Assessment of the anti-rheumatoid arthritis activity of Gastrodia elata (tian-ma) and Radix aconitic lateralis preparata (fu-zi) via network pharmacology and untargeted metabolomics analyses.

AIM: Gastrodia elata and Radix aconiti lateralis preparrata are respectively named as Tian-Ma and Fu-Zi (TF) in Chinese. We explored the active components against rheumatoid arthritis (RA) from an extensively used couplet of Chinese herbs, Gastrodia elata and Radix aconiti lateralis preparata (TF) via untargeted metabolomics and network pharmacological approaches. METHODS: Water extracts of TF were mixed at ratios 1:1, 3:2 and 2:3 (w/w). Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was then utilized as metabolomics screening. Human Metabolome (http://www.hmdb.ca/) and Lipidmaps (http://www.lipidmaps.org/) databases were used to annotate detected compounds. Further identification of vital genes and important pathways associated with the anti-RA properties of the TF preparations was done via network pharmacology, and verified by real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Four key compounds involved in unsaturated fatty acid biosynthesis and isoflavonoid biosynthesis were identified through metabolomics analyses. Three key components of TF associated with anti-RA activity were linoleic acid, daidzein, and daidzin. Results of RT-qPCR revealed that all 3 tested TF couplets (1:1, 3:2, and 2:3) markedly suppressed the transcription of PTGS2. These results were consistent with our network pharmacological predictions. CONCLUSIONS: The anti-RA properties of Tian-Ma and Fu-Zi are associated with the inhibition of arachidonic acid metabolism pathway.

HPLC profile and antiedematogenic activity of Ximenia americana L. (Olacaceae) in mice models of skin inflammation.

The aim of this study was to evaluate the anti-edematogenic activity of X. americana L. (HEXA) hydroethanolic extract in ear edema models (acute and chronic) induced by croton oil and by different phlogistic agents (arachidonic acid, capsaicin, phenol and histamine), identifying the possible anti-edematogenic mechanism. HEXA demonstrated a significant anti-edematogenic effect at concentrations of 100-500 µg/ear in ear edema induced by croton oil with higher inhibition of edema of 39.37. However, the concentrations of 100 and 200 µg/ear were taken as a standard, demonstrating the effect in the chronic model induced by croton oil with inhibition of 61.62% and 48.74%. In the AA-induced ear edema model, HEXA showed inhibition of: 24.45% and 32.31%; capsaicin inhibition of 72.72% and 47.57%; phenol inhibition of 34% and 20.1%; and histamine inhibition of 31.8% and 21.62%. Then, the results were showed that HEXA demonstrated an anti-edematogenic effect in acute and chronic inflammation models, demonstrating a probable mechanism of action by the inhibition or modulation of key mediators of the inflammatory process. The chemical profile and presence of flavonoids guaranteeing a profile of activity similar to natural drugs that act or modulate the production of mediators of inflammations.

Anti-platelet effects of vitamin supplements in age-related macular degeneration: an in vitro study.

OBJECTIVE: The purpose of this experimental study was to investigate the role of vitamin supplements (Ocuvite, Vitalux Omega, and Nutrof Total) as possible inhibitors of the onset of age-related macular degeneration (AMD). MATERIALS AND METHODS: The anti-aggregating effect of each vitamin was determined against four accumulative factors namely, platelet activating factor (PAF), adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), and arachidonic acid (AA) in the platelet rich plasma (PRP) of healthy volunteers. RESULTS: Ocuvite, Vitalux Omega, and Nutrof Total were more potent inhibitors against PAF and ADP compared to TRAP and AA. Among the three vitamins, Nutrof Total displayed more potent inhibitions against TRAP and AA, while against PAF and ADP all the three vitamins revealed similar IC50 values. CONCLUSIONS: The vitamins Ocuvite, Vitalux Omega, and Nutrof Total have anti-aggregating effects and therefore can be used against AMD in healthy volunteers.

In vivo anti-inflammatory and anti-platelet aggregation activities of longissiminone A, isolated from Usnea longissima.

Secondary metabolite, longissiminone A (1) was isolated from a lichen, Usnea longissima. It was screened for its' in vivo anti-inflammatroy and anti-platelet aggregation activities. Compound 1 showed moderate in vivo anti-inflammatory activity as well as moderately active against the aggregation induced by arachidonic acid at different doses.

