Synthesis and neuroprotective effects of new genipin derivatives against glutamate-induced oxidative damage.

Glutamate-induced oxidative stress is well-known to play a crucial role in the development of neurodegenerative diseases, such as stroke. Genipin, a natural iridoid compound, has demonstrated potential neuroprotective properties but is unstable in physiological conditions. The present study aimed to develop new derivatives of genipin that exhibit improved stability and activity for the treatment of stroke. Nineteen new derivatives were thus designed and synthesized. Their neuroprotective effect against glutamate-induced injury was evaluated in HT22 cells. Among the newly synthesized derivatives, 3e demonstrated significantly greater neuroprotection and improved stability compared to genipin. Specifically, 0.01 µM of 3e was found to effectively attenuate glutamate-induced oxidative damage by inhibiting ROS over-accumulation, reducing MDA content, and restoring the endogenous antioxidative system. Further investigation revealed that 3e inhibited oxidative stress by downregulating the phosphorylation levels of p38 MAPK and activating Nrf2 and HO-1 proteins. These results suggested that 3e has the potential to serve as a promising candidate for the treatment of stroke by protecting against glutamate-induced oxidative stress.

Chemical composition and anti-inflammatory activities of Castanopsis honey.

Castanopsis is diffusely spread in tropical and subtropical regions and is an important nectar source plant in China. The Castanopsis honey (CH) is characterized by its bitter taste. However, its composition and functions remain unclear. In this study, the physicochemical parameters, chemical composition, and antioxidant capacity of CH were comprehensively investigated, with the anti-inflammatory effects of the Castanopsis honey extract (CHE) evaluated based on the RAW 264.7 cell inflammatory model. The results revealed a high level of quality in CH based on the quality standards. Among a total of 84 compounds identified in CH, 5 high response compounds and 29 phenols were further quantified by UPLC-Q/TOF-MS. The high content of phenylethylamine (117.58 ± 64.81 mg kg-1) was identified as a potential marker of CH. Furthermore, the CH showed evident antioxidant activities, and the anti-inflammatory activities of CHE were observed to inhibit the release of nitric oxide (NO) and reduce the content of tumor necrosis factor alpha (TNF-α) and improve the content of interleukin-10 (IL-10) by regulating the NF-κB pathway. Our study indicates that CH has sound physicochemical properties and biological activities with a high level of quality, providing strong experimental evidence to support the further economic and agricultural development and application of CH.

Identification of Sitogluside as a Potential Skin-Pigmentation-Reducing Agent through Network Pharmacology.

Many traditional Chinese medicines (TCMs) with skin-whitening properties have been recorded in the Ben-Cao-Gang-Mu and in folk prescriptions, and some literature confirms that their extracts do have the potential to inhibit pigmentation. However, no systematic studies have identified the specific regulatory mechanisms of the potential active ingredients. The aim of this study was to screen the ingredients in TCMs that inhibit skin pigmentation through a network pharmacology system and to explore underlying mechanisms. We identified 148 potential active ingredients from 14 TCMs, and based on the average "degree" of the topological parameters, the top five TCMs (Fructus Ligustri Lucidi, Hedysarum multijugum Maxim., Ampelopsis japonica, Pseudobulbus Cremastrae Seu Pleiones, and Paeoniae Radix Alba) that were most likely to cause skin-whitening through anti-inflammatory processes were selected. Sitogluside, the most common ingredient in the top five TCMs, inhibits melanogenesis in human melanoma cells (MNT1) and murine melanoma cells (B16F0) and decreases skin pigmentation in zebrafish. Furthermore, mechanistic research revealed that sitogluside is capable of downregulating tyrosinase (TYR) expression by inhibiting the ERK and p38 pathways and inhibiting TYR activity. These results demonstrate that network pharmacology is an effective tool for the discovery of natural compounds with skin-whitening properties and determination of their possible mechanisms. Sitogluside is a novel skin-whitening active ingredient with dual regulatory effects that inhibit TYR expression and activity.

