Evaluation of circulating miR-216a and miR-217 as biomarkers of pancreatic damage in the L-arginine-induced acute pancreatitis mouse model.

We investigated the usefulness of circulating miR-216a-5p and miR-217-5p that are pancreas-enriched micro RNAs (miRNAs) as biomarkers of acute pancreatic damage, and compared them with conventional pancreatic biomarkers in L-arginine-induced acute pancreatitis mouse model. As the results, amylase and lipase levels apparently increased and peaked on Day 3 when acute pancreatitis including acinar cell degeneration/necrosis and inflammatory cell infiltration reached its peak. In contrast, miR-216a-5p and miR-217-5p increased from Day 1 when histopathological findings in the acinar cells were limited to decreased zymogen granules, and the increases in ratios were much higher than those of amylase and lipase. The miRNAs remained at high levels until Day 5 when the pseudo-tubular complex and replacement of inflammatory cells and fibrotic cells were apparent instead of necrosis, whereas amylase and lipase levels decreased to the control levels. Furthermore, we examined the relationship between biomarker levels and histopathological degeneration/necrosis scores in the acinar cells. miR-216a-5p and miR-217-5p levels increased depending on the score of degeneration/necrosis, and all individual miRNAs exceeded the control levels from a score of 2 (focal necrosis), whereas all individual amylase and lipase levels exceeded the control levels at scores of 4 (lobular necrosis) and 3 (sublobular necrosis), respectively. In conclusion, we demonstrated that circulating miR-216a-5p and miR-217-5p could detect pancreatic damage earlier with greater magnitude, and the sensitivity to detect acinar cell degeneration/necrosis was superior to that of conventional biomarkers in the L-arginine-induced acute pancreatitis mouse model.

A 90-day toxicity study of tripeptide, arginine-alanine-lysine.

There is a growing global interest in using peptides in the health industry for pharmaceuticals, cosmetics, and natural food products. Peptides contain two or more linked amino acids, whereas more than 50 amino acids are classified as polypeptides. Although there is a growing level of interest in the use of peptides in the health and wellness industry, there is a lack of literature pertaining to a specific tripeptide derived from arginine, alanine, and lysine (RAK) that is of interest for human dietary use. Therefore, a 90-day repeated-dose toxicity study was performed in rats to evaluate the subchronic oral toxicity of RAK. Eighty Han:WIST rats were administered RAK by gavage at doses of 0, 250, 500, or 1000 mg/kg bw/day. There were no mortalities or other treatment related effects, and no target organs were identified. A no-observed-adverse-effect-level (NOAEL) of 1000 mg/kg bw/day, the highest dose tested, was determined. This study will contribute to the body of research in regard to the safety of the use of RAK.

Chronic Exposure to Environmentally Relevant Concentrations of Microcystin-Leucine Arginine Causes Lung Barrier Damage through PP2A Activity Inhibition and Claudin1 Ubiquitination.

Microcystin-leucine arginine (MC-LR), ubiquitous in water and food, is a threat to public health. In the present study, after C57BL/6J mice were fed with environmental concentrations of MC-LR (0, 1, 30, 60, 90, and 120 µg/L) for 6, 9, and 12 months, it was found that MC-LR could enter into mouse lung tissues and cause microstructural damage, as shown by western blotting and HE staining. Electron microscopy examination showed that MC-LR could damage the lung barrier by disruption of the tight junctions, which was confirmed by the decreased expression of tight junction markers, including Occludin, Claudin1, and ZO-1. In addition, MC-LR also increased the ubiquitination of Claudin1, indicating that MC-LR could disrupt tight junctions by promoting the degradation of Claudin1. Furthermore, MC-LR increased the levels of TNF-α and IL-6 in mouse lung tissues, leading to pneumonia. Importantly, pretreatment with PP2A activator D-erythro-sphingosine (DES) was found to significantly alleviate MC-LR-induced decrease of Occludin and Claudin1 by inhibiting the P-AKT/Snail pathway in vitro. Together, this study revealed that chronic exposure to MC-LR causes lung barrier damage, which involves PP2A activity inhibition and enhancement of Claudin1 ubiquitination. This study broadens the awareness of the toxic effects of MC-LR on the respiratory system, which has deep implications for public health.

