Group IVA Phospholipase A2 Research Articles

Group IVA Phospholipase A2 in Collagen-Producing Cells Promotes High-Fat Diet-Induced Infiltration of Inflammatory Cells into the Liver by Upregulating the Expression of MCP-1.

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic inflammation and fibrosis due to excessive fat accumulation. Monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that infiltrates inflammatory cells into the liver during the development of NASH. Our previous studies demonstrated that a systemic deficiency of group IVA phospholipase A2 (IVA-PLA2), an enzyme that contributes to the production of lipid inflammatory mediators, protects mice against high-fat diet-induced hepatic fibrosis and markedly suppresses the CCl4-induced expression of MCP-1 in the liver. However, it remains unclear which cell types harboring IVA-PLA2 are involved in the elevated production of MCP-1. Hence, the present study assessed the types of cells responsible for IVA-PLA2-mediated production of MCP-1 using cultured hepatic stellate cells, endothelial cells, macrophages, and hepatocytes, as well as cell-type specific IVA-PLA2 deficient mice fed a high-fat diet. A relatively specific inhibitor of IVA-PLA2 markedly suppressed the expression of MCP-1 mRNA in cultured hepatic stellate cells, but the suppression of MCP-1 expression was partial in endothelial cells and not observed in monocytes/macrophages or hepatocytes. In contrast, a deficiency of IVA-PLA2 in collagen-producing cells (hepatic stellate cells), but not in other types of cells, reduced the high-fat diet-induced expression of MCP-1 and inflammatory cell infiltration in the liver. Our results suggest that IVA-PLA2 in hepatic stellate cells is critical for hepatic inflammation in the high-fat diet-induced development of NASH. This supports a potential therapeutic approach for NASH using a IVA-PLA2 inhibitor targeting hepatic stellate cells.

The roles of HCV core protein and its binding host factor in virus assembly and release

Hepatitis C virus (HCV) is a well-known virus that causes liver diseases such as liver cirrhosis and hepatocellular carcinoma. For several decades, numerous studies have been conducted to unravel the life cycle and molecular mechanisms of this virus with the aim of developing strategies to combat diseases caused by its infection. In this review, we summarize HCV assembly to budding, focusing on one of the structural proteins, the core, a viral capsid that binds both the viral genome and host membrane, along with the core-interacting host partners. The HCV core matures in the endoplasmic reticulum (ER), localizes at the lipid droplet (LD), and shuttles between the LD and ER to form viral particles. This process is controlled by many host factors known to binds core proteins, such as diacylglycerol acyltransferase-1 (DGAT-1), Rab18, μ subunit of the clathrin adaptor protein complex 2 (AP2M1), nuclear pore complex protein 98 (Nup98), Cortactin, group IVA phospholipase A2 (PLA2G4A) etc. Virion budding is thought to involve contributions from endosomal sorting complexes required for transport (ESCRT), similar to other envelope viruses. We delved into potential perspectives to enhance our understanding of the HCV mechanism by drawing insights from existing studies.

Eight Zhes Decoction ameliorates the lipid dysfunction of nonalcoholic fatty liver disease using integrated lipidomics, network pharmacology and pharmacokinetics

Nonalcoholic fatty liver disease (NAFLD) has developed into the most common chronic liver disease and can lead to liver cancer. Our laboratory previously developed a novel prescription for NAFLD, “Eight Zhes Decoction” (EZD), which has shown good curative effects in clinical practice. However, the pharmacodynamic material basis and mechanism have not yet been revealed. A strategy integrating lipidomics, network pharmacology and pharmacokinetics was used to reveal the active components and mechanisms of EZD against NAFLD. The histopathological results showed that EZD attenuated the degrees of collagen deposition and steatosis in the livers of nonalcoholic steatofibrosis model mice. Furthermore, glycerophospholipid metabolism, arachidonic acid metabolism, glycerolipid metabolism and linoleic acid metabolism with phospholipase A2 group IVA (PLA2G4A) and cytochrome P450 as the core targets and 12,13-cis-epoxyoctadecenoic acid, 12(S)-hydroxyeicosatetraenoic acid, leukotriene B4, prostaglandin E2, phosphatidylcholines (PCs) and triacylglycerols (TGs) as the main lipids were found to be involved in the treatment of NAFLD by EZD. Importantly, naringenin, artemetin, canadine, and bicuculline were identified as the active ingredients of EZD against NAFLD; in particular, naringenin reduces PC consumption by inhibiting the expression of PLA2G4A and thus promotes sufficient synthesis of very-low-density lipoprotein to transport excess TGs in the liver. This research provides valuable data and theoretical support for the application of EZD against NAFLD.