Monofloral Triadica Cochinchinensis Honey Polyphenols Improve Alcohol-Induced Liver Disease by Regulating the Gut Microbiota of Mice.

Honey produced from medicinal plants holds great promise for human health. Increasing evidence suggests that the gut microbiota plays an important role in liver pathology after alcohol intake. The aim of this study was to identify the polyphenol composition of triadica cochinchinensis honey (TCH), and to study the potential effect of honey polyphenols on the regulation of gut microbes in mice with alcohol-induced liver injury and the improvement of alcohol-induced liver disease. For these purposes, a total of 190 compounds were identified and 27 of them were quantified by ultraperformance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UPLC-Q/TOF-MS) and we successfully established a mouse model of alcohol-induced liver injury. The results show that TCH polyphenols can significantly restore the levels of ALT and AST, and TCH intervention can significantly improve the pathological changes of liver tissue in alcohol-exposed mice. Additionally, a significant decrease was observed in Firmicutes/Bacteroidetes after TCH treatment. Moreover, KEGG pathways of ATP-binding cassette (ABC) transporters, two-component system and biosynthesis of amino acids enriched the most differentially expressed genes after TCH intervention for 8 weeks. Our results may have important implications for the use of TCH as a functional food component with potential therapeutic utility against alcohol-induced liver disease.

Cruciferous vegetables (Brassica oleracea) confer cytoprotective effects in Drosophila intestines.

Varieties and cultivars of the cruciferous vegetable Brassica oleracea are widely presumed to elicit positive influences on mammalian health and disease, particularly related to their indole and sulforaphane content. However, there is a considerable gap in knowledge regarding the mechanisms whereby these plant-derived molecules elicit their beneficial effects on the host. In this study, we examined the chemical variation between B. oleracea varieties and evaluated their capacity to both activate Nrf2 in the Drosophila intestine and elicit cytoprotection. Ten types of edible B. oleracea were purchased and B. macrocarpa was wild collected. Fresh material was dried, extracted by double maceration and green kale was also subjected to anaerobic fermentation before processing. Untargeted metabolomics was used to perform Principal Component Analysis. Targeted mass spectral analysis determined the presence of six indole species and quantified indole. Extracts were tested for their capacity to activate Nrf2 in the Drosophila intestine in third instar Drosophila larvae. Cytoprotective effects were evaluated using a paraquat-induced oxidative stress gut injury model. A "Smurf" assay was used to determine protective capacity against a chemically induced leaky gut. Extracts of Brussels sprouts and broccoli activated Nrf2 and protected against paraquat-induced damage and leaky gut. Lacto-fermented kale showed a cytoprotective effect, increasing survival by 20% over the non-fermented extract, but did not protect against leaky gut. The protective effects observed do not directly correlate with indole content, suggesting involvement of multiple compounds and a synergistic mechanism.

Peanut selenium distribution, concentration, speciation, and effects on proteins after exogenous selenium biofortification.

Fortification of Se is vital importance for both nutritional demand and prevention of Se-deficiency-related diseases. To better understand t selenium distribution, concentration, speciation, its effects on proteins, and cytotoxic activity, the biofortification of exogenous Se in peanut was conducted in this study. Our data have shown that foliar spraying of Se-riched fertilizer was more efficient for biotransformation of inorganic Se to organic Se by peanut plant. Besides, the Se content in peanut was increased in a dose-dependent manner. Our present study also confirmed that SeCys2, MeSeCys, and SeMet were the main Se speciation within peanut proteins. Moreover, the secondary structure and thermostability of peanut protein were altered as a result of the Se treatments, and these alterations could be attributed to the replacements of cysteine and methionine by selenocysteine and selenomethionine, respectively. The Se-enriched peanut protein could significantly inhibit the growth of Caco-2 and HepG2 in a concentration-dependent manner.

In Situ Adsorption of Mixed Anionic/Cationic Collectors in a Spodumene-Feldspar Flotation System: Implications for Collector Design.

