PGG2 Research Articles

"Elucidating the immunomodulatory role of endocannabinoids in intervertebral disc degeneration".

The endocannabinoid system (ECS) has been well-established to play a crucial role in the regulation of several physiological processes as well as many inflammatory disease conditions. However, its role in intervertebral disc degeneration has been least explored. We aim to investigate the immunomodulatory role of endocannabinoids in regulating IVD health. The study population included 20 healthy volunteers (controls) and 40 patients with disc degeneration (disease group) (20 Modic and 20 Non Modic). 16S metagenome sequencing of the V3-V4 region was performed for the DNA extracted from NP tissue samples of both control and disease groups. Sequencing was carried out using the Novaseq 6000 platform using 250bp paired-end chemistry. A global metabolic profile was obtained using the uHPLC system coupled with Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. Our study revealed a higher prevalence of gram-negative bacteria, particularly opportunistic pathogens like Pseudomonas, in diseased discs (71-81%) compared to healthy controls (54%). Further investigation using metabolomics identified significant changes in the lipid profiles of diseased discs. We found that the signalling molecules of the ECS, 2-arachidonylglycerol (2-AG) and N-arachidonoylethanolamine (AEA), were significantly lower in diseased discs compared to controls (Log2FC -2.62 for 2-AG and -3.15 for AEA). Conversely, pro-inflammatory metabolites like LTA4, HPETE, HETE, and Prostaglandin G2 were elevated in diseased discs, with a Log2 fold increase greater than 2.5. The study reveals that the endocannabinoid metabolites (2-AG and AEA) of the ECS could be a significant molecule influencing susceptibility to infection and inflammation within the intervertebral discs, which could be a potential target for improving disc health. Diagnostic: individual cross-sectional studies with consistently applied reference standard and blinding.

A hypothesis linking the renin-angiotensin, kallikrein-kinin systems, and disseminated coagulation in COVID-19

In this article we present a hypothesis based on the relationship between four physiological systems: the renin-angiotensin system (RAS), the kallikrein-kinin system (KKS), the arachidonic acid (AA) cascade, and platelet aggregation/coagulation − in the context of COVID-19. The central point of our proposition relies on ACE2 levels, which reduce following SARS-CoV-2 entry in the cell. The following cascade of events. Triggered by this reduction, involves the increase in Ang II levels and its consequent binding to AT1-R, initiating the release of pro-inflammatory cytokines such as TNF-α, IL-1, IL-10, and IL-12. These pro-inflammatory cytokines activate immune cells, including mast cells, which release heparin, thus activating Coagulation factor XII and increasing pre-kallikrein (PK) production. Consequently, there is an increase in bradykinin (BK) levels. BK, via its receptor BKB2-R, induces Ca2 + release increasing secreted phospholipase A2 (sPLA2) levels. Concomitantly, both the imbalance in Ang II/AT1-R and BK/BKB2-R signaling contribute to sPLA2 activation, inducing the release of arachidonic acid (AA) from cell membrane phospholipids, by cyclooxygenase-2 (COX-2) cleavage to produce prostaglandin G2 (PGG2), and later prostaglandin H2 (PGH2). At least, PGH2 is converted to thromboxane A2 (TXA2), which is involved in the platelet aggregation process. Platelet aggregation further increases TXA2 levels, initiating a positive feedback loop leading to further platelet activation and clot formation. Ultimately, this cascade of events may contribute to the development of a pre-thrombotic state and increase the risk of mortality, in COVID-19. We believe that this integrated approach could provide a deeper understanding of the underlying mechanisms of COVID-19 and guide future therapeutic strategies.

Metabolome-microbiome insights into therapeutic impact of 8-O-acetylharpagide against breast cancer in a murine model.

