A Study of the Mechanisms and Characteristics of Fluorescence Enhancement for the Detection of Formononetin and Ononin.

In this work, the origins for the spectral difference between two isoflavones, formononetin (F) and ononin (FG), are revealed via a comparison study of the fluorescence molecular structure. The fluorescence enhancement of FG in hot alkaline conditions is reported for the first time. For F, there is almost no fluorescence under acidic conditions, but when the pH is >4.8, its fluorescence begins to increase due to the deprotonation of 7-OH. Under a pH between 9.3 and 12.0, the anionic form of F produces a strong and stable fluorescence. The fluorescence quantum yield (Yf) of F is measured to be 0.042. FG shows only weak fluorescence in aqueous solutions under a wide range of pH until it is placed in hot alkaline solutions, which is attributed to the cleavage reaction of the γ-pyrone ring in FG. The Yf of FG is determined to be 0.020. Based on the fluorescence sensitization methods of F and FG, the quantitative analysis and detection of two substances can be realized. The limit of the detections for F and FG are 2.60 ng·mL-1 and 9.30 ng·mL-1, respectively. The linear detection ranges of F and FG are 11.7~1860 ng·mL-1 and 14.6~2920 ng·mL-1, respectively. Although the structural relationship between F and FG is glycoside and aglycone, under hot alkaline conditions, the final products after the cleavage and hydrolysis reactions are essentially different. The different fluorescence characteristics between F and FG pave a way for further identification and a quantitative analysis of the corresponding components in Chinese herbal medicine.

Laurel Attenuates Dexamethasone-Induced Skeletal Muscle Atrophy In Vitro and in a Rat Model.

Prevention of muscle atrophy contributes to improved quality of life and life expectancy. In this study, we investigated the effects of laurel, selected from 34 spices and herbs, on dexamethasone (DEX)-induced skeletal muscle atrophy and deciphered the underlying mechanisms. Co-treatment of C2C12 myotubes with laurel for 12 h inhibited the DEX-induced expression of intracellular ubiquitin ligases-muscle atrophy F-box (atrogin-1/MAFbx) and muscle RING finger 1 (MuRF1)-and reduction in myotube diameter. Male Wistar rats were supplemented with 2% laurel for 17 days, with DEX-induced skeletal muscle atrophy occurring in the last 3 days. Laurel supplementation inhibited the mRNA expression of MuRF1, regulated DNA damage and development 1 (Redd1), and forkhead box class O 1 (Foxo1) in the muscles of rats. Mechanistically, we evaluated the effects of laurel on the cellular proteolysis machinery-namely, the ubiquitin/proteasome system and autophagy-and the mTOR signaling pathway, which regulates protein synthesis. These data indicated that the amelioration of DEX-induced skeletal muscle atrophy induced by laurel, is mainly mediated by the transcriptional inhibition of downstream factors of the ubiquitin-proteasome system. Thus, laurel may be a potential food ingredient that prevents muscle atrophy.

iTRAQ-Based Quantitative Proteomics Reveals the Energy Metabolism Alterations Induced by Chlorogenic Acid in HepG2 Cells.

Epidemiological studies have suggested that coffee consumption is associated with a decrease in the risk of developing obesity and diabetes; however, the detailed mechanisms underlying these effects of coffee consumption remain poorly understood. In this study, we examined the effects of chlorogenic acid on energy metabolism in vitro. Hepatocellular carcinoma G2 (HepG2) cells were cultured in a medium containing chlorogenic acid. Chlorogenic acid increased the activity of mitochondrial enzymes, including citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase (MDH), which are involved in the tricarboxylic acid (TCA) cycle. Proteome analysis using the isobaric tags for the relative and absolute quantitation (iTRAQ) method revealed the upregulation of proteins involved in the glycolytic system, electron transport system, and ATP synthesis in mitochondria. Therefore, we propose a notable mechanism whereby chlorogenic acid enhances energy metabolism, including the TCA cycle, glycolytic system, electron transport, and ATP synthesis. This mechanism provides important insights into understanding the beneficial effects of coffee consumption.

Restoration of Intestinal Mucosa in Euphorbia kansui-treated Severe Acute Pancreatitis Rats based on HMGB1/MFG-E8 Expression.