Herein, we investigated the effects of mixed collectors with varying alkyl chain lengths and ligand types on the hydrophobicity of the spodumene-feldspar flotation system. Various collector-mineral interactions were compared using in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy with two-dimensional correlation spectroscopy (2D-COS), in situ microcalorimetry, and X-ray photoelectron spectroscopy (XPS). The highest flotation separation performance can be achieved at a molar ratio of 6:1 and pH 8-9. The in situ microcalorimetry results revealed that the difference in the adsorption reaction heat of the mixed collector is larger than that of the single anionic collector. Moreover, the inconformity between the magnitude of adsorption reaction heat and the results observed for flotation recovery indicates that the heat of the reaction presumably involves the adsorption configurations of the collectors and the amounts adsorbed. In in situ ATR-FTIR with 2D-COS, it can be observed that octanohydroxamic acid/dodecylamine (OHA/DDA) is adsorbed much more intensely onto feldspar than onto spodumene due to the availability of more space on feldspar for the subsequent sorption of DDA after the prior bidentate chemisorption of OHA under alkaline conditions, whereas the sodium oleate (NaOL)/DDA adsorption sequence at pH 4-5 was the reverse of that at pH 8-9. Lastly, XPS was employed to provide further supplemental evidence for the bonding between these two minerals and single anionic/mixed collectors at the optimal pH of 8-9. In this study, the powerful in situ detection technologies can establish a new platform for exploring the underlying mechanism of new reagents at the solid-liquid interface. Moreover, the in-depth understanding related to the adsorption behavior of the mixed collector is beneficial for facilitating the selection and design of efficient and environmentally friendly flotation collectors with improved selectivity.

Black rice-derived anthocyanins inhibit HER-2-positive breast cancer epithelial-mesenchymal transition-mediated metastasis in vitro by suppressing FAK signaling.

This study aimed to investigate the role of focal adhesion kinase (FAK) signaling in the inhibitory effects of black rice anthocyanins (BRACs) on human epidermal growth factor receptor-2 (HER-2)-positive human breast cancer cell metastasis, using the MCF-10A, MCF-7 and MDA-MB-453 cells. BRACs exerted an anti-metastatic effect on the HER-2-positive breast cancer cells. The effects of BRACs on the proliferation of the MDA-MB-453 cells were examined by cell counting kit-8 assay. A wound-healing assay was used to examine the effects of BRACs on the migration of the breast cancer cells. BRACs interrupted migration and invasion. BRACs decreased the migration distance of the HER-2-positive human breast cancer cells, MDA-MB-453, by 37% compared with the cells in the untreated group. They also reduced the number of invading MDA-MB-453 cells by 68%. In addition, BRACs exerted an inhibitory effect on epithelial-mesenchymal transition. Western blot analysis revealed that BRACs decreased the phosphorylation of FAK, cSrc and p130Cas. The FAK inhibitor, Y15, was also used to further evaluate the role of FAK signaling in the anti-metastatic effects of BRACs on MDA-MB-453 cells. The results of western blot analysis revealed that BRACs increased the expression of the epithelial marker, E-cadherin, and decreased the expression of the mesenchymal markers, fibronectin and vimentin, in the MDA-MB­453 cells. In addition, BRACs decreased the interaction between HER-2 and FAK, FAK and cSrc, cSrc and p130Cas, and between FAK and p130Cas. These results suggest that BRACs suppress the metastasis of HER-2-positive breast cancer in vitro, and that the cSrc/FAK/p130Cas pathway plays a vital role in this inhibitory effect.

[Chemical constituents of Physalis pubescens].

