Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3'-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids.

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3'-hydroxylase (LaeC3'H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3'H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.

Differential response of phenylpropanoid pathway as linked to hormonal change in two Brassica napus cultivars contrasting drought tolerance.

Oilseed rape (Brassica napus L.) is a significant agro-economic crop with a wide range of uses. Drought is the most frequent unfavourable environmental stressor restraining its growth and development worldwide. This study was conducted to characterize the drought-responsive phenylpropanoid pathway and its link to hormonal changes in two cultivars, drought-resistant "Saturnin" and drought-susceptible "Mosa." Drought susceptibility in cv. Mosa was confirmed by its lower water use efficiency and higher lipid peroxidation levels with reactive oxygen species (ROS) accumulation. In cv. Saturnin, higher salicylic acid (SA) levels and expression of dehydration-responsive element binding 2 (DREB2) and non-expressor of pathogenesis-related gene 1 (NPR1) led to an upregulation of production of anthocyanin pigment 1 (PAP1) and phenylpropanoid pathway-related gene (CHS, F5H and COMT1) expression, increasing hydroxycinnamic acid and flavonoid compound concentrations. However, in cv. Mosa, higher increases in the activity of lignifying enzymes (polyphenol oxidase, coniferyl alcohol peroxidase, syringaldazine peroxidase, guaiacol peroxidase) and expression of the lignin synthesis-related gene cinnamyl alcohol dehydrogenase 2 (CAD2) were found along with greater increases in abscisic acid (ABA) levels and upregulation of ABA-responsive element binding 2 (AREB2) and basic helix-loop-helix transcription factor MYC2. These results indicate that drought-induced SA-mediated activation of the hydroxycinnamic acid and flavonoid pathways contributes to drought resistance, whereas ABA-mediated lignification contributes to drought susceptibility.

Analysis of Inhibitory Behaviour of Ferulic Acid and p-Coumaric Acid on Saccharomyces Cerevisiae Cells.

Metabolomics has been widely used to identify changes in relevant differential metabolites. The metabolites of Saccharomyces cerevisiae cells supplemented with ferulic acid and p-coumaric acid were prepared and extracted. Untargeted metabolomics analysis of saccharomyces cerevisiae metabolites was performed. In addition, GNPS, Respect and MassBank databases were used to search and compare the information in the whole database. It was found that 100 and 92 different metabolites were significantly changed (P value < 0.05,VIP value > 1,) in Saccharomyces cerevisiae cells treated with ferulic acid and p-coumaric acid respectively. Including isothiocyanate, L-threonine, adenosine, glycerin phospholipid choline, niacinamide and palmitic acid. These metabolites with significant differences were enriched by KEGG pathway using MetPA database.

Physiological and genomic characterization of Lactiplantibacillus plantarum isolated from Indri indri in Madagascar.

AIMS: Indri indri is a lemur of Madagascar which is critically endangered. The analysis of the microbial ecology of the intestine offers tools to improve conservation efforts. This study aimed to achieve a functional genomic analysis of three Lactiplantibacillus plantarum isolates from indris. METHODS AND RESULTS: Samples were obtained from 18 indri; 3 isolates of Lp. plantarum were obtained from two individuals. The three isolates were closely related to each other, with <10 single nucleotide polymorphisms, suggesting that the two individuals shared diet-associated microbes. The genomes of the three isolates were compared to 96 reference strains of Lp. plantarum. The three isolates of Lp. plantarum were not phenotypically resistant to antibiotics but shared all 17 genes related to antimicrobial resistance that are part of the core genome of Lp. plantarum. The genomes of the three indri isolates of Lp. plantarum also encoded for the 6 core genome genes coding for enzymes related to metabolism of hydroxybenzoic and hydroxycinnamic acids. The phenotype for metabolism of hydroxycinnamic acids by indri isolates of Lp. plantarum matched the genotype. CONCLUSIONS: Multiple antimicrobial resistance genes and gene coding for metabolism of phenolic compounds were identified in the genomes of the indri isolates, suggesting that Lp. plantarum maintains antimicrobial resistance in defense of antimicrobial plant secondary pathogens and that their metabolism by intestinal bacteria aids digestion of plant material by primate hosts.