Chlorogenic Acid, a Component of Oenanthe javanica (Blume) DC., Attenuates Oxidative Damage and Prostaglandin E2 Production Due to Particulate Matter 10 in HaCaT Keratinocytes

Oenanthe javanica (OJ) is a perennial herb that grows wildly or is cultivated in Asia, and it is used as food or in traditional medicine. The antioxidant and anti-inflammatory effects of OJ-derived materials have been extensively explored previously, but their effects on the cytotoxicity of air pollution are currently unknown. Therefore, the present study aimed to evaluate the effect of the hot water extract of OJ on atmospheric particulate matter 10 (PM10)-induced cytotoxicity and oxidative damage in human HaCaT keratinocytes, and to identify its active ingredient and mechanism of action. When the hot water extract of OJ was divided into methylene chloride, ethyl acetate (EA), n-butanol (BA), and water fractions, caffeic acid was enriched in the EA fraction and chlorogenic acid was enriched in the BA fraction. PM10 increased reactive oxygen species (ROS) production, lipid peroxidation, protein carbonylation, and inflammatory prostaglandin (PG) E2 production in cells. The BA fraction reduced the PM10-induced ROS production in cells more effectively than the total extract and other solvent fractions. Chlorogenic acid was more effective in reducing ROS levels than caffeic acid and N-acetyl cysteine (NAC). Chlorogenic acid attenuated the increase in lipid peroxidation and the PG E2 production of cells due to PM10 exposure. Of the genes involved in PG E2 production, phospholipase A2 group IVA (PLA2G4A), Prostaglandin-endoperoxide synthase 1 (PTGS1), and 2 (PTGS2) were transcriptionally up-regulated by PM10, whereas phospholipase A2 group IIA (PLA2G2A) was down-regulated and prostaglandin E synthetase 1 (PTGES1) and 2 (PTGES2) were a little altered. The PM10-induced increase in PLA2G4A mRNA was alleviated by chlorogenic acid and NAC. Accordingly, PM10 increased the expression levels of cytosolic phospholipase A2 (cPLA2) protein and its phosphorylated form, which were attenuated by chlorogenic acid and NAC. Thus, chlorogenic acid may attenuate the PM10-induced PG E2 production through the suppression of PLA2G4A mRNA and cPLA2 protein expressions. This study suggests that chlorogenic acid contained in OJ extract may help alleviate the oxidative damage to and inflammatory responses of the skin cells due to exposure to air pollutants.

Deficiency of Group IVA Phospholipase A2 in Collagen-Producing Cells Alleviates the Aggravated Hepatic Fibrosis in High-Fat Diet-Fed Mice after Returning to a Normal Diet.

Hepatic fibrosis, a primary feature of non-alcoholic steatohepatitis (NASH), develops with inflammation and subsequent activation of hepatic stellate cells (HSCs), the main extracellular matrix-producing cells. Currently, no approved pharmacotherapy is available to treat hepatic fibrosis, even under dietary intervention. The activation of cultured HSCs has been shown to be attenuated by pharmacological inhibition of group IVA phospholipase A2 (IVA-PLA2), an enzyme initiating the generation of lipid proinflammatory mediators. We examined the potential utility of IVA-PLA2 of HSCs as a therapeutic target for hepatic fibrosis in NASH under dietary modification using collagen-producing cell-specific IVA-PLA2-conditional knockout mice fed a high-fat diet and then returned to a normal one. Apparent hepatic fibrosis and the accumulation of hepatic lipid droplets developed in the IVA-PLA2-conditional knockout mice on a high-fat diet for nine weeks to a similar degree as in control mice. Most of the lipid droplets disappeared five weeks after switching the diet back to a normal one in both genetic mice. In contrast, the hepatic fibrosis in control mice still progressed even after changing back to a normal diet. However, deficiency of IVA-PLA2 in collagen-producing cells alleviated the aggravated hepatic fibrosis under dietary modification. Our results revealed that the protective effects of an HSC-specific IVA-PLA2 deficiency on fibrogenesis appear after switching the diet from a high-fat one back to a normal one, supporting the promising beneficial effects of the inhibition of IVA-PLA2 on progressive hepatic fibrosis under dietary intervention in NASH treatment.

Metabolomic mechanism and pharmacodynamic material basis of Buxue Yimu pills in the treatment of anaemia in women of reproductive age.