Chemical constituents of 95% ethanol extract of the dried persistent calyx of Physalis pubescens were investigated. By chromatography on a silica gel column and reverse-phase preparative HPLC, 10 compounds were isolated from the dichloromethane fraction. Based on the MS and 1D/2D NMR data, these compounds were identified as 5-O-(E-feruloyl) blumenol (1), isovanillin (2), (E) -ethyl 3-(4-hydroxyphenyl) acrylate (3), 4-hydroxybenzaldehyde(4), 4-methylphenol (5), (E) -methyl cinnamate (6), 7,3',4' trimethoxyquercetin (7), 5,3', 5'-trihydroxy-3,7,4'-trimethoxyflavone(8), danielone (9), and 5,5'-diisobutoxy-2,2'-bifuran (10).

A high-resolution peak fractionation approach for streamlined screening of nuclear-factor-E2-related factor-2 activators in Salvia miltiorrhiza.

Generation of a high-purity fraction library for efficiently screening active compounds from natural products is challenging because of their chemical diversity and complex matrices. In this work, a strategy combining high-resolution peak fractionation (HRPF) with a cell-based assay was proposed for target screening of bioactive constituents from natural products. In this approach, peak fractionation was conducted under chromatographic conditions optimized for high-resolution separation of the natural product extract. The HRPF approach was automatically performed according to the predefinition of certain peaks based on their retention times from a reference chromatographic profile. The corresponding HRPF database was collected with a parallel mass spectrometer to ensure purity and characterize the structures of compounds in the various fractions. Using this approach, a set of 75 peak fractions on the microgram scale was generated from 4mg of the extract of Salvia miltiorrhiza. After screening by an ARE-luciferase reporter gene assay, 20 diterpene quinones were selected and identified, and 16 of these compounds were reported to possess novel Nrf2 activation activity. Compared with conventional fixed-time interval fractionation, the HRPF approach could significantly improve the efficiency of bioactive compound discovery and facilitate the uncovering of minor active components.

Direct assessment of phytochemicals inherent in plant tissues using extractive electrospray ionization mass spectrometry.

An ambient pressure ionization mass spectrometric strategy called internal extractive electrospray ionization mass spectrometry (iEESI-MS) has been developed and applied for direct profiling of labile phytochemicals inherent in various native plant tissues, including leaves, roots, and fruits. By passing the electrospray solvent through the plant tissue, a variety of phytochemicals, such as amino acids, sugars (e.g., glucose, sucrose, polysaccharides, etc.), and alkaloids, were continuously extracted from the sample interior, driven toward the natural/cut electro-spraying tip, and vaporized into gaseous ions for mass spectrometric interrogation. Phytochemical patterns obtained by iEESI-MS permit a rapid differentiation between various species of ginkgo plant and strawberry maturity stages, as well as characterization of physiological/pathologic conditions of chlorophytum comosum. Our experimental results further demonstrate that the established iEESI-MS approach is potentially useful for direct phytochemomics studies with minimal biodegradation, allowing elucidation of plant metabolism with high speed, specificity, and simplicity of analysis.

Enzymatic production of zero-trans plastic fat rich in α-linolenic acid and medium-chain fatty acids from highly hydrogenated soybean oil, Cinnamomum camphora seed oil, and perilla oil by lipozyme TL IM.

In the present study, zero-trans α-linolenic acid (ALA) and medium-chain fatty acids (MCFA)-enriched plastic fats were synthesized through enzymatic interesterification reactions from highly hydrogenated soybean oil (HSO), Cinnamomum camphora seed oil (CCSO), and perilla oil (PO). The reactions were performed by incubating the blending mixtures of HSO, CCSO, and PO at different weight ratios (60:40:100, 70:30:100, 80:20:100) using 10% (total weight of substrate) of Lipozyme TL IM at 65 °C for 8 h. After reaction, the physical properties (fatty acids profile, TAG composition, solid fat content, slip melting point, contents of tocopherol, polymorphic forms, and microstructures) of the interesterified products and their physical blends were determined, respectively. Results showed that the fatty acid compositions of the interesterified products and physical blends had no significant changes, while the content of MCFA in both interesterified products and physical blends increased to 8.58-18.72%. Several new types of TAG species were observed in interesterified products (SSL/SLS, PLO/LLS, and OLLn/LnLO/LOLn). It should be mentioned that no trans fatty acids (TFA) were detected in all products. As the temperature increased, the solid fat content (SFC) of interesterified products was obviously lower than that of physical blends. The SFCs of interesterified products (60:40:100, 70:30:100, and 80:20:100, HSO:CCSO:PO) at 25 °C were 6.5%, 14.6%, and 16.5%, respectively, whereas the counterparts of physical blends were 32.5%, 38.5%, and 43.5%, respectively. Meanwhile, interesterified products showed more ß' polymorphs than physical blends, in which ß' polymorph is a favorite form for production of margarine and shortening. Such zero-trans ALA and MCFA-enriched fats may have desirable physical and nutritional properties for shortenings and margarines.