The Therapeutic Potential of Hydroxycinnamic Acid Derivatives in Parkinson's Disease: Focus on In Vivo Research Advancements.

Hydroxycinnamic acid derivatives (HCDs) are polyphenols that are abundant in cereals, coffee, tea, wine, fruits, vegetables, and other plant-based foods. To aid in the clinical prevention and treatment of Parkinson's disease (PD), we evaluated in vivo investigations of the pharmacological properties of HCDs relevant to PD, and their pharmacokinetic and safety aspects. An extensive search of published journals was conducted using several literature databases, including PubMed, Google Scholar, and the Web of Science. The search terms included "hydroxycinnamic acid derivatives," "ferulic acid," "caffeic acid," "sinapic acid," "p-coumaric acid," "Parkinson's disease," and combinations of these keywords. As of April 2023, 455 preclinical studies were retrieved, of which 364 were in vivo studies; we included 17 of these articles on the pharmaceutics of HCDs in PD. Available evidence supports the protective effects of HCDs in PD due to their anti-inflammatory, antioxidant, as well as antiapoptotic physiological activities. Studies have identified possible molecular targets and pathways for the protective actions of HCDs in PD. However, the paucity of studies on these compounds in PD, and the risk of toxicity induced with high-dose applications, limits their use. Thus, multifaceted studies of HCDs in vitro and in vivo are needed.

Molecular identification of a laccase that catalyzes the oxidative coupling of a hydroxycinnamic acid amide for hordatine biosynthesis in barley.

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound accumulated at high levels in young barley (Hordeum vulgare) seedlings. The enzyme responsible for the oxidative dimerization of pCA, which is the final step of the hordatine biosynthetic pathway, has not been identified. In this study, we first verified the presence of this enzyme activity in the crude extract of barley seedlings. Because the enzyme activity was not dependent on H2 O2 , the responsible enzyme was not peroxidase, which was previously implicated in HA biosynthesis. The analysis of the dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected HA in the wheat lines carrying the distal part of the 2H short arm. This chromosomal region contains two laccase genes (HvLAC1 and HvLAC2) that are highly expressed at the seedling stage and may encode enzymes that oxidize pCA during the formation of HA. Changes in the HvLAC transcript levels coincided with the changes in the HA biosynthesis-related enzyme activities in the crude extract and the HA content in barley seedlings. Moreover, HvLAC genes were heterologously expressed in Nicotiana benthamiana leaves and in bamboo (Phyllostachys nigra) suspension cells and HA biosynthetic activities were detected in the crude extract of transformed N. benthamiana leaves and bamboo suspension cells. The HA formed by the enzymatic reaction had the same stereo-configuration as the naturally occurring HA. These results demonstrate that HvLAC enzymes mediate the oxidative coupling of pCA during HA biosynthesis.

Conversion of hydroxycinnamic acids by Furfurilactobacillus milii in sorghum fermentations: Impact on profile of phenolic compounds in sorghum and on ecological fitness of Ff. milii.

The conversion of phenolic compounds by lactobacilli in food fermentations contributes to food quality. The metabolism of phenolics by lactobacilli has been elucidated in the past years but information on the contribution of specific enzymes in food fermentations remains scarce. This study aimed to address this gap by disruption of genes coding for the hydroxycimmanic acid reductase Par1, the hydroxycinnamic acid decarboxylase Pad, the hydrocinnamic esterase EstR, and strains with disruption of all three genes in Furfurilactobacillus milii FUA3583. The conversion of phenolics by Ff. milii and its isogenic mutants in sorghum fermentations was studied by LC-UV and LC-UV-MS/MS analyses. Ff. milii FUA3583 converted hydroxycinnamic acids predominantly with Par1. Vinylphenols were detected only in mutants lacking par1. A phenotype for the estR defective mutant was not identified. The formation of pyrano-3-deoxyanthocyanidins was observed only after fermentation with strains expressing Pad. Specifically, formation of these compounds was low with Ff. milii FUA3583, substantially increased in the Par1 mutant and abolished in all mutants with disrupted pad. Competition experiments with Ff. milii FUA3583 and its isogenic mutants demonstrated that expression of one of the two metabolic pathways for hydroxycinnamic acids increases the ecological fitness of the strain. Disruption of EstR in a Δpar1Δpar2Δpad background improved ecological fitness, indirectly demonstrating a phenotype of the esterase in Ff. milii. The documentation of the functionality of genes coding for conversion of hydroxycinnamic acids may support the selection of starter cultures for improved quality of fermented cereal products.

