the anticolitis effect of Ononin by establishing a colitis model in mice induced by dextran sulfate sodium (DSS). Male C57BL/6 mice were provided DSS, then treated with Ononin (10, 20, 40 mg/kg) or 5-ASA (40 mg/kg). The colitis symptoms were observed, the disease activity index (DAI) score were recorded daily, and colonic inflammation was evaluted by histopathological scoring. The expression of cytokines, inflammatory mediators, and mitophagy/NLRP3 inflammasome-related proteins were measured. Ononin significantly alleviated weight loss and colon shortening in mice with colitis (p < .01). Moreover, Ononin decreased the production of inflammatory cytokines and mediators associated with colitis (p < .05). In addition, Ononin inhibited macrophages infiltration and reduced caspase-1 activation in colitis mice. Caspase-1 activation is closely related to the NLRP3 inflammasome. Therefore, we investigated the effect of Ononin on NLRP3 inflammasome in vitro. The relevant results confirmed that Ononin inhibited NLRP3 inflammasome activation and inhibited mitochondrial damage (p < .05). Further studies revealed that Ononin inhibited mitochondrial damage through triggering mitophagy (p < .05). Ononin alleviates DSS-inducedcolitis by activating mitophagy to inhibit NLRP3 inflammasome.

Anemoside B4 protects against chronic relapsing colitis in mice by modulating inflammatory response, colonic transcriptome and the gut microbiota

BackgroundAnemoside B4 (AB4) is reported to prevent acute colitis when given via intraperitoneal injection by two recent studies. However, whether oral AB4 protects against chronic colitis which resembles the clinical phenotype of ulcerative colitis (UC) and its mechanism of action are largely unknown. PurposeTo systemically investigate the effects of oral AB4 against chronic colitis and illustrate the underlying mechanism of action. MethodsThe preventive, therapeutic, and dose-dependent effects of AB4 against UC were examined in mice with acute or chronic relapsing colitis induced by dextran sulfate sodium (DSS). The inflammatory responses, colonic transcriptome, and 16S rDNA sequencing of the intestinal content of mice were analyzed. ResultsOral administration of AB4 alleviated disease severity and colon shortening in mice with chronic relapsing colitis in a dose-dependent manner. The effects of AB4 were comparable to those of two positive-control compounds: tofacitinib and berberine. Unlike tofacitinib, AB4 did not have a deleterious effect on DSS-induced splenic swelling and anemia. Furthermore, AB4 inhibited the inflammatory responses of colitis, as evidenced by in-vivo, ex-vivo, and in-vitro studies. Transcriptomics revealed that AB4 treatment reversed the DSS-mediated decrease in the expression of colonic Pelo, B3gat2 and Mir8010. In addition, AB4 reversed DSS-induced alterations in the intestinal microbiome in mice. Through fecal microbiota transplantation, we proved that AB4 partially exerted its anti-colitis effects by modulating the gut microbiota. ConclusionsWe demonstrated for the first time that AB4 has dose-dependent therapeutic effects against chronic relapsing colitis by modulating the inflammatory response, colonic gene expression, and intestinal microbiota.

Abstract P1041: Redox Regulator MnTnBuOE-PyP5+ Protects Cardiomyocyte Against Oxidative Stress Mediated Myocardial Ischemia/reperfusion Injury

Background: Much evidence suggests that oxidative stress plays a major role in the pathogenesis of myocardial ischemia/reperfusion (I/R) injury and is a potential target for therapeutic interventions. We tested the hypothesis that the novel redox regulator, MnTnBUOE-2-PyP 5+ (BMX-001), mediates cardioprotection through reduction of oxidative stress in myocardial tissue following I/R injury. Methods and Results: BMX-001 (2mg/Kg, IP) or vehicle was administered in mice 4 hours before myocardial ischemia. Mice were then subjected to I/R injury by ligating the left anterior descending artery for 1 hour followed by 24 hours of reperfusion. Following 24 hours of reperfusion, infarct size, cardiomyocyte apoptosis, and SOD2 level were determined, and echocardiography was performed. Our study revealed that the redox regulator provided protection against I/R-induced injury in mice as evident by a significant reduction in infarct size, plasma LDH level, and oxidative stress. In addition, BMX-001 administration in mice markedly increased FoxO3a and SOD2 levels. BMX-001 treatment attenuated I/R induced cardiomyocyte apoptosis in-vivo and in-vitro as evident by a reduction in the number of TUNEL positive cells and improved cell viability. Echocardiography at 24 hours of reperfusion revealed improved ejection fraction and fractional shortening in mice treated with BMX-001. Furthermore, in-vitro treatment of BMX-001 (10μM) 24 hours before hypoxia/reoxygenation in H9c2 cells improved oxidative stress as evident by a reduction in cardiolipin peroxidation, mitochondrial superoxide level, and 4-HNE adducted proteins. Conclusion: Pre-treatment with redox regulator BMX-001 showed a beneficial effect in attenuating cardiomyocyte death, mitochondrial superoxide, and cardiolipin peroxidation following I/R injury in H9c2 cells. Pre-treatment with SOD2 mimetic improved heart function, decreased infarct size, and cardiomyocyte apoptosis in mice following I/R injury. Therefore, BMX-001 can be a novel cardioprotective agent against oxidative stress-induced myocardial I/R injury.