Immunoprecipitation coupled with HPLC-MS/MS to discover the aromatase ligands from Glycyrrhiza uralensis.

Affinity chromatography, applied to discover the enzyme inhibitors, needs special column with target protein and its carrier. Selection of stationary phase and mobile phase needs careful considerations due to the characteristics of proteins. In this study, a method immunoprecipitation (IP) coupled with HPLC-DAD-MS was developed to discover the aromatase ligands from Glycyrrhiza uralensis. An SB-C18 column was employed to separate target compounds without special consideration in mobile phase. Twenty-one compounds, including isolated compounds 4, 7, 8, 10, 11, 13, 15, 18-20, 23 and non-isolated compounds A-J, were found to have good affinity to aromatase by LC-MS. Seven of them (7, 15, 18, 19, 23, D, E) were detected to bind with aromatase in MCF-7 cells by IP coupled with HPLC-MS/MS. Bioassays disclosed aromatase inhibitory activities of the isolated compounds mentioned above, verifying the efficiency of IP coupled with HPLC-MS/MS as a method to screen aromatase ligands.

Quinone reductase (QR) inducers from Andrographis paniculata and identification of molecular target of andrographolide.

In the present study, it was demonstrated that the petroleum extract of Andrographis paniculata (AP) had quinone reductase (QR) inducing activity, which might be attributed to the modification of key cysteine residues in Keap1 by Michael addition acceptors (MAAs) in it. To screen MAAs in AP, glutathione (GSH) was employed, and a LC/MS/MS method was implied. Three compounds, andrographoside, andrographolide, 14-deoxy-14,15-dehydroandrographolide were revealed could well conjugated with GSH. Then, andrographolide along with 4 new and 14 known compounds were isolated to conduct QR induction evaluation, and the CD (the concentration required to double the activity of QR) value of andrographolide is 1.43µM. The QR induce activity of andrographolide might be attributed to its targeting multiple cysteine residues in Keap1, therefore, the alkylation of Keap1 by andrographolide was further studied and the result showed that four cysteine residues: Cys77, Cys151, Cys273 and Cys368 were alkylated, which indicated that Keap1 is a potential target for the QR induce activity of andrographolide.

Trace analysis in complex mixtures using a high-component filtering strategy with liquid chromatography-mass spectrometry.

Trace constituents are widely present in complex mixtures, and trace analysis is challenging because of the unpredictable matrix. In this work, a high-component filtering strategy was developed for improved analysis of trace constituents in complex sample by liquid chromatography-mass spectrometry (LC-MS). Using a specifically designed chromatographic apparatus, the high-abundant fractions were filtered prior to LC-MS analysis. The samples complexity was reduced and the sample-loading amount for the rest low-level fractions can be considerably increased. The application of this approach was illustrated with an analytically challenging sample, a traditional Chinese herbal medicine named Compound Danshen Sample. We observed that the loss rate for 12 analytes during the filtering procedure ranged from 6.54 to 26.11%, but showed a stable repeatability with RSD<3.79%. The proposed filtering method with quadrupole time-of-flight mass spectrometritry (Q-TOF/MS) enhanced the detection capacity, offering a comprehensive characterization of 133 compounds in Compound Danshen Samples. The quantification sensitivity was also improved in trace analysis, allowing six low compounds that cannot be quantified by the traditional methods to be tested by the filtering method. It can be predicted that the qualitative and quantitative trace analysis will be greatly improved when the loading samples is increased resulting from the filtration of high-level targets. The proposed strategy is promising to monitor trace constituents in diverse complex mixtures in the analytical field of pharmaceutics, metabonomics and environments.