Male reproductive toxicity induced by Microcystin-leucine-arginine (MC-LR).

Microcystin-LR (MC-LR) has been identified to pose an increasing threat to the male reproductive system in vivo and in vitro studies with the objects like mammal animals, amphibians, aquatic organisms, etc. This review demonstrates the latest research advances of the male reproductive toxicity induced by MC-LR in detail, which mainly consists of two aspects, namely pathological injuries to testis and prostate, as well as the endocrine disruption. Apart from the direct toxicity to the male reproductive system, we also underline the transgenerational reproductive toxicity that prenatal exposure may pass on to male offspring. This review also demonstrates the interactive effects between MC-LR and other compounds, including synergistic effects with some toxicants and antagonistic effects with some medicine or chemical modification. In terms of the mechanisms of MC-LR-induced toxicity, we mainly focus on the epigenetic modification and non-coding RNAs (ncRNAs)-related mechanisms which have provided a new perspective.

Chlorogenic acid reduces inflammation in murine model of acute pancreatitis.

BACKGROUND: The pathogenesis of acute pancreatitis (AP) initiation and progression is still unknown, and effective treatment is limited to supportive care. Many phytochemicals have the potential to alleviate AP symptoms and may be a useful and effective supplement to standard AP treatment. The objective of the study was to examine the potential role of chlorogenic acid (CGA), a polyphenol known for anti-inflammatory effect, in the treatment of experimental AP in mice. METHODS: Two intraperitoneal (ip) injections of L-arginine (dosage 400 mg/100 g BW) were given 1 h apart to generate the AP murine model. Mice were separated into two experimental groups after 12 h from the first L-arginine injection: AP mice treated with CGA (oral gavage (po) every 12 h; 20 mg/kg BW) and non-treated AP mice (po vehicle, 5% dimethyl sulfoxide every 12 h). Every 12 h, control mice were given an equivalent volume of vehicle. At 72 h, mice were slaughtered. Histology, as well as myeloperoxidase (MPO) and amylase activity assays, were performed on pancreatic tissues. RESULTS: In murine mouse model of AP po administration of CGA decreased MPO vs. AP (40.40 ± 2.10 U vs. 7.39 ± 0.34; p < 0.001) as well as amylase activity vs. AP (1444 ± 56 mU/mL vs. 3340 ± 144 mU/mL, Fig. 2B; p < 0.001). When comparing CGA mice to AP mice, histological research demonstrated that the severity of AP was reduced following CGA treatment. CONCLUSIONS: The current study found that CGA might have anti-inflammatory effect on L-arginine-induced pancreatitis. Dietary intervention with CGA may be advised as a supportive treatment for AP, according to our findings.

Repeated exposure to microcystin-leucine-arginine potentiates excitotoxicity induced by a low dose of kainate.

Microcystin-leucine-arginine (MLCR) is a cyanobacterial toxin, and has been demonstrated to cause neurotoxicity. In addition, MCLR has been identified as an inhibitor of protein phosphatase (PP)1 and PP2A, which are known to regulate the phosphorylation of various molecules related to synaptic excitability. Thus, in the present study, we examined whether MCLR exposure affects seizures induced by a low dose of kainic acid (KA; 0.05 µg, i.c.v.) administration. KA-induced seizure occurrence and seizure score significantly increased after repeated exposure to MCLR (2.5 or 5.0 µg/kg, i.p., once a day for 10 days), but not after acute MCLR exposure (2.5 or 5.0 µg/kg, i.p., 2 h and 30 min prior to KA administration), and hippocampal neuronal loss was consistently facilitated by repeated exposure to MCLR. In addition, repeated MCLR significantly elevated the membrane expression of kainate receptor GluK2 subunits, p-pan-protein kinase C (PKC), and p-extracellular signal-related kinase (ERK) at 1 h after KA. However, KA-induced membrane expression of Ca2+/calmodulin-dependent kinase II (CaMKII) was significantly reduced by repeated MCLR exposure. Consistent with the enhanced seizures and neurodegeneration, MCLR exposure significantly potentiated KA-induced oxidative stress and microglial activation, which was accompanied by increased expression of p-ERK and p-PKCδ in the hippocampus. The combined results suggest that repeated MCLR exposure potentiates KA-induced excitotoxicity in the hippocampus by increasing membrane GluK2 expression and enhancing oxidative stress and neuroinflammation through the modulation of p-CaMKII, p-PKC, and p-ERK.