Objective: To explore the pharmacological basis and mechanism of Buxue Yimu pills (BYP) in the treatment of anaemia in women from the perspective of metabolomics and network analysis. Materials and Methods: Forty-six women of reproductive age with haemoglobin 70-110g/L were recruited. Blood samples were collected before and after 4weeks of oral BYP treatment to assess the changes in haemoglobin, coagulation function, and iron metabolism indices. An integrated analysis of metabolomics (liquid chromatography mass spectrometry) and network analysis was performed to identify the potential pharmacodynamic mechanisms of BYP. Results: After BYP treatment, the haemoglobin level of patients significantly increased from 93.67 ± 9.77g/L to 109.28 ± 12.62g/L (p < 0.01), while no significant changes were found in iron metabolism and coagulation-related indicators. A total of 22 differential metabolites were identified after metabolomics analysis, which were mainly related to the inhibition of inflammation and oxidative stress. Integrating pharmacodynamics and metabolomics, a network of drug-active components-targets-metabolic pathways-metabolomics was established. Acetylcholinesterase, phospholipase A2 group IIA, and phospholipase A2 group IVA may be the most promising therapeutic targets. Conclusion: BYP can inhibit inflammation and oxidative stress as well as promote haematopoiesis, potentially improving anaemia.

Evaluation of clinical significances and anti-tumor effects with several prognostic factors in patients with acute myeloid leukemia

Backgrounds: Acute myeloid leukemia (AML) is a malignant tumor of the hemopoietic system in adults. Although multiple signaling pathways are associated with this disease, its pathogenesis has not yet been fully elucidated. Herein, we aimed to explore the novel prognostic, diagnostic and therapeutic targets in patients with AML. MethodsGene expression, overall survival (OS) and diagnosis were analyzed by bioinformatics software with The Cancer Genome Atlas (TCGA) database and our cohort population (52 healthy subjects and 98 AML patients). ResultsThree prognostic factors T-cell leukemia translocation-altered gene (TCTA), sideroflexin 3 (SFXN3) and phospholipase A2 group IVA (PLA2G4A) were closely linked to poor prognosis in patients with AML based on the TCGA database and our cohort population. Multivariate COX regression analysis indicated that age (>60 years), up-regulation of SFXN3 and PLA2G4A were the independent prognostic factors associated with poor OS. Intriguingly, TCTA, SFXN3 and PLA2G4A presented the remarkable diagnostic value to distinguish AML from healthy subjects. Knockdown of SFXN3 led to inhibition of AML cell growth in vitro and in vivo and prolonged the survival time of nude mouse. ConclusionA three-gene panel may play pivotal pathogenic roles in AML, represents potential prognostic and diagnostic indicators and therapeutic targets in the patients with AML. In addition, SFXN3 may be a potential therapeutic target of AML.

Integration analysis of metabolome and transcriptome reveals the effect of exogenous supplementation with mixtures of vitamins ADE, zinc, and selenium on follicular growth and granulosa cells molecular metabolism in donkeys (Equus asinus).

Vitamins and microelements play essential roles in mammalian ovarian physiology, including follicle development, ovulation, and synthesis and secretion of hormones and growth factors. However, it is nevertheless elusive to what extent exogenous supplementation with mixtures of vitamins ADE, zinc (Zn), and selenium (Se) affects follicular growth and granulosa cells (GCs) molecular function. We herein investigated their effect on follicular growth and GCs physiological function. We showed that follicular growth and ovulation time was accelerated and shortened with the increases of vitamins ADE, Zn, and Se doses by continually monitoring and recording (one estrus cycle of about 21 days) with an ultrasound scanner. Integrated omics analysis showed that there was a sophisticated network relationship, correlation expression, and enrichment pathways of the genes and metabolites highly related to organic acids and their derivatives and lipid-like molecules. Quantitative real-time PCR (qPCR) results showed that vitamin D receptor (VDR), transient receptor potential cation channel subfamily m member 6 (TRPM6), transient receptor potential cation channel subfamily v member 6 (TRPV6), solute carrier family 5 member 1 (SLC5A1), arachidonate 5-lipoxygenase (ALOX5), steroidogenic acute regulatory protein (STAR), prostaglandin-endoperoxide synthase 2 (PTGS2), and insulin like growth factor 1 (IGF-1) had a strong correlation between the transcriptome data. Combined multi-omics analysis revealed that the protein digestion and absorption, ABC transporters, biosynthesis of amino acids, aminoacyl-tRNA biosynthesis, mineral absorption, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, arginine biosynthesis, and ovarian steroidogenesis were significantly enriched. We focused on the gene-metabolite interactions in ovarian steroidogenesis, founding that insulin receptor (INSR), phospholipase a2 group IVA (PLA2G4A), adenylate cyclase 6 (ADCY6), cytochrome p450 family 1 subfamily b member 1 (CYP1B1), protein kinase camp-activated catalytic subunit beta (PRKACB), cytochrome p450 family 17 subfamily a member 1 (CYP17A1), and phospholipase a2 group IVF (PLA2G4F) were negatively correlated with β-estradiol (E2), progesterone (P4), and testosterone (T) (P < 0.05). while ALOX5 was a positive correlation with E2, P4, and T (P < 0.05); cytochrome p450 family 19 subfamily a member 1 (CYP19A1) was a negative correlation with cholesterol (P < 0.01). In mineral absorption, our findings further demonstrated that there was a positive correlation between solute carrier family 26 member 6 (SLC26A6), SLC5A1, and solute carrier family 6 member 19 (SLC6A19) with Glycine and L-methionine. Solute carrier family 40 member 1 (SLC40A1) was a negative correlation with Glycine and L-methionine (P < 0.01). TRPV6 and ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) were positively associated with Glycine (P < 0.05); while ATPase Na+/K+ transporting subunit beta 3 (ATP1B3) and cytochrome b reductase 1 (CYBRD1) were negatively related to L-methionine (P < 0.05). These outcomes suggested that the vitamins ADE, Zn, and Se of mixtures play an important role in the synthesis and secretion of steroid hormones and mineral absorption metabolism pathway through effects on the expression of the key genes and metabolites in GCs. Meanwhile, these also are required for physiological function and metabolism of GCs. Collectively, our outcomes shed new light on the underlying mechanisms of their effect on follicular growth and GCs molecular physiological function, helping explore valuable biomarkers.

