ABSTRACT
Background: Urate-lowering treatment (ULT) to target with xanthine oxidase inhibitors (XOIs) paradoxically causes early increase in gouty arthritis flares. Because delayed reduction in flare burden is mechanistically unclear, we tested for ULT inflammation responsiveness markers. Methods: Unbiased proteomics analyzed blood samples (baseline, 48 weeks ULT) in two, independent ULT out trial cohorts (n = 19, n = 30). STRING-db and multivariate analyses supplemented determinations of altered proteins via Wilcoxon matched pairs signed rank testing in XOI ULT responders. Mechanistic studies characterized proteomes of cultured XOI-treated murine bone marrow macrophages (BMDMs). Results: At 48 weeks ULT, serum urate normalized in all gout patients, and flares declined, with significantly altered proteins (p < 0.05) in clustering and proteome networks in sera and peripheral blood mononuclear cells. Serum proteome changes included decreased complement C8 heterotrimer C8A and C8G chains and chemokine PPBP/CXCL7, and increased urate crystal phagocytosis inhibitor sCD44. In both cohorts, a treatment-emergent serum interactome included key gouty inflammation mediators (C5, IL-1B, CXCL8, IL6). Last, febuxostat inhibited complement activation pathway proteins in cultured BMDMs. Conclusions: Reduced gout flares are kinked with a XOI-treatment emergent complement- and inflammation-regulatory serum protein interactome. Serum and leukocyte proteomes could help identify onset of anti-inflammatory responsiveness to ULT in gout. Trial registration: ClinicalTrials.gov Identifier: NCT02579096, posted October 19, 2015.
ABSTRACT
Gout is caused by the deposition of monosodium urate crystals (MSUc) in the joints, triggering a unique inflammatory and metabolic response in macrophages. Here, we present a protocol to generate MSUc for in vitro and in vivo studies in mouse and human cells. We describe steps for dissolving uric acid followed by crystallizing, purifying, evaluating, and analyzing MSUc. We then detail procedures for stimulating human/mouse-derived macrophages and determining endotoxin levels in MSUc preparation.
Subject(s)
Crystallization , Gout , Macrophages , Uric Acid , Uric Acid/metabolism , Uric Acid/chemistry , Animals , Humans , Mice , Macrophages/metabolism , Gout/metabolismABSTRACT
OBJECTIVES: Cellular NAD+ declines in inflammatory states associated with increased activity of the leukocyte-expressed NADase CD38. In this study, we tested the potential role of therapeutically targeting CD38 and NAD+ in gout. METHODS: We studied cultured mouse wild type and CD38 knockout (KO) murine bone marrow derived macrophages (BMDMs) stimulated by monosodium urate (MSU) crystals and used the air pouch gouty inflammation model. RESULTS: MSU crystals induced CD38 in BMDMs in vitro, associated with NAD+ depletion, and IL-1ß and CXCL1 release, effects reversed by pharmacologic CD38 inhibitors (apigenin, 78c). Mouse air pouch inflammatory responses to MSU crystals were blunted by CD38 KO and apigenin. Pharmacologic CD38 inhibition suppressed MSU crystal-induced NLRP3 inflammasome activation and increased anti-inflammatory SIRT3-SOD2 activity in macrophages. BMDM RNA-seq analysis of differentially expressed genes (DEGs) revealed CD38 to control multiple MSU crystal-modulated inflammation pathways. Top DEGs included the circadian rhythm modulator GRP176, and the metalloreductase STEAP4 that mediates iron homeostasis, and promotes oxidative stress and NF-κB activation when it is overexpressed. CONCLUSIONS: CD38 and NAD+ depletion are druggable targets controlling the MSU crystal- induced inflammation program. Targeting CD38 and NAD+ are potentially novel selective molecular approaches to limit gouty arthritis.