Targeting HMGB1/TLR4/NF-κB signaling pathway by protocatechuic acid protects against l-arginine induced acute pancreatitis and multiple organs injury in rats.

Acute pancreatitis (AP) is a common pancreatic inflammation associated with substantial morbidity and mortality. AP may be mild or severe which can spread systemically causing multiple organs failure (MOF) and even death. In the current study, protocatechuic acid (PCA), a natural phenolic acid, was investigated for its possible protective potential against L-arginine induced AP and multiple organs injury (MOI) in rats. AP was induced by L-arginine (500 mg/100 g, ip). Two dose levels of PCA were tested (50 and 100 mg/kg, oral, 10 days before L-arginine injection). PCA successfully protected against L-arginine induced AP and MOI that was manifested by normalizing pancreatic, hepatic, pulmonary, and renal tissue architecture and restoring the normal values of pancreatic enzymes (amylase and lipase), serum total protein, liver enzymes (alanine transaminase (ALT) and aspartate transaminase (AST)) and kidney function biomarkers (blood urea nitrogen (BUN) and serum creatinine (Cr)) that were significantly elevated upon L-arginine administration. Additionally, PCA restored balanced oxidant/antioxidants status that was disrupted by L-arginine and normalized pancreatic levels of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) content. Moreover, PCA significantly decreased L-arginine induced elevation in pancreatic high motility group box protein 1 (HMGB1), toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NF-κB), tumor necrosis factor- α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) expression. PCA significantly ameliorated L-arginine-induced AP and MOI through its anti-inflammatory and antioxidant effects. HMGB1/TLR4/NF-κB was the major pathway involved in the observed protective potential.

Maternal resveratrol therapy protected adult rat offspring against hypertension programmed by combined exposures to asymmetric dimethylarginine and trimethylamine-N-oxide.

Resveratrol, a phytochemical, has shown antioxidant properties and potential benefits in hypertension. Asymmetric dimethylarginine (ADMA)-related nitric oxide deficiency and gut microbiota-derived metabolite trimethylamine-N-oxide (TMAO) have been linked to hypertension. We aimed to test whether maternal resveratrol therapy would protect adult offspring against hypertension programmed by prenatal exposure to ADMA and TMAO. Pregnant Sprague-Dawley rats received ADMA 10 mg/kg/day (A), TMAO 0.65 mg/hr (T), ADMA+TMAO (AT), or vesicle (CV). One group of ADMA+TMAO-exposed rats received 50 mg/L of resveratrol in drinking water during pregnancy and lactation periods (ATR). Male offspring (n = 8/group) were assigned to five groups: CV, A, T, AT, and ATR. Rats were killed at 12 weeks of age. ADMA exposure caused the elevation of blood pressure in 12-week-old male offspring, which was exacerbated by TMAO exposure. Treatment with resveratrol rescued hypertension programmed by combined ADMA and TMAO exposure. This was accompanied by alterations in the compositions of gut microbiota and increased fecal butyrate levels. Both the abundance of the butyrate-producing genera Lachnospiraceae and Ruminococcaceae were augmented by resveratrol. Meanwhile, resveratrol therapy significantly increased the abundance of the Cyanobiaceae and Erysipelotrichaceae families. Moreover, the protective effects of resveratrol were related to the mediation of the renin-angiotensin system . Our data provide new insights into the protective mechanisms of resveratrol against hypertension programmed by ADMA and TMAO, including regulation of gut microbiota and their metabolites, the renin-angiotensin system, and nitric oxide pathway. Resveratrol might be a potential reprogramming strategy to protect against the hypertension of developmental origins.

Enhancement in corneal permeability of riboflavin using cyclodextrin derivates complexes as a previous step to transepithelial cross-linking.

