Double deficiency of cathepsin B and L in the mouse pancreas alters trypsin activity without affecting acute pancreatitis severity

BackgroundPremature intracellular trypsinogen activation has long been considered a key initiator of acute pancreatitis (AP). Cathepsin B (CTSB) activates trypsinogen, while cathepsin L (CTSL) inactivates trypsin(ogen), and both proteins play a role in the onset of AP.MethodsAP was induced by 7 hourly intraperitoneal injections of cerulein (50 μg/kg) in wild-type and pancreas-specific conditional Ctsb knockout (Ctsb^Δpan), Ctsl knockout (Ctsl^Δpan), and Ctsb;Ctsl double-knockout (Ctsb^Δpan;Ctsl^Δpan) mice. Pancreatic samples were collected and analyzed by histology, immunohistochemistry, real-time PCR, and immunoblots. Trypsin activity was measured in pancreatic homogenates. Peripheral blood was collected, and serum amylase activity was measured.ResultsDouble deletion of Ctsb and Cstl did not affect pancreatic development or mouse growth. After 7 times cerulein injections, double Ctsb and Ctsl deficiency in mouse pancreases increased trypsin activity to the same extent as that in Ctsl-deficient mice, while Ctsb deficiency decreased trypsin activity but did not affect the severity of AP. Ctsb^Δpan;Ctsl^Δpan mice had comparable serum amylase activity and histopathological changes and displayed similar levels of proinflammatory cytokines, apoptosis, and autophagy activity compared with wild-type, Ctsb^Δpan, and Ctsl^Δpan mice.ConclusionDouble deletion of Ctsb and Ctsl in the mouse pancreas altered intrapancreatic trypsin activity but did not affect disease severity and inflammatory response after cerulein-induced AP.     

Mechanism of action of Orthosiphon stamineus against non-alcoholic fatty liver disease: Insights from systems pharmacology and molecular docking approaches

Non-alcoholic fatty liver disease (NAFLD) is one of the most common complications of a metabolic syndrome caused by excessive accumulation of fat in the liver. Orthosiphon stamineus also known as Orthosiphon aristatus is a medicinal plant with possible potential beneficial effects on various metabolic disorders. This study aims to investigate the in vitro inhibitory effects of O. stamineus on hepatic fat accumulation and to further use the computational systems pharmacology approach to identify the pharmacokinetic properties of the bioactive compounds of O. stamineus and to predict their molecular mechanisms against NAFLD. Methods: The effects of an ethanolic extract of O. stamineus leaves on cytotoxicity, fat accumulation and antioxidant activity were assessed using HepG2 cells. The bioactive compounds of O. stamineus were identified using LC/MS and two bioinformatics databases, namely the Traditional Chinese Medicine Integrated Database (TCMID) and the Bioinformatics Analysis Tool for the Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM). Pathway enrichment analysis was performed on the predicted targets of the bioactive compounds to provide a systematic overview of the molecular mechanism of action, while molecular docking was used to validate the predicted targets. Results: A total of 27 bioactive compounds corresponding to 50 potential NAFLD-related targets were identified. O. stamineus exerts its anti-NAFLD effects by modulating a variety of cellular processes, including oxidative stress, mitochondrial β-oxidation, inflammatory signalling pathways, insulin signalling, and fatty acid homeostasis pathways. O. stamineus is significantly targeting many oxidative stress regulators, including JNK, mammalian target of rapamycin (mTOR), NFKB1, PPAR, and AKT1. Molecular docking analysis confirmed the expected high affinity for the potential targets, while the in vitro assay indicates the ability of O. stamineus to inhibit hepatic fat accumulation. Conclusion: Using the computational systems pharmacology approach, the potentially beneficial effect of O. stamineus in NAFLD was indicated through the combination of multiple compounds, multiple targets, and multicellular components.     

