Fluorescence nanoparticles from instant coffee accumulated in lysosome and induced lysosome-dependent cell death via necroptosis-like pathway.

Fluorescence nanoparticles (FNs) are a type of nano-dots generated during baking process, and their safety on organism is unclear and little is known to their cytotoxicity. In this study, the FNs from instant coffee were purified and characterized. The FNs with an average size about 2.08 nm emitted bright blue fluorescence with lifetime about 2.74 ns. The element and functional groups were analyzed by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy, respectively. The results indicated that these FNs were internalized in lysosomes and induced apoptosis of normal rat kidney (NRK) and Caco-2 cells. While, the pan-caspase inhibitor, Z-VAD-FMK didn't decrease the rate of apoptosis and cell death of the FNs-treated NRK and Caco-2 cells. These internalized FNs enlarged lysosomes, decreased lysosomal enzyme degradation activity and increased lysosomal pH value. Partial co-localization of receptor-interacting serine-threonine kinase 3 (RIPK3) to lysosomes in FNs-treated cells was observed, and the amount of RIPK1 and RIPK3 increased after treatment with FNs. The results demonstrated that the FNs from instant coffee induced lysosomal membrane permeabilization and initiated necroptosis.

Food-borne nanocarriers from roast beef patties for iron delivery.

Food-borne nanoparticles (FNs) produced during thermal processing of food may have potential as nanocarriers for Fe(ii) supplements. In this paper, the FNs in beef patties roasted for different times (15, 30, and 45 minutes) and the binding between FNs and ferrous ions were studied. The size of FNs decreased from 7.5 to 3.0 nm with the increase of baking time, and the FNs emitted bright blue fluorescence under ultraviolet light irradiation. The combination of FNs with ferrous ions was by means of the amino, hydroxyl and carboxyl functional groups on the particles. Cell viability study showed that the Fe(ii)-FNs increased the apoptotic rate, but significantly decreased the necrosis rate, which led to an increase in the number of living cells. In addition, the Fe(ii)-FNs can easily enter the Caco-2 cytoplasm, but not the cellular nucleus. The FNs derived from beef patties with an ultra-small size, high water solubility and plenty of functional groups might be good candidates as nanocarriers for Fe(ii) delivery.

Fluorescent nanoparticles in the popular pizza: properties, biodistribution and cytotoxicity.

Food-borne nanoparticles that are generated during the thermal processing of various consumed foods are of great concern due to their unique properties. In this study, the presence of fluorescent nanoparticles (FNPs) in pizza, their biodistribution and cytotoxicity were investigated. The spherical FNPs have a diameter of about 3.33 nm. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy analysis revealed that they contained 68.21% C, 27.44% O, 2.75% N and 1.60% S, and the functional groups on their surface included -OH, -COOH, C[double bond, length as m-dash]C, -NH2 and C[double bond, length as m-dash]O. In vitro and in vivo biodistribution of pizza FNPs was evaluated using normal rat kidney (NRK) cells, onion epidermal cells, Caenorhabditis elegans and mice. The fluorescence microscopy images clearly indicate that the pizza FNPs appear to be localized within the cytoplasm. However, the FNPs remained restricted to the extracellular space of the onion epithelium and did not enter the onion cell cytoplasm because of the cell wall. The FNPs were swallowed by the Caenorhabditis elegans worms when exposed to food OP50 and distributed within the pharynx, intestine and anus. Obvious fluorescence of the FNPs in the stomach, intestine, liver, lung and kidney was observed for the FNPs in mouse organs, but not the brain, heart, and spleen. Furthermore, the produced FNPs were found to cause cell cycle arrest at the G0/G1 phase in NRK cells, and resulted in cell apoptosis at high doses. The outcome of this research offers an important insight into the nature of thermal processing-induced nanoparticles and their in vivo and in vitro biological effects.

Mogroside V inhibits LPS-induced COX-2 expression/ROS production and overexpression of HO-1 by blocking phosphorylation of AKT1 in RAW264.7 cells.

Momordica grosvenori is a valuable edible plant with medicinal purposes, and it is widely used in medicated diets and traditional Chinese medicine in Asia. Mogroside V (MV), the main bioactive component from M. grosvenori, is commonly used as a natural sweetener. M. grosvenori extracts have been reported to exert potent anti-inflammatory property, however the underlying molecular mechanism still remains unknown. In the present study, the biological effect of MV in inflammation was investigated in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The ELISA and western blot analysis results showed that MV significantly inhibited LPS-induced prostaglandin E2 (PGE2) production and cyclooxygenase-2 (COX-2) expression. MV markedly decreased the phosphorylation of IκB-α, increased IκB-α, and reduced nuclear p-65 and C/EBPδ. Furthermore, MV attenuated LPS-induced phosphorylation of MAPKs and AKT1, and only the phosphorylation status of AKT1 was found to be consistent with the expression trend of COX-2. Moreover, MV reduced ROS level and restored overexpressed HO-1 and AP-1 to basal level, which can be markedly reversed by AKT1 inhibitor LY294002. These results revealed that AKT1 plays a key role in LPS-induced COX-2 expression, and acts as a mediator to keep the redox balance in LPS-stimulated RAW264.7 cells. MV exerts anti-inflammatory property by blocking AKT1-mediated NF-κB and C/EBPδ activation, ROS generation and AP-1/ HO-1 expression. Therefore, the present study indicated that MV has a significant chemopreventive effect on the inflammatory lesions and suggested that AKT1 is a potential specific target of MV for relieving inflammation.