Nuciferine Regulates Immune Function and Gut Microbiota in DSS-Induced Ulcerative Colitis.

Nuciferine, a major aporphine alkaloid obtained from the leaves of Nelumbo nucifera, exhibits anti-cancer and anti-inflammatory properties; however, its protective effects against inflammatory bowel diseases (IBD) has never been explored. In this study, an ulcerative colitis (UC) model was established in BALb/c mice by the continuous administration of 5% dextran sulfate sodium (DSS) in drinking water for 1 week. From day 8 to day 14, the DSS-treated mice were divided into a high-dose and a low-dose nuciferine treatment group and were intraperitoneally injected with the corresponding dose of the drug. Body weight loss, disease activity index (DAI), and colon length were measured. Histological changes were observed using hematoxylin and eosin staining. T lymphocyte proliferation was assessed by MTT assay. The ratio of CD3+, CD4+, CD8+, Th1, Th2, Th17, and Treg cells were estimated by flow cytometry. Finally, 16S rRNA sequencing was performed to compare the composition and relative abundance of the gut microbiota among the different treatment groups. The results showed that nuciferine treatment led to a significant improvement in symptoms, such as histological injury and colon shortening in mice with DSS-induced UC. Nuciferine treatment improved the Th1/Th2 and Treg/Th17 balance in the DSS-induced IBD model, as well as the composition of the intestinal microflora. At the phylum level, compared with the control group, the abundance of Firmicutes and Actinobacteriota was decreased in the model group, whereas that of Bacteroidetes increased. Meanwhile, at the genus level, compared with the control group, the numbers of the genera Lachnospiraceae_Clostridium, Bilophila and Halomonas reduced in the model group, while those of Bacteroides, Parabacteroides, and Paraprevotella increased. Notably, nuciferine administration reversed this DSS-induced gut dysbiosis. These results indicated that nuciferine modulates gut microbiota homeostasis and immune function in mice with DSS-induced UC.

Therapeutic Targeting of Nrf2 Signaling by Maggot Extracts Ameliorates Inflammation-Associated Intestinal Fibrosis in Chronic DSS-Induced Colitis.

Intestinal fibrosis is induced by excessive myofibroblast proliferation and collagen deposition, which has been regarded as a general pathological feature in inflammatory bowel disease (IBD). Therefore, identifying clinical markers and targets to treat and prevent intestinal fibrosis is urgently needed. The traditional Chinese medicine maggot, commonly known as “wu gu chong”, has been shown to reduce oxidative stress and alleviate inflammation in chronic colitis. This study investigated the mechanisms underlying the effects of maggot extract (ME) on inflammation-associated intestinal fibrosis in TGF-β1-stimulated human intestinal fibroblasts (CCD-18Co cells) and dextran sodium sulphate (DSS)-induced chronic colitis murine model. To assess the severity of inflammation and fibrosis, histological and macroscopic evaluation were carried out. The results showed that ME was a significant inhibitor of body weight loss and colon length shortening in mice with chronic colitis. In addition, ME suppressed the intestinal fibrosis by downregulating TGF-β1/SMADs pathway via upregulation of Nrf2 expression at both protein and mRNA levels. ME markedly increased the expression of Nrf2, thus resulting in a higher level of HO-1. After treatment with Nrf2 inhibitor (ML385) or siRNA-Nrf2 for deactivating Nrf2 pathway, the protective effects of ME were abolished both in vitro and in vivo. Moreover, the histopathological results for the major organs of DSS mice treated with ME showed no signs of clinically important abnormalities. Treatment with ME had no effect on the viability of CCD-18Co cells, suggesting its low in vitro cytotoxicity. Furthermore, ME could mediate intestine health by keeping the balance of the gut microbes through the enhancement of beneficial microbes and suppression of pathogenic microbes. In conclusion, this is the first ever report demonstrating that ME ameliorates inflammation-associated intestinal fibrosis by suppressing TGF-β1/SMAD pathway via upregulation of Nrf2 expression. Our findings highlight the potential of Nrf2 as an effective therapeutic target for alleviating intestinal fibrosis.