BACKGROUND: The pathogenesis of Severe Acute Pancreatitis (SAP) is mediated substantially by dysfunctions in the intestinal barrier. Euphorbia kansui (EK) is a medicinal plant used widely in traditional Chinese medicine to treat inflammation; however, its efficacy and mechanism of action in SAP treatment are not yet well understood. OBJECTIVE: To investigate the role of EK in intestinal barrier tissue repair and in the pathogenesis and development of SAP. METHODS: The rat SAP model was established by a retrograde injection of sodium taurocholate into the pancreatic bile duct. The SAP model group and the SAP + EK treatment groups were divided into 6 subgroups according to timing: 2, 6, 12, 24, 48, or 72h after inducing SAP. The progression of the SAP rats and of the rats receiving the EK treatment was evaluated using the ascites volume, serum amylase and plasma endotoxin levels, and histological grading of intestinal mucosal damage. In addition, serum inflammatory factor contents were measured using Enzyme-Linked Immunosorbent Assay (ELISA) tests and apoptotic cells in damaged ileum tissue were detected using TUNEL staining. Apoptosis markers and other signaling proteins in intestinal mucosal cells were detected by immunohistochemical assays and then validated by combining these data with quantitative polymerase chain reactions and western blotting. RESULTS: Compared with the results of the SAP model rats, the results of the rats that received EK treatment demonstrated that EK could effectively reduce the ascites volume and serum amylase and plasma endotoxin levels. EK treatment also greatly reduced the abnormal intestinal morphological alterations in the rat SAP model and significantly downregulated the serum contents of Interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α. EK treatment inhibited the elevation of capapse-3, inhibited the decrease of the Bcl-2 protein, and decreased the number of apoptotic cells in rat ileum tissue. Finally, EK treatment abrogated the increase of HMGB1 and the suppression of MFG-E8 protein expression in the SAP + EK rat ileum tissue. CONCLUSION: EK suppresses SAP pathogenesis by restoring the intestinal barrier function and modulating the HMGB1/MFG-E8 signaling axis.

Oridonin Sensitizes Hepatocellular Carcinoma to the Anticancer Effect of Sorafenib by Targeting the Akt Pathway.

BACKGROUND: Oridonin is the core bioactive component of Rabdosia rubescens, a traditional Chinese herbal medicine used in the treatment of hepatoma. Sorafenib, a targeted therapeutic agent for advanced hepatocellular carcinoma (HCC), has recently been shown to exert limited clinical effects. However, few studies have focused on the synergistic effect of these two drugs on hepatocellular carcinoma. METHODS: We treated different HCC cell lines with different concentrations of oridonin and sorafenib and assessed the viability by using MTT assays and examined proliferation, migration, invasion and apoptosis after cotreatment of HepG2 cells with 20 µM oridonin and 5 µM sorafenib via colony formation assays, Transwell assays and flow cytometry. Regulatory effects were measured by Western blotting. The in vivo synergistic effect was confirmed through xenograft tumor models, and tumor tissues were analyzed by immunohistochemistry. RESULTS: The inhibitory effects of oridonin and sorafenib cotreatment on HCC cells were stronger than those of either drug alone. In addition, combined treatment with the two drugs synergistically inhibited epithelial-mesenchymal transition and the Akt pathway but not NF-κB or MAPK signaling. Akt phosphorylation by SC79 reversed the inhibitory effects of the combined treatment. Synergistic inhibition was equally observed in vivo. CONCLUSION: Oridonin combined with sorafenib synergistically inhibited proliferation, migration, invasion, and epithelial-mesenchymal transition and induced apoptosis by targeting the Akt pathway but not NF-κB or MAPK signaling.

Differential effects of olive oil, soybean oil, corn oil and lard oil on carbon tetrachloride-induced liver fibrosis in mice.

