Danshen injection induces autophagy in podocytes to alleviate nephrotic syndrome via the PI3K/AKT/mTOR pathway.

BACKGROUND: Danshen injection (DSI) is an agent extracted from the Salvia miltiorrhiza Bunge, a natural drug commonly used to alleviate kidney diseases. However, the material basis and therapeutic effects of DSI on nephrotic syndrome (NS) remain unclear. PURPOSE: To investigate the material basis of DSI and the therapeutic effects and underlying mechanisms of NS. METHODS: NS models were established using adriamycin-induced BALB/c mice and lipopolysaccharide-induced mouse podocytes (MPC-5). Following DSI and prednisone administration, kidney coefficients, 24 h urine protein, blood urea nitrogen, and serum creatinine levels were tested. Histomorphology was observed by periodic acid-Schiff staining and hematoxylin and eosin staining of the kidney sections. The glomerular basement membrane and autophagosomes of the kidneys were observed using transmission electron microscopy. Nephrin and desmin levels in the glomeruli were tested using immunohistochemistry. The viability of MPC-5 cells was tested using cell counting kit-8 after chloroquine and rapamycin administration in combination with DSI. The in vivo and in vitro protein levels of phosphatidylinositol 3-kinase (PI3K), AKT, phosphorylated AKT (Ser473), mammalian target of rapamycin (mTOR), microtubule-associated protein light chain 3 (LC3), beclin1, cleaved caspase-3, and caspase-3 were detected using western blotting. RESULTS: Our results showed that DSI contained nine main components: caffeic acid, danshensu, lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, and 3, 4-Dihydroxybenzaldehyde. In in vivo studies, the NS mice showed renal function and pathological impairment. Podocytes were damaged, with decreased levels of autophagy and apoptosis, accompanied by inhibition of the PI3K/AKT/mTOR signaling. DSI administration resulted in improved renal function and pathology in NS mice, with the activation of autophagy and PI3K/AKT/mTOR signaling in the kidneys. Additionally, podocytes were less damaged and intracellular autophagosomes were markedly increased. In vitro studies have shown that DSI activated MPC-5 autophagy and reduced apoptosis via the PI3K/AKT/mTOR pathway. CONCLUSION: Collectively, this study demonstrated that DSI activated podocyte autophagy and reduced apoptosis via the PI3K/AKT/mTOR signaling, ultimately attenuating NS. Our study clarified the main components of DSI and elucidated its therapeutic effects and potential mechanisms for NS, providing new targets and agents for the clinical treatment of NS.

Oxysophocarpine inhibits airway inflammation and mucus hypersecretion through JNK/AP-1 pathway in vivo and in vitro.

Asthma is a high-incidence disease in the world. Oxysophocarpine (OSC), a quinolizidine alkaloid displays various pharmacological functions including anti-inflammation, neuroprotective, anti-virus and antioxidant. Here, we established mice and cell asthmatic model to explore the effects of OSC for asthma treatment. Mice were sensitized and challenged with ovalbumin (OVA) and treated with OSC before challenge. Enzyme-linked immuno sorbent assay (ELISA), hematoxylin and eosin (H&E), periodic acid-schiff (PAS), tolonium chloride staining and immunohistochemical assay were performed. OSC treatment inhibited inflammatory cell infiltration and mucus secretion in the airway, reduced IgE level in mouse serum and decreased IL-4, IL-5 production in bronchoalveolar lavage fluid (BALF). OSC also reduced the spleen index to regulate immune function. Meanwhile, NCI-H292 cells were induced by lipopolysaccharide (LPS) to simulate airway epithelial injury. OSC pretreatment decreased the IL-6 and IL-8 cytokine levels, mucin 5 AC expression, and mucin 5 AC mRNA level in the cell model. Further, OSC suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1, Fos and Jun). These findings revealed that OSC alleviated bronchial asthma associated with JNK/AP-1 signaling pathway.

Glycoproteomics enabled by tagging sialic acid- or galactose-terminated glycans.