Five New Triterpenoid Saponins from the Rhizomes of Panacis majoris and Their Antiplatelet Aggregation Activity.

Five new triterpenoid saponins (1-5) and four known triterpenoid saponins, ginsenoside Re5 (6), majonoside R1 (7), 24(R)-majonoside R1 (8), and ginsenoside Rf (9), were isolated from the rhizomes of Panacis majoris. The structures of new compounds were elucidated as (20S,24S,25R*)-6-O-[ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranosyl]-dammar-20,24-epoxy-3ß,6α,12ß,25,26-pentaol (1), (20S,24R,25R)-6-O-[ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranosyl]-dammar-20,24-epoxy-3ß,6α,12ß,25,26-pentaol (2), (20S)-6-O-[ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranosyl]-dammar-20,25-epoxy-3ß,6α,12ß,24α-tetraol (3), 6-O-[ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranosyl]-dammar-3ß,6α,12ß,20S,24R,25-hexaol (4), and 6-O-[ß-D-glucop-yranosyl-(1 → 2)-ß-D-glucopyranosyl]-dammar-25(26)-ene-3ß,6α,12ß,20S,24R-pentaol (5) on the basis of extensive spectral analysis and chemical methods. Ginsenoside Re5 was isolated from the plant for the first time. The similarities of the nine compounds lie in the fact that their aglycones are conjoined with the same glucopyranose moieties, the same linkage of the glycosyl chains, and the same glycosylation sites, while they have a varied C-17 side chain. Compounds 3 and 5 exhibited moderate antiplatelet aggregation activities induced by adenosine diphosphate with IC50 values of 23.24 and 18.43 µM, respectively. Compound 5 displayed moderate inhibition of arachidonic acid-induced platelet aggregation with an IC50 value of 30.11 µM.

Arachidonic Acid Metabolite as a Novel Therapeutic Target in Breast Cancer Metastasis.

Metastatic breast cancer (BC) (also referred to as stage IV) spreads beyond the breast to the bones, lungs, liver, or brain and is a major contributor to the deaths of cancer patients. Interestingly, metastasis is a result of stroma-coordinated hallmarks such as invasion and migration of the tumor cells from the primary niche, regrowth of the invading tumor cells in the distant organs, proliferation, vascularization, and immune suppression. Targeted therapies, when used as monotherapies or combination therapies, have shown limited success in decreasing the established metastatic growth and improving survival. Thus, novel therapeutic targets are warranted to improve the metastasis outcomes. We have been actively investigating the cytochrome P450 4 (CYP4) family of enzymes that can biosynthesize 20-hydroxyeicosatetraenoic acid (20-HETE), an important signaling eicosanoid involved in the regulation of vascular tone and angiogenesis. We have shown that 20-HETE can activate several intracellular protein kinases, pro-inflammatory mediators, and chemokines in cancer. This review article is focused on understanding the role of the arachidonic acid metabolic pathway in BC metastasis with an emphasis on 20-HETE as a novel therapeutic target to decrease BC metastasis. We have discussed all the significant investigational mechanisms and put forward studies showing how 20-HETE can promote angiogenesis and metastasis, and how its inhibition could affect the metastatic niches. Potential adjuvant therapies targeting the tumor microenvironment showing anti-tumor properties against BC and its lung metastasis are discussed at the end. This review will highlight the importance of exploring tumor-inherent and stromal-inherent metabolic pathways in the development of novel therapeutics for treating BC metastasis.

Group X Secreted Phospholipase A2 Releases ω3 Polyunsaturated Fatty Acids, Suppresses Colitis, and Promotes Sperm Fertility.

Within the secreted phospholipase A2(sPLA2) family, group X sPLA2(sPLA2-X) has the highest capacity to hydrolyze cellular membranes and has long been thought to promote inflammation by releasing arachidonic acid, a precursor of pro-inflammatory eicosanoids. Unexpectedly, we found that transgenic mice globally overexpressing human sPLA2-X (PLA2G10-Tg) displayed striking immunosuppressive and lean phenotypes with lymphopenia and increased M2-like macrophages, accompanied by marked elevation of free ω3 polyunsaturated fatty acids (PUFAs) and their metabolites. Studies usingPla2g10-deficient mice revealed that endogenous sPLA2-X, which is highly expressed in the colon epithelium and spermatozoa, mobilized ω3 PUFAs or their metabolites to protect against dextran sulfate-induced colitis and to promote fertilization, respectively. In colitis, sPLA2-X deficiency increased colorectal expression of Th17 cytokines, and ω3 PUFAs attenuated their production by lamina propria cells partly through the fatty acid receptor GPR120. In comparison, cytosolic phospholipase A2(cPLA2α) protects from colitis by mobilizing ω6 arachidonic acid metabolites, including prostaglandin E2 Thus, our results underscore a previously unrecognized role of sPLA2-X as an ω3 PUFA mobilizerin vivo, segregated mobilization of ω3 and ω6 PUFA metabolites by sPLA2-X and cPLA2α, respectively, in protection against colitis, and the novel role of a particular sPLA2-X-driven PUFA in fertilization.