Olive oil could attenuate carbon tetrachloride (CCl4) induced liver fibrosis (LF) in mouse model. The present study aimed to evaluate the effects of other common oils on CCl4 induced LF. Healthy male ICR mice were administered with CCl4 intraperitoneally at 2.5 ml/kg twice a week for total 3 weeks. Mice were pre-treated with olive oil, soybean oil, corn oil or lard oil. After treatment, histopathological changes were observed using Masson trichrome staining, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), hydroxyproline (HYP) and triglyceride (TG) were measured by commercial kits. The expression of LF related genes was detected by quantitative real-time PCR. We found that soybean oil or olive oil significantly reduced ALT and AST levels in serum, and MDA, HYP and TG levels in the liver, compared with corn oil or lard oil. Moreover, Masson trichrome staining and real-time PCR showed that the mice treated with CCl4 dissolved in soybean oil or olive oil had less fibrosis and apoptosis in the liver comparted to the mice treated with CCl4 dissolved in corn oil or lard oil. In conclusion, soybean oil but not corn or lard oil exerts protective effects against CCl4 induced LF in mice, possibly due to its antioxidant activity.

Remarkable Structural Diversity of N-Glycan Sulfation on Influenza Vaccines.

Sulfated N-glycans are biologically important structures derived from enzymatically post-glycosylational modifications of glycoproteins in many therapeutic biologics. The high-throughput analysis of sulfated N-glycomes remains a daunting technical challenge, because of negatively charged heterogeneous composition, large molecular structures, lability of sulfate attachments, and a lack of highly selective enrichment methods. Using liquid chromatography-mass spectrometry, we have analyzed the N-glycans of influenza viral hemagglutinin and neuraminidase from several subtypes of influenza vaccines, and utilized the existing resource to establish an N-glycan library consisting of 927 N-glycan structures and 387 sulfated N-glycan compositions. With the aid of database for data mining, 1380 unique N-glycopeptides were identified and manually validated by de novo glycopeptide sequencing, of which 514 were sulfated at the site-specific locations. We report here a mass spectrometric method that is able to identify and distinguish the isobaric structures of complex and hybrid N-glycans flanked by a terminal sulfation sequon on Gal-GlcNAc and GalNAc-GlcNAc of sulfated-3-Gal, sulfated-6-GlcNAc, and sulfated-4-GalNAc. The database-aided glycoproteomic analyses enable rapid determination of new sulfated-N-glycan structures in large sets of influenza vaccines, including those highly branched nonsialyl sulfo-N-glycans bearing lactosaminic extensions in both complex and hybrid N-glycans that especially interact with sulfotransferases. The novel findings highlight the tremendous structural diversity of sulfated N-glycans and strongly suggest potential functional importance of N-glycan sulfation of influenza glycoproteins.

Cardioprotective Effects of Puerarin-V on Isoproterenol-Induced Myocardial Infarction Mice Is Associated with Regulation of PPAR-Υ/NF-κB Pathway.

Puerarin is a well-known traditional Chinese medicine which has been used for the treatment of cardiovascular diseases. Recently, a new advantageous crystal form of puerarin, puerarin-V, has been developed. However, the cardioprotective effects of puerarin-V on myocardial infarction (MI) heart failure are still unclear. In this research, we aim to evaluate the cardioprotective effects of puerarin-V on the isoproterenol (ISO)-induced MI mice and elucidate the underlying mechanisms. To induce MI in C57BL/6 mice, ISO was administered at 40 mg/kg subcutaneously every 12 h for three times in total. The mice were randomly divided into nine groups: (1) control; (2) ISO; (3) ISO + puerarin injection; (4⁻9) ISO + puerarin-V at different doses and timings. After treatment, cardiac function was evaluated by electrocardiogram (ECG), biochemical and histochemical analysis. In vitro inflammatory responses and apoptosis were evaluated in human coronary artery endothelial cells (HCAECs) challenged by lipopolysaccharide (LPS). LPS-induced PPAR-Υ/NF-κB and subsequently activation of cytokines were assessed by the western blot and real-time polymerase chain reaction (PCR). Administration of puerarin-V significantly inhibits the typical ST segment depression compared with that in MI mice. Further, puerarin-V treatment significantly improves ventricular wall infarction, decreases the incidence of mortality, and inhibits the levels of myocardial injury markers. Moreover, puerarin-V treatment reduces the inflammatory milieu in the heart of MI mice, thereby blocking the upregulation of proinflammatory cytokines (TNF-α, IL-1ß and IL-6). The beneficial effects of puerarin-V might be associated with the normalization in gene expression of PPAR-Υ and PPAR-Υ/NF-κB /ΙκB-α/ΙΚΚα/ß phosphorylation. In the in vitro experiment, treatment with puerarin-V (0.3, 1 and 3 µM) significantly reduces cell death and suppresses the inflammation cytokines expression. Likewise, puerarin-V exhibits similar mechanisms. The cardioprotective effects of puerarin-V treatment on MI mice in the pre + post-ISO group seem to be more prominent compared to those in the post-ISO group. Puerarin-V exerts cardioprotective effects against ISO-induced MI in mice, which may be related to the activation of PPAR-γ and the inhibition of NF-κB signaling in vivo and in vitro. Taken together, our research provides a new therapeutic option for the treatment of MI in clinic.

