Chronic Mouse Model Research Articles

A comparative study of two chronic obstructive pulmonary disease mouse models established by different methods

Objective: To compare chronic obstructive pulmonary disease (COPD) mouse models established by two different methods-cigarette smoke (CS) exposure alone and CS exposure combined with airway instillation of bacterial LPS. Methods: Male C57 mice were randomly divided into control group(CTL group), CS exposure group (CS group) and intra-tracheal LPS instillation combined with CS exposure group (LPS+CS group) according to the random number table, with 8 rats in each group. After the models were established, we measured the lung function and collected the bronchoalveolar lavage fluid (BALF) to detect the number of inflammatory cells and the expression of mucin and inflammatory mediators. HE and PAS staining were performed to observe the pathological changes in airway and lung tissue and to detect the goblet cells in airway, respectively. Results: Total lung capacity (TLC), functional residual capacity (FRC) and airway resistance (RI) of the CS and LPS+CS groups were higher than those of the CTL group, while the FEV(50)/FVC of these 2 groups was lower (P<0.05). Moreover, both RI and FEV(50)/FVC in the LPS+CS group were higher compared with the CS group (P<0.05). HE staining of lung tissue showed that the average alveolar intercept and thickness of small airway wall in the CS and LPS+CS groups were higher compared to the CTL group. In addition, the average alveolar intercept in the LPS+CS group was lower than that in the CS group [(47.86±2.82) μm and (61.94±7.68) μm respectively, P<0.05], but the area of bronchial inflammation of LPS+CS group was higher. The number of total white blood cells, neutrophils, lymphocytes, macrophages and the level of interleukin-6 (IL-6) in BALF of CS and LPS+CS groups were higher than those of CTL group (all P<0.05). Furthermore, the number of neutrophils and IL-6 level in BALF of LPS+CS group were higher in comparison with CS group, while the number of macrophages in BALF of LPS+CS group was lower (P<0.05). PAS staining of lung tissue indicated that the number of goblet cells in large airways of CS and LPS+CS groups increased more significantly compared to the CTL group, and the number of goblet cells in the LPS+CS group was higher than that in the CS group [(0.16±0.02) and (0.09±0.02) respectively, P<0.05]. The expression levels of Muc5ac and Muc5b in BALF of LPS+CS and CS groups were also higher than those of CTL group (P<0.05), and the level of Muc5ac in BALF of LPS+CS group was higher compared with CS group[(2.69±0.72) and (2.19±0.29) respectively, P<0.05]. Conclusions: Combined exposure of LPS and CS for establishing a COPD mouse model could better simulate the pathological characteristics of human COPD during the acute exacerbation period. The COPD mouse model established by CS exposure alone was able to better imitate the basic features of human COPD in the stable period. Researchers could choose a more appropriate modeling method according to different purposes.

Dermal exposure determines the outcome of repeated airway exposure in a long-term chemical-induced asthma-like mouse model

BackgroundExposure to diisocyanates is an important cause of occupational asthma (OA) in the industrialized world. Since OA occurs after long-term exposure to diisocyanates, we developed a chronic mouse model of chemical-induced asthma where toluene diisocyanate (TDI) was administered at two different exposure sites. ObjectivesEvaluating the effect of long-term respiratory isocyanate exposure - with or without prior dermal exposure- on sensitization, inflammatory responses and airway hyperreactivity (AHR). MethodsOn days 1 and 8, BALB/c mice were dermally treated (20 μl/ear) with 0.5% 2,4-toluene diisocyanate TDI or the vehicle acetone olive oil (AOO) (3:2). Starting from day 15, mice received intranasal instillations with 0.1% TDI of vehicle five times in a week, for five successive weeks. One day after the last instillation airway hyperreactivity (AHR) to methacholine was assessed, followed by an evaluation of pulmonary inflammation and structural lung changes. Immune-related parameters were assessed in the lungs (BAL and tissue), blood, cervical- and auricular lymph nodes. ResultsMice repeatedly intranasally exposed to TDI showed systemic sensitization and a mixed Th1/Th2 type immune response, without the presence of AHR. However, when mice are first dermally sensitized with TDI, followed by repeated intranasal TDI challenges, this results in a pronounced Th2 response and AHR. ConclusionDermal exposure to TDI determines airway hyperreactivity after repeated airway exposure to TDI.