Antiplatelet Effects of Flavonoids Mediated by Inhibition of Arachidonic Acid Based Pathway.

Flavonoids, important components of human diet, have been claimed to possess a significant antiplatelet potential, in particular due to their effects on the arachidonic acid cascade. Due to variable and incomplete results, this study was aimed at delivering a detailed analysis of the effects of 29 structurally relevant, mainly natural flavonoids on three consecutive steps of the arachidonic acid cascade.Only the isoflavonoids genistein and daidzein were shown to possess a marked cyclooxygenase-1 inhibitory activity, which was higher than that of acetylsalicylic acid using the isolated ovine enzyme, and physiologically relevant, although lower than acetylsalicylic acid in human platelets. None of the tested flavonoids possesses an effect on thromboxane synthase in a clinically achievable concentration. Contrarily, many flavonoids, particularly those possessing an isolated 7-hydroxyl group and/or a 4'-hydroxyl group, acted as antagonists on thromboxane receptors. Interestingly, the substitution of the free 7-hydroxyl group by glucose might not abolish the activity.In conclusion, the consumption of few flavonoids in a diet, particularly of the isoflavonoids genistein and daidzein, may positively influence platelet aggregation.

Anti-inflammatory Activity of Magnesium Isoglycyrrhizinate Through Inhibition of Phospholipase A2/Arachidonic Acid Pathway.

Glycyrrhiza glabra (licorice) has been known to possess various pharmacological properties including anti-inflammatory, antioxidants, antiviral, and hepatoprotective activities. Magnesium isoglycyrrhizinate (MgIG), a magnesium salt of 18-α glycyrrhizic acid stereoisomer, is clinically used for the treatment of inflammatory liver diseases. However, the mechanism by which MgIG exerts its anti-inflammatory effects remains unknown. In the present study, we investigated the inhibitory potential of MgIG in phospholipase A2 (PLA2)/arachidonic acid (AA) pathway and release of the pathway-generated inflammatory lipid mediators in RAW264.7 macrophages. Results revealed that MgIG suppressed LPS-induced activation of PLA2 and production of AA metabolites such as prostaglandin E2 (PGE2), prostacyclin (PGI2), thromboxane 2 (TXB2), and leukotrienes (LTB4) in macrophages. Furthermore, LPS-induced AA-metabolizing enzymes including COX-2, COX-1, 5-LOX, TXB synthase, and PGI2 synthase were significantly inhibited by MgIG. Taken together, our data suggest that modulation of cyclooxygenase (COXs) and 5-lipoxygenase (LOX) pathways in AA metabolism could be a novel mechanism for the anti-inflammatory effects of MgIG.

Dual inhibition of arachidonic acid pathway by mulberry leaf extract.

The present work investigates the anti-inflammatory, analgesic and antipyretic activity of methanolic extract of mulberry leaves of variety S-1, S-13 and S-146. The S-146 extract was further evaluated for its efficacy against adjuvant arthritis in albino rats followed by inhibitory potential for COX 1, COX 2 and 5 LOX. The HPLC analysis enumerated the presence of morin, reversterol, scopoletin and 7-hydroxy coumarin as the major constituents. The anti-inflammatory, antipyretic and analgesic activity observed in the present experiment could be accredited to the dual inhibition in the AA pathway. The inhibition of COX and LOX enzymes could be imparted to the presence of resveraterol, morin, scopoletin and 7-hydroxy coumarin.

Molecular and behavioral characterization of adolescent protein kinase C following high dose ethanol exposure.