Subject(s)
ADP-ribosyl Cyclase 1 , Inflammation , Macrophages , Mice, Inbred C57BL , Mice, Knockout , NAD , Uric Acid , Animals , ADP-ribosyl Cyclase 1/genetics , ADP-ribosyl Cyclase 1/metabolism , Macrophages/drug effects , Macrophages/metabolism , Inflammation/drug therapy , Mice , NAD/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Male , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Cells, Cultured , Arthritis, Gouty/drug therapy , Arthritis, Gouty/metabolism , Arthritis, Gouty/genetics , Inflammasomes/metabolism , Inflammasomes/drug effectsABSTRACT
Background: Effective xanthine oxidoreductase inhibition (XOI) urate-lowering treatment (ULT) to target significantly reduces gout flare burden and synovitis between 1-2 years therapy, without clearing all monosodium urate crystal deposits. Paradoxically, treat to target ULT is associated with increased flare activity for at least 1 year in duration on average, before gout flare burden decreases. Since XOI has anti-inflammatory effects, we tested for biomarkers of sustained, effective ULT that alters gouty inflammation. Methods: We characterized the proteome of febuxostat-treated murine bone marrow macrophages. Blood samples (baseline and 48 weeks ULT) were analyzed by unbiased proteomics in febuxostat and allopurinol ULT responders from two, independent, racially and ethnically distinct comparative effectiveness trial cohorts (n=19, n=30). STRING-db and multivariate analyses supplemented determinations of significantly altered proteins via Wilcoxon matched pairs signed rank testing. Results: The proteome of cultured IL-1b-stimulated macrophages revealed febuxostat-induced anti-inflammatory changes, including for classical and alternative pathway complement activation pathways. At 48 weeks ULT, with altered purine metabolism confirmed by serum metabolomics, serum urate dropped >30%, to normal (<6.8 mg/dL) in all the studied patients. Overall, flares declined from baseline. Treated gout patient sera and peripheral blood mononuclear cells (PBMCs) showed significantly altered proteins (p<0.05) in clustering and proteome networks. CRP was not a useful therapy response biomarker. By comparison, significant serum proteome changes included decreased complement C8 heterotrimer C8A and C8G chains essential for C5b-9 membrane attack complex assembly and function; increase in the NLRP3 inflammasome activation promoter vimentin; increased urate crystal phagocytosis inhibitor sCD44; increased gouty inflammation pro-resolving mediator TGFB1; decreased phagocyte-recruiting chemokine PPBP/CXCL7, and increased monocyte/macrophage-expressed keratin-related proteins (KRT9,14,16) further validated by PBMC proteomics. STRING-db analyses of significantly altered serum proteins from both cohorts revealed a tight interactome network including central mediators of gouty inflammation (eg, IL-1B, CXCL8, IL6, C5). Conclusions: Rewiring of inflammation mediators in a tight serum protein interactome was a biomarker of sustained XOI-based ULT that effectively reduced serum urate and gout flares. Monitoring of the serum and PBMC proteome, including for changes in the complement pathway could help determine onset and targets of anti-inflammatory changes in response to effective, sustained XOI-based ULT.Trial Registration: ClinicalTrials.gov Identifier: NCT02579096.
ABSTRACT
Concerns over human health risks associated with chemical contaminants (micropollutants) in drinking waters are rising due to the increased use of reclaimed water or water supplies impacted by upstream wastewater discharges. Ultraviolet (UV)-driven advanced oxidation processes (UV-AOPs) using radiation sources that emit at 254 nm have been developed as advanced treatments to degrade contaminants, while those UV-AOPs can be improved towards higher radical yields and lower byproduct formation. Several previous studies have suggested that Far-UVC radiation (200-230 nm) is a promising radiance source to drive UV-AOPs because the direct photolysis of micropollutants and production of reactive species from oxidant precursors can both be improved. In this study, we summarize from the literature the photodecay rate constants of five micropollutants by direct UV photolysis, which are higher at 222 than 254 nm. We experimentally determine the molar absorption coefficients at 222 and 254 nm of eight oxidants commonly used in water treatment and present the quantum yields of the oxidant photodecay. Our experimental results also show that the concentrations of HO·, Cl·, and ClO· generated in the UV/chlorine AOP can be increased by 5.15-, 15.76-, and 2.86-fold, respectively, by switching the UV wavelength from 254 to 222 nm. We also point out the challenges of applying Far-UVC for micropollutant abatement in water treatment, including the strong light screening effect of matrix components (e.g., carbonate, nitrate, bromide, and dissolved organic matter), the formation of byproducts via new reaction pathways, and the needs to improve the energy efficiency of the Far-UVC radiation sources.