Tu1765 – Histone 3 Lysine 27 Acetylation Profiling Reveals Its Role in the Chronic Dss-Induced Colitis Mouse Model

Tu1765 – Histone 3 Lysine 27 Acetylation Profiling Reveals Its Role in the Chronic Dss-Induced Colitis Mouse Model

697 – Intestinal Permeability is an Important Factor in the Development of Alcoholic Liver Disease in a Chronic and Binge Mouse Model of Alcoholic Steatohepatitis

697 – Intestinal Permeability is an Important Factor in the Development of Alcoholic Liver Disease in a Chronic and Binge Mouse Model of Alcoholic Steatohepatitis

The effects and mechanisms of myeloid differentiation protein 2 on intestinal mucosal permeability in mice with chronic colitis.

The present study was designed to investigate the mechanism of myeloid differentiation protein 2 (MD2) on intestinal mucosa destruction in mice with chronic colitis. Briefly, a chronic colitis mouse model was established by the administration of dextran sulfate sodium (DSS) in transgenic mice of MD2 overexpression (Transgenic, MD2-Tg) and C57BL/6 wild-type mice (MD2-WT). In addition, Caco-2 cells were cultured to form a monolayer cell model in vitro. The small interfering RNA was utilized to silence the MD2 gene in Caco-2 cells, and tumor necrosis factor-α (TNF-α) was used to establish the model of intestinal mucosal inflammation. After DSS induction, the intestinal mucosal tissue inflammation was more severe in MD2-Tg mice than MD2-WT. In addition, the intestinal mucosa was severely damaged, the intestinal mucosal permeability was increased, bacterial translocation was obvious, and the expression levels of MD2, MyD88, Toll-like receptor 4 (TLR4), and HMGB1 in mucosal tissues were significantly increased, while the expression levels of tight junction proteins, occludin, and claudin-1 were significantly lower in MD2-Tg mice compared with those in MD2-WT mice. TNF-α could induce inflammatory apoptosis in Caco-2 cell models. After MD2 silencing, the apoptotic level was decreased, the value of transepithelial electrical resistance was increased, the permeability of intestinal mucosa was decreased, the cellular expression levels of MD2, MyD88, TLR4, and HMGB1 were decreased, while the expression levels of tight junction proteins, occludin and claudin-1 were increased. MD2 could aggravate the destruction of intestinal mucosa in chronic colitis through the HMGB1-TLR4-MyD88 pathway.

Chronic stress augments esophageal inflammation, and alters the expression of transient receptor potential vanilloid1 and protease‑activated receptor 2 in a murine model.

Stress is a pivotal factor for inflammation, reactive oxygen species (ROS) production and formation of visceral hypersensitivity (VH) in the process of gastroesophageal reflux disease (GERD). In the present study, the effects of stress on esophageal inflammation, oxidative stress and VH were investigated in a chronic restraint stress mouse model. C57BL/6J male mice were subjected to 2weeks of intermittent restraint stress, and histopathological analysis revealed that stress induced esophageal inflammation and fibrosis, while no distinct changes were detected in non‑stressed control mice. In addition, increased NADPH oxidase 4 expression was observed in the plasma and esophagus of stressed mice, indicating accumulation of ROS. The expression levels of antioxidants, including Mn‑superoxide dismutase (MnSOD), Cu/Zn‑SOD, catalase and glutathione peroxidase, were also analyzed using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). In addition, transient receptor potential vanilloid1 (TRPV‑1) and protease‑activated receptor2 (PAR‑2), which are crucial receptors for VH, were measured by immunohistochemistry and RT‑qPCR. The results demonstrated that stress markedly reduced antioxidant expression, while it significantly upregulated TRPV‑1 and PAR‑2 expression levels in the mouse esophagus. Finally, 2weeks of restraint stress significantly increased the esophageal and plasma levels of inflammatory cytokines, including interleukin (IL)‑6, IL‑8, interferon‑γ and tumor necrosis factor‑α. Taken together, the present study results indicated that stress‑induced esophageal inflammation and ROS generation involves VH.