Iridoid glycosides extract, which is the main active extract of Ajuga decumbens Thunb, has been proved to have anti-breast cancer activity in previous studies. However, it is still unknown whether 8-O-acetylharpagide, a main active compound in the extract, has anti-breast cancer activity. In this study, 4T1 breast cancer mice model was first successfully established. Then the anti-breast cancer effect of 8-O-acetylharpagide was systematically investigated. Feces were collected for metabolomics and 16S rRNA analysis to assess the potential mechanism. The results showed that 8-O-acetylharpagide was effective in reducing 4T1 mouse tumor volume and weight compared with the model group. Metabolome analysis revealed 12 potential metabolite biomarkers in feces, mainly involved in primary bile acid biosynthesis and arachidonic acid metabolism. The 16S rRNA sequencing results demonstrated that 8-O-acetylharpagide modulated the abundance of the intestinal flora in 4T1 mice. Spearman correlation analysis showed that calcitriol and prostaglandin G2 strongly correlated with Akkermansia, Firmicutes and Muribaculum. Overall, the active compound 8-O-acetylharpagide could inhibit significantly breast cancer growth in 4T1 breast cancer model mice. The mechanism of the anti-breast cancer effect of 8-O-acetylharpagide may be related to the regulation of primary bile acid biosynthesis and arachidonic acid metabolism and modulation of the abundance of Akkermansia and Firmicutes.

Effect of chronic deltamethrin exposure on brain transcriptome and metabolome of juvenile crucian carp.

Deltamethrin (Del), a widely administered pyrethroid insecticide, has been established as a common contaminant of the freshwater environment and detected in many freshwater ecosystems. In this study, we investigated the changes in brain transcriptome and metabolome of crucian carp after exposure to 0.6 μg/L Del for 28 days. Elevated MDA levels and inhibition of SOD activity indicate damage to the antioxidant system. Moreover, a total of 70 differential metabolites (DMs) were identified using the liquid chromatography-mass spectrometry, including 32 upregulated and 38 downregulated DMs in the Del-exposed group. The DMs associated with chronic Del exposure were enriched in steroid hormone biosynthesis, fatty acid metabolism, and glycerophospholipid metabolism for prostaglandin G2, 5-oxoeicosatetraenoic acid, progesterone, androsterone, etiocholanolone, and hydrocortisone. Transcriptomics analysis revealed that chronic Del exposure caused lipid metabolism disorder, endocrine disruption, and proinflammatory immune response by upregulating the pla2g4, cox2, log5, ptgis, lcn, and cbr expression. Importantly, the integrative analysis of transcriptomics and metabolomics indicated that the arachidonic acid metabolism pathway and steroid hormone biosynthesis were decisive processes in the brain tissue of crucian carp after Del exposure. Furthermore, Del exposure perturbed the tight junction, HIF-1 signaling pathway, and thyroid hormone signaling pathway. Overall, transcriptome and metabolome data of our study offer a new insight to assess the risk of chronic Del exposure in fish brains.

In-vitro and computational analysis of Urolithin-A for anti-inflammatory activity on Cyclooxygenase 2 (COX-2)

Cyclooxygenase 2 (COX-2) participates in the inflammation process by converting arachidonic acid into prostaglandin G2 which increases inflammation, pain and fever. COX-2 has an active site and a heme pocket and blocking these sites stops the inflammation. Urolithin A is metabolite of ellagitannin produced from humans and animals gut microbes. In the current study, Urolithin A showed good pharmacokinetic properties. Molecular docking of the complex of Urolithin A and COX-2 revealed the ligand affinity of −7.97 kcal/mol with the ligand binding sites at TYR355, PHE518, ILE517 and GLN192 with the 4-H bonds at a distance of 2.8 Å, 2.3 Å, 2.5 Å and 1.9 Å. The RMSD plot for Urolithin A and COX-2 complex was observed to be constant throughout the duration of dynamics. A total of 3 pair of hydrogen bonds was largely observed on average of 3 simulation positions for dynamics duration of 500 ns. The MMPBSA analysis showed that active site amino acids had a binding energy of –22.0368 kJ/mol indicating that throughout the simulation the protein of target was bounded by Urolithin A. In-silico results were validated by biological assays. Urolithin A strongly revealed to exhibit anti-inflammatory effect on COX-2 with an IC50 value of 44.04 µg/mL. The anti-inflammatory capability was also depicted through reduction of protein denaturation that showed 37.6 ± 0.1 % and 43.2 ± 0.07 % reduction of protein denaturation for BSA and egg albumin respectively at 500 µg/mL. The present study, suggests Urolithin A to be an effective anti-inflammatory compound for therapeutic use.