Enzymatic interesterification of palm stearin with Cinnamomum camphora seed oil to produce zero-trans medium-chain triacylglycerols-enriched plastic fat.

It is known that Cinnamomum camphora seed oil (CCSO) is rich in medium-chain fatty acids (MCFAs) or medium-chain triacylglycerols (MCTs). The purpose of the present study was to produce zero-trans MCTs-enriched plastic fat from a lipid mixture (500 g) of palm stearin (PS) and CCSO at 3 weight ratios (PS:CCSO 60:40, 70:30, 80:20, wt/wt) by using lipase (Lipozyme TL IM, 10% of total substrate) as a catalyst at 65 °C for 8 h. The major fatty acids of the products were palmitic acid (C16:0, 42.68% to 53.42%), oleic acid (C18:1, 22.41% to 23.46%), and MCFAs (8.67% to 18.73%). Alpha-tocopherol (0.48 to 2.51 mg/100 g), γ-tocopherol (1.70 to 3.88 mg/100 g), and δ-tocopherol (2.08 to 3.95 mg/100 g) were detected in the interesterified products. The physical properties including solid fat content (SFC), slip melting point (SMP), and crystal polymorphism of the products were evaluated for possible application in shortening or margarine. Results showed that the SFCs of interesterified products at 25 °C were 9% (60:40, PS:CCSO), 18.50% (70:30, PS:CCSO), and 29.2% (80:20, PS:CCSO), respectively. The ß' crystal form was found in most of the interesterified products. Furthermore, no trans fatty acids were detected in the products. Such zero-trans MCT-enriched fats may have a potential functionality for shortenings and margarines which may become a new type of nutritional plastic fat for daily diet.

[Effects of retained needling and non-retained needling on regulation of the cardiac autonomic nerve].

OBJECTIVE: To investigate the mechanism of retained needling and non-retained needling on the physiologic regulating effect of the cardiac autonomic nerve in healthy persons. METHODS: Sixty volunteers were randomly divided into a retained needling group and a non-retained needling group, 30 cases in each group. They were all received perpendicular needling at Neiguan (PC 6) and Jianshi (PC 5) retaining for 20 min in the retained nee dling group and without retaining in the non-retained needling group. The heart rate variability (HRV) indices, i.e., R-R interval, low frequency (LF), high frequency (HF) and LF/HF were observed. RESULTS: Compared with before acupuncture, the R-R interval were significant increased during the stimulation period and during the post-stimulation period in the retained needling group, especially during the stimulation period (all P < 0.01), and during the post-stimulation period of 5 min and 10 min in the non-retained needling group (P < 0.01, P < 0.05), then returned to pre-acupuncture level quickly, and the R-R interval in the retained needling group was significant ly higher than that in the non retained needling group at 10 min (P < 0.05). At 5 min, the HF were significantly higher, and the LF/HF were significantly lower than those before acupuncture (P < 0.01, P < 0.05), and there was no obvious change in LF in both groups. CONCLUSION: Retained needling and non-retained needling at Neiguan (PC 6) and Jianshi (PC 5) can both excite cardiac vagus nerve briefly and then the acupuncture effects reduce grad ually until disappearance, while no obvious changes can be found with cardiac sympathetic activity. Both of needling can reduce heart rate and the action time of heart rate decrease by retained needling is longer than non-retained needling.

A deuterium-labelling mass spectrometry-tandem diode-array detector screening method for rapid discovery of naturally occurring electrophiles.