Corneal cross-linking has been described as an effective treatment to slow the progression of keratoconus. The standard protocol entails corneal epithelial removal to allow the diffusion of riboflavin into the stroma. Although, de-epithelization can generate risks or complications that transepithelial cross-linking tries to solve or avoid. Different formulations were developed after verifying that hydroxypropyl-ß-cyclodextrin (HPßCD) and sulfobuthylether-ß-cyclodextrin (SBEßCD) in a 20% concentration, increased the solubility of practically insoluble in water drugs such as riboflavin from 0.12 mg/mL to 0.35 mg/mL and 0.29 mg/mL respectively. These values were higher when chitosan and arginine were added to the formulation, showing solubility of 0.78 mg/mL when HPßCD concentration was not modified. Ex vivo corneal permeability was measured after having kept in contact bovine corneas with intact epithelium for 5 h with the 0.1 mg/mL riboflavin solution, the formulations developed and a reproduced nanoemulsion from another work. Riboflavin's permeability was increased when cyclodextrins, chitosan, and arginine were part of the formulations, compared to the control drug solution. The best permeability coefficient was reached when riboflavin was combined with 40% (w/v) HPßCD, 0.5% (w/w) arginine, and 0.5% (w/w) chitosan. After having carried out toxicity studies as bovine corneal opacity and permeability (BCOP) and Hens Egg Test - Chorioallantoic Membrane Test (HET-CAM) it was verified that both, the active ingredients and the excipients of the different formulations were not harmful without generating irritation, loss of transparency or corneal permeability alterations. The results show the great potential of the ocular developed solution for their use in transepithelial cross-linking for keratoconus treatment.

Suppression of L-arginine-induced acute necrotizing pancreatitis in rats by metformin associated with the inhibition of myeloperoxidase and activation of interleukin-10 / Supresión de la pancreatitis necrotizante aguda inducida por L-arginina en ratas por metformina asociada con la inhibición de mieloperoxidasa y activación de interleucina-10

SUMMARY: Acute pancreatitis is a frequent life-threatening inflammatory disease of the pancreas characterized by severe abdominal pain that lasts for days to weeks. We sought to determine whether the antidiabetic and anti-inflammatory drug, metformin can substantially protect against acute pancreatitis in an animal model of L-arginine-induced acute pancreatitis, and whether this is associated with the augmentation of the anti-inflammatory cytokine interleukin-10 (IL-10) and inhibition of the enzyme that promotes tissue damage, myeloperoxidase (MPO). Rats were either injected with two doses of the amino acid L-arginine (2.5 gm/kg; i.p., at one-hour intervals) before being sacrificed after 48 hours (model group) or were pretreated with metformin (50 mg/kg) daily for two weeks prior to L- arginine injections and continued receiving metformin until the end of the experiment (protective group). Using microscopic examination of the pancreas and blood chemistry, we observed that L-arginine induced acute pancreatic injury. This is demonstrated by an enlarged pancreas with patchy areas of haemorrhage, vacuolated cytoplasm and pyknotic nuclei in the acini, disorganized lobular architecture with infiltration of inflammatory cells within the interlobular connective tissue (CT) septa, and the presence of congested blood vessels that were substantially ameliorated by metformin. Metformin also significantly (p<0.05) inhibited L-arginine-induced MPO, lactate dehydrogenase (LDH), and the inflammatory biomarker tumor necrosis factor alpha (TNF-α). Whereas, metformin significantly (p<0.05) increased IL-10 levels that were inhibited by pancreatitis induction. We further demonstrated a significant (p<0.001) correlation between the scoring of the degree of pancreatic lobules damage tissue damage and the blood levels of TNF-α, IL-10, LDH, and MPO. Thus, metformin effectively protects against L-arginine-induced acute pancreatitis, which is associated with the inhibition of MPO and augmentation of IL-10.