Subject(s)
Water Pollutants, Chemical , Water Purification , Humans , Wastewater , Oxidation-Reduction , Chlorine , Oxidants , Water Purification/methods , Ultraviolet Rays , Hydrogen PeroxideABSTRACT
OBJECTIVE: To evaluate the neurodevelopmental outcomes at 5.5 years of age in children who were previously randomized to cow milk-based infant formula (control) or similar formula (milk fat globule membrane + lactoferrin) with added sources of bovine milk fat globule membrane and bovine lactoferrin through 12 months of age. DESIGN: Children who completed study feeding were invited to participate in follow-up assessments: cognitive development across multiple domains (primary outcome; Wechsler Preschool & Primary Scale of Intelligence, 4th Edition), inhibitory control/rule learning (Stroop Task), flexibility/rule learning (Dimensional Change Card Sort), and behavior/emotion (Child Behavior Checklist). RESULTS: Of 292 eligible participants (control: 148, milk fat globule membrane + lactoferrin: 144), 116 enrolled and completed assessments (control: 59, milk fat globule membrane + LF: 57). There were no group demographic differences except family income (milk fat globule membrane + lactoferrin significantly higher). Wechsler Preschool & Primary Scale of Intelligence, 4th Edition composite scores (mean ± standard error) for Visual Spatial (100.6 ± 1.7 vs 95.3 ± 1.7; P = .027), Processing Speed (107.1 ± 1.4 vs 100.0 ± 1.4; P < .001), and Full-Scale IQ (98.7 ± 1.4 vs 93.5 ± 1.5; P = .012) were significantly higher for milk fat globule membrane + lactoferrin versus control, even after controlling for demographic/socioeconomic factors. Stroop Task scores were significantly higher in milk fat globule membrane + lactoferrin versus control (P < .001). Higher Dimensional Change Card Sort scores (P = .013) in the border phase (most complex/challenging) were detected, and more children passed the border phase (32% vs 12%; P = .039) for milk fat globule membrane versus control. No group differences in Child Behavior Checklist score were detected. CONCLUSIONS: Children who received infant formula to 12 months of age with added bovine milk fat globule membrane and bovine lactoferrin versus standard formula demonstrated improved cognitive outcomes in multiple domains at 5.5 years of age, including measures of intelligence and executive function. TRIAL REGISTRATION: Clinicaltrials.gov: https://clinicaltrials.gov/ct2/show/NCT04442477.
Subject(s)
Infant Formula , Lactoferrin , Child , Child, Preschool , Female , Humans , Infant , Glycolipids , Glycoproteins , Lactoferrin/pharmacologyABSTRACT
Krypton chloride (KrCl*) excimer ultraviolet (UV) light may provide advantages for contaminant degradation compared to conventional low-pressure (LP) UV. Direct and indirect photolysis as well as UV/hydrogen peroxide-driven advanced oxidation (AOP) of two chemical contaminants were investigated in laboratory grade water (LGW) and treated secondary effluent (SE) for LPUV and filtered KrCl* excimer lamps emitting at 254 and 222 nm, respectively. Carbamazepine (CBZ) and N-nitrosodimethylamine (NDMA) were chosen because of their unique molar absorption coefficient profiles, quantum yields (QYs) at 254 nm, and reaction rate constants with hydroxyl radical. Quantum yields and molar absorption coefficients at 222 nm for both CBZ and NDMA were determined, with measured molar absorption coefficients of 26â¯422 and 8170 M-1 cm-1, respectively, and QYs of 1.95 × 10-2 and 6.68 × 10-1 mol Einstein-1, respectively. The 222 nm irradiation of CBZ in SE improved degradation compared to that in LGW, likely through promotion of in situ radical formation. AOP conditions improved degradation of CBZ in LGW for both UV LP and KrCl* sources but did not improve NDMA decay. In SE, photolysis of CBZ resulted in decay similar to that of AOP, likely due to the in situ generation of radicals. Overall, the KrCl* 222 nm source significantly improves contaminant degradation compared to that of 254 nm LPUV.
Subject(s)
Water Pollutants, Chemical , Water Purification , Dimethylnitrosamine , Water Pollutants, Chemical/metabolism , Oxidation-Reduction , Carbamazepine , Ultraviolet Rays , Photolysis , Hydrogen Peroxide , Water Purification/methodsABSTRACT
During early embryogenesis, the transcription factor SOX17 contributes to hepato-pancreato-biliary system formation and vascular-hematopoietic emergence. To better understand Sox17 function in the developing endoderm and endothelium, we developed a dual-color temporal lineage-tracing strategy in mice combined with single-cell RNA sequencing to analyze 6934 cells from Sox17-expressing lineages at embryonic days 9.0-9.5. Our analyses showed 19 distinct cellular clusters combined from all 3 germ layers. Differential gene expression, trajectory and RNA-velocity analyses of endothelial cells revealed a heterogenous population of uncommitted and specialized endothelial subtypes, including 2 hemogenic populations that arise from different origins. Similarly, analyses of posterior foregut endoderm revealed subsets of hepatic, pancreatic, and biliary progenitors with overlapping developmental potency. Calculated gene-regulatory networks predict gene regulons that are dominated by cell type-specific transcription factors unique to each lineage. Vastly different Sox17 regulons found in endoderm versus endothelial cells support the differential interactions of SOX17 with other regulatory factors thereby enabling lineage-specific regulatory actions.