In this paper, we present two complementary strategies for enrichment of glycoproteins on living cells that combine the desirable attributes of "robust enrichment" afforded by covalent-labeling techniques and "specificity for glycoproteins" typically provided by lectin or antibody affinity reagents. Our strategy involves the selective introduction of aldehydes either into sialic acids by periodate oxidation (periodate oxidation and aniline-catalyzed oxime ligation (PAL)) or into terminal galactose and N-acetylgalactosamine residues by galactose oxidase (galactose oxidase and aniline-catalyzed oxime ligation (GAL)), followed by aniline-catalyzed oxime ligation with aminooxy-biotin to biotinylate the glycans of glycoprotein subpopulations with high efficiency and cell viability. As expected, the two methods exhibit reciprocal tagging efficiencies when applied to fully sialylated cells compared with sialic acid-deficient cells. To assess the utility of these labeling methods for glycoproteomics, we enriched the PAL- and GAL-labeled (biotinylated) glycoproteome by adsorption onto immobilized streptavidin. Glycoprotein identities (IDs) and N-glycosylation site information were then obtained by liquid chromatography-tandem mass spectrometry on total tryptic peptides and on peptides subsequently released from N-glycans still bound to the beads using peptide N-glycosidase F. A total of 175 unique N-glycosylation sites were identified, belonging to 108 nonredundant glycoproteins. Of the 108 glycoproteins, 48 were identified by both methods of labeling and the remainder was identified using PAL on sialylated cells (40) or GAL on sialic acid-deficient cells (20). Our results demonstrate that PAL and GAL can be employed as complementary methods of chemical tagging for targeted proteomics of glycoprotein subpopulations and identification of glycosylation sites of proteins on cells with an altered sialylation status.

Saccharomyces cerevisiae: the effect of different forms and concentrations of iodine on uptake and yeast growth.

The essentiality of iodine for humans, especially in the early stages of life, is well recognized. The chemical forms of iodine in food supplements, infant formulae and iodated salt are either iodide (KI) or iodate (KIO(3)). Because there are no or rare data about iodine uptake by yeasts, we investigated the influence of different sources of iodine, as KI, KIO(3) and periodate (KIO(4)), on its uptake in and growth of the model yeast Saccharomyces cerevisiae. KIO(3) inhibited the growth of the yeast the most and already at a 400 microM initial concentration in the growth medium; the OD was reduced by 23% in comparison with the control, where no KIO(3) was added. The uptake of different iodine sources by the yeast S. cerevisiae was minimal, in total <1%. Tracer experiments with radioactive (131)I added as KI showed that the yeast S. cerevisiae does not have the ability to transform KI into volatile species. We investigated the specificity of iodine uptake added as KIO(3) in the presence of Na(2)SeO(4) or ZnCl(2) or K(2)CrO(4) in the growth medium, and it was found that chromate had the most influence on reduction of KIO(3) uptake.

Isolation, purification and structural investigation of a water-soluble polysaccharide from Solanum lyratum Thunb.

Polysaccharides, extracted from the herbs of Chinese natural Solanum lyratum Thunb (SLT), which is a traditional Chinese medicine with hot water and 0.04 M sodium hydroxide successively, were fractionated and purified by ion-exchange and gel-filtration chromatography. According to methylation, periodate oxidation, NMR spectroscopy, partial and graded acid and enzymic hydrolysis analysis, the results indicated the D-glucan to be linear and to contain both (1-->3)- and (1-->4)-linkages. The anomeric NMR and IR spectra measurements confirmed that the sugar residues were beta-glycosidically linked.

Glycoinositolphosphosphingolipids (basidiolipids) of higher mushrooms.