Phytotoxic Mechanism of Nanoparticles: Destruction of Chloroplasts and Vascular Bundles and Alteration of Nutrient Absorption.

This study focused on determining the phytotoxic mechanism of CeO2 nanoparticles (NPs): destroying chloroplasts and vascular bundles and altering absorption of nutrients on conventional and Bt-transgenic cottons. Experiments were designed with three concentrations of CeO2 NPs including: 0, 100 and 500 mg·L(-1), and each treatment was three replications. Results indicate that absorbed CeO2 nanoparticles significantly reduced the Zn, Mg, Fe, and P levels in xylem sap compared with the control group and decreased indole-3-acetic acid (IAA) and abscisic acid (ABA) concentrations in the roots of conventional cotton. Transmission electron microscopy (TEM) images revealed that CeO2 NPs were absorbed into the roots and subsequently transported to the stems and leaves of both conventional and Bt-transgenic cotton plants via xylem sap. In addition, the majority of aggregated CeO2 NPs were attached to the external surface of chloroplasts, which were swollen and ruptured, especially in Bt-transgenic cotton. The vascular bundles were destroyed by CeO2 nanoparticles, and more damage was observed in transgenic cotton than conventional cotton.

20-Hydroxyeicosatetraenoic acid impairs endothelial insulin signaling by inducing phosphorylation of the insulin receptor substrate-1 at Ser616.

20-Hydroxyeicosatetraenoic acid (20-HETE) induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs) or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser616; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr632 and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser1177 site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser616 is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.

Surface modifications of influenza proteins upon virus inactivation by ß-propiolactone.

Inactivation of intact influenza viruses using formaldehyde or ß-propiolactone (BPL) is essential for vaccine production and safety. The extent of chemical modifications of such reagents on viral proteins needs to be extensively investigated to better control the reactions and quality of vaccines. We have evaluated the effect of BPL inactivation on two candidate re-assortant vaccines (NIBRG-121xp and NYMC-X181A) derived from A/California/07/2009 pandemic influenza viruses using high-resolution FT-ICR MS-based proteomic approaches. We report here an ultra performance LC MS/MS method for determining full-length protein sequences of hemagglutinin and neuraminidase through protein delipidation, various enzymatic digestions, and subsequent mass spectrometric analyses of the proteolytic peptides. We also demonstrate the ability to reliably identify hundreds of unique sites modified by propiolactone on the surface of glycoprotein antigens. The location of these modifications correlated with changes to protein folding, conformation, and stability, but demonstrated no effect on protein disulfide linkages. In some cases, these modifications resulted in suppression of protein function, an effect that correlated with the degree of change of the modified amino acids' side chain length and polarity.

Bioactivity determination of native and variant forms of therapeutic interferons.

The traditional antiviral assays for the determination of interferon potency are reported to have considerable variability between and within assays. Although several reporter gene assays based on interferon-inducible promoter activities have been reported, data from comprehensive validation studies are lacking and few studies have been conducted to analyze the variant forms of interferons, which could have undesirable clinical implications. Here, a reporter gene assay employing a HEK293 cell line stably transfected with luciferase gene under the control of interferon-stimulated response element promoter was developed and validated. The assay was found to be more sensitive, with a larger detection range than the antiviral assay. Several cytokines tested did not interfere with the test, suggesting the assay possesses a certain degree of selectivity. Moreover, the robustness of the assay was demonstrated by minimal variations in the results generated by different analysts and cell passage number (up to 52 passages). Finally, the method was employed to analyze several interferon variants (interferon-α 2a) and we found that the aggregated form has completely lost its potency; while a modest loss of bioactivity in oxidized interferon was observed (approx. 23%), the deamidated form essentially retained its activity.