Structures and diversities of bacterial communities in oil-contaminated soil at shale gas well site assessed by high-throughput sequencing.

Currently, there is limited understanding of the structures and variabilities of bacterial communities in oil-contaminated soil within shale gas development. The Changning shale gas well site in Sichuan province was focused, and high-throughput sequencing was used to investigate the structures of bacterial communities and functions of bacteria in soil with different degrees of oil pollution. Furthermore, the influences of the environmental factors including pH, moisture content, organic matter, total nitrogen, total phosphorus, oil, and the biological toxicity of the soil on the structures of bacterial communities were analyzed. The results revealed that Proteobacteria and Firmicutes predominated in the oil-contaminated soil. α-Proteobacteria and γ-Proteobacteria were the main classes under the Proteobacteria phylum. Bacilli was the main class in the Firmicutes phylum. Notably, more bacteria were only found in CN-5 which was the soil near the storage pond for abandoned drilling mud, including Marinobacter, Balneola, Novispirillum, Castellaniella, and Alishewanella. These bacteria exhibited resilience to higher toxicity and demonstrated proficiency in oil degradation. The functions including carbohydrate transport and metabolism, energy metabolism, replication, recombination and repair replication, signal transduction mechanisms, and amino acid transport and metabolism responded differently to varying concentrations of oil. The disparities in bacterial genus composition across samples stemmed from a complex play of pH, moisture content, organic matter, total nitrogen, total phosphorus, oil concentration, and biological toxicity. Notably, bacterial richness correlated positively with moisture content, while bacterial diversity showed a significant positive correlation with pH. Acidobacteria exhibited a significant positive correlation with moisture content. Litorivivens and Luteimonas displayed a significant negative correlation with pH, while Rhizobium exhibited a significant negative correlation with moisture content. Pseudomonas, Proteiniphilum, and Halomonas exhibited positive correlations not only with organic matter but also with oil concentration. Total nitrogen exhibited a significant positive correlation with Taonella and Sideroxydans. On the other hand, total phosphorus showed a significant negative correlation with Sphingomonas. Furthermore, Sphingomonas, Gp6, and Ramlibacter displayed significant negative correlations with biological toxicity. The differential functions exhibited no significant correlation with environmental factors but displayed a significant positive correlation with the Proteobacteria phylum. Aridibacter demonstrated a significant positive correlation with cell motility and cellular processes and signaling. Conversely, Pseudomonas, Proteiniphilum, and Halomonas were negatively correlated with differential functions, particularly in amino acid metabolism, carbohydrate metabolism, and membrane transport. Compared with previous research, more factors were considered in this research when studying structural changes in bacterial communities, such as physicochemical properties and biological toxicity of soil. In addition, the correlations of differential functions of communities with environmental factors, bacterial phyla, and genera were investigated.

Ramulus Cinnamomi essential oil exerts an anti-inflammatory effect on RAW264.7 cells through N-acylethanolamine acid amidase inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Ramulus Cinnamomi, the dried twig of Cinnamomum cassia (L.) J.Presl., is a traditional Chinese medicine (TCM) with anti-inflammatory effects. The medicinal functions of Ramulus Cinnamomi essential oil (RCEO) have been confirmed, although the potential mechanisms by which RCEO exerts its anti-inflammatory effects have not been fully elucidated. AIM OF THE STUDY: To investigate whether N-acylethanolamine acid amidase (NAAA) mediates the anti-inflammatory effects of RCEO. MATERIALS AND METHODS: RCEO was extracted by steam distillation of Ramulus Cinnamomi, and NAAA activity was detected using HEK293 cells overexpressing NAAA. N-Palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), both of which are NAAA endogenous substrates, were detected by liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The anti-inflammatory effects of RCEO were analyzed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the cell viability was measured with a Cell Counting Kit-8 (CCK-8) kit. The nitric oxide (NO) in the cell supernatant was measured using the Griess method. The level of tumor necrosis factor-α (TNF-α) in the RAW264.7 cell supernatant was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The chemical composition of RCEO was assessed by gas chromatography-mass spectroscopy (GC-MS). The molecular docking study for (E)-cinnamaldehyde and NAAA was performed by using Discovery Studio 2019 software (DS2019). RESULTS: We established a cell model for evaluating NAAA activity, and we found that RCEO inhibited the NAAA activity with an IC50 of 5.64 ± 0.62 µg/mL. RCEO significantly elevated PEA and OEA levels in NAAA-overexpressing HEK293 cells, suggesting that RCEO might prevent the degradation of cellular PEA and OEA by inhibiting the NAAA activity in NAAA-overexpressing HEK293 cells. In addition, RCEO also decreased NO and TNF-α cytokines in lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, the GC-MS assay revealed that more than 93 components were identified in RCEO, of which (E)-cinnamaldehyde accounted for 64.88%. Further experiments showed that (E)-cinnamaldehyde and O-methoxycinnamaldehyde inhibited NAAA activity with an IC50 of 3.21 ± 0.03 and 9.62 ± 0.30 µg/mL, respectively, which may represent key components of RCEO that inhibit NAAA activity. Meanwhile, docking assays revealed that (E)-cinnamaldehyde occupies the catalytic cavity of NAAA and engages in a hydrogen bond interaction with the TRP181 and hydrophobic-related interactions with LEU152 of human NAAA. CONCLUSIONS: RCEO showed anti-inflammatory effects by inhibiting NAAA activity and elevating cellular PEA and OEA levels in NAAA-overexpressing HEK293 cells. (E)-cinnamaldehyde and O-methoxycinnamaldehyde, two components in RCEO, were identified as the main contributors of the anti-inflammatory effects of RCEO by modulating cellular PEA levels through NAAA inhibition.

Effects of fatty acid-ethanol amine (FA-EA) derivatives on lipid accumulation and inflammation.

This study aimed to investigate the effect of fatty acid-ethanol amine (FA-EA) derivatives (L1-L10) on the mitigation of intracellular lipid accumulation and downregulation of pro-inflammatory cytokines in vitro. First, the series of FA-EA derivatives were synthesized and characterized. Then, their cytotoxic, intracellular lipid accumulation and inhibition of pro-inflammatory cytokines were evaluated. The oil red O staining experiment showed that the tested compounds L4, L6, L8, L9, and L10 could reduce intracellular lipid accumulation induced by palmitic acid (PA). Moreover, ω-3/ω-6 PUFA-EA derivatives showed inhibitory effect on the production of pro-inflammatory cytokines in lipopolysaccharide (LPS) -stimulated RAW 264.7 cells. ω-3/ω-6 PUFA-EA derivatives at a concentrations of 10 µM could significantly decrease mRNA levels of IL-6, IL-1ß, and TNF-α, inhibit NO production, and alleviate the protein expression of IL-1ß in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These data suggest that ω-3 PUFA-EA derivatives can be beneficial for further pharmaceutical development to treat chronic low-grade inflammation diseases such as obesity.