Cyclooxygenases and platelet functions.

Cyclooxygenase (COX) isozymes, i.e., COX-1 and COX-2, are encoded by separate genes and are involved in the generation of the same products, prostaglandin (PG)G2 and PGH2 from arachidonic acid (AA) by the COX and peroxidase activities of the enzymes, respectively. PGH2 is then transformed into prostanoids in a tissue-dependent fashion due to the different expression of downstream synthases. Platelets present almost exclusively COX-1, which generates large amounts of thromboxane (TX)A2, a proaggregatory and vasoconstrictor mediator. This prostanoid plays a central role in atherothrombosis, as shown by the benefit of the antiplatelet agent low-dose aspirin, a preferential inhibitor of platelet COX-1. Recent findings have shown the relevant role played by platelets and TXA2 in developing chronic inflammation associated with several diseases, including tissue fibrosis and cancer. COX-2 is induced in response to inflammatory and mitogenic stimuli to generate PGE2 and PGI2 (prostacyclin), in inflammatory cells. However, PGI2 is constitutively expressed in vascular cells in vivo and plays a crucial role in protecting the cardiovascular systems due to its antiplatelet and vasodilator effects. Here, platelets' role in regulating COX-2 expression in cells of the inflammatory microenvironment is described. Thus, the selective inhibition of platelet COX-1-dependent TXA2 by low-dose aspirin prevents COX-2 induction in stromal cells leading to antifibrotic and antitumor effects. The biosynthesis and functions of other prostanoids, such as PGD2, and isoprostanes, are reported. In addition to aspirin, which inhibits platelet COX-1 activity, possible strategies to affect platelet functions by influencing platelet prostanoid receptors or synthases are discussed.

Identification of crucial genes and metabolites regulating the eggshell brownness in chicken

BackgroundProtoporphyrin IX (Pp IX) is the primary pigment for brown eggshells. However, the regulatory mechanisms directing Pp IX synthesis, transport, and genetic regulation during eggshell calcification in chickens remain obscure. In this study, we investigated the mechanism of brown eggshell formation at different times following oviposition, using White Leghorn hens (WS group), Rhode Island Red light brown eggshell line hens (LBS group) and Rhode Island Red dark brown eggshell line hens (DBS group).ResultsAt 4, 16 and 22 h following oviposition, Pp IX concentrations in LBS and DBS groups were significantly higher in shell glands than in liver (P < 0.05). Pp IX concentrations in shell glands of LBS and DBS groups at 16 and 22 h following oviposition were significantly higher than WS group (P < 0.05). In comparative transcriptome analysis, δ-aminolevulinate synthase 1 (ALAS1), solute carrier family 25 member 38 (SLC25A38), ATP binding cassette subfamily G member 2 (ABCG2) and feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), which were associated with Pp IX synthesis, were identified as differentially expressed genes (DEGs). RT-qPCR results showed that the expression level of ALAS1 in shell glands was significantly higher in DBS group than in WS group at 16 and 22 h following oviposition (P < 0.05). In addition, four single nucleotide polymorphisms (SNPs) in ALAS1 gene that were significantly associated with eggshell brownness were identified. By identifying the differential metabolites in LBS and DBS groups, we found 11-hydroxy-E4-neuroprostane in shell glands and 15-dehydro-prostaglandin E1(1-) and prostaglandin G2 2-glyceryl ester in uterine fluid were related to eggshell pigment secretion.ConclusionsIn this study, the regulatory mechanisms of eggshell brownness were studied comprehensively by different eggshell color and time following oviposition. Results show that Pp IX is synthesized de novo and stored in shell gland, and ALAS1 is a key gene regulating Pp IX synthesis in the shell gland. We found three transporters in Pp IX pathway and three metabolites in shell glands and uterine fluid that may influence eggshell browning.