Effect of forsythoside A on the transcriptional profile of bovine mammary epithelial cells challenged with lipoteichoic acid.

Mastitis is a common disease of the dairy cattle, which affects the development of the dairy industry and leads to huge economic losses. Forsythoside A (FTA) has anti-inflammatory, antioxidant, antiviral and anti-apoptotic effects. However, the therapeutic effect and molecular mechanism of FTA on dairy cow mastitis remain unclear. In this study, bovine mammary epithelial cells (BMECs) were stimulated with lipoteichoic acid (LTA), a key virulence factor of Staphylococcus aureus (S. aureus), to construct in vitro models, and then treated with FTA. Subsequently, the differentially expressed genes (DEGs) in different groups were determined by RNA sequencing (RNA-Seq) analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyse the possible function of the DEGs, real-time quantitative PCR (RT-qPCR) was used to verify whether the expression levels of these DEGs were consistent with RNA-Seq results. The results showed that cell division cycle 20B (CDC20B), endothelial cell surface expressed chemotaxis and apoptosis regulator (ECSCR), complement factor H-related 5 (CFHR5) and phospholipase A2 group IVA (PLA2G4A) were down-regulated after FTA treatment. In contrast, Kruppel-like factor 15 (KLF15) and Metallothionein 1E (MT1E) were up-regulated. These DEGs are involved in processes such as apoptosis, inflammation and development of cancer. This study provides valuable insights into the transcriptome changes in BMECs after FTA treatment. Further analysis may help identify the underlying molecular mechanisms.

Mitochondrial-bound hexokinase 1 can affect the glucolipid metabolism and reactive oxygen species production in goose fatty liver

To investigate the functions of hexokinase 1 (HK1) in the formation of goose fatty liver, a total of 40 healthy 63-day-old male Landes geese were selected and randomly assigned to a control group and an overfeeding treatment. In addition, the overexpression or RNA interference assay of HK1, and transcriptome analysis after HK1 overexpression were performed in the goose primary hepatocytes. Data showed that the mRNA expression of hepatic HK1 was upregulated in overfed treatment compared to control on the 19 days of overfeeding. The expression of HK1 was increased in 50 mM glucose treatment in hepatocytes. Moreover, overexpression of HK1 tended to increase the relative lipid accumulation level and had weakened fluorescence intensity of reactive oxygen species (ROS), while knockdown of HK1 resulted in a tendency of relative lipid content decrease and had enhanced fluorescence intensity of ROS in cells in comparison to the control. The verification of transcriptome analysis indicated that the expression of ceruloplasmin (CP), acyl-CoA dehydrogenase medium-chain (ACADM), phosphoglucomutase 2 (PGM2), and phospholipase A2 group IVA (PLA2G4A) was significantly induced by HK1 overexpression, while that of enoyl-CoA hydratase, short chain 1 (ECHS1), cytochrome P450 family 2 subfamily C member 19 (CYP2C19), carnitine palmitoyltransferase 1 A (CPT1A), and oxidative stress-induced growth inhibitor 1 (OSGIN1) was inhibited. In summary, HK1 could promote fat deposition by affecting the process of glucolipid metabolism in the formation of goose fatty liver. Additionally, HK1 might decrease the ROS level in hepatocytes by regulating the expression of redox-related genes. HIGHLIGHTS The HK1 gene may promote the goose fatty liver production. The HK1 gene may be used as a research target to decrease the reactive oxygen species level in fatty liver.