A suite of phenotypic assays to ensure pipeline diversity when prioritizing drug-like Cryptosporidium growth inhibitors

Cryptosporidiosis is a leading cause of life-threatening diarrhea in children, and the only currently approved drug is ineffective in malnourished children and immunocompromised people. Large-scale phenotypic screens are ongoing to identify anticryptosporidial compounds, but optimal approaches to prioritize inhibitors and establish a mechanistically diverse drug development pipeline are unknown. Here, we present a panel of medium-throughput mode of action assays that enable testing of compounds in several stages of the Cryptosporidium life cycle. Phenotypic profiles are given for thirty-nine anticryptosporidials. Using a clustering algorithm, the compounds sort by phenotypic profile into distinct groups of inhibitors that are either chemical analogs (i.e. same molecular mechanism of action (MMOA)) or known to have similar MMOA. Furthermore, compounds belonging to multiple phenotypic clusters are efficacious in a chronic mouse model of cryptosporidiosis. This suite of phenotypic assays should ensure a drug development pipeline with diverse MMOA without the need to identify underlying mechanisms.

SUN-LB082 Sexual Dimorphism of Phosphoethanolamine in HPA-Axis Response to LXR Agonist 25-Hydroxycholesterol: Neuroendocrine Implications for Major Depressive Disorder

Major depression is a chronic illness and a major component of disease burden, with high prevalence in women than in men. Plasma phosphoethanolamine (PEA) levels are significantly decreased in major depressive disorder (MDD) patients compared to healthy controls. Additionally, in the chronic ACTH treatment MDD mouse model, plasma and frontal cortex PEA were significantly decreased compared to vehicle treated controls. However, the reasons behind MDD prevalence in females, the neuroendocrine mechanisms by which PEA changes in vivo, and its role in the pathobiology of MDD is not yet understood. In cells, LXR agonist 25-hydroxycholesterol increased PEA levels: To understand the sexually dimorphic PEA function in the neuroendocrine circuitry of MDD, basal in vivo central and peripheral PEA levels, as well as post 25-hydroxylcholesterol treatment PEA levels in blood plasma, adrenal gland, and brain nuclei implicated in MDD were studied in mice. Both targeted and untargeted metabolomics analysis using capillary electrophoresis-mass spectrometry were applied to measure PEA and various metabolite levels in the respective tissues. Here we report that compared to vehicle control females, vehicle treated male mice had about 50% higher basal plasma PEA levels than females. In the brain areas implicated in MDD, male mice displayed significantly increased basal PEA levels compared to female basal levels by about 3 fold. Specifically, basal PEA levels in males were higher than females in the frontal cortex, striatum, hippocampus, hypothalamus, and the amygdala. Moreover, while acute LXR agonist, 25-hydroxycholesterol decreased plasma PEA levels, chronic LXR agonism significantly increased plasma PEA levels in both male and female mice. Hypothalamic PEA decreased by 20% in females and 80% in males after LXR agonist treatment. Moreover, basal adrenal PEA was 45% significantly higher in females than in males; and 25-hydroxycholesterol significantly increased female adrenal PEA by 35% over basal levels, with no change in males. Metabolomics analysis further revealed significant changes in levels of several metabolites in the plasma, brains, and adrenals of 25-hydroxycholesterol treated mice in a sexually dimorphic manner. These findings underscore the importance of the PEA-LXR system in explaining the sexual dimorphic differential coping strategy of the two sexes to stressful stimuli; and may serve as a novel potential diagnostic and therapeutic target for major depressive disorder. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Neuroprotective effects of lithium on a chronic MPTP mouse model of Parkinson's disease via regulation of α‑synuclein methylation.

