Paeonol promotes longevity and fitness in Caenorhabditis elegans through activating the DAF-16/FOXO and SKN-1/Nrf2 transcription factors.

Paeonol, as one of the most abundant plant-derived polyphenols, has multiple bioactivities including anti-inflammatory, anti-tumor, and anti-cardiovascular diseases. Nevertheless, the anti-aging effects and related mechanisms of paeonol are rarely reported. In this study, we found that paeonol significantly prolonged the mean lifespan of Caenorhabditis elegans (C. elegans) by 28.49% at a dose of 200 µM. Moreover, paeonol promoted the health of C. elegans by increasing the body bending and pharyngeal pumping rates and reducing the lipofuscin accumulation level. Meanwhile, paeonol induced the expression of stress-related genes or proteins by activating the transcription factors DAF-16/FOXO, SKN-1/Nrf2, and HSF-1, which in turn enhanced oxidative and thermal stress tolerance. The mechanism behind the anti-aging effect of paeonol occurred by down-regulating the insulin/IGF-1 signaling (IIS) pathway. Our findings shed new light on the application of paeonol for longevity promotion and human health.

Identification of key phenolic compounds for alleviating gouty inflammation in edible chrysanthemums based on spectrum-effect relationship analyses.

Edible chrysanthemum is a common food resource for tea and functional foods with potential benefits for human health. Studies have indicated that chrysanthemum has the potential effect on inflammatory diseases, while the effects on gouty inflammation remain underexplored. The present study aimed to investigate the anti-gout activity and characterize the active ingredients of chrysanthemums by using metabolite profiles, in vitro experiments, and spectrum-effect analysis. Results showed that 'Boju' (BJ), 'Hangbaiju' (HBJ), and 'Huaiju' (HJ) exhibited regulatory effects on monosodium urate (MSU)-induced inflammation. At the dose of 50 µg/mL, the inhibitory rates of IL-1ß secretion were 24.53 %, 14.36 %, and 38.10 %, respectively. A total of 32 phenolic compounds were identified or preliminarily assigned in UPLC-Q/TOF-MS analysis. And seven phenolics related to anti-gout activity were identified by spectrum-effect relationships. According to ADME (absorption, distribution, metabolism, excretion) evaluation and experiments verification, luteolin, acacetin-7-O-glucoside, and apigenin-7-O-glucoside were critical constituents potentially associated with the reduction of inflammation in gout. Additionally, these phenolics might be suitable as quality control indicators. This study clarified the anti-gout properties of different cultivars of chrysanthemums and active compounds, providing a theoretical basis for its scientific utilization in functional foods.

Vitexin and isovitexin delayed ageing and enhanced stress-resistance through the activation of the SKN-1/Nrf2 signaling pathway.

Vitexin and isovitexin, as potential SKN-1/Nrf2 (SKN-1 is a homologous protein of mammalian Nrf2) activators, extended lifespan and promoted healthspan in Caenorhabditis elegans. This study aims to elucidate the role of SKN-1/Nrf2 in vitexin and isovitexin-induced anti-aging and stress-resistance. Vitexin and isovitexin upregulated antioxidant gene and protein expressions, reduced ROS accumulation, and increased SKN-1 accumulation in the nucleus. They prolonged lifespan and clear ROS during stressful conditions in a skn-1-dependent manner. skn-1 was also found to be necessary for these compounds-induced longevity under normal conditions. They were also witnessed to retard cellular senescence and scavenge ROS in senescent cells by directly binding to the pocket of Keap1 to promote the dissociation and activation of Nrf2. This study showed that SKN-1/Nrf2 signaling was vital to delaying ageing and enhancing anti-stress capacity with vitexin and isovitexin. The findings provide new insights into apigenin C-glycosides activating the SKN-1/Nrf2 pathway and demonstrate their potential as candidates for innovative strategies in chemoprophylaxis against ageing and oxidative-related diseases.

Red raspberry supplementation mitigates alcohol-induced liver injury associated with gut microbiota alteration and intestinal barrier dysfunction in mice.