Development of an Enzyme-Based Thin-Layer Chromatographic Assay for the Detection of Cyclooxygenase-2 Inhibitors

The search for new anti-inflammatory drugs with less side effects requires simple, fast and reliable screening methods. In this context, we have developed a sensitive thin-layer chromatographic (TLC) assay on silica gel plates to detect cyclooxygenase-2 (COX-2) inhibition. COX-2 catalyzes two sequential enzymatic reactions: a first oxygenation step that converts arachidonic acid into prostaglandin G2, and a subsequent reduction of prostaglandin G2 into prostaglandin H2. Our test is based on the co-oxidation during this peroxidation step of a co-substrate, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), leading to a blue-grey product. As a consequence, COX-2 inhibitors appear on the TLC plate after revelation as clear spots against the colored background. Parameters such as concentrations of enzyme, substrate, and chromogenic reagent have been optimized. The limit of detection was found to be below the microgram for standard COX-2 inhibitors such as celecoxib or ibuprofen. The developed TLC assay was also conclusive when applied to 60 various natural pure compounds and some complex natural extracts. Results demonstrated a COX-2 inhibitory activity mostly for triterpene and sterol derivatives. This COX-2 TLC assay appears as a suitable low-cost and reliable strategy for the screening of natural extracts to discover new anti-inflammatory compounds.

Cyclooxygenase-2 inhibition affects the expression of down syndrom cell adhesion molecule via interaction with metabotropic glutamate receptor 5.

Since we previously discovered that simultaneous inhibition of cyclooxygenase-2 (COX-2; a highly inducible enzyme, crucial for the conversion of arachidonic acid to prostaglandin G2, which plays a predominant role in the CNS) by NS398 and metabotropic glutamate receptor 5 (mGluR5) through the use of its antagonist [3-((2-methyl-4-thiazolyl)ethyl)pyridine; MTEP] alters mouse behavior (e.g., affects spatial learning, and induces/intensifies the antidepressant effect), our aim was to discover the mechanism responsible for these changes. Down syndrome cell adhesion molecule (DSCAM), a member of the immunoglobulin cell adhesion molecule (Ig-CAM) superfamily, is involved in developing the central and peripheral nervous system by influencing cell adhesion mechanisms necessary for synaptic activity and plasticity. Since COX-2 has been implicated in several neuropsychiatric diseases (e.g., major depressive disorder) resulting from neuroplasticity disorders, and on the other hand, its expression is regulated by synaptic activity, we hypothesized that cognitive changes after administration of COX-2 inhibitor and mGluR5 antagonist might be a consequence of impaired DSCAM expression. Importantly, DSCAM deficiency leads to dysregulation of glutamatergic transmission and plasticity. In previous studies, we have demonstrated glutamatergic changes after NS398 and MTEP administration, further supporting the validity of our hypothesis. Due to the different effects observed in behavioral tests, this study used the prefrontal cortex (PFC) and hippocampus (HC) of C57BL/6J mice, which received NS398 and MTEP alone, or in combination, for 7 or 14 days. Among many properties, we also previously investigated the antidepressant potential of these compounds, so we used imipramine (a tricyclic antidepressant) as the reference drug. DSCAM mRNA expression was determined by qRT-PCR. Our results indicate that DSCAM expression after administration of MTEP and NS398 and imipramine along with NS398 is structure- and time-dependent.