The pathological process of Parkinson's disease (PD) is closely associated with the death of nigral neurons, for which an effective treatment has yet to be found. Lithium, one of the most widely certified anticonvulsant and mood‑stabilizing agents, exhibits evident neuroprotective effects in the treatment of epilepsy and bipolar disorder. In the present study, the neuroprotective mechanisms by which lithium acts on a chronic 1‑methyl‑4‑phenyl‑1,2,3,6‑tetrahydropyridine (MPTP) mouse model of PD were investigated by employing animal behavioral tests, immunohistochemistry, RT‑PCR, and western blotting. The results revealed that, in open field tests, lithium treatment counteracted the reduction in movement distance as well as activity time induced by MPTP administration. The compound could also prolong the drop time of MPTP‑treated mice in rotarod tests. Moreover, lithium treatment corrected the loss of nigral neurons, the increase of α‑synuclein (SNCA) in substantia nigra as well as in the striatum of MPTP‑treated mice, and decreased the methylation of SNCA intron 1 in DNA from the same regions. Furthermore, marked changes were observed in the expression of miRNAs including miR‑148a, a potential inhibitor of DNMT1, in the MPTP‑treated mice. These results suggested that the early application of lithium was important for alleviating the behavioral deficits experienced in the PD model, and that the neuroprotective action of lithium was achieved through a lithium‑triggered miRNA regulation mechanism. Essentially, our findings indicated that lithium may be beneficial in the prevention and treatment of PD through the regulation of α‑synuclein methylation.

The antifibrotic and anti-inflammatory effects of icariin on the kidney in a unilateral ureteral obstruction mouse model

BackgroundThe pathology change of renal tubulointerstitial fibrosis is a critical feature of chronic kidney disease (CKD), regardless of the primary insults. The infiltration of inflammatory cells and the consecutive secretion of profibrotic factors are frequently and conspicuously observed during the development of renal fibrosis. Icariin, an active polyphenol of the Epimedium genus, has been found to alleviate the symptoms of chronic diseases like diabetes, neurodegeneration, and heart and renal diseases. The effect and mechanism of icariin on the prevention of CKD-associated renal fibrosis still needed clarification. PurposeThe aims of this study were to investigate whether icariin treatment improves the development of CKD-associated renal fibrosis and its possible mechanism. MethodsAn experimental unilateral ureteral obstruction (UUO)-induced chronic renal fibrosis mouse model was used. Mice were orally administered with icariin (20 mg/kg/day) for 3 consecutive days before and 14 consecutive days after UUO surgery. ResultsThe pathological changes, collagen deposition, and protein expressions of profibrotic factors (transforming growth factor-β and connective tissue growth factor) and fibrotic markers (α-smooth muscle actin and fibronectin), which were significantly elevated in the kidneys of UUO mice, could be significantly reversed by icariin treatment. Icariin treatment also significantly inhibited the increased Smad2/3 and decreased E-cadherin protein expressions in the kidneys of UUO mice. Icariin treatment prominently mitigated the protein expression of proinflammatory factors like nuclear factor-κB, cyclooxygenase-2, interleukin 1-β and prooxidative enzyme (NADPH oxidase-4), and it increased the protein expression of antioxidative enzymes (superoxide dismutase and catalase). ConclusionIcariin treatment protects against CKD-associated renal fibrosis via its antifibrotic and anti-inflammatory properties. Icariin may serve as a therapeutic agent in the prevention of CKD-associated renal fibrosis.

Neuroplasticity and Neuroprotective Effect of Treadmill Training in the Chronic Mouse Model of Parkinson's Disease.

