Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins from Plasmodium falciparum and Plasmodium vivax have been cloned and expressed for a variety of structural and enzymatic studies. Recombinant plasmepsins possess kinetic similarity to the native enzymes, indicating their suitability for target-based antimalarial drug development. We developed an automated assay of P. falciparum plasmepsin II and P. vivax plasmepsin to quickly screen compounds in the Walter Reed chemical database. A low-molecular-mass (346 Da) diphenylurea derivative (WR268961) was found to inhibit plasmepsins with a K i of 1 to 6 M. This compound appears to be selective for plasmepsin, since it is a poor inhibitor of the human aspartic protease cathepsin D (K i greater than 280 M). WR268961 inhibited the growth of P. falciparum strains W2 and D6, with 50% inhibitory concentrations ranging from 0.03 to 0.16 g/ml, but was much less toxic to mammalian cells. The Walter Reed chemical database contains over 1,500 compounds with a diphenylurea core structure, 9 of which inhibit the plasmepsins, with K i values ranging from 0.05 to 0.68 M. These nine compounds show specificity for the plasmepsins over human cathepsin D, but they are poor inhibitors of P. falciparum growth in vitro. Computational docking experiments indicate how diphenylurea compounds bind to the plasmepsin active site and inhibit the enzyme.Malaria, the most severe parasitic disease, infects nearly 300 million people and kills more than a million each year (28). Plasmodium falciparum and Plasmodium vivax are the two malaria species responsible for the most infections and deaths. Although several very effective antimalarial drugs have been used to control this disease, P. falciparum has developed resistance to nearly all available antimalarial drugs (27). Recently, P. vivax from Southeast Asia has developed resistance to the most widely used antimalarial drug, chloroquine. The search for novel antimalarial drugs against specific parasitic targets is thus an urgent task to pursue. In the last decade, many potential targets for new antimalarial drugs have been discovered, such as dihydropteroate synthase, hemoglobin degradation enzymes, and shikimate pathway enzymes (17). Our work focuses on the discovery of new inhibitors of hemoglobin degradation enzymes called plasmepsins.Malarial parasites invade human erythrocytes in the asexual stage of infection. While residing in erythrocytes, the parasites rely on human hemoglobin as a food source, digesting it with a series of proteases. The aspartic proteases, called plasmepsins, are critical for hemoglobin degradation and are thus logical targets for antimalarial drug development (14,19,25). At least four plasmepsins have been identified and cloned from P. falciparum (26; R. Banerjee and D. E. Goldberg, Mol. Parasite Meet., MBL, Woods Hole, Mass., 1999). Active recombinant plasmepsin II has been successfully obtained in large enough quantities (3, 10) to facilit...

Ferulic acid (FA) has been demonstrated to have a remarkable antioxidant activity, the mechanism of FA of protecting human umbilical vein endothelial cells (HUVECs) from radiation induced oxidative stress was investigated in the present study. The oxidative protection of FA was assessed by cellular glutathione (GSH) content, nicotinamide adenine dinucleotide phosphate (NADPH) levels, and reactive oxygen species (ROS) analysis. Nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation was detected using Western blotting. The upstream signaling pathway involved in FA mediated Nrf2 activation was determined by signaling inhibitors. FA significantly increased the transcription of antioxidant related genes such as GCLC (glutamate-cysteine ligase catalytic subunit), GCLM (glutamate-cysteine ligase regulatory subunit), NQO1 (NADPH quinone oxidoreductase-1) and heme oxygenase-1 (HO-1) mRNA in radiated cells, and these changes involved in a significant increase of the intracellular GSH content and the expression of NAPDH. FA evidently promoted Nrf2 translocation into nuclei and increased the intracellular GSH and NADPH levels in radiated cells. Phosphatidylinositol 3-kinase (PI3K) and extracellular signal regulated kinase (ERK) pathways were associated with FA-induced Nrf2 activation. The results suggested that FA-induced Nrf2 activation play key role in cytoprotective effect of FA against oxidative stress via PI3K and ERK signaling pathways.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver diseases. This study sought to evaluate the insulin-sensitizing effect of paeoniflorin (PF) on high-fat diet-induced NAFLD and possible molecular mechanisms. Male Sprague Dawley rats were fed a high-fat diet (HFD) for 10 weeks to establish the NAFLD model, and PF (20 mg/kg/d) was gavaged to the NAFLD rats for another four weeks. Our results demonstrated that HFD resulted in hepatocellular ballooning, micro-/macrovesicular steatosis, and oxidative stress in the liver, accompanied by increased serum total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels and homeostasis model of insulin resistance (HOMA-IR) index. PF treatment improved the biochemical and histopathological changes in NAFLD rats. Moreover, we also found that PF could inhibit lipid ectopic deposition via regulating lipid metabolism (inhibiting lipid synthesis of cholesterol and de novo pathway), and exert insulin sensitizing effect by regulating the insulin signaling pathway IRS/Akt/GSK3β and anti-oxidation. The study findings suggest that PF has therapeutic potential against NAFLD and that it acts through multiple signaling pathways.