Alcoholic liver disease (ALD) is still a global health concern. Long-term alcohol intake alters the gut microbiota diversity and metabolic activity, and causes intestinal barrier dysfunction, leading to the development of ALD. This research explored the protective effects and underlying mechanisms of red raspberry (RR) on alcohol-related disorders in mice. Male C57BL/6J mice were fed a standard diet or a standard diet supplemented with 2%, 4%, and 8% weight/weight RR. Meanwhile, mice were administered 35% (v/v) ethanol (EtOH, 10 mL per kg body weight) intragastrically once daily for six weeks, except the control group mice. The results showed that RR supplementation decreased liver injury markers (alanine and aspartate transaminases) in the serum, reduced triglyceride level in the liver and downregulated hepatic cytochrome P450 2E1 mRNA expression in mice administered EtOH. In addition, EtOH-mediated oxidative stress in the liver was attenuated by RR supplementation through decreased hepatic malondialdehyde content and increased antioxidant (glutathione, glutathione peroxidase, and catalase) levels and activities in mice exposed to EtOH. Moreover, RR supplementation reversed EtOH-induced alteration in the cecal microbial composition at the phylum, order, genus, and species levels and improved the intestinal barrier function associated with the inhibition of the NF-κB/MLCK pathway, which was accompanied by upregulation of tight junctions (zonula occludens 1, occludin, claudin-1, and claudin-4) and E-cadherin mRNA and protein expressions. Accordingly, RR supplementation resulted in a decreased level of endotoxins in the serum and attenuation of the inflammatory response in the liver, illustrated by a significant decrease in tumor necrosis factor-alpha, interleukin (IL)-1ß, and IL-6 levels. Overall, RR supplementation alleviated the adverse effects of chronic alcohol intake in C57BL/6J mice and could be a potential supplement for improving ALD.

Longevity-promoting properties of ginger extract in Caenorhabditis elegans via the insulin/IGF-1 signaling pathway.

Ginger is a traditional medicinal and edible plant with multiple health-promoting properties. Nevertheless, the effects and potential mechanism of ginger on antiaging remain unknown. The aim of this study was to comprehend the antiaging effects and potential mechanism of ginger in Caenorhabditis elegans (C. elegans). The current findings showed that the lifespan of C. elegans was prolonged by 23.16% with the supplementation of 60 µg mL-1 ginger extract (GE), and the extension of lifespan was mainly attributed to the major bioactive compounds in GE, 6-, 8-, 10-gingerol and 6-, 8-, 10-shogaol. Subsequently, GE promoted healthy aging by improving nematode movement and attenuating lipofuscin accumulation, and enhanced stress tolerance by up-regulating the expression of stress-related genes and activating DAF-16 and SKN-1. Moreover, lifespan assays of relative mutants revealed that GE mediated extension of lifespan via the insulin/IGF-1 signaling (IIS) pathway. In summary, GE endowed nematodes (C. elegans) with longevity and stress resistance in an IIS pathway dependent manner.

Vitexin and Isovitexin Act through Inhibition of Insulin Receptor to Promote Longevity and Fitness in Caenorhabditis elegans.

SCOPE: Vitexin and isovitexin are natural plant nutraceuticals for human health and longevity. This research investigates the underlying mechanisms of vitexin and isovitexin on aging and health. The vital role of DAF-2/insulin-like growth factor-1 receptor (IGFR) is illustrated in the insulin/insulin-like growth signaling pathway (IIS) modulated by vitexin and isovitexin. METHODS AND RESULTS: In vitro, in vivo models and molecular docking methods are performed to explore the antiaging mechanism of vitexin and isovitexin. Vitexin and isovitexin (50 and 100 µM) extended the lifespan of Caenorhabditis elegans. The declines of pharyngeal pumping and body bending rates, and the increase of intestinal lipofuscin accumulation, three markers of aging, are postponed by these compounds. They inhibit IIS pathway in a daf-16-dependent manner, subsequently increasing the expressions of DAF-16 downstream protein and gene in nematodes. Molecular docking studies demonstrate that these compounds mightinhibit insulin signal by binding to the crucial amino acid residue ARG1003 in the pocket of IGFR. Western blot indicates that IGFR, PI3K, and AKT kinase expressions in senescent cells are decreased after vitexin and isovitexin treatment. CONCLUSION: Vitexin and isovitexin may inhibit IIS pathway by occupying adenosine-triphosphate binding site pocket of IGFR, subsequently decreasing IGFR expression, thereby promoting longevity and fitness.