Conversion of Phenolic Acids in Canola Fermentation: Impact on Antimicrobial Activity against Salmonella enterica and Campylobacter jejuni.

Canola meal (CM) is commonly used in poultry feeds. CM has a high protein content but also contains high levels of antimicrobial phenolic acids. Lactic acid bacteria can alter CM phenolic composition during fermentation and influence its antimicrobial activity against pathogens. Fermented CM was analyzed for phenolic composition using tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). Sinapic acid and derivatives were the major phenolic acids in CM. Growth of lactobacilli in CM was attenuated when compared to cereal substrates. Glucosides and esters of sinapic acid were extensively hydrolyzed during fermentation with Lactiplantibacillus plantarum and Furfurilactobacillus milii. Lp. plantarum transformed hydroxycinnamic acids to dihydro, 4-vinyl, and 4-ethyl derivatives, Ff. milii reduced hydroxycinnamic acids to dihydroderivatives, but Limosilactobacillus reuteri did not convert hydroxycinnamic acids. The minimum inhibitory concentration of phenolic extracts was assessed with lactobacilli, Salmonella, and Campylobacter jejuni as indicator strains. Fermentation of CM with Lp. plantarum or Ff. milii increased the antimicrobial activity of phenolic extracts against Salmonella enterica and Campylobacter jejuni. Fermentation with Lm. reuteri TMW1.656 but not fermentation with Lm. reuteri TMW1.656ΔrtcN increased the antimicrobial activity of extracts owing to the production of reutericyclin. This study demonstrates that fermentation of CM with lactobacilli converts hydroxycinammic esters and may increase the antimicrobial activity of phenolic compounds in CM against pathogens.

Arrested Acetylene-Induced Pulmonary and Testicular Toxicity in Rats Through Treatment With Polyphenols.

Occupational exposure to potentially harmful substances is one of the dangers associated with industrial jobs. This study evaluated the modulatory influence of selected dietary polyphenols on the pulmonotoxic and testiculotoxic effects of crude acetylene, an industrial gas used in welding metals. Wistar rats were exposed to 58 000 ppm acetylene, 20 min daily for 30 days, in a 36 L glass inhalation chamber. Some acetylene-exposed animals were treated concurrently with 30 mg/kg quercetin, rutin, caffeic acid, ferulic acid, or coumaric acid. At the end of the treatment sessions, the levels of superoxide dismutase, reduced glutathione, glutathione peroxidase, lactate dehydrogenase, and hormonal markers in rats exposed to acetylene were significantly decreased, with a concomitant increase in lipid peroxidation, nitric oxide level, cholesterol concentration, and histopathological abnormalities. These damaging biochemical and histopathological changes were significantly ameliorated in animals administered the polyphenols. Quercetin showed greater ameliorative activity than rutin while the phenolic acids exhibited increasing levels of ameliorative activity in the order: caffeic acid > ferulic acid > coumaric acid. These results indicate that inhalation of crude acetylene is deleterious to the lungs and testes, and polyphenols provide protection against these detrimental effects.

Programmable Polyproteams of Tyrosine Ammonia Lyases as Cross-Linked Enzymes for Synthesizing p-Coumaric Acid.