RESUMEN: La pancreatitis aguda es una enfermedad inflamatoria del páncreas que amenaza la vida y se caracteriza por un dolor abdominal intenso que dura de días a semanas. Buscamos determinar si la metformina, fármaco antidiabético y antiinflamatorio, puede proteger contra la pancreatitis aguda en un modelo animal de pancreatitis aguda inducida por L-arginina. Además se estudió la asociación con el aumento de la citocina antiinflamatoria interleucina-10. (IL-10) e inhibición de la enzima que promueve el daño tisular, mieloperoxidasa (MPO). Las ratas se inyectaron con dos dosis del aminoácido L-arginina (2,5 g / kg; ip, a intervalos de una hora) antes de ser sacrificadas des- pués de 48 horas (grupo modelo) o se pre trataron con metformina (50 mg / kg) durante dos semanas antes del tratamiento de L- arginina y continuaron recibiendo metformina hasta el final del experimento (grupo protector). Mediante el examen microscópico del páncreas y la química sanguínea, se observó que la L- arginina inducía una lesión pancreática aguda. Se observó un aumento significativo de tamaño del páncreas con áreas hemorrágicas, citoplasma vacuolado y núcleos picnóticos en los acinos, arquitectura desorganizada con infiltración de células inflamatorias dentro de los tabiques del tejido conjuntivo interlobulillar (TC) y la presencia de vasos sanguíneos congestionados mejorados por metformina. Se observó que la metformina inhibió significativamente (p <0,05) la MPO inducida por L- arginina, la lactato deshidrogenasa (LDH) y el factor de necrosis tumoral alfa (TNF-α). Además, demostramos una correlación significativa (p <0,001) entre la puntuación del grado de daño tisular de los lóbulos pancreáticos y los niveles sanguíneos de TNF-α, IL-10, LDH y MPO. Por tanto, la metformina protege eficazmente contra la pancreatitis aguda inducida por L-arginina, que se asocia con la inhibición de MPO y el aumento de IL-10.

Effects of Chronic Exposure to Microcystin-LR on the Gut Microbiota of Male Mice.

Increasing evidence indicates that environmental pollutants can change human gut microbiota. Microcystin-leucine arginine (MC-LR), considered a major hazard to mammals, is one of the important contaminants. However, little is known about the long-term influence of MC-LR on gut microbial communities. We aimed to investigate the effect of MC-LR on gut microbiota composition and functions by conducting a chronic exposure of male mice to MC-LR via the oral route. Using 16S rRNA gene sequencing analysis on cecum samples of mice, our results showed that significant changes of species diversity were observed in the gut microbiota of MC-LR-exposed mice. In addition, comparative analysis of the microbial communities showed that the reduction of the Actinobacteria and Saccharibacteria populations was detected in MC-LR-exposed mice. Collectively, our study highlighted the significant effects of MC-LR on the shift of gut microbial communities which could contribute to the development of metabolic syndromes.

Asymmetrical dimethylarginine induces dysfunction of insulin signal transduction via endoplasmic reticulum stress in the liver of diabetic rats.

AIMS: Endoplasmic reticulum stress (ERS) as an emerging factor is involved in insulin resistance (IR), which is the pathological basis of diabetes mellitus. Accumulation of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase is associated with IR, but the underlying mechanisms have not been elucidated. This study was to reveal the important role of ADMA in IR and determine whether endogenous ADMA accumulation contributes to hepatic IR via ERS in diabetic rats and hepatocytes. MATERIALS AND METHODS: Diabetic rat model was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg). Phosphorylation of insulin receptor substrate 1 (IRS1) and protein kinase B (Akt) was detected to evaluate IR. The protein kinase PKR-like ER kinase (PERK) and eukaryotic initiation factor 2α kinase (eIF2α) phosphorylation, x-box binding protein-1 (XBP-1) splicing, glucose-regulated protein 78 (GRP78) and C/EBP homologues protein (CHOP) expressions were measured to assess ERS. KEY FINDINGS: Endogenous ADMA content was significantly increased and positively correlated with either IR as evidenced by increased IRS1 at serine and reduced Akt phosphorylation or ERS as indicated by upregulations of PERK and eIF2α phosphorylation, XBP-1 splicing, GRP78 and CHOP expressions in the liver of diabetic rats compared with control rats. Exogenous ADMA directly caused IR and ERS in dose- and time-dependent manners in primary mouse hepatocytes. Pretreatment with ERS inhibitor 4-phenylbutyrate or ADMA antagonist L-arginine not only improved ADMA-associated or -induced hepatic IR but also attenuated ADMA-associated or -induced ERS in diabetic rats or hepatocytes. SIGNIFICANCE: These findings indicate that endogenous ADMA accumulation contributes to hepatic IR via ERS in diabetic rats.