Subject(s)
Embryonic Development , Endothelial Cells , Gene Expression Regulation, Developmental , Gene Regulatory Networks , SOXF Transcription Factors , Animals , Mice , Cell Differentiation , Cell Lineage/genetics , Endoderm/metabolism , Endothelial Cells/metabolism , HMGB Proteins/genetics , HMGB Proteins/metabolism , Sequence Analysis, RNA , SOXF Transcription Factors/genetics , SOXF Transcription Factors/metabolism , Transcription Factors/metabolism , Embryonic Development/geneticsABSTRACT
OBJECTIVE: In gout, hyperuricemia promotes urate crystal deposition, which stimulates the NLRP3 inflammasome and interleukin-1ß (IL-1ß)-mediated arthritis. Incident gout without background hyperuricemia is rarely reported. To identify hyperuricemia-independent mechanisms driving gout incidence and progression, we characterized erosive urate crystalline inflammatory arthritis in a young female patient with normouricemia diagnosed as having sufficient and weighted classification criteria for gout according to the American College of Rheumatology (ACR)/EULAR gout classification criteria (the proband). METHODS: We conducted whole-genome sequencing, quantitative proteomics, whole-blood RNA-sequencing analysis using serum samples from the proband. We used a mouse model of IL-1ß-induced knee synovitis to characterize proband candidate genes, biomarkers, and pathogenic mechanisms of gout. RESULTS: Lubricin level was attenuated in human proband serum and associated with elevated acute-phase reactants and inflammatory whole-blood transcripts and transcriptional pathways. The proband had predicted damaging gene variants of NLRP3 and of inter-α trypsin inhibitor heavy chain 3, an inhibitor of lubricin-degrading cathepsin G. Changes in the proband's serum protein interactome network supported enhanced lubricin degradation, with cathepsin G activity increased relative to its inhibitors, SERPINB6 and thrombospondin 1. Activation of Toll-like receptor 2 (TLR-2) suppressed levels of lubricin mRNA and lubricin release in cultured human synovial fibroblasts (P < 0.01). Lubricin blunted urate crystal precipitation and IL-1ß induction of xanthine oxidase and urate in cultured macrophages (P < 0.001). In lubricin-deficient mice, injection of IL-1ß in knees increased xanthine oxidase-positive synovial resident M1 macrophages (P < 0.05). CONCLUSION: Our findings linked normouricemic erosive gout to attenuated lubricin, with impaired control of cathepsin G activity, compounded by deleterious NLRP3 variants. Lubricin suppressed monosodium urate crystallization and blunted IL-1ß-induced increases in xanthine oxidase and urate in macrophages. The collective activities of articular lubricin that could limit incident and erosive gouty arthritis independently of hyperuricemia are subject to disruption by inflammation, activated cathepsin G, and synovial fibroblast TLR-2 signaling.
Subject(s)
Arthritis, Gouty , Gout , Hyperuricemia , Female , Humans , Mice , Animals , Toll-Like Receptor 2/genetics , Cathepsin G/adverse effects , Uric Acid , NLR Family, Pyrin Domain-Containing 3 Protein , Xanthine Oxidase , Gout/genetics , Inflammation/metabolism , Interleukin-1beta/metabolismABSTRACT
OBJECTIVE: This study was undertaken to determine the role of CD38, which can function as an enzyme to degrade NAD+ , in osteoarthritis (OA) development. METHODS: Human knee cartilage from normal donors and OA donors were examined for CD38 expression. "Gain-of-function," through overexpression of CD38 via transient transfection, and "loss-of-function," through pharmacologic inhibition of CD38, approaches were used to assess the effects of CD38 on intracellular NAD+ :NADH ratio and catabolic activity in chondrocytes. We also initiated joint injury-induced OA by surgical destabilization of the medial meniscus (DMM) in CD38 knockout mice and wild-type (WT; C57BL/6) mice and in WT male mice in the presence or absence of apigenin treatment. Cartilage degradation, synovial inflammation, subchondral bone changes, and pain behavior were evaluated after DMM surgery. We also examined expression of CD38 and the neuropeptide calcitonin gene-related peptide (CGRP) in knee sections from these mice. RESULTS: CD38 expression was up-regulated in human knee OA cartilage and in chondrocytes stimulated with the proinflammatory cytokine interleukin-1ß (IL-1ß). Overexpression of CD38 in chondrocytes resulted in reduced cellular NAD+ :NADH ratio and augmented catabolic responses to IL-1ß. These effects were reversed by pharmacologic inhibition of CD38. Cartilage degradation and synovial inflammation, associated with increased CD38 expression in cartilage and synovium, osteophyte formation and subchondral bone sclerosis, and pain-like behavior linked to increased CGRP expression in the synovium were observed in WT mice after joint injury. Such effects were significantly reduced in mice deficient in CD38 through either genetic knockout or pharmacologic inhibition. CONCLUSION: CD38 deficiency exerts OA disease-modifying effects. Inhibition of CD38 has the potential to be a novel therapeutic approach for OA treatment.