Ideal immobilization with enhanced biocatalyst activity and thermostability enables natural enzymes to serve as a powerful tool to yield synthetically useful chemicals in industry. Such an enzymatic method strategy becomes easier and more convenient with the use of genetic and protein engineering. Here, we developed a covalent programmable polyproteam of tyrosine ammonia lyases (TAL-CLEs) by fusing SpyTag and SpyCatcher peptides into the N-terminal and C-terminal of the TAL, respectively. The resulting circular enzymes were clear after the spontaneous isopeptide bonds formed between the SpyTag and SpyCatcher. Furthermore, the catalytic performance of the TAL-CLEs was measured via a synthesis sample of p-Coumaric acid. Our TAL-CLEs showed excellent catalytic efficiency, with 98.31 ± 1.14% yield of the target product-which is 4.15 ± 0.08 times higher than that of traditional glutaraldehyde-mediated enzyme aggregates. They also showed over four times as much enzyme-activity as wild-type TAL does and demonstrated good reusability, and so may become a good candidate for industrial enzymes.

Improving accessibility and bioactivity of raw, germinated and enzymatic-treated spelt (Triticum spelta L.) seed antioxidants by fermentation.

Lactic acid, alcoholic, combined and spontaneous fermentation of raw, germinated and enzymatic-treated spelt seeds significantly improved the content of extractable and bound phenolics and considerably increased the extractable:bound ratio, and therefore positively affected the accessibility of the spelt antioxidants. The highest extractable and bound individual phenolic contents and in vitro antioxidant activities of extracts were obtained following fermentation of germinated spelt seeds with Saccharomyces cerevisiae, while for enzymatic-treated seeds, Lactobacillus plantarum (alone or with S. cerevisiae) was the most effective. For extractable phenolics, trans-ferulic acid increased the most in yeast-fermented germinated seeds (2922%); for bound phenolics, cis-ferulic acid showed the greatest relative increase in yeast-fermented raw spelt seeds (466%). Spontaneous fermentation of germinated and enzymatic-treated samples decreased intracellular oxidation most effectively, probably due to apigenin derivatives. Cellular uptake of bound hydroxycinnamic acids was significantly higher than that of extractable hydroxycinnamic acids; however, the latter were more efficient in vivo antioxidants.

Cool Temperature Enhances Growth, Ferulic Acid and Flavonoid Biosynthesis While Inhibiting Polysaccharide Biosynthesis in Angelica sinensis.

Angelica sinensis, a perennial herb that produces ferulic acid and phthalides for the treatment of cardio-cerebrovascular diseases, prefers growing at an altitude of 1800-3000 m. Geographical models have predicted that high altitude, cool temperature and sunshade play determining roles in geo-authentic formation. Although the roles of altitude and light in yield and quality have been investigated, the role of temperature in regulating growth, metabolites biosynthesis and gene expression is still unclear. In this study, growth characteristics, metabolites contents and related genes expression were investigated by exposing A. sinensis to cooler (15 °C) and normal temperatures (22 °C). The results showed that plant biomass, the contents of ferulic acid and flavonoids and the expression levels of genes related to the biosynthesis of ferulic acid (PAL1, 4CLL4, 4CLL9, C3H, HCT, CCOAMT and CCR) and flavonoids (CHS and CHI) were enhanced at 15 °C compared to 22 °C. The contents of ligustilide and volatile oils exhibited slight increases, while polysaccharide contents decreased in response to cooler temperature. Based on gene expression levels, ferulic acid biosynthesis probably depends on the CCOAMT pathway and not the COMT pathway. It can be concluded that cool temperature enhances plant growth, ferulic acid and flavonoid accumulation but inhibits polysaccharide biosynthesis in A. sinensis. These findings authenticate that cool temperature plays a determining role in the formation of geo-authentic and also provide a strong foundation for regulating metabolites production of A. sinensis.

Influence of 8-week daily consumption of a new product combining green coffee hydroxycinnamates and beta-glucans on polyphenol bioavailability in subjects with overweight and obesity.