A Simplified Direct O2 Consumption-Based Assay to Test COX Inhibition

Background: Cyclooxygenase is a well-known oxidoreductase that catalyzes the uptake of two moles of O2 by arachidonic acid (AA), producing the hydroperoxide Prostaglandin G2 (PGG2), then reduced to the prostaglandin precursor Prostaglandin H2 (PGH2). O2 consumption during such reactions is a measure of cyclooxygenase activity. O2 involved is generally measured by indirect methods, accomplished in the presence of the substrate AA and/or inhibitors. Methods: We developed a new simplified and easy to be carried out protocol for O2 consumption measurement by using disrupted HEK293-derived adherent cells, stably transfected either with COX-1 or COX-2 genes, as a source of the COX enzymes. The Clark electrode is used to measure the O2 concentration variation during the enzyme-catalyzed reactions. Results and Discussion: The novel assay was validated by determining the IC50 values of the known inhibitors such as indomethacin, ibuprofen, SC560, and celecoxib. Indomethacin and ibuprofen are two traditional non-steroidal anti-inflammatory drugs (tNSAIDs). SC560 is a commercially available reference compound used for COX-1 inhibition investigations. Celecoxib is a clinically used COXIBs. The assay was also applied to measure the kinetics and IC50 of mofezolac and P6. Mofezolac is the most potent selective COX-1 inhibitor, and active principle ingredient of Disopain® used to treat rheumatoid arthritis in Japan. P6, uncovered by us, is used together with mofezolac as a reference in in vitro and in vivo COX inhibition investigations and as a scaffold for structure-inhibition activity relationship studies. Conclusion: The obtained results showed the suitability of the newly developed assay to measure COXs activity in the presence of inhibitors as well as the kinetics of the inhibition (i.e., Vmax and Km).

Physiological defense and metabolic strategy of Pistia stratiotes in response to zinc-cadmium co-pollution

Pistia stratiotes is a cadmium (Cd) hyperaccumulating plant with strong bioaccumulation and translocation capacity for Cd. A hydroponic experiment was used to evaluate the combined effect of Zinc (Zn) and Cd at different concentrations on leaf growth and metabolism of P. stratiotes. This study revealed the physiological defense and metabolic strategy of responses to Zn–Cd co-pollution. With the Zn50Cd1, Zn50Cd10, Zn100Cd1, and Zn100Cd10 treatments for 9 d, the relative crown diameter, relative leave number, and ramet number of the plant had no significant difference with the control. Under the compound treatments containing Zn50Cd50 and Zn100Cd50, the activity of the glyoxalase system and amino acid metabolism in the leaves were inhibited. The leaf photosynthetic apparatus increased heat dissipation to reduce the damage to the photosystem II (PS II) reaction center caused by excess excitation energy under Zn–Cd stress. This safeguarded the balance between the absorption and utilization of light energy. Compared to the control, the Zn and Cd co-pollution for 9 d had no effect on the reduced glutathione (GSH) and oxidized glutathione (GSSG) contents. There was no effect on the dehydroascorbate reductase (DHAR) and glutathione reductase (GR) activities, but there was increased ascorbate peroxidase (APX) activity and oxidized ascorbic acid (DHA) content. These increased the antioxidant capacity of the ascorbate-glutathione (AsA-GSH) cycle. The treated plants also had increased levels of carnosol and substances related to lipid metabolism including 9, 10-Dihydroxystearate, Prostaglandin G2, Sphingosine, and 13-L-Hydroperoxylinoleic acid, maintaining the cell stability and resistance to the Zn–Cd stress.

Genome-Wide Analysis Indicates a Complete Prostaglandin Pathway from Synthesis to Inactivation in Pacific White Shrimp, Litopenaeus vannamei.

Prostaglandins (PGs) play many essential roles in the development, immunity, metabolism, and reproduction of animals. In vertebrates, arachidonic acid (ARA) is generally converted to prostaglandin G2 (PGG2) and H2 (PGH2) by cyclooxygenase (COX); then, various biologically active PGs are produced through different downstream prostaglandin synthases (PGSs), while PGs are inactivated by 15-hydroxyprostaglandin dehydrogenase (PGDH). However, there is very limited knowledge of the PG biochemical pathways in invertebrates, particularly for crustaceans. In this study, nine genes involved in the prostaglandin pathway, including a COX, seven PGSs (PGES, PGES2, PGDS1/2, PGFS, AKR1C3, and TXA2S), and a PGDH were identified based on the Pacific white shrimp (Litopenaeus vannamei) genome, indicating a more complete PG pathway from synthesis to inactivation in crustaceans than in insects and mollusks. The homologous genes are conserved in amino acid sequences and structural domains, similar to those of related species. The expression patterns of these genes were further analyzed in a variety of tissues and developmental processes by RNA sequencing and quantitative real-time PCR. The mRNA expression of PGES was relatively stable in various tissues, while other genes were specifically expressed in distant tissues. During embryo development to post-larvae, COX, PGDS1, GDS2, and AKR1C3 expressions increased significantly, and increasing trends were also observed on PGES, PGDS2, and AKR1C3 at the post-molting stage. During the ovarian maturation, decreasing trends were found on PGES1, PGDS2, and PGDH in the hepatopancreas, but all gene expressions remained relatively stable in ovaries. In conclusion, this study provides basic knowledge for the synthesis and inactivation pathway of PG in crustaceans, which may contribute to the understanding of their regulatory mechanism in ontogenetic development and reproduction.