Possible mechanisms mediating the protective effect of cilostazol in L-arginine induced acute pancreatitis in rats: role of cGMP, cAMP, and HO-1.

Acute pancreatitis (AP) is an inflammatory disorder with a high mortality rate. Cilostazol is a selective phosphodiesterase-3 inhibitor drug that is commonly used as an antiplatelet, antithrombotic, and vasodilator drug. It exhibits antioxidant, anti-inflammatory, and anti-apoptotic activities, but its effect on AP has not been fully elucidated yet. The present study aimed to investigate the effects of cilostazol on L-arginine-induced AP and the possible protective mechanisms. A rat model of AP was established by a single i.p. injection of 3-g/kg L-arginine on day 13 of the experiment. The treated groups received a single daily oral dose of either 100 or 300 mg/kg/day for 14 consecutive days. Rats with AP showed histopathological changes of pancreatic tissue injury together with increased serum amylase enzyme activity and decreased serum insulin, pancreatic adiponectin, and cGMP levels. Moreover, AP rats showed increased pancreatic inflammatory biomarker (TNF-α, VCAM-1, and MPO) levels with decreased anti-inflammatory IL-10 levels. In addition, oxidative stress biomarkers (MDA and NO) were increased in AP with decreased antioxidant SOD activity and GSH level. Moreover, HO-1 immunostaining was increased in the AP group. Cilostazol pretreatment reversed the histopathological change; decreased the amylase activity and the levels of TNF-α, VCAM-1, and MPO; and increased the levels of insulin, adiponectin, cGMP, cAMP, and IL-10. Moreover, cilostazol decreased MDA and NO but increased SOD and GSH. Lastly, cilostazol increased the HO-1 immunostaining more than in the AP group. These data suggest that cilostazol protects against L-arginine-induced AP, which may be related to an increase in cGMP, cAMP, and upregulation of HO-1 with subsequent anti-inflammatory and antioxidant properties.

Asymmetric dimethylarginine exacerbates cognitive dysfunction associated with cerebrovascular pathology.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor and uncoupler of nitric oxide synthase, has gained attention as a risk factor for cardiac disease, metabolic syndrome, and cerebrovascular disease. In this study, we investigated the role of systemic ADMA overburden in cerebromicrovascular pathology associated with cognitive dysfunction using APPSwDI transgenic mice expressing human ß-amyloid precursor protein Swedish (Tg-SwDI), a model of cerebrovascular ß-amyloidosis. To induce systemic overburden of ADMA, Tg-SwDI mice were treated with a daily dose of exogenous ADMA. ADMA treatment resulted in elevated ADMA levels in the blood and brain of Tg-SwDI mice. ADMA treatment induced the brain nitrosative stress and inflammation as well as enhanced the brain Aß deposition and cognitive impairment in Tg-SwDI mice. However, ADMA treatment had no such effects on wild type mice. ADMA treatment also exacerbated brain microvascular pathology in Tg-SwDI mice as observed by increased blood-brain barrier dysfunction, loss of tight junction proteins, increased endothelial stress fibers, and decreased microvessel density in the brain. In addition, similar observations were made in cultured human brain microvessel endothelial cells, where ADMA in the presence of VEGF-induced endothelial cell signaling for F-actin stress fiber inducing endothelial barrier dysfunction. Overall, these data document the potential role of ADMA in the cognitive pathology under conditions of cerebrovascular ß-amyloidosis.

Arginine in C9ORF72 Dipolypeptides Mediates Promiscuous Proteome Binding and Multiple Modes of Toxicity.

C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a mouse neuronal-like cell line (Neuro2a) to demonstrate that the Arg residues in the most toxic DPRS, PR and GR, leads to a promiscuous binding to the proteome compared with a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats appeared to robustly stall on ribosomes during translation suggesting Arg-rich peptide domains can electrostatically jam the ribosome exit tunnel during synthesis. Poly-GR also recruited arginine methylases, induced hypomethylation of endogenous proteins, and induced a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation that may explain the dysfunction of biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.

