Effectiveness of Narciclasine in Suppressing the Inflammatory Response in Sepsis: Molecular Docking and In Silico Studies.

Narciclasine is an alkaloid belonging to the Amaryllidaceae family which has been reported to have many beneficial properties. Especially its anticancer properties have been widely reported. Here, we have focused on its potential use in suppressing the inflammatory response in sepsis using in silico methods. Lipopolysaccharide (LPS) is an endotoxin which is present in the outer membrane of gram-negative bacteria and is a crucial player in the pathogenesis of gram-negative sepsis. Activation of toll-like receptor 4 (TLR4) signaling by LPS is an important event in the pathogenesis of gram-negative sepsis. This initiates a downstream signaling pathway comprising of several adaptor proteins such as toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), myeloid differentiation primary response protein 88 (MyD88), interleukin-1 receptor-associated kinase (IRAK)-1, IRAK-4, interferon regulatory factor 3 (IRF-3), tumor necrosis factor receptor-associated factor 6 (TRAF-6) leading to nuclear factor kappa B (NF-κß) activation resulting in elevated production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6. S100 calcium binding proteins A8/A9 (S100A8/A9) have been found to be an agonist of TLR4, and it amplifies the inflammatory response in sepsis. Molecular docking studies of narciclasine with target proteins associated with the LPS-TLR4 pathway showed that it has good binding affinity and stable interactions with the targets studied. Molecular dynamics (MD) simulation studies over 100 ns showed that most of the ligand-target complexes were stable. The structures of all the targets except TRAF-6 were retrieved from the Protein Data Bank (PDB) database. Homology modeling was done to predict the 3-dimensional structure of TRAF-6. MD simulation of narciclasine-TRAF-6 complex showed that the structure is stable. Metapocket was used for active site prediction in the target proteins. Toxicity analysis by admetSAR revealed that narciclasine was readily biodegradable and exhibited minimum toxicity. These results indicate that narciclasine has effective anti-inflammatory properties which could be useful in suppressing the inflammatory response in sepsis.

Effect of zinc supplementation on relative expression of immune response genes in neonates with sepsis: A preliminary study.

Background & objectives: Zinc alters gene expression mainly by binding to a site on the transcription factor. Genome-wide expression studies have shown early repression of genes related to zinc and immunity in adult patients with sepsis. The present study was conducted to evaluate the role of zinc supplementation on relative expression of immune response genes in neonatal sepsis. Methods: In the present study, a sample of convenience of 22 neonates each was selected from the zinc supplemented and control groups using random numbers for expression of immune-related genes by zinc supplementation. These neonates with sepsis were earlier randomized into two groups: with and without zinc supplementation in addition to standard antibiotics and supportive care. Relative expression of immune response genes were analyzed for 22 neonates in each group using quantitative real-time PCR for calprotectin (S100A8/A9), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), toll-like receptor-4 (TLR-4), cluster of differentiation 14 (CD14) and lipopolysaccharide-binding protein (LBP) genes. Results: An increase in serum zinc levels was observed in zinc-supplemented group compared to controls. S100A8 gene showed downregulation by three-fold (P <0.001) and S100A9 gene showed upregulation by two-fold (P <0.05) in zinc group compared to controls. CD14 gene showed upregulation by one-fold in zinc-supplemented group compared to controls (P <0.05). No significant fold changes were observed with respect to TNF-α, IL-6, LBP and TLR-4 genes between the two groups. Interpretation & conclusions: The results of our preliminary study showed that the zinc supplementation might modulates the relative expression of immune-related genes involved in sepsis pathway among neonates. However, studies with larger sample size are needed to be done to provide a better picture on the outcome by gene expression in neonatal sepsis by zinc supplementation.

Narciclasine improves outcome in sepsis among neonatal rats via inhibition of calprotectin and alleviating inflammatory responses.

Sepsis is associated with exacerbated inflammatory response which subsequently results in multiple organ dysfunction. Sepsis accounts for high mortality and morbidity among newborns worldwide. Narciclasine is a plant alkaloid which has shown to possess anti-inflammatory properties. In this study we investigated the effect and mechanism of action of narciclasine in neonatal sepsis rat models. The excessive release of S100A8/A9 or calprotectin in neonatal sepsis could be detrimental as it could exacerbate the inflammatory responses. We found that narciclasine significantly reduced the plasma levels of S100A8/A9 and also suppressed its expression in the liver and lung. The systemic and local bacterial load was also reduced in the narciclasine treated rats. The systemic and local production of pro-inflammatory cytokines in plasma and organs (liver and lungs) was significantly reduced in the narciclasine treated rats. The histopathological studies showed that narciclasine prevents the organ damage associated with sepsis and improved the survival of neonatal rats. Sepsis increased the phosphorylated NF-κß p65 protein expression in the liver. Narciclasine suppressed the phosphorylation of NF-κß p65 and the degradation of NF-κß inhibitory protein alpha. It could also suppress the expression of adaptor proteins of the toll like receptor signaling pathway viz., myeloid differentiation factor 88 (MyD88), Interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor associated factor 6 (TRAF6). These results suggest that narciclasine protects against sepsis in neonatal rats through the inhibition of calprotectin, pro-inflammatory cytokines and suppression of NF-κß signaling pathway.

Predicting Severity of Acute Kidney Injury in Term Neonates with Perinatal Asphyxia Using Urinary Neutrophil Gelatinase Associated Lipocalin.

OBJECTIVE: To evaluate the utility of urinary Neutrophil Gelatinase Associated Lipocalin (NGAL) as a biomarker for predicting Acute Kidney Injury (AKI) and its severity among neonates with perinatal asphyxia. METHODS: This descriptive study included 120 term neonates with perinatal asphyxia. Renal parameters of neonates were monitored and AKI was ascertained as per Acute Kidney Injury Network criteria. Urinary NGAL was estimated and correlated with severity of AKI. RESULTS: Among the 120 neonates with perinatal asphyxia, 55(46 %) had AKI. The median urinary NGAL level was 165 ng/ml (88.8-245.8) in neonates with AKI compared to 58.97(42.8-74.7) in those without AKI. The median NGAL was 134.45(112.2-162.5), 301.2(255.5-361.2), 416.2(412.2-465.5) in AKI stages 1, 2 and 3 respectively. An NGAL cut off value of 86.82 ng/ml had 87 % sensitivity and 87.7 % specificity in predicting AKI. CONCLUSIONS: Urinary NGAL is a useful biomarker for predicting AKI and its severity among neonates with perinatal asphyxia.