Endothelial group IVA phospholipase A2 promotes hepatic fibrosis with sinusoidal capillarization in the early stage of non-alcoholic steatohepatitis in mice

AimNon-alcoholic steatohepatitis (NASH) is characterized by steatosis, inflammatory responses and fibrosis. Our previous studies provided evidence that group IVA phospholipase A2 (IVA-PLA2), a key PLA2 isozyme in the arachidonic acid cascade, is involved in the development of NASH. However, which types of cells are critical for the IVA-PLA2-dependent onset and progression of NASH is unclear. We elucidated the effects of the cell-type-specific deficiency of IVA-PLA2 in mice on the development of NASH. Main methodsCell-type-specific IVA-PLA2-conditional knockout (cKO) mice and littermate controls were fed a choline-deficient, L-amino-acid-defined, high-fat diet with 0.1% methionine as a NASH model. The degree of hepatic fibrosis was evaluated by staining with picric acid-Sirius red, and the number of activated hepatic stellate cells was determined by immunoblotting and immunostaining for α-smooth muscle actin. Sinusoidal capillarization was analyzed by scanning electron microscopy. Key findingsThe deposition of collagen and number of activated hepatic stellate cells were markedly reduced in endothelial cell/liver sinusoidal endothelial cell (EC/LSEC)-specific IVA-PLA2 cKO mice but not in hepatocyte-, monocyte/macrophage-, or hepatic stellate cell-specific IVA-PLA2 cKO mice. In addition, EC/LSEC-specific IVA-PLA2-deficient mice showed more fenestrae than control mice fed a CDAHFD, indicating suppression of sinusoidal capillarization. SignificanceThese results suggest that ECs/LSECs contribute to the IVA-PLA2-dependent onset and/or progression of NASH. Endothelial IVA-PLA2 is a promising factor for promoting sinusoidal capillarization and the ensuing HSC activation and fibrosis; thus IVA-PLA2 in ECs/LSECs is a potential therapeutic target for NASH.

Analysis of Structural Variants Reveal Novel Selective Regions in the Genome of Meishan Pigs by Whole Genome Sequencing.

Structural variants (SVs) represent essential forms of genetic variation, and they are associated with various phenotypic traits in a wide range of important livestock species. However, the distribution of SVs in the pig genome has not been fully characterized, and the function of SVs in the economic traits of pig has rarely been studied, especially for most domestic pig breeds. Meishan pig is one of the most famous Chinese domestic pig breeds, with excellent reproductive performance. Here, to explore the genome characters of Meishan pig, we construct an SV map of porcine using whole-genome sequencing data and report 33,698 SVs in 305 individuals of 55 globally distributed pig breeds. We perform selective signature analysis using these SVs, and a number of candidate variants are successfully identified. Especially for the Meishan pig, 64 novel significant selection regions are detected in its genome. A 140-bp deletion in the Indoleamine 2,3-Dioxygenase 2 (IDO2) gene, is shown to be associated with reproduction traits in Meishan pig. In addition, we detect two duplications only existing in Meishan pig. Moreover, the two duplications are separately located in cytochrome P450 family 2 subfamily J member 2 (CYP2J2) gene and phospholipase A2 group IVA (PLA2G4A) gene, which are related to the reproduction trait. Our study provides new insights into the role of selection in SVs' evolution and how SVs contribute to phenotypic variation in pigs.

Dysregulation of endocannabinoid concentrations in human subcutaneous adipose tissue in obesity and modulation by omega-3 polyunsaturated fatty acids.