Because electrophiles regulate many signalling pathways in cells, by modifying cysteine residues in proteins, they have a wide range of biological activity. In this study, a deuterium-labelling mass spectrometry-tandem diode-array detector (MS-DAD) screening method was established for rapid discovery of naturally occurring electrophiles. Glutathione (GSH) was used as a probe and incubated with natural product extracts. To distinguish different types of electrophile, incubation was performed in two reaction solvents, H(2)O and D(2)O. Ten types of naturally occurring electrophile were chosen, on the basis of their properties, to undergo the screening assay. By using this screening method, we successfully discovered the bioactive electrophile 4-hydroxyderricin in an ethanol extract of Angelica keiskei. This electrophile had potent NAD(P)H:quinone oxidoreductase 1 (NQO1)-inducing activity at a concentration of 20 µmol L(-1).

Sinapine detection in radish taproot using surface desorption atmospheric pressure chemical ionization mass spectrometry.

Plant research and natural product detection are of sustainable interests. Benefited by direct detection with no sample preparation, sinapine, a bioactive chemical usually found in various seeds of Brassica plants, has been unambiguously detected in radish taproot (Raphanus sativus) tissue using a liquid-assisted surface desorption atmospheric pressure chemical ionization mass spectrometry (DAPCI-MS). A methanol aqueous solution (1:1) was nebulized by a nitrogen sheath gas toward the corona discharge, resulting in charged ambient small droplets, which affected the radish tissue for desorption/ionization of analytes on the tissue surface. Thus, sinapine was directly detected and identified by tandem DAPCI-MS experiments without sample pretreatment. The typical relative standard deviation (RSD) of this method for sinapine detection was 5-8% for six measurements (S/N=3). The dynamic response range was 10(-12)-10(-7) g/cm2 for sinapine on the radish skin surface. The discovery of sinapine in radish taproot was validated by using HPLC-UV methods. The data demonstrated that DAPCI assisted by solvent enhanced the overall efficiency of the desorption/ionization process, enabling sensitive detection of bioactive compounds in plant tissue.

Flagellin administration protects gut mucosal tissue from irradiation-induced apoptosis via MKP-7 activity.

BACKGROUND AND AIMS: Radiotherapy for neoplastic disease is associated with significant adverse enteric effects associated with excessive cell death. Ionising radiation induces cell death by a mechanism that is dependent on JNK (c-jun N-terminal kinase) pathway signalling. Additionally, it is known that cells exposed to extracellular bacterial products such as flagellin, pleiotropically activate a number of innate immune pathways, including that of JNK. The JNK pathway controls its own activity by inducing the transcription of mitogen-activated protein kinase phosphatase-7 (MKP-7) which directly targets phosphorylated JNK, thus functioning as a negative feedback loop. Previously, it has been shown that flagellin limits ionising radiation-induced mortality in mice, but the cellular mechanism of protection remained unknown. METHODS: Wild-type C57BL/6 or tlr5(-/-) C57BL/6 were injected with flagellin 2 h before exposure to irradiation, and their intestines were examined for apoptosis. Candidate proteins mediating cytoprotection from irradiation were identified by expression profiling. One of these candidates, MKP-7, was cloned and packaged into adenovirus particles, used to infect cultured cells, and examined for the extent to which its activity reduced cellular apoptosis by flow cytometry or immunoblot analysis. RESULTS: Flagellin pretreatment protected mice from radiation-induced intestinal mucosal injury and apoptosis via a Toll-like receptor 5 (TLR5)-dependent mechanism. Expression profiling of flagellin-treated mice showed upregulation of MKP-7, an inducible repressor of the JNK pathway. MKP-7 expression reached a maximum at 2 h after flagellin treatment, coinciding with suppression of phosphorylated JNK and JNK pathway inhibition. Furthermore, constitutive MKP-7 expression protected cultured cells from radiation-induced apoptosis. CONCLUSIONS: Flagellin is a promising adjuvant for suppressing ionising radiation-induced injury. MKP-7 activity exhibits cytoprotective effects, and is thus a candidate cellular molecule for limiting the damaging effect of radiotherapy on the gastreointestinal system.