Vascular and renal telomere shortening in mice exposed to chronic intermittent hypoxia.

Obstructive sleep apnea (OSA) is a chronic condition characterized by chronic intermittent hypoxia (IH) and is associated with cardiovascular (CVD) and chronic kidney diseases (CKD). Patients with OSA have increased biomarkers of aging such as telomere shortening. We used PCR to report shortened telomere lengths in aortic and renal tissues from mice exposed to 8 weeks of IH. Our data indicate that IH, a hallmark of OSA, accelerates vascular and renal aging that may contribute to OSA-induced CVD and CKD.

High fat diet induces change in mitochondria respiration, activation of K-ATP channel and action potential shortening in mice

Obesity is associated with an increased risk of atrial fibrillation (AF). This epidemiologic observation may not be only due to shared co-morbidities but also from direct impacts of metabolic disorders on myocardium. To examine the impact of high fat diet (HFD) induced obesity on electrical properties in mice atria. Eight weeks old C57Bl/6 J mice were subjected to 4 months of HFD (60% fat) or normal diet (ND, 4% fat). Rapid burst atrial pacing were delivered using a trans-esophageal probe to induced AF in anesthetized animals. Action potential (AP) were recorded in left atrium using glass microelectrode technique. Potassium currents were recorded in isolated atrial myocytes using whole cell or perforated patch-clamp configurations. Mitochondrial respiration was studied in saponin-permeabilized atrial muscle fibers using a Clarke electrode. HFD mice showed higher atrial vulnerability to AF as indicated by longer AF episodes. APs were shorter in HFD versus ND mice (AP duration measured at 90% of repolarization: 49.3 ± 1.6 msec in HFD vs. 56.7 ± 2.3 msec in ND, P < 0.01). Glibenclamide, a KATP channel blocker, reversed AP shortening whereas cromakalim, a KATP channel activator, had no effect. Only in perforated patch-clamp configuration, without intracellular dialysis, KATP component of potassium current was enhanced in HFD mice (at +70 mV: 0.41 ± 0.12 pA/pF in HFD vs. 0.13 ± 0.08 pA/pF in ND, P < 0.05). There was no change in mitochondria respiration capacity between ND and HFD, however the ability of palmitoyl-CoA to support mitochondrial respiration was higher in HFD (25.99 ± 4.11% vs. 46.9 ± 3.96% in ND, P = 0.001). In addition, HADHA activity was enhanced in HFD mice. Obesity in mice is associated with atrial AP shortening which appears to result from increased activity of KATP-regulated currents and be associated with a metabolic shift. Shortening of AP duration could contribute to higher vulnerability to AF during obesity.

Abstract 613: Disturbed Flow-induced Telomeric Repeat Binding Factor 2 (terf2)-interacting Protein (terf2ip) K240 Sumoylation is Critical for Endothelial Senescence but Not Inflammation

Upregulation of both inflammation and senescence (Sen) of endothelial cells (ECs) in the disturbed flow area within the aortic arch is well established, but the exact mechanisms and the relationship between inflammation and senescence remain unclear. TERF2IP is involved in regulation of DNA double strand break (DSB) repair of telomeres (TLs) as a component of the shelterin complex and in IκB kinase (IKK)-NF-kB signaling in the cytosol. TERF2IP and TERF2 form a stable heterodimer to protect the duplex region of TLs. Recent studies showed that TERF2IP or TERF2 deficiency caused TL shortening in mice. We studied how TERF2IP SUMOylation is regulated in ECs and what role it may play in EC inflammation and Sen. We found that disturbed flow (d-flow) activated p90RSK and increased TERF2IP S205 phosphorylation and subsequently K240 SUMOylation. Overexpression of dominant negative p90RSK inhibited d-flow-induced TERF2IP S205 phosphorylation and K240 SUMOylation, and also inhibited EC inflammation and Sen provoked by d-flow. TERF2IP S205 phosphorylation induced TERF2IP nuclear export, which then caused EC inflammation and Sen. In contrast, TERF2IP K240 SUMOylation had no effect on TERF2IP nuclear export, and the TERF2IP K240R SUMOylation site mutant failed to inhibit d-flow-induced NF-kB activation. Interestingly, this point mutation inhibited d-flow-induced Sen with reduced d-flow-induced TL shortening, 8-OHdG level, and apoptosis. Overexpression of a de-SUMOylation enzyme SENP2 and SENP2 T368 mutant significantly inhibited d-flow-induced TERF2IP SUMOylation, suggesting that SENP2 regulates nuclear TERF2IP SUMOylation. Lastly, EC Sen increased when SENP2 was specifically knocked out in ECs in mice, suggesting that TERF2IP K240 SUMOylation may play a role in d-flow-induced EC Sen. These data suggest that TERF2IP post-translational modifications play roles in regulating EC inflammation and Sen, which are differentially regulated by S205 phosphorylation and K240 SUMOylation. TERF2IP K240R knock-in mice will be used to determine the role of TERF2IP K240 SUMOylation in d-flow-induced atherogenesis. We suggest that TERF2IP SUMOylation promotes TL dysfunction, but it does not interfere with TERF2IP-mediated IKK-NF-kB activation in the cytosol.