Artemisia selengensis Turcz. leaf extract promotes longevity and stress resistance in Caenorhabditis elegans.

BACKGROUND: Artemisia selengensis Turcz. (AST) is a common edible and medicinal herb possessing extensive biological activities and various health-promoting functions. However, the anti-aging effects of AST have been neglected. This work evaluated the effects of AST leaf extract (ASTE) on stress tolerance and longevity in Caenorhabditis elegans. RESULTS: ASTE treatment enhanced stress resistance and significantly extended the lifespan of C. elegans. Moreover, ASTE prolonged the healthspan by increasing body bending and pharyngeal pumping rates, and by reducing the intestinal lipofuscin level and accumulation of intracellular reactive oxygen species (ROS). Caffeoylquinic acids in ASTE, especially dicaffeoylquinic acids, were the major components responsible for these benefits. The mechanism underlying the anti-aging effect of ASTE occurred by activating insulin/insulin-like growth factor, SIR-2.1 signaling and mitochondrial dysfunction pathways, which in turn induced the activity of the transcription factors DAF-16/FOXO and SKN-1/Nrf2. CONCLUSION: These findings provide direct evidence for the anti-aging effects of AST and reveal its potential on promoting healthy aging. © 2022 Society of Chemical Industry.

Capsaicinoid-Glucosides of Fresh Hot Pepper Promotes Stress Resistance and Longevity in Caenorhabditis elegans.

In this study, capsaicin-glucoside and dihydro-capsaicin-glucoside derived from fresh hot-red pepper were isolated and identified using UPLC-ESI-Q-TOF-MS/PDA. Synchronized worms were treated with capsaicinoid-glucosides (CG), and then lifespan and stress resistance were examined. The 50 µg/ml concentration of CG-intake could effectively protect the Caenorhabditis elegans (C. elegans) against stresses factors including oxidation and heat as well as reactive oxygen species (ROS), thereby enhancing the survival of CG-treated worms under stress. Enhancing stress resistance in CG-treated worms could be due to the increased expressions of stress-related genes in C. elegans such as daf-16, skn-1 and their downstream target genes (sod-3, hsp-16.2, gst-4 and gcs-1). Lifespan study of different C. elegans strains and RT-PCR showed that the CG-mediated lifespan extension was dependent on DAF-16/FOXO and SKN-1/Nrf2 transcription factors. The study is a step forward in exploring the stress resistance and anti-aging properties of this beneficial extract. Thus, this study will be useful in formulating remedies for stresses factors and age associated disorders.

Small berries as health-promoting ingredients: a review on anti-aging effects and mechanisms in Caenorhabditis elegans.

Aging is an inevitable, irreversible, and complex process of damage accumulation and functional decline, increasing the risk of various chronic diseases. However, for now no drug can delay aging process nor cure aging-related diseases. Nutritional intervention is considered as a key and effective strategy to promote healthy aging and improve life quality. Small berries, as one of the most common and popular fruits, have been demonstrated to improve cognitive function and possess neuroprotective activities. However, the anti-aging effects of small berries have not been systematically elucidated yet. This review mainly focuses on small berries' anti-aging activity studies involving small berry types, active components, the utilized model organism Caenorhabditis elegans (C. elegans), related signaling pathways, and molecular mechanisms. The purpose of this review is to propose effective strategies to evaluate the anti-aging effects of small berries and provide guidance for the development of anti-aging supplements from small berries.

A promising strategy for investigating the anti-aging effect of natural compounds: a case study of caffeoylquinic acids.