Subject(s)
Cartilage, Articular , Osteoarthritis, Knee , Mice , Male , Humans , Animals , NAD/metabolism , NAD/pharmacology , Calcitonin Gene-Related Peptide/metabolism , Calcitonin Gene-Related Peptide/pharmacology , Cartilage, Articular/metabolism , Disease Models, Animal , Mice, Inbred C57BL , Osteoarthritis, Knee/metabolism , Inflammation/metabolism , Pain/metabolism , Mice, KnockoutABSTRACT
OBJECTIVE: Linked metabolic and cardiovascular comorbidities are prevalent in hyperuricemia and gout. For mechanistic insight into impact on inflammatory processes and cardiometabolic risk factors of xanthine oxidase inhibitor urate-lowering therapy (ULT) titration to target, we performed a prospective study of gout serum metabolomes from a ULT trial. METHODS: Sera of gout patients meeting the 2015 ACR/EULAR gout classification criteria (n = 20) and with hyperuricemia were studied at time zero and weeks 12 and 24 of febuxostat or allopurinol dose titration ULT. Ultrahigh performance liquid chromatography-tandem mass spectroscopy acquired the serum spectra. Data were assessed using the Metabolon and Metaboloanalyst software. Lipolysis validation assays were done in febuxostat and/or colchicine-treated 3T3-L1 differentiated adipocytes. RESULTS: Serum urate decreased from time zero (8.21 ±1.139 SD) at weeks 12 (5.965 ± 1.734 SD) and 24 (5.655 ±1.763 SD). Top metabolites generated by changes in nucleotide and certain amino acid metabolism and polyamine pathways were enriched at 12 and 24 weeks ULT, respectively. Decreases in multiple fatty acid metabolites were observed at 24 weeks, linked with obesity. In cultured adipocytes, febuxostat significantly decreased while colchicine increased the lipolytic response to ß-adrenergic-agonism or TNF. CONCLUSION: Metabolomic profiles linked xanthine oxidase inhibitor-based ULT titration to target with reduced serum free fatty acids. In vitro validation studies revealed that febuxostat, but not colchicine, reduced lipolysis in cultured adipocytes. Since soluble urate, xanthine oxidase inhibitor treatment, and free fatty acids modulate inflammation, our findings suggest that by suppressing lipolysis, ULT could regulate inflammation in gout and comorbid metabolic and cardiovascular disease.
Subject(s)
Gout , Hyperuricemia , Adipocytes , Allopurinol/therapeutic use , Colchicine , Enzyme Inhibitors , Fatty Acids, Nonesterified/therapeutic use , Febuxostat/pharmacology , Febuxostat/therapeutic use , Gout Suppressants/adverse effects , Humans , Inflammation/drug therapy , Lipolysis , Prospective Studies , Uric Acid , Xanthine Oxidase/therapeutic useABSTRACT
Elevated serum urate (hyperuricemia) promotes crystalline monosodium urate tissue deposits and gout, with associated inflammation and increased mortality. To identify modifiers of uric acid pathologies, we performed a fly Genome-Wide Association Study (GWAS) on purine metabolites using the Drosophila Genetic Reference Panel strains. We tested the candidate genes using the Drosophila melanogaster model of hyperuricemia and uric acid crystallization ("concretion formation") in the kidney-like Malpighian tubule. Medusa (mda) activity increased urate levels and inflammatory response programming. Conversely, whole-body mda knockdown decreased purine synthesis precursor phosphoribosyl pyrophosphate, uric acid, and guanosine levels; limited formation of aggregated uric acid concretions; and was sufficient to rescue lifespan reduction in the fly hyperuricemia and gout model. Levels of mda homolog FAM214A were elevated in inflammatory M1- and reduced in anti-inflammatory M2-differentiated mouse bone marrow macrophages, and influenced intracellular uric acid levels in human HepG2 transformed hepatocytes. In conclusion, mda/FAM214A acts in a conserved manner to regulate purine metabolism, promotes disease driven by hyperuricemia and associated tissue inflammation, and provides a potential novel target for uric acid-driven pathologies.