Obesity is believed to be associated with a dysregulated endocannabinoid system which may reflect enhanced inflammation. However, reports of this in human white adipose tissue (WAT) are limited and inconclusive. Marine long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) have anti-inflammatory actions and therefore may improve obesity-associated adipose tissue inflammation. Therefore, fatty acid (FA) concentrations, endocannabinoid concentrations, and gene expression were assessed in subcutaneous WAT (scWAT) biopsies from healthy normal weight individuals (BMI 18.5-25 kg/m2) and individuals living with metabolically healthy obesity (BMI 30-40 kg/m2) prior to and following a 12-week intervention with 3 g fish oil/day (1.1 g eicosapentaenoic acid (EPA) + 0.8 g DHA) or 3 g corn oil/day (placebo). WAT from individuals living with metabolically healthy obesity had higher n-6 PUFAs and EPA, higher concentrations of two endocannabinoids (anandamide (AEA) and eicosapentaenoyl ethanolamide (EPEA)), higher expression of phospholipase A2 Group IID (PLA2G2D) and phospholipase A2 Group IVA (PLA2G4A), and lower expression of CNR1. In response to fish oil intervention, WAT EPA increased to a similar extent in both BMI groups, and WAT DHA increased by a greater extent in normal weight individuals. WAT EPEA and docosahexaenoyl ethanolamide (DHEA) increased in normal weight individuals only and WAT 2-arachidonyl glycerol (2-AG) decreased in individuals living with metabolically healthy obesity only. Altered WAT fatty acid, endocannabinoid, and gene expression profiles in metabolically healthy obesity at baseline may be linked. WAT incorporates n-3 PUFAs when their intake is increased which affects the endocannabinoid system; however, effects appear greater in normal weight individuals than in those living with metabolically healthy obesity.

The Contribution of Cytosolic Group IVA and Calcium-Independent Group VIA Phospholipase A2s to Adrenic Acid Mobilization in Murine Macrophages.

Adrenic acid (AA), the 2-carbon elongation product of arachidonic acid, is present at significant levels in membrane phospholipids of mouse peritoneal macrophages. Despite its abundance and structural similarity to arachidonic acid, very little is known about the molecular mechanisms governing adrenic acid mobilization in cells of the innate immune system. This contrasts with the wide availability of data on arachidonic acid mobilization. In this work, we used mass-spectrometry-based lipidomic procedures to define the profiles of macrophage phospholipids that contain adrenic acid and their behavior during receptor activation. We identified the phospholipid sources from which adrenic acid is mobilized, and compared the data with arachidonic acid mobilization. Taking advantage of the use of selective inhibitors, we also showed that cytosolic group IVA phospholipase A2 is involved in the release of both adrenic and arachidonic acids. Importantly, calcium independent group VIA phospholipase A2 spared arachidonate-containing phospholipids and hydrolyzed only those that contain adrenic acid. These results identify separate mechanisms for regulating the utilization of adrenic and arachidonic acids, and suggest that the two fatty acids may serve non-redundant functions in cells.

Inhibition of Group IVA Phospholipase A2 as a Novel Therapeutic Strategy for Nonalcoholic Steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a lifestyle-related disease characterized by hepatic fibrosis with the accumulation of fat and inflammation and can progress to cirrhosis or hepatocellular carcinoma. However, effective pharmacotherapeutic strategies for hepatic fibrosis in NASH remain to be established. Among the initiators of inflammation, we have been investigating the possible involvement of group IVA phospholipase A2 (IVA-PLA2), which catalyzes the initial step in the generation of lipid mediators, including eicosanoids and lysophospholipids, in the progression of hepatic fibrosis. We have recently demonstrated that a lack of IVA-PLA2 alleviates hepatic fibrosis in NASH model mice fed a high-fat and high-cholesterol diet and in CCl4-treated mice. CCl4-induced hepatic fibrosis was also prevented by the administration of an orally active, specific IVA-PLA2 inhibitor even after hepatic fibrosis had developed. Based on these findings suggesting that IVA-PLA2 mediates the cellular responses contributing to the progression of hepatic fibrosis, we have been exploring which types of cells in the liver are involved in IVA-PLA2-mediated hepatic fibrosis using cell-specific IVA-PLA2 knockout mice. The preliminary experimental results suggest that IVA-PLA2 in endothelial cells, but not monocyte-derived cells, plays a role, in part, in the hepatic stellate cell-mediated progression of hepatic fibrosis. In this paper, we discuss the possibility that IVA-PLA2 and/or its related molecules are candidate pharmacotherapeutic targets for NASH treatment.

Cytosolic group IVA phospholipase A2 inhibitors, AVX001 and AVX002, ameliorate collagen-induced arthritis