Assessment of Polyunsaturated Fatty Acids on COVID-19-Associated Risk Reduction.

Pooled evidence conveys the association between polyunsaturated fatty acids and infectious disease. SARS-CoV-2, an enveloped mRNA virus, was also reported to interact with polyunsaturated fatty acids. The present review explores the possible mode of action, immunology, and consequences of these polyunsaturated fatty acids during the viral infection. Polyunsaturated fatty acids control protein complex formation in lipid rafts associated with the function of two SARS-CoV-2 entry gateways: angiotensin-converting enzyme-2 and cellular protease transmembrane protease serine-2. Therefore, the viral entry can be mitigated by modulating polyunsaturated fatty acids contents in the body. α-Linolenic acid is the precursor of two clinically important eicosanoids eicosapentaenoic acid and docosahexaenoic acid, the members of ω-3 fats. Resolvins, protectins, and maresins derived from docosahexaenoic acid suppress inflammation and augment phagocytosis that lessens microbial loads. Prostaglandins of 3 series, leukotrienes of 5 series, and thromboxane A3 from eicosapentaenoic acid exhibit anti-inflammatory, vasodilatory, and platelet anti-aggregatory effects that may also contribute to the control of pre-existing pulmonary and cardiac diseases. In contrast, ω-6 linoleic acid-derived arachidonic acid increases the prostaglandin G2, lipoxins A4 and B4, and thromboxane A2. These cytokines are pro-inflammatory and enhance the immune response but aggravate the COVID-19 severity. Therefore, the rational intake of ω-3-enriched foods or supplements might lessen the complications in COVID-19 and might be a preventive measure.Graphic

Non-invasive monitoring of saliva can be used to identify oxidative stress biomarkers in preterm and term newborn infants.

The aim of this study was to appraise the feasibility and reproducibility of applying a validated analytical method to determine salivary oxidative stress biomarkers in newborn infants. Prospective observational single-centre study was carried out in level III neonatal intensive care unit. Eligible patients were preterm infants and healthy full-term newborn infants. Salivary samples were analysed in the chromatographic system. A total of 23 premature newborn infants and 13 full-term newborns were included. We analysed salivary levels of oxidative stress biomarkers for 5-F2t isoprostane, 15-E2t isoprostane, prostaglandin E2 and prostaglandin F2α. The multivariate predictive model showed a positive association between female and 5-F2t isoprostonae, and between female sex and prostglandin F2α. In addition, we found a positive association between gestational age and levels of prostaglandin E2 . Furthermore, in the premature group, we found a positive association between the inspired fraction of oxygen and levels of prostaglandin G2 . We identified and determined lipid peroxidation biomarkers in term and preterm newborn infants' saliva using specific and validated mass spectrometry technology.

Mechanisms exploration of Xiaojin Pills on lung cancer based on metabolomics and network pharmacology.