RATIONALE: Ethanol is commonly used and abused during adolescence. Although adolescents display differential behavioral responses to ethanol, the mechanisms by which this occurs are not known. The protein kinase C (PKC) pathway has been implicated in mediating many ethanol-related effects in adults, as well as gamma-aminobutyric acid (GABA(A)) receptor regulation. OBJECTIVES: The present study was designed to characterize cortical PKC isoform and GABA(A) receptor subunit expression during adolescence relative to adults as well as assess PKC involvement in ethanol action. RESULTS: Novel PKC isoforms were elevated, while PKCγ was lower during mid-adolescence relative to adults. Whole-cell lysate and synaptosomal preparations correlated for all isoforms except PKCδ. In parallel, synaptosomal GABAA receptor subunit expression was also developmentally regulated, with GABA(A)R δ and α4 being lower while α1 and γ2 were higher or similar, respectively, in adolescents compared to adults. Following acute ethanol exposure, synaptosomal novel and atypical PKC isoform expression was decreased only in adolescents. Behaviorally, inhibiting PKC with calphostin C, significantly increased ethanol-induced loss of righting reflex (LORR) in adolescents but not adults, whereas activating PKC with phorbol dibutyrate was ineffective in adolescents but decreased LORR duration in adults. Further investigation revealed that inhibiting the cytosolic phospholipase A2/arachidonic acid (cPLA2/AA) pathway increased LORR duration in adolescents, but was ineffective in adults. CONCLUSIONS: These data indicate that PKC isoforms are variably regulated during adolescence and may contribute to adolescent ethanol-related behavior. Furthermore, age-related differences in the cPLA2/AA pathway may contribute to ethanol's age-related effects on novel and atypical PKC isoform expression and behavior.

A target analogue imprinted polymer for the recognition of antiplatelet active ingredients in Radix Salviae Miltiorrhizae by LC/MS/MS.

The purpose of this study was to prepare a propyl gallate (PrG) molecular imprinted polymer as a cartridge stuffing material to isolate antiplatelet active ingredients. A macroporous polymer was synthesized utilizing ethylene glycol dimethacrylate (EDMA) as the crosslinking agent, PrG as the template molecule and 4-vinylpyridine (4-Vpy) as the functional monomer. Subsequently, PrG was removed by washing with methanol-glacial acetic acid (9:1, v/v). The molecular imprinted polymer recognized an active ingredient, protocatechuic acid, from a crude extract of the Chinese herbal medicine, Radix Salviae Miltiorrhizae (Danshen), using an on-line column switching solid phase extraction process. Pharmacological experiments showed that protocatechuic acid inhibits arachidonic acid (10 mg/kg) induced aggregation of rat platelets in vivo. This study provides an example of an application of separation-analysis technique for screening potentially bioactive compounds.

The antiplatelet activity of camel urine.

BACKGROUND: For centuries, camel urine has been used for medicinal purposes and anecdotally proclaimed as a cure for a wide range of diseases. However, the apparent therapeutic actions of camel urine have yet to be subjected to rigorous scientific scrutiny. Recent preliminary studies from the authors' laboratory have indicated that camel urine possesses potent antiplatelet activity, not found in human or bovine urines, suggesting a possible role for camel urine in inhibiting platelet function. The goal of the current study was to characterize the antiplatelet activity of camel urine against normal human platelets based on agonist-induced aggregation and platelet function analyzer (PFA-100) closure time. MATERIALS AND METHODS: Urine was collected from healthy virgin, pregnant, and lactating camels aged 2-10 years. Platelet-rich plasma (PRP) was prepared from blood collected from healthy individuals' blood into citrated anticoagulant. Agonist-induced aggregometry using donor PRP and PFA-100 closure times in whole blood were carried out in the presence and absence of added camel urine. The responses of platelets to multiple doses of camel urine were also assessed. The experimental procedure was repeated in human and bovine urines. RESULTS: Camel urine completely inhibited arachidonic acid (AA) and adnosine diphosphate (ADP)-induced aggregation of human platelets in a dose-dependent manner. PFA-100 closure time using human whole blood was prolonged following the addition of camel urine in a dose-dependent manner. Virgin camel urine was less effective in inhibiting ADP-induced aggregation as compared to urine from lactating and pregnant camels; however, all three showed comparable inhibitory activity. Neither human nor bovine urine exhibited antiplatelet activity. CONCLUSIONS: Camel urine has potent antiplatelet activity against ADP-induced (clopidogrel-like) and AA-induced (aspirin-like) platelet aggregation; neither human nor bovine urine exhibited such properties. These novel results provide the first scientific evidence of the mechanism of the presumed therapeutic properties of camel urine.