Physical training confers protection to dopaminergic neurons in rodent models of parkinsonism produced by neurotoxins. The sparing effect of physical training on dopaminergic neurons can be tested with training applied during chronic MPTP treatment, while the neurorestorative effect when training is applied after completing such treatment. In this study, the effect of the onset of training respective to chronic MPTP treatment was specifically addressed. Three groups of mice were injected with 10 doses of MPTP (12.5 mg/kg/injection) over 5 weeks. The first group remained sedentary; the second one underwent early onset training, which started 1 week before commencing MPTP treatment, continued throughout 5 weeks of treatment and 4 weeks thereafter; the third group underwent late-onset training of the same length and intensity as the former group, except that it started immediately after the end of MPTP treatment. Two groups served as controls: a saline-injected group that remained sedentary and saline-injected group, which underwent the same training as the early and late-onset training groups. Both early and late-onset physical training saved almost all nigral and VTA dopaminergic neurons, prevented inflammatory response, and increased the BDNF and GDNF levels to a similar extent. From these results one may conclude that early and late-onset training schedules were equipotent in their neuroprotective effect and that the mechanism of neuroprotection was similar. The sparing effect of early onset training may be satisfactorily explained by assuming that the increased level of BDNF and GDNF prevented the degeneration of dopaminergic neurons. To explain a similar number of dopaminergic neurons detected at the end of the early and late-onset training, one should additionally assume that the former training schedule induced neurogenesis. Results of this study support the view that physical activity may be neuroprotective even at a more advanced stage of PD and justify starting physical activity at any point of the disease.

Role of Neuro‐immune Interactions in a Cigarette Smoke‐induced Chronic Airway Inflammation Mouse Model

Chronic obstructive pulmonary disease characterized by progressive persistent airway inflammation &amp; airway resistance is caused mainly by cigarette smoke‐triggered inflammatory cascades &amp; tissue damage. The airways are densely innervated by capsaicin‐sensitive sensory nerves expressing Transient Receptor Potential Vanilloid 1 (TRPV1) ion channels, which play an important regulatory role in inflammation through the release of sensory neuropeptides. Pro‐inflammatory peptides, such as tachykinins (substance P, neurokinin A) encoded by the TAC1 gene induce neurogenic inflammation via NK1 &amp; NK2 receptor activation in the innervated area. The overall significance of these mediators depends on the pathophysiological mechanisms involved in certain inflammatory processes. Therefore, we established a predictive mouse model of cigarette smoke‐induced chronic bronchitis &amp; consequent airway hyperresponsiveness to investigate the roles of TRPV1 channel &amp; tachikinins.We exposed TRPV1−/− &amp; TAC1−/− gene‐deleted mice and their C57Bl/6 wildtype counterparts to cigarette smoke in a whole body smoke exposure chamber for 3 months. Bronchial responsiveness &amp; enhanced pause (Penh) correlating with airway resistance to inhalation of 22 mM carbachol were measured with unrestrained whole body plethysmography weekly. At the end of each month inflammatory cell profile and count from the bronchoalveolar lavage fluid (BALF), semiquantitative histopathological scoring of the lung, myeloperoxidase (MPO) activity with spectrophotometry &amp; IL‐1β concentrations with ELISA were performed.In wildtype mice mild bronchial hyperresponsiveness developed in the 1st month, then it decreased &amp; started to increase after 10 weeks. The inflammatory cell peak in BALF was observed in the 2nd month, correlating with the histology. In the 1st month significant perivascular/peribronchial edema, in the 2nd month remarkable inflammatory cell accumulation, then increasing atelectasy, emphysema, mucus production &amp; irregular bronchiolar mucosa were observed. Lung IL‐1β concentration gradually increased, but MPO activity only transiently elevated in the 2nd month. Hyperreactivity occured earlier &amp; increased significantly in TRPV1−/−, but did not change in TAC1−/− mice. Remarkably more severe inflammatory histopathological parameters, higher BALF cell counts, MPO activity a&amp; IL‐1β production were observed in TRPV1‐deficient mice. Meanwhile, some inflammatory alterations in the 1st month, &amp; BALF cell counts in the 2nd month were significantly decreased in the TAC1 gene‐deleted mice.Neurogenic inflammatory mechanisms are not crucial in chronic cigarette smoke‐induced inflammation &amp; hyperresponsiveness. The overall role of TRPV1 receptor is protective in smoking‐induced chronic bronchitis.Support or Funding InformationÚNKP‐18‐3 New National Excellence Program of the Ministry of Human CapacitiesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FRI-465-The INCRNA H19 is an oncogenic driver of HCC in chronic inflammation-mediated mouse model

FRI-465-The INCRNA H19 is an oncogenic driver of HCC in chronic inflammation-mediated mouse model

Effect of Lactobacillus rhamnosus GG immunopathologic changes in chronic mouse asthma model.