This study was designed to evaluate the pharmacological activity and therapeutic mechanism of Xiaojin Pills (XJW) on lung cancer. Mice were orally administered with Xiaojin Pills for 21 days. Tumour samples were collected to evaluate the antilung cancer effect, and blood samples were collected to identify differential metabolites with metabolomics. Through the analysis of network pharmacology, the active ingredients and targets related to XJW therapy for lung cancer were filtered. Different expression of seven metabolites related to seven pathways, including Arachidonic acid metabolism, Citrate cycle, tryptophan metabolism, glyoxylate and dicarboxylate metabolism, arginine and proline metabolism, primary bile acid biosynthesis and nicotinate and nicotinamide metabolism, were demonstrated to explain the efficacy of XJW in the treatment of lung cancer. Furthermore, a total of 19 active ingredients (ursolic acid, α-thujone, pelargonidin, succinic acid, boswellic acid, muscone, daidzein, xanthorrhizol, isoeugenol, oleic acid, β-caryophyllene, vanillin, β-sitosterol, lupeol, palmitic acid, eugenol, methylbutenol, β-elemene and quercetin) acted directly on 9 targets (CAT, PTGS2, PTGS1, CTH, ABTA, ALT1, ME2, AGXT and AGXT 2) and regulated 3 out of 7 metabolites (3-Hydroxyanthranilic acid, Pyruvate and Prostaglandin G2). Through metabolomics and network pharmacology analyses, this study demonstrated that the major metabolites of XJW in treating lung cancer were regulated by multitarget and multicomponent interaction network.

The Structural Environment Around the Heme-Binding Site in Cyclooxygenase and Its Implication

Cyclooxygenases (COX-1 and COX-2) have a cyclooxygenase activity involved in prostaglandin G2 (PGG2) formation and an associated peroxidase (POX) activity for the reduction of PGG2 to PGH2, which are functionally interdependent. Peroxide-mediated oxidation of the heme group at the POX active site forming an oxyferryl heme radical cation (known as Compund I) initiates the whole catalytic cycle. The heme in COX proteins is ferriprotoporphyrin IX, much of which is lost upon isolation, but can be replaced to convert apo- to holo-enzyme.

Cycle Network Model of Prostaglandin H Synthase-1.

The kinetic model of Prostaglandin H Synthase-1 (PGHS-1) was developed to investigate its complex network kinetics and non-steroidal anti-inflammatory drugs (NSAIDs) efficacy in different in vitro and in vivo conditions. To correctly describe the complex mechanism of PGHS-1 catalysis, we developed a microscopic approach to modelling of intricate network dynamics of 35 intraenzyme reactions among 24 intermediate states of the enzyme. The developed model quantitatively describes interconnection between cyclooxygenase and peroxidase enzyme activities; substrate (arachidonic acid, AA) and reducing cosubstrate competitive consumption; enzyme self-inactivation; autocatalytic role of AA; enzyme activation threshold; and synthesis of intermediate prostaglandin G2 (PGG2) and final prostaglandin H2 (PGH2) products under wide experimental conditions. In the paper, we provide a detailed description of the enzyme catalytic cycle, model calibration based on a series of in vitro kinetic data, and model validation using experimental data on the regulatory properties of PGHS-1. The validated model of PGHS-1 with a unified set of kinetic parameters is applicable for in silico screening and prediction of the inhibition effects of NSAIDs and their combination on the balance of pro-thrombotic (thromboxane) and anti-thrombotic (prostacyclin) prostaglandin biosynthesis in platelets and endothelial cells expressing PGHS-1.

Toxicology of paraquat and pharmacology of the protective effect of 5-hydroxy-1-methylhydantoin on lung injury caused by paraquat based on metabolomics