Caffeoylquinic acids, as plant-derived polyphenols, exhibit multiple biological activities such as antioxidant, anti-inflammatory, and neuroprotective activities. However, only limited information about their effect on longevity is available. In the current study, molecular docking was employed to explore the interactions between six representative caffeoylquinic acids and the insulin-like growth factor-1 receptor (IGFR), which is an important target protein for longevity. The results indicated that all six compounds were embedded well in the active pocket of IGFR, and that 3,5-diCQA exhibited the strongest affinity to IGFR. Moreover, ASP1153, GLU1080, ASP1086, and ARG1003 were the key amino acid residues during the interaction of these 6 compounds with IGFR. Furthermore, the lifespan extension effect of caffeoylquinic acids was evaluated in a Caenorhabditis elegans (C. elegans) model. The results revealed that all the caffeoylquinic acids significantly extended the lifespan of wild-type worms, of which 3,5-diCQA was the most potent compound. Meanwhile, 3,5-diCQA enhanced the healthspan by increasing the body bending and pharyngeal pumping rates and reducing the intestinal lipofuscin level. Further studies demonstrated that 3,5-diCQA induced longevity effects by downregulating the insulin/insulin-like growth factor signaling (IIS) pathway. This study suggested that the combination of molecular docking and genetic analysis of specific worm mutants could be a promising strategy to reveal the anti-aging mechanisms of small molecule natural compounds.

Flavonoids from the mung bean coat promote longevity and fitness in Caenorhabditis elegans.

Mung beans possess health benefits related to their bioactive ingredients, mainly flavonoids, which are highly concentrated in the coat. However, the anti-aging effects of mung beans are rarely reported. In this work, we found that mung bean coat extract (MBCE), rich in vitexin and isovitexin, extended the lifespan and promoted the health of Caenorhabditis elegans (C. elegans) without any disadvantages. Moreover, MBCE enhanced the resistance to heat and oxidation of C. elegans by reducing the accumulation of intracellular reactive oxygen species and up-regulating the expression of stress-resistant genes or proteins. Further studies demonstrated that MBCE improved longevity, stress-resistance and fitness by mediating the mitochondrial function, mimicking calorie restriction, and altering histone modification. These findings provide direct evidence for the anti-aging effects of mung beans and new insights into the innovations and applications of mung beans for the healthcare industry.

Inactivation, Aggregation and Conformational Changes of Polyphenol Oxidase from Quince (Cydonia oblonga Miller) Juice Subjected to Thermal and High-Pressure Carbon Dioxide Treatment.

Polyphenol oxidase (PPO) causes the browning reaction in fruits and vegetables and deteriorates the quality. Thermal treatment for enzyme inactivation may result in defects as opposed to high pressure CO2 (HPCD) processing. In this study, the changes in activity, dissociation, aggregation and conformation of purified PPO from thermal and HPCD treated juice were investigated. HPCD exhibited inactivation of PPO at 55⁻65 °C whereas thermal processing alone at the same temperature resulted in PPO still showing activity. Under thermal treatment at 25 and 65 °C, the browning degree was higher (0.39 and 0.24) than for HPCD-treated juice (0.23 and 0.12). Fluorescence and circular dichroism spectral results indicated that HPCD induced large decreases in intensities, revealing a rearrangement of the secondary structure and destruction of the native configuration of the PPO molecule. The particle size distribution (PSD) pattern revealed structural modification leading to initial dissociation and subsequent aggregation of PPO after HPCD treatment. Polyacrylamide gel electrophoresis (PAGE) analysis exhibited that molecular size of protein was 40 kDa. In conclusion, the HPCD method was found to be more effective than thermal treatment to inactivate PPO. Structural modifications provided better insights into the phenomena of activation and inactivation of PPO.