Aristolactam I (ALI) is an active component derived from some Traditional Chinese medicines (TCMs), and also the important metabolite of aristolochic acid. Long-term administration of medicine-containing ALI was reported to be related to aristolochic acid nephropathy (AAN), which was attributed to ALI-induced nephrotoxicity. However, the toxic mechanism of action involved is still unclear. Recently, pathogenic ferroptosis mediated lipid peroxidation was demonstrated to cause kidney injury. Therefore, this study explored the role of ferroptosis induced by mitochondrial iron overload in ALI-induced nephrotoxicity, aiming to identify the possible toxic mechanism of ALI-induced chronic nephropathy. Our results showed that ALI inhibited HK-2 cell activity in a dose-dependent manner and significantly suppressed glutathione (GSH) levels, accompanying by significant increases in intracellular 4-hydroxynonenal (4-HNE) and intracellular iron ions. Moreover, the ALI-mediated cytotoxicity could be reversed by deferoxamine mesylate (DFO). Compared with other inhibitors, Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, obviously alleviated ALI-induced cytotoxicity. Furthermore, we have shown that ALI could remarkably increase the levels of superoxide anion and ferrous ions in mitochondria, and induce mitochondrial damage and condensed mitochondrial membrane density, the morphological characteristics of ferroptosis, all of which could be reversed by DFO. Interestingly, ALI dose-dependently inhibited these protein contents of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutathione peroxidase 4 (GPX4), which could be partly rescued by Tin-protoporphyrin IX (SnPP) and mitoTEMPO co-treatment. In conclusion, our results demonstrated that mitochondrial iron overload-mediated antioxidant system inhibition would assist ALI-induced ferroptosis in renal tubular epithelial cells, and Nrf2-HO-1/GPX4 antioxidative system could be an important intervention target to prevent medicine containing ALI-induced nephropathy.

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Alternate partial root-zone irrigation has profound impacts on the crop uptake of residual nitrogen originated from the previous season.

In an attempt to find new types of anti-hypoxic agents from herbs, we identified 5-hydroxymethyl-2-furfural (5-HMF) as a natural agent that fulfills the criterion. 5-HMF, the final product of carbohydrate metabolism, has favorable biological effects such as anti-oxidant activity and inhibiting sickling of red blood cells. The role of 5-HMF in hypoxia, however, is not yet. Our pilot results showed that pretreatment with 5-HMF markedly increased both the survival time and the survival rate of mice under hypoxic stress. The present study was aimed to further investigate the protective role of 5-HMF and the underlying mechanisms in hypoxic injury using ECV304 cells as an in vitro model. ECV304 cells pretreated with or without 5-HMF for 1 h were exposed to hypoxic condition (0.3% O 2 ) for 24 h and then cell apoptosis, necrosis, the changes of mitochondrial membrane potential (MMP) and the expressions of phosphorylation-extracellular signal-regulated kinase (p-ERK) were investigated. Pretreatment with 5-HMF markedly attenuated hypoxia-induced cell necrosis and apoptosis at late stage (p<0.01). Furthermore, pretreatment with 5-HMF rescued both the decline of the MMP and the increase of p-ERK protein under hypoxia. In a word, these results indicated that 5-HMF had protective effects against hypoxic injury in ECV304 cells, and its effects on MMP and p-ERK may be involved in the mechanism.