Inverse correlation between the expression of AMPK/SIRT1 and NAMPT in psoriatic skin: A pilot study

PurposeEpidermal hyperplasia and the involvement of immune cells characterize the clinical picture of psoriasis. Among the several factors involved, attention has been focused on sirtuin 1 (SIRT1) - a deacetylase endowed with a variety of functions including the control of metabolic and inflammatory processes-, and on nicotinamide phosphoribosyltransferase (NAMPT), important for SIRT1 activation and involved in inflammatory events. The aim of the study was to analyze changes of SIRT1 and NAMPT expression in psoriatic skin.Patients and methodsSamples from healthy controls and psoriatic patients were subjected to immunohistochemical analysis.ResultsA strong downregulation of SIRT1 expression was observed in skin samples from psoriatic patients compared to healthy controls. This was accompanied by a parallel reduction of adenosine monophosphate-activated kinase (AMPK) expression and, more strikingly, by the disappearance of cells immunolabeled for its active, phosphorylated form (pAMPK). In both cases, analysis of the distribution of immunopositive cells revealed a shift towards reduced intensity of staining. In contrast, NAMPT expression was upregulated in psoriatic samples in line with its pro-inflammatory role. This was again more visible with an intensity-based distribution analysis that evidenced a shift towards more intensely immunostained cell populations.ConclusionsThe present data correlate in the same samples the expression of SIRT1, pAMPK/AMPK and NAMPT in psoriasis and open the way for novel pharmacological targets in the treatment of the disease.     

Inflamación y adecuación de la hemodiálisis: ¿están los niveles de proteína C reactiva influidos por la dosis de diálisis recibida?

IntroductionChronic inflammation and the underlying cardiovascular comorbidity are still current problems in chronic hemodialysis patients. There are few studies comparing the “dialysis dose” (Kt/V) with the degree of inflammation in the patient. Our main objective was to determine whether there is a relationship between serum C-reactive protein (CRP) levels and the Kt/V using ionic dialysance.MethodsMulticenter cross-sectional study. A total of 536 prevalent chronic hemodialysis patients were included. CRP levels, neutrophil-lymphocyte ratio and platelet-lymphocyte ratio were collected. Kt was obtained by ionic dialysance and urea distribution volume was calculated from the Watson's formula. The sample was divided into 2 groups, taking the median CRP as the cut-off point. Dialysis adequacy obtained in each group was compared. Finally, a logistic regression model was carried out to determine the variables with the greatest influence.ResultsMedian CRP was 4.10 mg/L (q25-q75: 1.67-10) and mean Kt/V was 1.48 ± 0.308. Kt/V was lower in the patients included in the high inflammation group (P = .01). In the multivariate logistic regression, the “high” levels of CRP were directly correlated with the Log neutrophil-lymphocyte ratio (P < .001) and inversely proportional with serum albumin values (P = .014), Kt/V (P = .037) and serum iron (P < .001).ConclusionThe poorer adequacy in terms of dialysis doses (lower Kt/V values) may contribute to a higher degree of inflammation in chronic hemodialysis patients.     

Inflammaging and Cannabinoids

Aging is a complex phenomenon associated with a wide spectrum of physical and physiological changes affecting every part of all metazoans, if they escape death prior to reaching maturity. Critical to survival, the immune system evolved as the principal component of response to injury and defense against pathogen invasions. Because how significantly immune system affects and is affected by aging, several neologisms now appear to encapsulate these reciprocal relationships, such as Immunosenescence. The central part of Immunosenescence is Inflammaging –a sustained, low-grade, sterile inflammation occurring after reaching reproductive prime. Once initiated, the impact of Inflammaging and its adverse effects determine the direction and magnitudes of further Inflammaging. In this article, we review the nature of this vicious cycle, we will propose that phytocannabinoids as immune regulators may possess the potential as effective adjunctive therapies to slow and, in certain cases, reverse the pathologic senescence to permit a more healthy aging.     