Subject(s)
Drosophila Proteins , Gout , Hyperuricemia , Animals , Humans , Mice , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Genome-Wide Association Study , Gout/genetics , Gout/complications , Gout/metabolism , Hyperuricemia/genetics , Hyperuricemia/complications , Hyperuricemia/metabolism , Inflammation/genetics , Inflammation/complications , Purines/metabolism , Uric Acid/urine , Drosophila Proteins/geneticsABSTRACT
Monosodium urate crystals (MSUc) induce inflammation in vivo without prior priming, raising the possibility of an initial cell-autonomous phase. Here, using genome-wide transcriptomic analysis and biochemical assays, we demonstrate that MSUc alone induce a metabolic-inflammatory transcriptional program in non-primed human and murine macrophages that is markedly distinct to that induced by LPS. Genes uniquely upregulated in response to MSUc belong to lipid and amino acid metabolism, glycolysis, and SLC transporters. This upregulation leads to a metabolic rewiring in sera from individuals and mice with acute gouty arthritis. Mechanistically, the initiating inflammatory-metabolic changes in acute gout flares are regulated through a persistent expression and increased binding of JUN to the promoter of target genes through JNK signaling-but not P38-in a process that is different than after LPS stimulation and independent of inflammasome activation. Finally, pharmacological JNK inhibition limits MSUc-induced inflammation in animal models of acute gouty inflammation.
Subject(s)
Arthritis, Gouty , Uric Acid , Animals , Arthritis, Gouty/chemically induced , Arthritis, Gouty/metabolism , Inflammation/metabolism , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Mice , Uric Acid/metabolismABSTRACT
Different infant diets have strong effects on child development and may engender variations in fecal microbiota and metabolites. The objective of this study was to evaluate the effect of an infant formula containing sn-2 palmitate on fecal microbiota and metabolites in healthy term infants. The study involved three groups as indicated below. Investigational: the group fed a formula containing high sn-2 palmitate for 16 weeks. Control: the group fed a formula using a regular vegetable oil for 16 weeks. Breastfed: the group fed breast milk for 16 weeks. Fecal samples were collected at 8 weeks (n = 35, 37, and 35, respectively) and 16 weeks (n = 30, 32, and 30, respectively) for the control, investigational, and breastfed infants. Microbiota data were obtained using 16S rRNA sequencing. Short-chain fatty acid (SCFA) analysis was performed using GC-MS, and untargeted metabolomics was conducted using LC-MS. The effect of the formula containing sn-2 palmitate was different from that of the control formula on microbiota and metabolites. Sn-2 palmitate promoted the proliferation of Bifidobacterium and reduced the abundance of Escherichia-Shigella at 8 weeks. Furthermore, it increased α-diversity and enhanced acetate content in feces at both 8 and 16 weeks. In the investigational group infants, the abundance of DL-tryptophan, indole-3-acrylic acid, acetyl-ß-methylcholine, L-methionine, and 2-hydroxyvaleric acid significantly increased at 8 weeks, while a notable increase in the abundance of 3-phenyllactic acid, palmitic acid, L-phenylalanine, and leucylproline was observed at 16 weeks. In addition, compared with that of the control infants, the intestinal microbiota and metabolites of sn-2 palmitate-supplemented infants were more similar to those of the breastfed infants. The study hopes to provide a scientific basis for the development of functional infant formulas in the future.
Subject(s)
Infant Formula/chemistry , Palmitates/chemistry , Bifidobacterium/isolation & purification , Double-Blind Method , Feces/microbiology , Female , Humans , Infant, Newborn , Male , Metabolome , MicrobiotaABSTRACT
OBJECTIVE: We investigated the effect of berberine, a natural plant product that can activate AMP-activated protein kinase (AMPK), on Osteoarthritis (OA) development and associated pain in mice. DESIGN: Human primary knee chondrocytes were utilized to investigate how AMPK is activated by berberine. Both global knockout (KO) of AMPKα1 and congenic wild type (WT) mice were subjected to the post-traumatic OA through destabilization of medial meniscus (DMM) surgery. Two weeks after surgery, the mice were randomly divided into two groups with one group receiving berberine chloride daily via drinking water and were sacrificed at 6 and 12 weeks after surgery. OA severity was assessed by histological and histomorphometric analyses of cartilage degradation, synovitis, and osteophyte formation. OA-associated pain behavior was also determined. Immunohistochemistry (IHC) analyses were carried out to examine changes in AMPK signaling. RESULTS: Berberine induced phosphorylation of AMPKα (Thr172) via liver kinase B1 (LKB1), the major upstream kinase of AMPK, in chondrocytes in vitro. Both WT and AMPKα1KO developed OA and associated pain post DMM surgery. However, treatment with berberine significantly reduced severity of OA and associated pain in WT but not AMPKα1KO mice. IHC analysis of WT DMM knee cartilage further revealed that berberine inhibited concomitant loss of expression and phosphorylation of AMPKα and expression of SIRT1 and SIRT3, suggesting an important role of activation of AMPK signaling in mediating beneficial effect of berberine. CONCLUSIONS: Berberine acts through AMPK to reduce joint structural damage and pain associated with post-traumatic OA in mice in vivo.