Plasma-derived exosomes contribute to pancreatitis-associated lung injury by triggering NLRP3-dependent pyroptosis in alveolar macrophages.

Progression of acute pancreatitis (AP) into a severe form usually results in a life-threatening condition with multiple organ dysfunction, and in particular acute lung injury (ALI), often contributes to the majority of AP-associated deaths. Increasing evidence has shown that uncontrolled activation of the immune system with rapid production of inflammatory cytokines play a dominant role in this process. As an intracellular inflammatory signaling platform, the NOD-like receptor protein 3 (NLRP3) inflammasome, is recently reported to be involved in the pathogenesis of AP progression, however, the relationship between NLRP3 inflammasome activation and AP-associated lung injury remains unclear yet. Here, we show that NLRP3 inflammasome activation and subsequent pyroptosis in alveolar macrophages (AMs) is responsible for the lung injury secondary to AP. In addition, plasma-derived exosomes from AP mice is capable of triggering NLRP3-dependent pyroptosis in AMs. Inhibition of exosome release or uptake in vivo by inhibitors substantially suppresses AMs pyroptosis and thereby alleviates AP-induced pulmonary lesion. Collectively, the current work reveals for the first time the involvement of NLRP3-dependent pyroptosis induced by plasma exosomes in the pathogenesis of AP-induced ALI, suggesting that the exosome-mediated NLRP3 inflammatory pathway is a potential therapeutic target for the treatment of lung injury during AP.

The Neuropeptide Y Y2 Receptor Is Coexpressed with Nppb in Primary Afferent Neurons and Y2 Activation Reduces Histaminergic and IL-31-Induced Itch.

Itch stimuli are detected by specialized primary afferents that convey the signal to the spinal cord, but how itch transmission is regulated is still not completely known. Here, we investigated the roles of the neuropeptide Y (NPY)/Y2 receptor system on scratch behavior. The inhibitory Y2 receptor is expressed on mouse primary afferents, and intrathecal administration of the Y2 agonist peptide YY (PYY)3-36 reduced scratch episode frequency and duration induced by compound 48/80, an effect that could be reversed by intrathecal preadministration of the Y2 antagonist BIIE0246. Also, scratch episode duration induced by histamine could be reduced by PYY3-36 In contrast, scratch behavior induced by α-methyl-5HT, protease-activated receptor-2-activating peptide SLIGRL, chloroquine, topical dust mite extract, or mechanical itch induced by von Frey filaments was unaffected by stimulation of Y2 Primary afferent neurons expressing the Npy2r gene were found to coexpress itch-associated markers such as natriuretic peptide precursor b, oncostatin M receptor, and interleukin (IL) 31 receptor A. Accordingly, intrathecal PYY3-36 reduced the scratch behavior induced by IL-31. Our findings imply that the NPY/Y2 system reduces histaminergic and IL-31-associated itch through presynaptic inhibition of a subpopulation of itch-associated primary afferents. SIGNIFICANCE STATEMENT: The spinal neuropeptide Y system dampens scratching behavior induced by histaminergic compounds and interleukin 31, a cytokine involved in atopic dermatitis, through interactions with the Y2 receptor. The Y2 receptor is expressed by primary afferent neurons that are rich in itch-associated neurotransmitters and receptors such as somatostatin, natriuretic peptide precursor b, and interleukin 31 receptors.

Versatile cationic liposomes for RIP3 overexpression in colon cancer therapy and RIP3 downregulation in acute pancreatitis therapy.

Because the induction of strong host antitumor responses plays a very important role in antitumor therapy, identifying effective approaches to elicit immunogenic cell death could have important implications. RIP3-dependent necroptotic cancer cells have been reported to release damage-associated molecular patterns and enhance antitumor immunity. In this study, hyaluronic acid-conjugated cationic liposomes (DOTAP/DOPE/PEG-DSPE/CHOL) (HA-P-LP) were prepared as a vector for mRIP3-pDNA overexpression in tumours. Compared with standard cationic liposomes, this vector markedly increased cellular gene internalisation in vitro, enhanced the tumour-targeting effect in vivo and exhibited a significant antitumor effect in combination with adjuvant chloroquine. Considering the dramatic increase in RIP3 under the pathological condition of pancreatitis and the correlation between pancreatitis and necroptosis, non-HA-conjugated liposomes with the same formulation loaded with shRNA mRIP3-pDNA effectively controlled the disease by decreasing the serum amylase concentration and inflammatory cell infiltration. The versatile cationic liposomes loaded with plasmids with opposing functions in this study provide a new concept and method for both tumour therapy and pancreatitis therapy.