Asthma is a heterogeneous inflammatory disorder of the airway. A Th2 response usually contributes to high levels of allergen-specific IgE and eosinophilic airway inflammation. Several findings have demonstrated that neutrophils, not eosinophils, are the major inflammatory cells in chronic asthma patients with steroid-resistance. Lactobacillus rhammosus GG (LGG) exhibits anti-inflammatory properties on OVA-induced acute airway inflammation. We hypothesized that orally administrated LGG should reduce airway remodeling in chronic experimental models. Female Balb/c mice were sensitized with OVA. LGG was used to investigate whether oral administrations of LGG inhibited OVA-induced airway inflammation in a chronic asthma model and the different intervention times between LGG pre-treatment and post-treatment groups. BALF was analyzed with Liu's stain and ELISA assay. Lung histopathology was assayed with HE, IHC and Masson's trichrome staining. Lung tissues were assayed with PCR (T-bet, GATA3, RORrt and Foxp3). Many cytokines were detected in the serum and BALF. LGG significantly decreased the number of infiltrating inflammatory cells. We also found that the oral LGG group suppressed not only Th2 cytokine, but also IL-17, TNF-α and HMGB1 in the BALF levels. However, GATA3 and RORrt decreased significantly in the RNA level in the LGG groups, but the T-bet and Foxp3 increased in the RNA level. LGG not only had anti-inflammatory effects on OVA-induced airway inflammation, but also improved airway remodeling and collagen expression in the chronic asthma mouse model. Moreover, LGG might be an additional or supplementary therapy for allergic airway diseases.

Poloxamer 188 rescues MPTP-induced lysosomal membrane integrity impairment in cellular and mouse models of Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Rupture of lysosome is a major cellular stress condition leading to cell death in PD. We have previously shown that environmental oxidative toxins could impair autophagic flux and lysosomal functions in PD. Poloxamer 188 (P188) is an amphipathic polymer which has cytoprotective effect in traumatic brain injury and stroke. But whether Dyrk1A could rescue lysosome malfunction-mediated DA neuron death and α-synuclein aggregation in PD is still unknown. In the present study, MPTP mice models and MPP+-treated SH-SY5Y cells were used for study, and we found that P188 rescued MPP+-induced lysosomal dysfunction and impaired autophagy flux in mild MPP+-treated SH-SY5Y cells. P188 administration significantly restored lysosomal membrane integrity and prevented cathepsins leakage from the lysosomes into the cytoplasm, which triggered caspase-dependent apoptotic cell death in sub-acute MPTP mouse model and MPP+-treated SH-SY5Y cells. Furthermore, P188 ameliorated α-synuclein accumulation and behavioral impairment in chronic MPTP mouse model with MPTP and probenecid treatment. P188 could alleviate MPTP-induced DA neurons damage by restoring lysosome function.

Triheptanoin alters [U-13C6]-glucose incorporation into glycolytic intermediates and increases TCA cycling by normalizing the activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase in a chronic epilepsy mouse model

Triheptanoin is anticonvulsant in several seizure models. Here, we investigated changes in glucose metabolism by triheptanoin interictally in the chronic stage of the pilocarpine mouse epilepsy model. After injection of [U-13C6]-glucose (i.p.), enrichments of 13C in intermediates of glycolysis and the tricarboxylic acid (TCA) cycle were quantified in hippocampal extracts and maximal activities of enzymes in each pathway were measured. The enrichment of 13C glucose in plasma was similar across all groups. Despite this, we observed reductions in incorporation of 13C in several glycolytic intermediates compared to control mice suggesting glucose utilization may be impaired and/or glycogenolysis increased in the untreated interictal hippocampus. Triheptanoin prevented the interictal reductions of 13C incorporation in most glycolytic intermediates, suggesting it increased glucose utilization or – as an additional astrocytic fuel – it decreased glycogen breakdown. In the TCA cycle metabolites, the incorporation of 13C was reduced in the interictal state. Triheptanoin restored the correlation between 13C enrichments of pyruvate relative to most of the TCA cycle intermediates in “epileptic” mice. Triheptanoin also prevented the reductions of hippocampal pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities. Decreased glycogen breakdown and increased glucose utilization and metabolism via the TCA cycle in epileptogenic brain areas may contribute to triheptanoin's anticonvulsant effects.