Paraquat (PQ) is a non-selective herbicide and is exceedingly toxic to humans. The mechanism of PQ toxicity is very complex and has not been clearly defined. There is no specific antidote for PQ poisoning. 5-hydroxy-1-methylhydantoin (HMH) is an intrinsic antioxidant and can protect against renal damage caused by PQ. The mechanism of PQ toxicology and the possible effects of HMH on PQ-induced lung injury were determined in this study. It was found that PQ decreased superoxide dismutase (SOD) activity and elevated the level of malondialdehyde (MDA), while HMH elevated SOD activity and decreased the level of MDA. Based on metabolomics, the citrate cycle, glutathione metabolism, taurine and hypotaurine metabolism, regulation of lipolysis in adipocytes, inflammatory mediator regulation of TRP channels, purine and pyrimidine metabolism, aldosterone synthesis and secretion, and phenylalanine metabolism were changed in the PQ group. Compared with the PQ group, the levels of N-acetyl-l-aspartic acid, L-glutamic acid, L-aspartic acid, mesaconic acid, adenosine 5′ monophosphate, methylmalonic acid, cytidine, phosphonoacetic acid, hypotaurine, glutathione (reduced) and cysteinylglycine increased, while the levels of corticosterone, xanthine, citric acid, prostaglandin G2, 4-pyridoxic acid and succinyl proline decreased in the HMH group. These metabolites revealed that HMH can alleviate inflammation caused by PQ and elevate the activity of intrinsic antioxidants. In conclusion, our results revealed PQ toxicology and the pharmacology underlying the protective effect of HMH on lung injury due to PQ. Toxicity caused by PQ results in lipid peroxidation and an increase in reactive oxygen species (ROS), nitric oxide (NO), damage to the biliary system, gastrointestinal system and nervous system, in addition to lungs, kidneys, and the liver. HMH is a good antioxidant and protects against lung injury caused by PQ. In summary, HMH efficiently reduced PQ-induced lung injury in mice.

Washed microbiota transplantation vs. manual fecal microbiota transplantation: clinical findings, animal studies and in vitro screening

Fecal microbiota transplantation (FMT) by manual preparation has been applied to treat diseases for thousands of years. However, this method still endures safety risks and challenges the psychological endurance and acceptance of doctors, patients and donors. Population evidence showed the washed microbiota preparation with microfiltration based on an automatic purification system followed by repeated centrifugation plus suspension for three times significantly reduced FMT-related adverse events. This washing preparation makes delivering a precise dose of the enriched microbiota feasible, instead of using the weight of stool. Intraperitoneal injection in mice with the fecal microbiota supernatant obtained after repeated centrifugation plus suspension for three times induced less toxic reaction than that by the first centrifugation following the microfiltration. The toxic reactions that include death, the change in the level of peripheral white blood cells, and the proliferation of germinal center in secondary lymphoid follicles in spleen were noted. The metagenomic next-generation sequencing (NGS) indicated the increasing types and amount of viruses could be washed out during the washing process. Metabolomics analysis indicated metabolites with pro-inflammatory effects in the fecal microbiota supernatant such as leukotriene B4, corticosterone, and prostaglandin G2 could be removed by repeated washing. Near-infrared absorption spectroscopy could be served as a rapid detection method to control the quality of the washing-process. In conclusion, this study for the first time provides evidence linking clinical findings and animal experiments to support that washed microbiota transplantation (WMT) is safer, more precise and more quality-controllable than the crude FMT by manual.

Metabolomic Profiles of Bovine Mammary Epithelial Cells Stimulated by Lipopolysaccharide

Bovine mammary epithelial cells (bMECs) are the main cells of the dairy cow mammary gland. In addition to their role in milk production, they are effector cells of mammary immunity. However, there is little information about changes in metabolites of bMECs when stimulated by lipopolysaccharide (LPS). This study describes a metabolomics analysis of the LPS-stimulated bMECs to provide a basis for the identification of potential diagnostic screening biomarkers and possible treatments for bovine mammary gland inflammation. In the present study, bMECs were challenged with 500 ng/mL LPS and samples were taken at 0 h, 12 h and 24 h post stimulation. Metabolic changes were investigated using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF MS) with univariate and multivariate statistical analyses. Clustering and metabolic pathway changes were established by MetaboAnalyst. Sixty-three differential metabolites were identified, including glycerophosphocholine, glycerol-3-phosphate, L-carnitine, L-aspartate, glutathione, prostaglandin G2, α-linolenic acid and linoleic acid. They were mainly involved in eight pathways, including D-glutamine and D-glutamic acid metabolism; linoleic acid metabolism; α-linolenic metabolism; and phospholipid metabolism. The results suggest that bMECs are able to regulate pro-inflammatory, anti-inflammatory, antioxidation and energy-producing related metabolites through lipid, antioxidation and energy metabolism in response to inflammatory stimuli.