Silencing XIST mitigated lipopolysaccharide (LPS)-induced inflammatory injury in human lung fibroblast WI-38 cells through modulating miR-30b-5p/CCL16 axis and TLR4/NF-κB signaling pathway.

BACKGROUND: Emerging evidence shows that long noncoding RNA (lncRNA) has been a novel insight in various diseases, including pneumonia. Even though lncRNA X-inactive-specific transcript (XIST) is well studied, its role in pneumonia remains to be largely unrevealed. METHODS: Expression of XIST, miRNA-30b-5p (miR-30b-5p), and CC chemokine ligand 16 (CCL16) was detected using reverse transcriptase quantitative polymerase chain reaction and western blotting; their interaction was confirmed by dual-luciferase reporter assay. Apoptosis, inflammation, and toll-like receptor 4 (TLR4)/NF-κB signaling pathway were measured using methyl thiazolyl tetrazolium assay, flow cytometry, western blotting, and enzyme-linked immunosorbent assay. RESULTS: Lipopolysaccharide (LPS) stimulation decreased cell viability and B cell lymphoma (Bcl)-2 expression, and increased cell apoptosis rate and expression of Bcl-2-associated X protein (Bax), cleaved-caspase-3, interleukin (IL)-6, IL-1ß, and tumor necrosis factor α (TNF-α) in WI-38 cells. Expression of XIST and CCL16 was upregulated in the serum of patients with pneumonia and LPS-induced WI-38 cells, respectively; silencing XIST and CCL16 could suppress LPS-induced apoptosis and inflammation in WI-38 cells, and this protection was abolished by miR-30b-5p downregulation. Moreover, XIST and CCL16 could physically bind to miR-30b-5p, and XIST regulated CCL16 expression via sponging miR-30b-5p. TLR4 and phosphorylated P65 (p-P65) and p-IκB-α were highly induced by LPS treatment, and this upregulation was diminished by blocking XIST, accompanied with CCL16 downregulation and miR-30b-5p upregulation. CONCLUSIONS: Silencing XIST could alleviate LPS-induced inflammatory injury in human lung fibroblast WI-38 cells through modulating miR-30b-5p/CCL16 axis and inhibiting TLR4/NF-κB signaling pathway.

Dynamic Change of Sedimental Microbial Community During Black Bloom-an In Situ Enclosure Simulation Study.

Black bloom is a worldwide environmental problem. Sediment microbes play important roles in the process of black bloom. The dynamic change of sedimental microbial community and their potential link between taste and odor compounds during black bloom was investigated in an in situ black bloom enclosure simulation experiment. Through high-throughput sequencing and analysis, pronounced shifts of sedimental microbial community were observed on the 3rd and 7th day in the black bloom group. Microbes in Cyanobacteria, Verrucomicrobia, Planctomycetes, and Actinobacteria were obviously increased, while microbes from the phyla OP8, Chloroflexi, and Acidobacteria were decreased significantly. RDA analysis revealed that the concentrations of chlorophyll a (Chla), total phosphorus (TP), and turbidity (NTU) in the water and the TP, TN concentrations in the sediment were the main environmental factors that affect the microbial community in the sediment. Correlation analysis revealed that microbes Dechloromonas sp. (OTU003567 and OTU000093), Desulfococcus sp. (OTU000911), Chromatiaceae (OTU001222), and Methanosaeta sp. (OTU004809) were positively correlated with the taste and odor substances in the sediment, such as dimethyl sulfide (DMS), ß-ionone, ß-cyclocitral and geosmin. The sedimental microbial community gradually recovered in the late phase of black bloom, indicating the stability and self-recovery ability of the sedimental microbial community during black bloom. Noteworthily, we observed many possible pathogens increased significantly during the black bloom, which alerts us to keep away from contaminated sediment when black bloom occurred.

Quantitatively evaluating detoxification of the hepatotoxic microcystin-LR through the glutathione (GSH) pathway in SD rats.

Glutathione (GSH) plays crucial roles in antioxidant defense and detoxification metabolism of microcystin-LR (MC-LR). However, the detoxification process of MC-LR in mammals remains largely unknown. This paper, for the first time, quantitatively analyzes MC-LR and its GSH pathway metabolites (MC-LR-GSH and MC-LR-Cys) in the liver of Sprague-Dawley (SD) rat after MC-LR exposure. Rats received intraperitoneal (i.p.) injection of 0.25 and 0.5 lethal dose 50 (LD50) of MC-LR with or without pretreatment of buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH synthesis. The contents of MC-LR-GSH were relatively low during the experiment; however, the ratio of MC-LR-Cys to MC-LR reached as high as 6.65 in 0.5 LD50 group. These results demonstrated that MC-LR-GSH could be converted to MC-LR-Cys efficiently, and this metabolic rule was in agreement with the data of aquatic animals previously reported. MC-LR contents were much higher in BSO + MC-LR-treated groups than in the single MC-LR-treated groups. Moreover, the ratio of MC-LR-Cys to MC-LR decreased significantly after BSO pretreatment, suggesting that the depletion of GSH induced by BSO reduced the detoxification of MCs. Moreover, MC-LR remarkably induced liver damage, and the effects were more pronounced in BSO pretreatment groups. In conclusion, this study verifies the role of GSH in the detoxification of MC-LR and furthers our understanding of the biochemical mechanism for SD rats to counteract toxic cyanobacteria.