BackgroundCytosolic phospholipase A2 group IVA (cPLA2α)-deficient mice are resistant to collagen-induced arthritis, suggesting that cPLA2α is an important therapeutic target. Here, the anti-inflammatory effects of the AVX001 and AVX002 cPLA2α inhibitors were investigated.MethodsIn vitro enzyme activity was assessed by a modified Dole assay. Effects on inhibiting IL-1β-induced release of arachidonic acid (AA) and prostaglandin E2 (PGE2) were measured using SW982 synoviocyte cells. In vivo effects were studied in prophylactic and therapetic murine collagen-induced arthritis models and compared to methotrexate (MTX) and Enbrel, commonly used anti-rheumatic drugs. The in vivo response to treatment was evaluated in terms of the arthritis index (AI), histopathology scores and by plasma levels of PGE2 following 14 and 21 days of treatment.ResultsBoth cPLA2α inhibitors are potent inhibitors of cPLA2α in vitro. In synoviocytes, AVX001 and AVX002 reduce, but do not block, release of AA or PGE2 synthesis. In both CIA models, the AI and progression of arthritis were significantly lower in the mice treated with AVX001, AVX002, Enbrel and MTX than in non- treated mice. Several histopathology parameters of joint damage were found to be significantly reduced by AVX001 and AVX002 in both prophylactic and therapeutic study modes; namely articular cavity and peripheral tissue inflammatory cell infiltration; capillary and synovial hyperplasia; articular cartilage surface damage; and periostal and endochondral ossification. In comparison, MTX did not significantly improve any histopathology parameters and Enbrel only improved ossification. Finally, as a biomarker of inflammation and as an indication that AVX001 and AVX002 blocked the cPLA2α target, we determined that plasma levels of PGE2 were significantly reduced in response to the AVX inhibitors and MTX, but not Enbrel.ConclusionsAVX001 and AVX002 display potent anti-inflammatory activity and disease-modifying properties in cellular and in vivo models. The in vivo effects of AVX001 and AVX002 were comparable to, or superior, to those of MTX and Enbrel. Taken together, this study suggests that cPLA2α inhibitors AVX001 and AVX002 are promising small molecule disease-modifying anti-rheumatic therapies.

Identification of the transgenic integration site in 2C T cell receptor transgenic mice

2C T cell receptor (TCR) transgenic mice have been long used to study the molecular basis of TCR binding to peptide/major compatibility complexes and the cytotoxicity mechanism of cytotoxic T lymphocytes (CTLs). To study the role of variable gene promoters in allelic exclusion, we previously constructed mutant mice in which the Vβ13 promoter was deleted (P13 mice). Introduction of 2C transgene into P13 mice accelerated the onset of systemic CD8 T cell lymphoma between 14 and 27weeks of age, although parental P13 mice appeared to be normal. This observation suggests that the lymphoma development may be linked to features of 2C transgene. To identify the integration site of 2C transgene, Southern blotting identified a 2C-specific DNA fragment by 3' region probe of 2C TCR α transgene, and digestion-circularization-polymerase chain reaction (DC-PCR) amplified the 2C-specific DNA fragment with inverse primers specific to the southern probe. Sequence analysis revealed that DC-PCR product contained the probe sequences and the junction sequences of integration site, indicating that 2C TCR α transgene is integrated into chromosome 1. Further genomic analysis revealed cytosolic phospholipase A2 group IVA (cPLA2) as the nearest gene to the integration site. cPLA2 expression was upregulated in the normal thymi and T cell lymphomas from 2C transgenic mice, although it was not altered in the lymph nodes of 2C transgenic mice. The result is the first report demonstrating the integration site of 2C TCR transgene, and will facilitate the proper use of 2C transgenic mice in studies of CTLs.

Analysis of hepatic transcriptome demonstrates altered lipid metabolism following Lactobacillus johnsonii BS15 prevention in chickens with subclinical necrotic enteritis

BackgroundSubclinical necrotic enteritis (SNE) widely outbreaks in chickens which inflicted growth-slowing, causing enormous social and economic burdens. To better understand the molecular underpinnings of SNE on lipid metabolism and explore novel preventative strategies against SNE, we studied the regulatory mechanism of a potential probiotic, Lactobacillus johnsonii BS15 on the lipid metabolism pathways involved in chickens with SNE.MethodsOne hundred eighty one-day-old chickens were randomly divided into three groups and arranged with basal diet (control and SNE group). Added with BS15 (1 × 106 cfu/g) or Man Rogosa Sharpe (MRS) liquid medium for 28 days. The hepatic gene expression of each group was then measured using high-throughput analysis methods (RNA-Seq). Quantitative real-time PCR (qRT-PCR) was used to detect the expression changes of the related genes.ResultsThe results showed that there are eleven lipid metabolic pathways were found during the prevention of BS15 treatment in SNE chickens by RNA-Seq, including the peroxisome proliferator-activated receptor (PPAR) signaling pathway and arachidonic acid metabolism. BS15 notably facilitated the expressions of fatty acid binding protein 2 (FABP2), acyl-CoA synthetase bubblegum family member 1 (ACSBG1), perilipin 1 (PLIN1) and perilipin 2 (PLIN2), which were involved in PPAR signaling pathway of SNE chickens. Besides, suppression of phospholipase A2 group IVA (PLA2G4A) in arachidonic acid metabolism was observed in SNE chickens after BS15 prevention. The expression patterns of FABP2, ACSBG1, PLIN1, PLIN2 and PLA24G in qRT-PCR validation were consistent with RNA-Seq results.ConclusionsThese findings indicate that SNE may affect the hepatic lipid metabolism of chickens. Meanwhile, BS15 pretreatment may provide a prospective natural prophylaxis strategy against SNE through improving the PPAR signaling pathway and arachidonic acid metabolism.