The basidiolipids of six mushroom species, i.e. the basidiomycetes Amanita virosa (engl., death cup), Calvatia exipuliformis (engl., puffball), Cantharellus cibarius (engl., chanterelle), Leccinum scabrum (engl., red birch boletus), Lentinus edodes (jap., Shiitake), and Pleurotus ostreatus (engl., oystermushroom), were isolated, and their chemical structures investigated. All glycolipids are structurally related to those of the Agaricales (engl., field mushroom). They are glycoinositolphosphosphingolipids, their ceramide moiety consisting of t18:0-trihydroxysphinganine and an alpha-hydroxy long-chain fatty acid. In contrast to a previous study [Jennemann, R., Bauer, B.L., Bertalanffy, H., Geyer, R., Gschwind, R.M., Selmer, T. & Wiegandt, H. (1999) Eur. J. Biochem. 259, 331--338], the glycoside anomery of the hexose (mannose) connected to the inositol of all investigated basidiomycete glycolipids, including the basidiolipids of Agaricus bisporus, was determined unequivocally to be alpha. Therefore, the root structure of all basidiolipids consists of alpha-DManp-2Ins1-[PO(4)]-Cer. In addition, for some mushroom species, the occurrence of an inositol substitution position variant, alpha-Manp-4Ins1-[PO(40]-Cer, is shown. The carbohydrate of chanterelle basidiolipids consists solely of mannose, i.e. Cc1, Man alpha-3 or -6Man alpha; Cc2, Man alpha-3(Man alpha-6)Man alpha-. All other species investigated show extension of the alpha-mannoside in the 6-position by beta-galactoside, which, in some instances, is alpha-fucosylated in 2-position (Fuc alpha-2)Gal beta-6Man alpha-. Further sugar chain elongation at the beta-galactoside may be in 3- and/or 6-position by alpha-galactoside, e.g. Ce4, Po2, Gal alpha-3-(Gal alpha-6)(Fuc alpha-2)Gal beta-6Man alpha-, whereas A. virosa, Av-3, has a more complex, highly alpha-fucosylated terminus, Gal alpha-3 (Fuc alpha-2)(Fuc alpha-6)Gal alpha-2(Gal alpha-3)Gal beta-6Man alpha-. L. edodes basidiolipids show further elongation by alpha-mannoside, e.g. Le3, Man alpha-2Man alpha-6Gal alpha-3(Fuc alpha-2)Gal beta-6Man alpha-, C. exipuliformis glycolipid by alpha-glucoside, i.e. Ce3, Glc alpha-6Gal beta-6Man alpha-. Basidiolipid Ls1 from L. scabrum, notably, has a 3-alpha-mannosylated alpha-fucose, i.e. Gal alpha-6(Man alpha-3Fuc alpha-2)Gal alpha-6Gal beta-6Man alpha-. In conclusion, basidiolipids, though identical in their ceramide constitution, display wide and systematic mushroom species dependent variabilities of their chemical structures.

Isolation and partial characterization of Fes p 4 allergen.

More than 75% of grass pollen-allergic patients produce specific IgE antibodies against group-4 allergens. Purification and characterization of different grass group-4 allergens should help to further understand their allergenicity. In this study, an attempt was made to isolate and characterize Fes p 4 allergen by several biochemical and immunochemical methods. Fes p 4 was purified by a combination of chromatographic techniques (gel permeation and ion exchange chromatography). Isolated protein revealed four main spots at a molecular weight of 60 kDa and a pI ranging from 8.7 to 9.1. Eight sera were selected from patients with positive result of skin prick test to the mixture of grass pollen extracts. ELISA inhibition technique was used to study Fes p 4-specific IgE in the patients' sera. ELISA to Festuca pratensis was inhibited up to 80% by F. pratensis pollen extract and up to 48% by Fes p 4. 2D-PAGE-immunoblot was used to identify allergenic and antigenic components of Fes p 4 with patients' IgE and monoclonal antibodies (MABs). Three components of purified protein expressed IgE binding ability. Two MABs which recognized unrelated regions on Phl p 4, bound three components of Fes p 4. The role of the carbohydrate moiety in allergenicity was examined with individual patient sera by using periodate-treated Fes p 4. Six out of eight patients reduced IgE binding to periodate-treated allergen. Isolated Fes R 4 glycoprotein consisted of four components, three of which were allergenic, and share common epitopes specific for grass group-4 homologs. The results of periodate oxidation of Fes p 4 suggest that the carbohydrate moiety is involved in IgE binding.