The endogenous inflammatory reflex inhibits the inflammatory response to different immune challenges in mice

The splanchnic anti-inflammatory pathway, the efferent arm of the endogenous inflammatory reflex, has been shown to suppress the acute inflammatory response of rats to systemic lipopolysaccharide (LPS). Here we show for the first time that this applies also to mice, and that the reflex may be engaged by a range of inflammatory stimuli. Experiments were performed on mice under deep anaesthesia. Half the animals were subjected to bilateral section of the splanchnic sympathetic nerves, to disconnect the splanchnic anti-inflammatory pathway, while the remainder underwent a sham operation. Mice were then challenged intravenously with one of three inflammatory stimuli: the toll-like receptor (TLR)-4 agonist, LPS (60 µg/kg), the TLR-3 agonist Polyinosinic:polycytidylic acid (Poly I:C, 1 mg/kg) or the TLR-2 and -6 agonist dipalmitoyl-S-glyceryl cysteine (Pam2cys, 34 µg/kg). Ninety minutes later, blood was sampled by cardiac puncture for serum cytokine analysis. The splanchnic anti-inflammatory reflex action was assessed by comparing cytokine levels between animals with cut versus those with intact splanchnic nerves. A consistent pattern emerged: Tumor necrosis factor (TNF) levels in response to all three challenges were raised by prior splanchnic nerve section, while levels of the anti-inflammatory cytokine interleukin 10 (IL-10) were reduced. The raised TNF:IL-10 ratio after splanchnic nerve section indicates an enhanced inflammatory state when the reflex is disabled. These findings show for the first time that the inflammatory reflex drives a coordinated anti-inflammatory action also in mice, and demonstrate that its anti-inflammatory action is engaged, in similar fashion, by inflammatory stimuli mimicking a range of bacterial and viral infections.     

Recent insights into the characteristics and role of peritoneal macrophages from ascites of cirrhotic patients

Macrophages are a diverse myeloid cell population involved in innate and adaptive immune responses, embryonic development, wound repair, and regulation of tissue homeostasis. These cells link the innate and adaptive immunities and are crucial in the development and sustainment of various inflammatory diseases. Macrophages are tissue-resident cells in steady-state conditions; however, they are also recruited from blood monocytes after local pathogen invasion or tissue injury. Peritoneal macrophages vary based on their cell complexity, phenotype, and functional capabilities. These cells regulate inflammation and control bacterial infections in the ascites of decompensated cirrhotic patients. Our recent work reported several phenotypic and functional characteristics of these cells under both healthy and pathological conditions. A direct association between cell size, CD14/CD16 expression, intracellular level of GATA-6, and expression of CD206 and HLA-DR activation/maturation markers, indicate that the large peritoneal macrophage CD14^highCD16^high subset constitutes the mature phenotype of human resident peritoneal macrophages during homeostasis. Moreover, elevated expression of CD14/CD16 is related to the phagocytic capacity. The novel large CD14^highCD16^high peritoneal subpopulation is increased in the ascites of cirrhotic patients and is highly sensitive to lipopolysaccharide (LPS)-induced activation, thereby exhibiting features of inflammatory priming. Thus, phosphorylation of ERK1/2, PKB/Akt, and c-Jun is remarkably increased in response to LPS in vitro, whereas that of p38 MAPK is reduced compared with the monocyte-derived macrophages from the blood of healthy controls. Furthermore, in vitro activated monocyte-derived macrophages from ascites of cirrhotic patients secreted significantly higher levels of IL-6, IL-10, and TNF-α and lower amounts of IL-1β and IL-12 than the corresponding cells from healthy donor’s blood. Based on these results, other authors have recently reported that the surface expression level of CD206 can be used to identify mature, resident, inflammatory peritoneal macrophages in patients with cirrhosis. Soluble CD206 is released from activated large peritoneal macrophages, and increased concentrations in patients with cirrhosis and spontaneous bacterial peritonitis (SBP) indicate reduced odds of survival for 90 d. Hence, the level of soluble CD206 in ascites might be used to identify patients with SBP at risk of death. In conclusion, peritoneal macrophages present in ascites of cirrhotic patients display multiple phenotypic modifications characterized by reduced ratio of cells expressing several membrane markers, together with an increase in the ratios of complex and intermediate subpopulations and a decrease in the classic-like subset. These modifications may lead to the identification of novel pharmaceutical targets for prevention and treatment of hepatic damage.