Adrenergic modulation of AMPK‑dependent autophagy by chronic stress enhances cell proliferation and survival in gastric cancer.

Epidemiological data show that chronic stress has adverse effects on the incidence and progression of cancer. As a critical target organ for stress hormones, the stomach is frequently subjected to stress-related injury. However, few reports regarding the association between stress and gastric cancer (GC) have been published. The present study aimed to investigate the effect of chronic stress on the growth and survival of GC, and the role of the autophagy process. A restraint-stress procedure over 21 days was used to establish a chronic stress mouse model. Subcutaneous xenografts and gastric orthotopic xenografts were established in BALB/c nude mice. Alzet osmotic minipumps containing either PBS or propranolol hydrochloride was inserted on the nape of the neck 7 days prior to the initiation of restraint stress. The presence of autophagosomes and autolysosomes were examined by electron microscopy. The stress hormone norepinephrine significantly enhanced the proliferation of GC cells. By inhibiting adrenoreceptor expression, it was demonstrated that β2-adrenergic receptor (ADRB2) was the specific β-adrenergic receptor subtype responsible for catecholamine release. In addition, it was demonstrated that the induction of autophagy was a novel consequence of β2-adrenergic activation in GC cells. This was demonstrated by the appearance of double-membrane vesicles, punctuate GFP-RFP-microtubule-associated protein 1 light chain 3 distribution in the cytoplasm and a corresponding increase in autophagic flux. Notably, norepinephrine-induced autophagy was shown to have a tumor-promoting role under conditions of chronic stress in vitro and in vivo. It was further demonstrated that, upon activation of cAMP-response element binding protein, chronic stress promoted autophagic flux through the adenosine 5′-monophosphate-activated protein kinase-unc-51 like autophagy activating kinase 1 (AMPK-ULK1) pathway. Tissue microarray analysis revealed a negative correlation between the expression of ADRB2 and autophagic marker p62/sequestosome-1 in GC tumor samples. Additionally, high protein levels of ADRB2 correlated positively with tumor, node, metastasis stage and poor prognosis in patients with GC. These results establish a novel pathway that chronic stress activates tumor-promoting autophagy to accelerate the progression of GC. The present study is the first, to the best of our knowledge, providing preclinical evidence that chronic stress serves a role in the progression of GC.

Injured liver-released miRNA-122 elicits acute pulmonary inflammation via activating alveolar macrophage TLR7 signaling pathway

Hepatic injury is often accompanied by pulmonary inflammation and tissue damage, but the underlying mechanism is not fully elucidated. Here we identify hepatic miR-122 as a mediator of pulmonary inflammation induced by various liver injuries. Analyses of acute and chronic liver injury mouse models confirm that liver dysfunction can cause pulmonary inflammation and tissue damage. Injured livers release large amounts of miR-122 in an exosome-independent manner into the circulation compared with normal livers. Circulating miR-122 is then preferentially transported to mouse lungs and taken up by alveolar macrophages, in which it binds Toll-like receptor 7 (TLR7) and activates inflammatory responses. Depleting miR-122 in mouse liver or plasma largely abolishes liver injury-induced pulmonary inflammation and tissue damage. Furthermore, alveolar macrophage activation by miR-122 is blocked by mutating the TLR7-binding GU-rich sequence on miR-122 or knocking out macrophage TLR7. Our findings reveal a causative role of hepatic miR-122 in liver injury-induced pulmonary dysfunction.