Penile alterations at early stage of type 1 diabetes in rats

ABSTRACT Objective Diabetes affects the erectile function significantly. However, the penile alterations in the early stage of diabetes in experimental animal models have not been well studied. We examined the changes of the penis and its main erectile components in diabetic rats. Materials and methods Male Sprague-Dawley rats were divided into 2 groups: streptozotocin (STZ)-induced diabetics and age-matched controls. Three or nine weeks after diabetes induction, the penis was removed for immunohistochemical staining of smooth muscle and neuronal nitric oxide synthase (nNOS) in midshaft penile tissues. The cross-sectional areas of the whole midshaft penis and the corpora cavernosa were quantified. The smooth muscle in the corpora cavernosa and nNOS in the dorsal nerves were quantified. Results The weight, but not the length, of the penis was lower in diabetics. The cross-sectional areas of the total midshaft penis and the corpora cavernosa were lower in diabetic rats compared with controls 9 weeks, but not 3 weeks after diabetes induction. The cross-sectional area of smooth muscle in the corpora cavernosa as percentage of the overall area of the corpora cavernosa was lower in diabetic rats than in controls 9 weeks, but not 3 weeks after diabetes induction. Percentage change of nNOS in dorsal nerves was similar at 3 weeks, and has a decreased trend at 9 weeks in diabetic rats compared with controls. Conclusions Diabetes causes temporal alterations in the penis, and the significant changes in STZ rat model begin 3-9 weeks after induction. Further studies on the reversibility of the observed changes are warranted.

Penile alterations at early stage of type 1 diabetes in rats.

OBJECTIVE: Diabetes affects the erectile function significantly. However, the penile alterations in the early stage of diabetes in experimental animal models have not been well studied. We examined the changes of the penis and its main erectile components in diabetic rats. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into 2 groups: streptozotocin (STZ)-induced diabetics and age-matched controls. Three or nine weeks after diabetes induction, the penis was removed for immunohistochemical staining of smooth muscle and neuronal nitric oxide synthase (nNOS) in midshaft penile tissues. The cross-sectional areas of the whole midshaft penis and the corpora cavernosa were quantified. The smooth muscle in the corpora cavernosa and nNOS in the dorsal nerves were quantified. RESULTS: The weight, but not the length, of the penis was lower in diabetics. The crosssectional areas of the total midshaft penis and the corpora cavernosa were lower in diabetic rats compared with controls 9 weeks, but not 3 weeks after diabetes induction. The cross-sectional area of smooth muscle in the corpora cavernosa as percentage of the overall area of the corpora cavernosa was lower in diabetic rats than in controls 9 weeks, but not 3 weeks after diabetes induction. Percentage change of nNOS in dorsal nerves was similar at 3 weeks, and has a decreased trend at 9 weeks in diabetic rats compared with controls. CONCLUSIONS: Diabetes causes temporal alterations in the penis, and the significant changes in STZ rat model begin 3-9 weeks after induction. Further studies on the reversibility of the observed changes are warranted.

Radiosensitization of non-small-cell lung cancer cells and xenografts by the interactive effects of pemetrexed and methoxyamine.

BACKGROUND AND PURPOSE: The anti-folate pemetrexed is a radiosensitizer. In pre-clinical models, pemetrexed is more effective along with the base-excision-repair inhibitor methoxyamine. We tested whether methoxyamine enhances pemetrexed-mediated radiosensitization of lung adenocarcinoma cells and xenografts. MATERIALS AND METHODS: A549 and H1299 cells were evaluated for cell cycle distribution by flow cytometry, radiosensitization by clonogenic assay, and DNA repair by neutral comet assay and repair protein activation. H460 cells were included in some studies. Xenografts in nude mice received drug(s) and/or radiation, and tumor growth was monitored by caliper and in vivo toxicity by animal weight. RESULTS: Exposure to pemetrexed/methoxyamine for 24 (H1299, H460) or 48 (A549)hours before irradiation resulted in accumulation of cells near the radiosensitive G1/S border; dose-enhancement factors of 1.62±0.19, 1.97±0.25, and 1.67±0.30, respectively; reduction of mean inactivation dose by 32%, 30%, and 46%, respectively; and significant reductions of SF2 and SF4 (p<0.05). Radiosensitization was associated with rapid DNA double-strand-break rejoining and increased levels of DNA-PKcs. Both tumor-growth rate and tumor-growth delay were significantly improved by adding methoxyamine to pemetrexed pre-irradiation (p<0.0001); no mice lost weight during treatment. CONCLUSIONS: Addition of methoxyamine to pemetrexed and fractionated radiotherapy may improve outcome for patients with locally advanced non-squamous non-small-cell lung cancer.

Bladder function in mice with inducible smooth muscle-specific deletion of the manganese superoxide dismutase gene.