Effect of Tumor Conditioned Media from Prostate Cancer Cells on Fibroblast Cell Morphology, Viability and Gene Expression

Cancer is a heterogeneous collection of neoplastic cells, surrounded by non‐malignant, stromal cells, and extracellular matrix (ECM), among other constituents. Cross‐talk between tumor and stromal cells forms a dynamic tumor microenvironment (TME) that mediates cell growth, proliferation, and metastasis. The TME can act to inhibit cancer growth and proliferation during the initial stages of carcinogenesis. However, cancer cells are able to evade these inhibitory signals, and subsequently reshape the TME, making it more amenable to their own growth. This gives rise to numerous cancer‐associated stromal cells, including cancer‐associated fibroblasts (CAFs). CAFs further modulate the TME by regulating the release of growth signals, cell death, ECM remodeling, angiogenesis, and epithelial‐mesenchymal transition. To study the ability of prostate cancer to alter the TME in vitro, we exposed human bone fibroblasts (HS‐5 cells) to tumor conditioned media isolated from a human neuroendocrine prostate cancer cell line (DU145) and assessed changes in cell morphology, viability (using MTT assay) and the expression of Group IIA secretory phospholipase A2 (PLA2G2A), as well as its regulatory proteins, glypican‐1 (GPC‐1) and Group IVA phospholipase A2 (PLA2G4A), using qPCR. Exposure of HS‐5 cells to TCM from DU145 cells altered cellular morphology to a more mesenchymal phenotype typically seen in active myofibroblasts. TCM also decreased MTT staining in a time‐dependent manner that was independent of the presence of serum in the TCM. Changes in cell morphology and viability correlated to increases in the expression of PLA2G2A, GPC‐1 and PLA2G4A. The effect of TCM was time‐ and serum‐dependent, and was differential for each gene studied. Alteration in cellular morphology and increases in PLA2G2A, GPC‐1 and PLA2G4A in HS‐5 cells correlated to increases in the expression of fibroblast activation protein‐α, which is a CAF marker. To elucidate the mechanisms mediating changes in HS‐5 cells exposed to TCM, we investigated the expression of GLi1, a transcription factor involved in the activation of the sonic hedgehog signaling pathway. We found that TCM increased GLi1 mRNA levels in HS‐5 cells, with greater increases being observed using TCM containing serum. Collectively, these data demonstrate that media from DU145 cells can induce morphological and genotypic changes in human fibroblasts similar to those observed in CAFs. As such, this model holds promise for identifying molecular mechanisms by which prostate cancer cells alter the TME and induce prostate cancer cell growth.Support or Funding InformationNational Institute of Biomedical Imaging and Bioengineering NIBIB (EB0160100)Department of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-543 Promotes Migration, Invasion and Epithelial-Mesenchymal Transition of Esophageal Cancer Cells by Targeting Phospholipase A2 Group IVA

Background/Aims: The aim of this study was to investigate the roles of miR-543 and phospholipase A2 group IVA (PLA2G4A) in cell mobility and the invasiveness cascade in esophageal squamous cell carcinoma (ESCC) and to validate the interactive relationship between miR-543 and PLA2G4A. Methods: Microarray analysis showed the different expression levels of PLA2G4A in two ESCC cell lines (KYSE30 and KYSE180). The expression levels of miR-543 and PLA2G4A in ESCC tissues were confirmed by qRT-PCR and Western blotting. The targeted relationship between miR-543 and PLA2G4A was studied and verified by a luciferase activity assay. Then, the invasion and metastasis ability of ESCC cell lines transfected with miR-543 mimics, miR-543 inhibitor, or PLA2G4A and miR-543 mimics were analyzed separately by Transwell migration and invasion assays. In addition, the roles of miR-543 and PLA2G4A in the expression of E-cadherin and vimentin were also investigated. Results: PLA2G4A up-regulated the level of E-cadherin and down-regulated the level of vimentin, which curbed ESCC cell mobility and invasion. In ESCC cells, the expression of miR-543 was significantly higher, whereas the expression of PLA2G4A was markedly lower. MiR-543 facilitated ESCC cell mobility and invasion by repressing PLA2G4A. Conclusions: MiR-543 enhanced the cell mobility and the invasiveness cascade in ESCC cells via the down-regulation of PLA2G4A expression.