Nutraceuticals based on plant extracts rich in polyphenols, as well as dietary fibres, are new means to fight overweight/obesity and associated diseases. However, to understand the potential effects of polyphenols on health it is critical to study their bioavailability and metabolic fate. Consumption of a green coffee phenolic extract (GCPE) in combination with oat beta-glucan (BG) could affect the pharmacokinetic profile of the main polyphenols present in coffee (hydroxycinnamates). Moreover, the regular intake of the combination could also induce changes. Nine overweight men and women consumed a novel nutraceutical product containing 300 mg of green coffee hydroxycinnamic acids and 2.5 g of BG twice a day for 8 weeks. A pharmacokinetic study was carried out in blood and urine samples taken before (baseline) and at week 8 after the nutraceutical intervention, collecting samples at different times in a 0-24 h interval. Faecal samples were also obtained at 0 and 24 h after the intake of the nutraceutical at baseline and week 8. Phenolic metabolites were analysed by LC-MS-QToF. Results showed that polyphenols were differentially absorbed and extensively metabolized throughout the gastrointestinal tract. An apparent reduction in the excretion of small intestinal metabolites was accompanied by a tendency to increase colonic metabolites after sustained intake (p = 0.052). In conclusion, continued consumption of the GCPE/BG nutraceutical appears to enhance the absorption of hydroxycinnamates by increasing the colonic bioavailability of their derived metabolites compared to baseline, although the regular intake of the nutraceutical did not modify the metabolite profile in any of the biological samples.

Nephroprotective effect of ferulic acid on gentamicin-induced nephrotoxicity in female rats.

Ferulic acid is a kind of phenolic compound that can be found in various fruits and vegetables. This study aims to investigate the effect of ferulic acid on nephrotoxicity induced by gentamicin (GM). In this study, rats were separated into 4 groups such that each containing 8 randomly selected rats: Control group, Ferulic Acid (FA) group, Gentamicin (GM) group and Gentamicin + Ferulic acid (GM + FA) group. Blood samples were collected after 24 hours following the 8-day trial period, and kidneys were taken out for histopathological evaluation. Serum urea, creatinine, uric acid and LDH analyses were performed in autoanalyzer while Malondialdehyde (MDA), Advanced Oxidized Protein Products (AOPP), Glutathione (GSH), Superoxide dismutase (SOD), Catalase (CAT), Interleukin 6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α) analyses were performed in ELISA, and kidney tissues were also examined histopathologically. Urea (p < .001), creatinine (p < .001), MDA (p < .01), AOPP (p < .001), IL-6 (p < .01) and TNF-α (p < .001) levels were found to be statistically and significantly lowered in GM + FA group when compared to GM group. As a result, ferulic acid has reduced the inflammation in nephrotoxicity induced by GM, causing decreased oxidative stress. In this study, anti-inflammatory features of ferulic acid have come to the forefront rather than the antioxidant features. It can be said that ferulic acid reduces nephrotoxic damage and has protective properties for kidneys.

Curcumin biosynthesis from ferulic acid by engineered Saccharomyces cerevisiae.

BACKGROUND AND GOAL: Curcumin is a polyphenolic compound found in Curcuma longa. This bioactive molecule has several reported health-benefit effects, being the anticarcinogenic activity among the most promising ones. However, curcumin extraction from natural sources is hampered by impure products obtained from harsh chemicals and limited by plant seasonality and high prices. Therefore, curcumin heterologous production emerged as an interesting alternative. Escherichia coli has been explored as chassis but the implementation of the pathway in Saccharomyces cerevisiae can have several advantages, including its generally regarded as safe status. Hence, S. cerevisiae was engineered for the first time to produce curcumin from its precursor ferulic acid. METHODS AND RESULTS: The enzymes 4-coumarate-CoA ligase (4CL1) from Arabidopsis thaliana or feruloyl-CoA synthetase (FerA) from Pseudomonas paucimobilis and type III polyketide synthases (PKSs) from Oryza sativa or C. longa were expressed in BY4741 strain. To avoid ferulic acid deviation, the gene FDC1 coding a ferulic acid decarboxylase was deleted. The maximum curcumin titer was obtained with FerA combined with C. longa PKSs (2.7 mg L-1 ). CONCLUSIONS AND IMPLICATIONS: Up to our knowledge, this is the first work reporting the expression of a feruloyl-CoA synthase and also curcuminoid biosynthetic enzymes in S. cerevisiae, and consequently, curcumin production.