Manganese superoxide dismutase (MnSOD) is considered a critical component of the antioxidant systems that protect against oxidative damage. We are interested in the role of oxidative stress in bladder detrusor smooth muscle (SM) in different disease states. In this study, we generated an inducible, SM-specific Sod2(-/-) mouse model to investigate the effects of MnSOD depletion on the function of the bladder. We crossbred floxed Sod2 (Sod2(lox/lox)) mice with mice containing heterozygous knock-in of a gene encoding a tamoxifen-activated Cre recombinase in the SM22α promoter locus [SM-CreER(T2)(ki)(Cre/+)]. We obtained Sod2(lox/lox),SM-CreER(T2)(ki)(Cre/+) mice and injected 8-wk-old males with 4-hydroxytamoxifen to induce Cre-mediated excision of the floxed Sod2 allele. Twelve weeks later, SM-specific deletion of Sod2 and depletion of MnSOD were confirmed by polymerase chain reaction, immunoblotting, and immunohistochemistry. SM-specific Sod2(-/-) mice exhibited normal growth with no gross abnormalities. A significant increase in nitrotyrosine concentration was found in bladder SM tissue of SM-specific Sod2(-/-) mice compared with both wild-type mice and Sod2(+/+), SM-CreER(T2)(ki)(Cre/+) mice treated with 4-hydroxytamoxifen. Assessment of 24-h micturition in SM-specific Sod2(-/-) mice revealed significantly higher voiding frequency compared with both wild-type and SM-specific Cre controls. Conscious cystometry revealed significantly shorter intercontraction intervals and lower functional bladder capacity in SM-specific Sod2(-/-) mice compared with wild-type mice. This novel model can be used for exploring the mechanistic role of oxidative stress in organs rich in SM in different pathological conditions.

Tissue specific dysregulated protein subnetworks in type 2 diabetic bladder urothelium and detrusor muscle.

Diabetes mellitus is well known to cause bladder dysfunction; however, the molecular mechanisms governing this process and the effects on individual tissue elements within the bladder are poorly understood, particularly in type 2 diabetes. A shotgun proteomics approach was applied to identify proteins differentially expressed between type 2 diabetic (TallyHo) and control (SWR/J) mice in the bladder smooth muscle and urothelium, separately. We were able to identify 1760 nonredundant proteins from the detrusor smooth muscle and 3169 nonredundant proteins from urothelium. Pathway and network analysis of significantly dysregulated proteins was conducted to investigate the molecular processes associated with diabetes. This pinpointed ERK1/2 signaling as a key regulatory node in the diabetes-induced pathophysiology for both tissue types. The detrusor muscle samples showed diabetes-induced increased tissue remodeling-type events such as Actin Cytoskeleton Signaling and Signaling by Rho Family GTPases. The diabetic urothelium samples exhibited oxidative stress responses, as seen in the suppression of protein expression for key players in the NRF2-Mediated Oxidative Stress Response pathway. These results suggest that diabetes induced elevated inflammatory responses, oxidative stress, and tissue remodeling are involved in the development of tissue specific diabetic bladder dysfunctions. Validation of signaling dysregulation as a function of diabetes was performed using Western blotting. These data illustrated changes in ERK1/2 phosphorylation as a function of diabetes, with significant decreases in diabetes-associated phosphorylation in urothelium, but the opposite effect in detrusor muscle. These data highlight the importance of understanding tissue specific effects of disease process in understanding pathophysiology in complex disease and pave the way for future studies to better understand important molecular targets in reversing bladder dysfunction.

Stem cell homing factor, CCL7, expression in mouse models of stress urinary incontinence.