Subject(s)
AMP-Activated Protein Kinases/physiology , Arthralgia/prevention & control , Berberine/administration & dosage , Osteoarthritis/prevention & control , AMP-Activated Protein Kinases/drug effects , Administration, Oral , Animals , Arthralgia/etiology , Berberine/pharmacology , Joints/injuries , Male , Mice , Osteoarthritis/etiologyABSTRACT
CONTEXT: Upper respiratory tract infection (URTI) is the most common illness in humans. Fermented milk containing probiotics can mitigate URTI symptoms. OBJECTIVE: This study tests the effect of fermented milk (Qingrun), a yogurt supplemented with Bifidobacterium animalis subsp. lactis Bl-04, on adults with URTIs who live in a haze-covered area in a randomized clinical trial. MATERIALS AND METHODS: A total of 136 subjects were enrolled in the study at the baseline and randomized to consume either control yogurt or Qingrun yogurt (250 g) once daily for 12 weeks. The duration and severity of URTI were evaluated by the Wisconsin Upper Respiratory Symptom Survey-24. Blood and faecal samples were collected at the baseline and post-intervention, to determine the changes of immune biomarkers. RESULTS: Qingrun yogurt significantly reduced the incidence of the common cold (OR, 0.38; 95% CI, 0.17-0.81; p = 0.013) and influenza-like illness (OR, 0.32; 95% CI, 0.11-0.97; p = 0.045). Compared to the control yogurt, Qingrun yogurt significantly reduced the duration (1.23 ± 2.73 vs. 4.78 ± 5.09 d) and severity score (3.58 ± 7.12 vs. 11.37 ± 11.73) of URTI. In addition, the post-intervention levels of interferon-γ (139.49 ± 59.49 vs. 113.45 ± 65.12 pg/mL) and secretory immunoglobulin A (529.19 ± 91.70 vs. 388.88 ± 53.83 mg/dL) significantly increased in the Qingrun group, compared with those in the control group. CONCLUSIONS: Qingrun yogurt showed a protective effect against URTI in adults, suggesting that the use of yogurt with probiotics could be a promising dietary supplement for mitigating URTI.
Subject(s)
Bifidobacterium , Dietary Supplements , Probiotics/therapeutic use , Respiratory Tract Infections/therapy , Adult , Air Pollution/adverse effects , China , Double-Blind Method , Female , Fermentation , Humans , Male , Middle Aged , Respiratory Tract Infections/etiology , Yogurt/microbiologyABSTRACT
Current drug delivery approaches for the treatment of cartilage disorders such as osteoarthritis (OA) remain inadequate to achieve sufficient drug penetration and retention in the dense cartilage matrix. Herein, we synthesize sub-30 nm lipid-polymer hybrid nanoparticles functionalized with collagen-targeting peptides for targeted drug delivery to the cartilage. The nanoparticles consist of a polymeric core for drug encapsulation and a lipid shell modified with a collagen-binding peptide. By combining these design features, the nanoparticles can penetrate deep and accumulate preferentially in the cartilage. Using MK-8722, an activator of 5'-adenosine monophosphate-activated protein kinase (AMPK), as a model drug, the nanoparticles can encapsulate the drug molecules in high capacity and release them in a sustained and controllable manner. When injected into the knee joints of the mice with collagenase-induced OA, the drug-loaded nanoparticles can effectively reduce cartilage damage and alleviate the disease severity. Overall, the ultrasmall targeted nanoparticles represent a promising delivery platform to overcome barriers of dense tissues for the treatment of various indications, including cartilage disorders.
ABSTRACT
Chondrocytes, the only cells in articular cartilage, are metabolically active and responsible for the turnover of extracellular matrix and maintenance of the tissue homeostasis. Changes in chondrocyte function can cause degradation of the matrix and loss of articular cartilage integrity, leading to development and progression of osteoarthritis (OA). These changes are exemplified by accumulated mitochondrial damage and dysfunction. Because mitochondria are the critical organelles to produce energy and play a key role in cellular processes, the approaches to assess mitochondrial function under both physiological and pathological conditions enable us to uncover the mechanisms on how dysfunction of mitochondria in chondrocytes mediates signaling pathways that are involved in disturbance of cartilage homeostasis. In this chapter, we describe the methods to evaluate mitochondrial biogenesis, activity and mitochondrial DNA (mtDNA) integrity in chondrocytes.