Ferulic Acid Metabolites Attenuate LPS-Induced Inflammatory Response in Enterocyte-like Cells.

Ferulic acid (FA) is a polyphenol pertaining to the class of hydroxycinnamic acids present in numerous foods of a plant origin. Its dietary consumption leads to the formation of several phase I and II metabolites in vivo, which represent the largest amount of ferulates in the circulation and in the intestine in comparison with FA itself. In this work, we evaluated their efficacy against the proinflammatory effects induced by lipopolysaccharide (LPS) in intestinal Caco-2 cell monolayers, as well as the mechanisms underlying their protective action. LPS-induced overexpression of proinflammatory enzymes such as inducible nitric oxide synthase (iNOS) and the consequent hyperproduction of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) were limited by physiological relevant concentrations (1 µM) of FA, its derivatives isoferulic acid (IFA) and dihydroferulic acid (DHFA), and their glucuronidated and sulfated metabolites, which acted upstream by limiting the activation of MAPK p38 and ERK and of Akt kinase, thus decreasing the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) translocation into the nucleus. Furthermore, the compounds were found to promote the expression of Nrf2, which may have contributed to the downregulation of NF-ĸB activity. The overall data show that phase I/II metabolites retain the efficacy of their dietary free form in contrasting inflammatory response.

Advanced Cancer Starvation Therapy by Simultaneous Deprivation of Lactate and Glucose Using a MOF Nanoplatform.

Recent investigations reveal that lactate is not a waste product but a major energy source for cells, especially in the mitochondria, which can support cellular survival under glucose shortage. Accordingly, the new understanding of lactate prompts to target it together with glucose to pursue a more efficient cancer starvation therapy. Herein, zeolitic imidazolate framework-8 (ZIF-8) nanoplatforms are used to co-deliver α-cyano-4-hydroxycinnamate (CHC) and glucose oxidase (GOx) and fulfill the task of simultaneous depriving of lactate and glucose, resulting in a new nanomedicine CHC/GOx@ZIF-8. The synthesis conditions are carefully optimized in order to yield monodisperse and uniform nanomedicines, which will ensure reliable and steady therapeutic properties. Compared with the strategies aiming at a single carbon source, improved starvation therapy efficacy is observed. Besides, more than boosting the energy shortage, CHC/GOx@ZIF-8 can block the lactate-fueled respiration and relieve solid tumor hypoxia, which will enhance GOx catalysis activity, depleting extra glucose, and producing more cytotoxic H2 O2 . By the synergistically enhanced anti-tumor effect, both in vitro and in vivo cancer-killing efficacies of CHC/GOx@ZIF-8 show twice enhancements than the GOx mediated therapy. The results demonstrate that the dual-depriving of lactate and glucose is a more advanced strategy for strengthening cancer starvation therapy.

Ferulic acid inhibits LPS-induced apoptosis in bovine mammary epithelial cells by regulating the NF-κB and Nrf2 signalling pathways to restore mitochondrial dynamics and ROS generation.

In bovine mammary epithelial cells (BMECs), a cascade of inflammatory reactions induced by lipopolysaccharide (LPS) has been shown to result in cell injury and apoptosis. The present study aims to reveal the protective effect of ferulic acid (FA) on LPS-induced BMEC apoptosis and explore its potential molecular mechanisms. First, we showed that FA had low cytotoxicity to BMECs and significantly decreased cell apoptosis and the proinflammatory response induced by LPS. Next, FA blocked LPS-induced oxidative stress by restoring the balance of the redox state and inhibiting mitochondrial dysfunction, the main contributor to LPS-induced apoptosis and ROS generation. Furthermore, the relief of inflammation and redox disturbance in the FA preconditioning group were accompanied by weaker NF-κB activation, enhanced Nrf2 activation and maintained cell viability compared to the LPS group. When BMECs were treated with FA alone, we observed that Nrf2 activation was induced before the inhibition of NF-κB activation and that the Keap1-Nrf2 relationship was disturbed. We concluded that FA prevented LPS-induced BMEC apoptosis by reversing the dominant relationship between NF-κB and Nrf2.