NLRP3 Inflammasome Regulates Development of Systemic Inflammatory Response and Compensatory Anti-Inflammatory Response Syndromes in Mice With Acute Pancreatitis.

BACKGROUND & AIMS: Pancreatitis starts with primarily sterile local inflammation that induces systemic inflammatory response syndrome, followed by compensatory anti-inflammatory response syndrome (CARS). We investigated the mechanisms of these processes in mice and human serum. METHODS: We induced severe acute pancreatitis by partial duct ligation with caerulein stimulation or intraperitoneal injection of l-arginine in mice with deletion of interleukin (IL)12B, NLRP3, or IL18 and in mice given MCC950, a small molecule inhibitor of the NLRP3-inflammasome. Pancreata were collected from mice and analyzed by histology, and cytokine levels were measured in serum samples. We measured activation of adaptive immune responses in mice with pancreatitis by flow cytometry analysis of T cells (CD25 and CD69) isolated from the spleen. Differentiation of T-helper (Th1) cells, Th2 cells, and T-regulatory cells was determined by nuclear staining for TBET, GATA3, and FOXP3. We performed transcriptome analysis of mouse lymph nodes and bone marrow-derived macrophages after incubation with acini. We measured levels of cytokines in serum samples from patients with mild and severe acute pancreatitis. RESULTS: Activation of the adaptive immune response in mice was initiated by macrophage-derived, caspase 1-processed cytokines and required activation of NLRP3 (confirmed in serum samples from patients with pancreatitis). Spleen cells from mice with pancreatitis had increases in Th2 cells but not in Th1 cells. Bone marrow-derived macrophages secreted IL1B and IL18, but not IL12, after co-incubation with pancreatic acini. T-cell activation and severity of acute pancreatitis did not differ significantly between IL12B-deficient and control mice. In contrast, NLRP3- or IL18-deficient mice had reduced activation of T cells and no increase in Th2 cell-mediated responses compared with control mice. The systemic type 2 immune response was mediated by macrophage-derived cytokines of the IL1 family. Specifically, IL18 induced a Th2 cell-mediated response in the absence of IL12. MCC950 significantly reduced neutrophil infiltration, T-cell activation, and disease severity in mice. CONCLUSIONS: In mice with severe pancreatitis, we found systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome developed in parallel. Infiltrating macrophages promote inflammation and simultaneously induce a Th2 cell-mediated response via IL18. Inhibition of NLRP3 reduces systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome and might be used to treat patients with severe pancreatitis.

Chrm3 protects against acinar cell necrosis by stabilizing caspase-8 expression in severe acute pancreatitis mice model.

Acinar cells in acute pancreatitis (AP) die through apoptosis and necrosis, the impacts of which are quite different. Early clinical interference strategies on preventing the progress of AP to severe acute pancreatitis (SAP) are the elimination of inflammation response and inhibition of necrosis. Muscarinic acetylcholine receptor M3 was encoded by Chrm3 gene. It is one of the best-characterized receptors of pancreatic ß cells and regulates insulin secretion, but its function in AP remains unclear. In this study, we explored the function of Chrm3 gene in the regulation of cell death in l-arginine-induced SAP animal models. We found that Chrm3 was upregulated in pancreatitis, and we further confirmed the localization of Chrm3 resided in both pancreatic islets and acinar cell membranes. The reduction of Chrm3 decreased the pathological lesion of SAP and reduced amylase activities in serum. Consistently, Chrm3 can suppress acinar cells necrosis markedly, but has no effect on regulating apoptosis after l-arginine treatment. It was shown that Chrm3 attenuated acinar cells necrosis at least in part by stabilizing caspase-8. Thus, this study indicates that Chrm3 is critical participants in SAP, and regulation of Chrm3 expression might be a useful therapeutic strategy for preventing pathologic necrosis.