Augmented improvement of cognition and memory by aripiprazole add-on for cilostazol treatment in the chronic cerebral hypoperfusion mouse model

Cerebrovascular dysfunction is associated with cognitive impairment in vascular dementia patients. This study aimed to explore augmented improvement of cognition and memory by aripiprazole add-on for cilostazol treatment in vascular dementia model. Male C57BL/6 mice were subjected to BCAS, and spatial probe and memory retention were examined using the Morris water maze (MWM) test. In the present study, the escape latency on the first day after 3rd week was 21.4 ± 4.0 s in sham-operated mice, and 76.3 ± 4.2 s in the vehicle-treated BCAS mice. In the spatial probe tests in the 3rd week, aripiprazole (1 mg/kg/day) showed time-dependently amelioration in spatial learning and memory impairments in contrast to 0.5 mg/kg/day. After treatment with 20 mg/kg/day of cilostazol for 3 weeks, the escape latency significantly decreased to 26.6 ± 5.8 s on the first day and further shortened to 21.6 ± 6.8 s on the fourth day. When the BCAS mice were concurrently treated with 0.5 mg/kg/day aripiprazole plus 20 mg/kg/day of cilostazol for 3 weeks, the escape latency was more shortened from 20.4 ± 1.2 s (1st day) to 14.9 ± 1.7 s on the 4th day of the 3-week trials. Furthermore, decreased spatial memory retention in BCAS mice was significantly alleviated by aripiprazole plus cilostazol cotreatment, indicating the benefit of aripiprazole add-on therapy. In line with these, significantly increased mBDNF and P-CREB levels and reduced apoptosis were identified in the BCAS mouse brain dentate gyrus by cotreatment as contrasted to each monotherapy. These results may provide the synergistic therapeutic avenues for augmented improvement of cognition and memory by cotreatment with aripiprazole plus cilostazol in cases of vascular dementia.

3375 Chronic inflammation promotes intestinal macrophages to become modulators of the Notch pathway

OBJECTIVES/SPECIFIC AIMS: The purpose of this research was to investigate how chronic inflammation promotes the generation of proinflammatory intestinal macrophages and if macrophages contribute to intestinal inflammation through Notch activation. METHODS/STUDY POPULATION: We utilized two animal models of chronic colitis, the chronic DSS-induced colitis mouse model and the spontaneous enterocolitis development in IL-10-deficient mice to investigate the role of chronic inflammation in the generation of proinflammatory intestinal macrophages and its influence in notch signaling. Bone marrow-derived monocytes were collected from each group and differentiated into macrophages (BMM) for gene and protein analysis. Ex vivo phenotypical and functional analysis of colonic macrophages was assessed as was the presence of goblet cells and mucosal T cells. In addition, we analyzed the development of goblet cell differentiation in colonoids in a co-culture system with proinflammatory macrophages. RESULTS/ANTICIPATED RESULTS: Our chronic inflammation models revealed an increase in proinflammatory macrophages present in the lamina propria and that these cells expressed significantly higher levels of notch ligand, Jagged1. Jagged 1 has been shown to enhance TH1 differentiation and T cells isolated from the mucosa of both chronic colitis models display strong TH1 skewing compared to controls. Chronic inflammation also contributes to intestinal barrier defects, enhanced permeability and bacterial translocation. We believe this enhanced intestinal permeability and subsequent bacterial translocation promote Jagged1 expression in intestinal macrophages. To support this concept, we show TLR stimulation induces the upregulation of Jagged1 in BMM. Additionally, the generation of BMM from our chronic DSS-induced colitis mice or age matched controls, revealed BMM derived from a host of chronic inflammation were skewed to a proinflammatory state prior to stimulation showing increased gene expression of several proinflammatory molecules including IL-1α, IL-1β, IL-12 and TNF-α. This would suggest monocytes migrating to the intestinal mucosa have more potential to become proinflammatory instead of traditional anti-inflammatory macrophages. Furthermore, proinflammatory notch ligand-positive macrophages co-cultured with colonoids, derived from unperturbed mice, significantly decreased the number of mucus producing goblet cells. In support of this observation, notch activation in intestinal stem cells promote absorptive (i.e. colonocytes) cell differentiation and prevents secretory cell (i.e. goblet cells) differentiation. DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, our results strongly suggest chronic inflammation modulates macrophages role in maintaining intestinal homeostasis through possible notch activation in both T cells and the intestinal epithelial barrier.