The Improvement Effect of Sodium Ferulate on the Formation of Pulmonary Fibrosis in Silicosis Mice Through the Neutrophil Alkaline Phosphatase 3 (NALP3)/Transforming Growth Factor-ß1 (TGF-ß1)/α-Smooth Muscle Actin (α-SMA) Pathway.

BACKGROUND Pneumoconiosis is a chronic progressive fibrotic interstitial pneumonia for which the pathogenesis and treatment remain unclear. Previous studies showed that sodium ferulate (SF) may have a therapeutic effect, and this study explored the mechanism underlying SF-related improvement. MATERIAL AND METHODS In this study, a silicosis mouse model and primary cultured mouse lung fibroblasts were established. Hematoxylin-eosin staining, western blot analysis, quantitative real-time polymerase chain reaction, and Masson staining were used to observe the lung injury, expression of vimentin, and the degree of pulmonary fibrosis. The extracted lung fibroblasts were identified by immunofluorescence. The expression of fibrosis-related genes encoding transforming growth factor-ß1 (TGF-ß1), neutrophil alkaline phosphatase 3 (NALP3), collagen-1, alpha-smooth muscle actin (alpha-SMA), and phosphorylated p38 (p-p38) and p38 proteins were detected by western blot. The effects of SF and the TGF-ß pathway agonist SRI-011381 on cell proliferation and the expression of fibrosis-related protein in mouse lung fibroblasts were measured by Cell Counting Kit-8, immunofluorescence, and western blot as needed. RESULTS SF reduced the lung lesions in silicosis mice and inhibited the expression of vimentin and fibrosis-related genes, while having no effect on body weight. Vimentin expression was positive in the extracted cells. In vitro experiments showed that SF inhibited the proliferation of lung fibroblasts and the expression of fibrosis-related proteins. In addition, SF partly reversed the opposite regulatory effect of SRI-011381 on lung fibroblasts. CONCLUSIONS SF inhibited lung injury and fibrosis in silicosis mice through the NALP3/TGF-ß1/alpha-SMA pathway.

Ferulic acid positively modulates the inflammatory response to septic liver injury through the GSK-3ß/NF-κB/CREB pathway.

AIMS: Ferulic acid (FA) is a component found in plants that has free radical scavenging and liver-protective properties. Acute liver injury (ALI) is a serious complication of sepsis and is closely associated with changes in the levels of inflammatory factors. This study was taken to examine the role of FA in cecal ligation and perforation (CLP)-induced murine ALI and lipopolysaccharide (LPS)-induced cellular ALI models. MATERIALS AND METHODS: An in vivo ALI model was established by performing CLP surgery on C57BL/6 mice. After the ALI model was established, mice were examined for liver injury, including HE staining to observe tissue sections, the percentage of liver/body weight and inflammatory factor levels. Myeloperoxidase (MPO), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured in liver or serum using commercial kits. An in vitro ALI model was established using LPS-stimulated RAW264.7 cells. Cell viability was measured by MTT method and the intracellular levels of IL-10, IL-1ß, IL-6, IL-12 and TNF-α inflammatory factors were measured using kits. The expression of GSK-3ß, NF-κB and CREB was measured by western blot or immunofluorescence. KEY FINDINGS: FA pretreatment significantly reduced liver/body weight ratio, decreased MPO, AST and ALT activity, alleviated the inflammatory responses and improved CLP-induced histopathological changes in liver. In addition, in vitro results showed that FA could dose-dependently increase the viability of RAW264.7 cells and decrease the levels of pro-inflammatory factors. SIGNIFICANCE: In conclusion, our data suggest that FA can ameliorate ALI-induced inflammation via the GSK-3ß/NF-κB/CREB pathway, suggesting that FA can be used to protect the liver against ALI.