OBJECTIVES: Animal models of vaginal distention (VD) have demonstrated increased expression of chemokine (C-C motif) ligand 7 (CCL7) In this study, we investigated the expression of CCL7 in mice models of simulated birth trauma-induced urinary incontinence using VD and pudendal nerve transection (PNT). METHODS: Forty-nine mice were divided into 6 groups: VD, sham VD, PNT, sham PNT, anesthesia, and age-matched controls. The urethra, vagina, and rectum were harvested for the expression of CCL7 immediately or 24 hours after assigned procedure. Venous sampling for quantification of serum CCL7 was also performed. An analysis of variance model was used to compare the relative expression of CCL7 in each group. RESULTS: Urethral CCL7 expression in the VD group was significantly higher than control group after 24 hours (P < 0.01). There was no difference in the urethral CCL7 expression in PNT, sham PNT, sham VD, or anesthesia groups compared with the controls. No statistically significant difference was noted in the vaginal and rectal expression of CCL7 between any of the groups except for sham PNT. Statistically significant differences were noted in the serum CCL7 expression in the VD, PNT, and sham PNT (P < 0.01 in all) groups after 24 hours compared with the control group. CONCLUSIONS: This study demonstrates overexpression of urethral CCL7 after VD but not PNT. This suggests that nerve injury does not contribute to the CCL7 overexpression. The overexpression of CCL7 in the serum of mice after VD suggests a translational potential where CCL7 measurement could be used as a surrogate for injury after delivery.

Expression of monocyte chemotactic protein 3 following simulated birth trauma in a murine model of obesity.

OBJECTIVE: To determine the effect of obesity on simulated birth trauma in leptin-deficient obese mice as measured by relative monocyte chemotactic protein 3 (MCP-3) expression. MATERIALS AND METHODS: A total of 25 wild-type and 25 obese C57BL/6 virgin female mice underwent 1 hour of vaginal distension (VD), sham VD, or anesthesia without VD. Pelvic organ tissues were then harvested either immediately or 24-hours post VD and subsequent real-time polymerase chain reaction analysis was performed. RESULTS: Urethral MCP-3 levels in wild-type mice were elevated from baseline at 0 hours with a return to baseline at 24 hours in both VD and sham VD groups. In obese mice, there was a 6-fold elevation in MCP-3 levels at 0 hours after sham VD vs control (P <.05), which then returned to baseline levels at 24 hours. After undergoing VD, MCP-3 levels increased to 6-fold baseline values (P = .002) at 0 hours, with continued elevation in MCP-3 levels to 15 times control levels (P = .0003) at 24 hours. CONCLUSIONS: MCP-3 is significantly over-expressed in the urethral tissues of both wild-type and obese mice immediately after any urethral manipulation. At 24 hours, the MCP-3 expression patterns become divergent between VD and sham VD in obese mice. With a greater degree of trauma, MCP-3 continued to rise at 24 hours, suggesting that the underlying obesity resulted in alterations in response to tissue injury, paralleling the degree of injury. Such associations warrant further investigation into the role of MCP-3 as a chemokine for stem cell migration, with implications for subsequent tissue repair mechanisms after birth trauma.

ITCH K63-ubiquitinates the NOD2 binding protein, RIP2, to influence inflammatory signaling pathways.

BACKGROUND: The inability to coordinate the signaling pathways that lead to proper cytokine responses characterizes the pathogenesis of inflammatory diseases such as Crohn's disease. The Crohn's disease susceptibility protein, NOD2, helps coordinate cytokine responses upon intracellular exposure to bacteria, and this signal coordination by NOD2 is accomplished, in part, through K63-linked polyubiquitin chains that create binding surfaces for the scaffolding of signaling complexes. RESULTS: In this work, we show that the NOD2 signaling partner, RIP2, is directly K63-polyubiquitinated by ITCH, an E3 ubiquitin ligase that when lost genetically causes widespread inflammatory disease at mucosal surfaces. We show that ITCH is responsible for RIP2 polyubiquitination in response to infection with listeria monocytogenes. We also show that NOD2 can bind polyubiquitinated RIP2 and that whereas ITCH E3 ligase activity is required for optimal NOD2:RIP2-induced p38 and JNK activation, ITCH inhibits NOD2:RIP2-induced nuclear factor kappa B (NFkappaB) activation. This effect can be seen independently at the whole-genome level by microarray analysis of muramyl dipeptide (MDP)-treated Itch(-/-) primary macrophages. CONCLUSIONS: These findings suggest that ITCH helps regulate NOD2-dependent signal transduction pathways and, as such, may be involved in the pathogenesis of NOD2-mediated inflammatory disease.