Gymnemic acid I triggers mechanistic target of rapamycin-mediated ß cells cytoprotection through the promotion of autophagy under high glucose stress.

Gymnemic acid I (GA I) is a bioactive component of Gymnema sylvestre. It is an Indian traditional medicinal herb which has antidiabetic effect. However, the molecular mechanism is remaining to be elucidated. Here, we showed that high glucose promoted the rate of apoptosis, GA I decreased the apoptosis under the high glucose stress. Our further study explored that GA I increased the number of autophagosome and the ratio of light chain 3-I (LC3-I)/LC3-II in MIN-6 cells under the normal or high glucose stress by the methods of western blot analysis and immunofluorescence. It induced autophagy flux and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase ß-1 (p70 S6K/S6K1), which is a substrate of mTOR. GA I decreased the rate of apoptosis and the activity of caspase-3 under the high glucose stress. The inhibition of apoptosis and caspase-3 activity by GA I were increased after treating with autophagy inhibitor in mouse islet ß cells MIN-6. Our data suggested that GA I-induced autophagy protected MIN-6 cells from apoptosis under high glucose stress via inhibition the phosphorylation activity of mTOR.

Organosulfur compounds induce cytoprotective autophagy against apoptosis by inhibiting mTOR phosphorylation activity in macrophages.

Organosulfur compounds (OSCs) are the bioactive components of garlic. Some OSCs have apoptotic or autophagy-inducing effects. Autophagy plays roles in both cytoprotection and apoptosis-related cell death, and the interaction between autophagy and apoptosis is important in the modulation of immune responses. The mechanism of an OSC-mediated effect via the interaction of autophagy and apoptosis is unknown. In this study, the effects of five OSC compounds on autophagy in the macrophage cell line RAW264.7 and primary macrophages were investigated. We found that S-allylcysteine (SAC), diallyl disulde (DADS) and diallyl tetrasulfide (DTS) treatment increased the number of autophagosomes of RAW264.7 cells, inhibited the phosphorylation of ribosomal protein S6 kinase beta-1 (p70S6K/S6K1) which is a substrate of mammalian target of rapamycin (mTOR), and significantly enhanced autophagy flux. The induction of autophagy by SAC, DADS and DTS was inhibited by stably knocking down the expression of autophagy-related gene 5 (ATG5) with short hairpin RNA (shRNA). Further experiments confirmed that SAC, DADS and DTS also induced apoptosis in RAW264.7 cells. The induction of apoptosis and Caspase 3 activity by SAC, DADS and DTS were increased by stably knocking down of ATG5 expression with shRNA in RAW264.7 cells or treating with 5 mM 3-MA in primary macrophages. Our results suggest that SAC, DADS and DTS induce both autophagy and apoptosis. The autophagy induction protects macrophages from apoptosis by inhibiting mTOR phosphorylation activity to maintain the mass of immune cells.

Xiaokeping-induced autophagy protects pancreatic ß-cells against apoptosis under high glucose stress.

Xiaokeping (XKP), a prescribed Traditional Chinese Medicine (TCM), has been used to treat patients with type Ⅱ diabetes mellitus for many years; however, the molecular mechanism of its effects is unknown. As the only insulin producer, the pancreatic ß cell plays an important role in diabetes. Whether XKP influences the viability of pancreatic ß cells remains to be substantiated. In the present study, autophagy/apoptosis analyses were used to evaluate the therapeutic effect of XKP on pancreatic ß-cells induced by high glucose levels and to investigate a potential causal molecular mechanism of XKP effect on the cells. The pancreatic ß-cell lines MIN-6 were divided into four groups: control, high glucose (33.3 mmol/L), high glucose with XKP, high glucose with XKP and 3-Methyladenine (3-MA). Immunofluorescence assay was employed to determine autophagosome formation and flow cytometry was used to determine apoptotic rates of the ß cells by the detecting expression of autophagy- and apoptosis-related proteins. High glucose increased the apoptotic rate of ß-cells from 5.37% to 23.24%; however addition of XKP mitigated the rate at 10.92%. Data indicate that autophagy of ß-cells was induced by XKP via the mammalian target of rapamycin (mTOR) pathway. Where the autophagy inhibitor 3-MA was added, the apoptotic rate was 23.94%, similar to the high glucose group rate. The results suggest a potential cytoprotective effect of XKP from high glucose toxicity by its induction of autophagy which may be linked to mTOR-mediated autophagy.

Synthesis and screening of 3-MA derivatives for autophagy inhibitors.

Autophagy is a conserved degradation process, which plays important pathophysiological roles. The lack of effective inhibitors of autophagy has been an obstacle in both basic research and understanding the physiological role of autophagy in disease manifestation. The most widely used inhibitor, 3-methyladenine (3-MA), is poorly soluble at room temperature and is effective only at high concentrations. In this study, we synthesized a library of small compounds by chemically modifying 3-MA and screened this library for autophagy inhibitors. Three 3-MA derivatives generated through this approach showed improved solubility and effectiveness in inhibiting autophagy. We demonstrated that chemical modification of an existing autophagy inhibitor is an effective method to generate improved autophagy inhibitors.