GUT MICROBIOTA DEPLETION PRESERVES HEART FUNCTION, SUPPRESSES CARDIAC FIBROSIS AND HYPERTROPHY IN A NON‐ISCHEMIC HEART FAILURE MOUSE MODEL

The gut is a major reservoir of T cells and diverse resident microbes, microbiota, which can influence immune responses in sites distant from the mucosal surfaces. Complete sterilization of the gut has proven to be beneficial in some experimental models of T cell mediated diseases, whereas partial recolonization post sterilization leads to microbial perturbations, a process called dysbiosis, and, worsens the outcome. Gut dysbiosis is thus recently becoming associated with the pathogenesis of several diseases, in part, by mechanisms in which certain bacteria promote T cell activation and enhance disease progression in a vicious cycle. The complex syndrome of heart failure (HF), a leading cause of morbidity and mortality affecting more than 24 million people worldwide, is recently becoming associated with gut dysbiosis and T cell mediated systemic inflammation in patients, but the mechanisms regulating this emerging gut‐heart axis, and whether T cell activation and heart infiltration play a role remains unclear. We have previously reported that T cells infiltrate the heart in patients with non‐ischemic HF, and using the transverse aortic constriction (TAC) mouse model of HF, demonstrated that T cells are critical regulators of adverse cardiac remodeling and HF. We hypothesized that sterilization of the gut by microbiota depletion prevents adverse cardiac remodeling and HF in a T cell dependent manner. C57/BL6 mice were orally treated with a well‐established cocktail of antibiotics and antifungal (ABX) and subjected to TAC or Sham surgery. ABX treatment started 1 week before TAC surgery and was terminated 4 weeks after TAC. In vivo transthoracic echocardiography and hemodynamics showed a preserved ejection fraction and fractional shortening in mice treated with ABX as compared to untreated mice. Furthermore, gut microbiota depletion with ABX resulted in decreased left ventricular interstitial and perivascular fibrosis, and decreased cardiac hypertrophy in response to TAC, as compared to non‐ABX treated TAC mice. These changes correlated with significant reduction of CD4 T cell activation in the mediastinal lymph nodes (mLNs) draining the heart, determined by FACS, as well as in the number of CD4 T cells infiltrated in the heart in ABX treated mice. Our findings indicate that ABX treatment results in distal effects in T cell activation occurring during TAC and protects from adverse cardiac remodeling, supporting the potential importance of gut microbiota in pressure overload induced HF. Future studies will determine whether dysbiosis post ABX treatment contributes to pathological cardiac remodeling and the mechanisms regulating the gut‐heart axis in non‐ischemic HF.Support or Funding InformationThis work was supported by the NIH‐RO1‐HL123658 grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutant cartilage oligomeric matrix protein (COMP) compromises bone integrity, joint function and the balance between adipogenesis and osteogenesis

Mutations in COMP (cartilage oligomeric matrix protein) cause severe long bone shortening in mice and humans. Previously, we showed that massive accumulation of misfolded COMP in the ER of growth plate chondrocytes in our MT-COMP mouse model of pseudoachondroplasia (PSACH) causes premature chondrocyte death and loss of linear growth. Premature chondrocyte death results from activation of oxidative stress and inflammation through the CHOP-ER pathway and is reduced by removing CHOP or by anti-inflammatory or antioxidant therapies. Although the mutant COMP chondrocyte pathologic mechanism is now recognized, the effect of mutant COMP on bone quality and joint health (laxity) is largely unknown. Applying multiple analytic approaches, we describe a novel mechanism by which the deleterious consequences of mutant COMP retention results in upregulation of miR-223 disturbing the adipogenesis - osteogenesis balance. This results in reduction in bone mineral density, bone quality, mechanical strength and subchondral bone thickness. These, in addition to abnormal patterns of ossification at the ends of the femoral bones likely contribute to precocious osteoarthritis (OA) of the hips and knees in the MT-COMP mouse and PSACH. Moreover, joint laxity is compromised by abnormally thin ligaments. Altogether, these novel findings align with the PSACH phenotype of delayed ossification and bone age, extreme joint laxity and joint erosion, and extend our understanding of the underlying processes that affect bone in PSACH. These results introduce a novel finding that miR-223 is involved in the ossification defect in MT-COMP mice making it a therapeutic target.