The role of glutathione detoxification pathway in MCLR-induced hepatotoxicity in SD rats.

In the present study, we investigated the role of glutathione (GSH) and its related enzymes in Sprague Dawley (SD) rats subjected to microcystin-leucine-arginine (MCLR)-induced hepatotoxicity. SD rats were intraperitoneally (i.p.) injected with MCLR after pretreating with or without buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH synthesis. The depletion of GSH with BSO enhanced MCLR-induced oxidative stress, resulting in more severe liver damage and higher MCLR accumulation. Similarly, the contents of malondialdehyde (MDA), total GSH (T-GSH), oxidized GSH (GSSG) and GSH were significantly enhanced in BSO pretreated rats following MCLR treatment. The study showed that the transcription of GSH-related enzymes such as glutathione-S-transferase (GST), γ-glutamylcysteine synthetase (γ-GCS), glutathione reductase (GR) varied in different ways (expect for glutathione peroxidase (GPx), whose gene expression was induced in all treated groups) with or without BSO pretreatment before MCLR exposure, suggesting an adaptative response of GSH-related enzymes at transcription level to combat enhancement of oxidative stress induced by MCLR when pretreated with BSO. These data suggested the tissues with low GSH concentration are highly vulnerable to MCLR toxicity and GSH was critical for the detoxification in MCLR-induced hepatotoxicity in vivo.

Quantitative liquid chromatography-tandem mass spectrometry method for determination of microcystin-RR and its glutathione and cysteine conjugates in fish plasma and bile.

A rapid and sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of microcystin-RR (MC-RR) and its glutathione and cysteine conjugates (MC-RR-GSH and MC-RR-Cys, respectively) in fish plasma and bile. The analytes were extracted using methanol, followed by an Oasis mixed-mode cation-exchange polymeric sorbent. The separation was performed on a reversed-phase Waters XBridge C18 column with the gradient mobile phase, consisting of water and acetonitrile (both acidified with 0.5‰ formic acid). Mean recoveries of MC-RR, MC-RR-GSH and MC-RR-Cys ranged from 80.7 to 93.7%, 81.1 to 93.1% and 80.3 to 93.2%, respectively, at three concentrations (0.2, 1.0 and 5.0 µg mL(-1)). Limits of detection (LODs) for MC-RR, MC-RR-GSH and MC-RR-Cys were 6, 12 and 9 ng mL(-1), respectively. Limits of quantification (LOQs) were 15, 30 and 22.5 ng mL(-1) for MC-RR, MC-RR-GSH and MC-RR-Cys, respectively. This method makes it feasible for the identification and quantification of MC-RR, MC-RR-GSH and MC-RR-Cys in limited and complex biological fluid samples (such as plasma and bile, typically 50 µL), which were previously excluded or difficult to study due to the relatively large sample volumes.

Simultaneous quantitative determination of microcystin-LR and its glutathione metabolites in rat liver by liquid chromatography-tandem mass spectrometry.

The roles of glutathione (GSH) and cysteine (Cys) in the detoxification of Microcystin-LR (MC-LR) have recently become a popular area of research. However, lacking analysis methods for MC-LR-GSH and MC-LR-Cys (two main GSH pathway metabolites) in mammals, elucidation of the detoxification mechanism and metabolic pathway of MC-LR in mammals is difficult. In this study, a novel method for the simultaneous quantitative analysis of MC-LR, MC-LR-GSH and MC-LR-Cys in rat liver was developed and validated. The analytes were simultaneously extracted from rat liver using 3M sodium chloride solution containing 0.01M EDTA-Na2-5% acetic acid, followed by solid-phase extraction (SPE) on Oasis HLB and silica cartridges and determination by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS). Under the optimized pretreatment conditions and instrument parameters, good recoveries of MC-LR, MC-LR-GSH and MC-LR-Cys were obtained at three concentrations (0.2, 1.0 and 2.5 µg g(-1) dry weight (DW)) with values ranging from 97.7 ± 4.2 to 98.7 ± 5.1%, 70.1 ± 4.8 to 71.1 ± 4.1% and 79.8 ± 3.5 to 81.4 ± 4.0%, respectively. The relative standard deviations (RSDs) of these compounds at 0.2, 1.0 and 2.5 µg g(-1) DW were between 4.3% and 6.9%. The limits of detection (LODs) were 0.005, 0.007 and 0.006 µg g(-1) DW and the limits of quantification (LOQs) were 0.017, 0.023 and 0.020 µg g(-1) DW for MC-LR, MC-LR-GSH and MC-LR-Cys, respectively. Furthermore, this method was successfully applied to both time- and dosage-effect studies of MC-LR, MC-LR-GSH and MC-LR-Cys in vivo.