Subject(s)
Chondrocytes/metabolism , DNA, Mitochondrial/genetics , DNA, Mitochondrial/isolation & purification , Mitochondria/genetics , Mitochondria/metabolism , Organelle Biogenesis , Adenosine Triphosphate , Animals , Biomarkers , Cartilage, Articular/metabolism , Gene Expression Regulation , Humans , Knee Joint , Mutation , Osteoarthritis/genetics , Osteoarthritis/metabolism , Osteoarthritis/pathology , Protein Processing, Post-Translational , Real-Time Polymerase Chain ReactionABSTRACT
BACKGROUND: With the frequent use of video display units, eye fatigue is becoming more common globally. An alternative nutritional strategy is needed to prevent the aggravation of eye fatigue symptoms. OBJECTIVES: The objective was to evaluate the protective effect of a novel botanical combination of lutein ester, zeaxanthin, and extracts of blackcurrant, chrysanthemum, and goji berry on adults with eye fatigue in a randomized, double-blind, placebo-controlled clinical trial. METHODS: We randomly allocated 360 participants into 4 groups to receive placebo and 3 doses of our formula (chewable tablets, containing 6 mg, 10 mg, or 14 mg of lutein) once daily for 90 d. Each participant had 3 visits at baseline (V1), 45 d (V2), and 90 d (V3) during the study. RESULTS: Intervention with the formula improved individual scores of eye fatigue symptoms, including eye soreness, blurred vision, dry eye, foreign body sensation, and tearing. Compared with placebo, the formula at all 3 doses significantly decreased the total score of eye fatigue symptoms and increased the visuognosis persistence time at both V2 and V3. According to the Schirmer test, both 10-mg and 14-mg lutein formula groups had improved tear secretion at V3 compared with the placebo. The keratography results indicated that the first tear break-up time, average tear break-up time, and tear meniscus height were significantly increased after formula intervention. The formula at all 3 doses significantly increased the macular pigment optical density at V2 and V3 compared with the placebo, whereas optical coherence tomography showed no significant difference in retinal thickness and retinal volume across all groups at both visits. CONCLUSIONS: Our botanical formula improves eye fatigue, dry eye, and macular function without changing the retinal structure, and thus it could serve as an effective nutritional strategy in improving eye fatigue without causing serious side effects.Clinical Trial Registry: chictr.org.cn (ChiCTR1800018987).
Subject(s)
Asthenopia/drug therapy , Chrysanthemum/chemistry , Dry Eye Syndromes/drug therapy , Plant Preparations/administration & dosage , Ribes/chemistry , Adult , Aged , Dietary Supplements/analysis , Double-Blind Method , Female , Humans , Lutein/administration & dosage , Lutein/analysis , Lycium/chemistry , Male , Middle Aged , Plant Preparations/analysis , Visual Acuity/drug effects , Young Adult , Zeaxanthins/administration & dosage , Zeaxanthins/analysisABSTRACT
OBJECTIVES: In this study, we aim to determine the effect of metformin on osteoarthritis (OA) development and progression. METHODS: Destabilisation of the medial meniscus (DMM) surgery was performed in 10-week-old wild type and AMP-activated protein kinase (AMPK)α1 knockout (KO) mice. Metformin (4 mg/day in drinking water) was given, commencing either 2 weeks before or 2 weeks after DMM surgery. Mice were sacrificed 6 and 12 weeks after DMM surgery. OA phenotype was analysed by micro-computerised tomography (µCT), histology and pain-related behaviour tests. AMPKα1 (catalytic alpha subunit of AMPK) expression was examined by immunohistochemistry and immunofluorescence analyses. The OA phenotype was also determined by µCT and MRI in non-human primates. RESULTS: Metformin upregulated phosphorylated and total AMPK expression in articular cartilage tissue. Mild and more severe cartilage degeneration was observed at 6 and 12 weeks after DMM surgery, evidenced by markedly increased Osteoarthritis Research Society International scores, as well as reduced cartilage areas. The administration of metformin, commencing either before or after DMM surgery, caused significant reduction in cartilage degradation. Prominent synovial hyperplasia and osteophyte formation were observed at both 6 and 12 weeks after DMM surgery; these were significantly inhibited by treatment with metformin either before or after DMM surgery. The protective effects of metformin on OA development were not observed in AMPKα1 KO mice, suggesting that the chondroprotective effect of metformin is mediated by AMPK signalling. In addition, we demonstrated that treatment with metformin could also protect from OA progression in a partial medial meniscectomy animal model in non-human primates. CONCLUSIONS: The present study suggests that metformin, administered shortly after joint injury, can limit OA development and progression in injury-induced OA animal models.