Chemical study and anti-inflammatory, analgesic and antioxidant activities of the leaves of Aristotelia chilensis (Mol.) Stuntz, Elaeocarpaceae.

OBJECTIVES: Aristotelia chilensis leaves (Elaeocarpaceae) are used in Chilean folk medicine to treat pain and inflammation. A bioguided study was carried out on serial extracts (hexane, dichloromethane, methanol, aqueous extract (INFU) and a crude mixture of alkaloids (ALK-MIX). All extracts were evaluated for (1) topical administration against both arachidonic acid and 12-deoxyphorbol-13-decanoate (TPA)-induced inflammation in mice and (2) per-os administration against inflammation by λ-carrageenan-induced paw oedema in guinea-pigs and (3) topical analgesia in tail flick and formalin models and per-os writhing test in mice. METHODS: Greater anti-inflammatory effects were obtained against TPA with dichloromethane extract and methanol extract (63.9 and 66.0%, respectively). INFU showed the most potent effect (56.2%) against arachidonic acid. Greater effects were obtained in the writhing test with hexane and dichloromethane extracts (89.2% both). In the topical analgesia models, all the extracts and ALK-MIX were active with exception of the hexane extract in the formalin assay. In tail flick test, ALK-MIX and the methanol extract were the most active (58.2 and 55.2%, respectively). In relation to the tail formalin assay, the methanol extract (74.1%) was the most active. Concerning antioxidant activity, both INFU and the methanol extract were the most active either in the inhibition of xanthine oxidase (52.9 and 62.7%, respectively) or in the DPPH free radical scavenging activity (EC50 (concentration that produced 50% of activity) = 12.1 and 9.7 µg/ml, respectively). KEY FINDINGS: Aristoteline, aristone, serratoline and hobartinol were isolated from ALK-MIX. Ursolic acid, friedelin and quercetin 5,3'-dimethyl ether were present in the dichloromethane extract while quercetin 3-O-ß-D-glucoside and kaempferol were present in the methanol extract. From INFU were isolated protopine, aristoteline and caffeic and ferulic acids. CONCLUSIONS: The effects of A. chilensis are herein demonstrated, validating its use in traditional medicine. Protopine is reported for the first time in Elaeocarpaceae.

Homoisoflavanone inhibits UVB-induced skin inflammation through reduced cyclooxygenase-2 expression and NF-kappaB nuclear localization.

BACKGROUND: Since the generation of reactive oxygen species (ROS) and release of inflammatory mediators play a major role in UVB-induced inflammation, vigorous attempts have been made for the pharmacological management of these molecules as well as for uncovering the molecular signaling pathways. Homoisoflavanone (5,7-dihydroxy-3-(3-hydroxy-4-methoxybenzyl)-chroman-4-one, HIF) extracted from Cremastra appendiculata has anti-angiogenic activities, but its effect on inflammation was unknown. OBJECTIVE: To investigate the anti-inflammatory effects of HIF on the skin and the underlying molecular mechanisms. METHODS: HaCaT cells were irradiated by UVB (10 mJ/cm(2)) with or without HIF. Prostaglandin E(2) (PGE(2)) level was measured by enzyme immunoassay. Activation of MAPK and production of cyclooxygenase-2 (COX-2) were determined by Western blot analysis. Localization of nuclear factor kappa B (NF-kappaB) was assessed by immunofluorescence microscopy. Hairless mice were stimulated with UVB or chemical stimulants to induce inflammatory responses in skin. RESULTS: Pretreatment with HIF inhibited the production of intracellular ROS induced by UVB irradiation in HaCaT cells. Further analysis revealed a decrease in the level of MAPK activation and down-regulation of COX-2 expression. In addition, HIF attenuated the nuclear localization of NF-kappaB, resulting in the suppression of inflammatory molecules such as IL-6, IL-8, and TNF-alpha. Finally, topical treatment with HIF inhibited ear edema induced by UVB, 12-O-tetradecanoylphorbol-13-acetate (TPA), arachidonic acid (AA), or croton oil. CONCLUSION: HIF has a strong protective effect against proinflammatory responses, implying the possibility of preventive application for inflammatory skin diseases.

Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis.

Arachidonic acid (AA) and its metabolites have recently generated a heightened interest due to growing evidence of their significant role in cancer biology. Thus, inhibitors of the AA cascade, first and foremost COX inhibitors, which have originally been of interest in the treatment of inflammatory conditions and certain types of cardiovascular disease, are now attracting attention as an arsenal against cancer. An increasing number of investigations support their role in cancer chemoprevention, although the precise molecular mechanisms that link levels of AA, and its metabolites, with cancer progression have still to be elucidated. This article provides an overview of the AA cascade and focuses on the roles of its inhibitors and their implication in cancer treatment. In particular, emphasis is placed on the inhibition of cell proliferation and neo-angiogenesis through inhibition of the enzymes COX-2, 5-LOX and CYP450. Downstream effects of inhibition of AA metabolites are analysed and the molecular mechanisms of action of a selected number of inhibitors of catalytic pathways reviewed. Lastly, the benefits of dietary omega-3 fatty acids and their mechanisms of action leading to reduced cancer risk and impeded cancer cell growth are mentioned. Finally, a proposal is put forward, suggesting a novel and integrated approach in viewing the molecular mechanisms and complex interactions responsible for the involvement of AA metabolites in carcinogenesis and the protective effects of omega-3 fatty acids in inflammation and tumour prevention.

[Ankylosing Spondylitis and convenient nutrition]. / Günstige Ernährung bei Morbus Bechterew.

The genetic setting and immunopathogenesis of patients with ankylosing spondylitis can not be changed by the diet. Therefore, an effective pharmacological therapy always is required. As adjuvant therapy the anti-inflammatory diet can help to reduce inflammatory processes, and to delay flares of the disease. The anti-inflammatory diet is consistent with a balanced diet and prevents infirmity provoked by malnutrition. Ankylosing spondylitis is an autoimmune disease and, as such, nutrients may mimic an antigen and cause flares of the disease and increase disease activity driven by antigen reaction. This nutrient-receptor interaction occurs in the individual patient, necessitating an individual dietary counselling for the patient with ankylosing spondylitis.

Antiplatelet and antithrombotic activities of Korean Red Ginseng.

The antiplatelet and antithrombotic activities of Korean Red Ginseng (KRG) were examined on rat carotid artery thrombosis in vivo, and platelet aggregation in vitro and ex vivo. Administration of KRG to rats not only prevented carotid artery thrombosis in vivo in a dose-dependent manner, but also significantly inhibited ADP- and collagen-induced platelet aggregation ex vivo, while failed to prolong coagulation times such as activated partial thromboplastin time (APTT) and prothrombin time (PT), indicating the antithrombotic effect of KRG might be due to its antiplatelet aggregation rather than anticoagulation effect. In line with the above observations, KRG inhibited U46619-, arachidonic acid-, collagen- and thrombin-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with IC50 values of 620 +/- 12, 823 +/- 22, 722 + 21 and 650 +/- 14 microg/mL, respectively. Accordingly, KRG also inhibited various agonists-induced platelet serotonin secretions as it suppressed platelet aggregation. These results suggest that KRG has a potent antithrombotic effect in vivo, which may be due to antiplatelet rather than anticoagulation activity, and KRG intake may be beneficial to the individuals with high risks of thrombotic and cardiovascular diseases.

Acyl-coenzyme A:cholesterol acyltransferase promotes oxidized LDL/oxysterol-induced apoptosis in macrophages.

7-Ketocholesterol (7KC) is a cytotoxic component of oxidized low density lipoproteins (OxLDLs) and induces apoptosis in macrophages by a mechanism involving the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we examined the role of ACAT in 7KC-induced and OxLDL-induced apoptosis in murine macrophages. An ACAT inhibitor, Sandoz 58-035, suppressed 7KC-induced apoptosis in P388D1 cells and both 7KC-induced and OxLDL-induced apoptosis in mouse peritoneal macrophages (MPMs). Furthermore, compared with wild-type MPMs, ACAT-1-deficient MPMs demonstrated significant resistance to both 7KC-induced and OxLDL-induced apoptosis. Macrophages treated with 7KC accumulated ACAT-derived [14C]cholesteryl and [3H]7-ketocholesteryl esters. Tandem LC-MS revealed that the 7KC esters contained primarily saturated and monounsaturated fatty acids. An inhibitor of cPLA2, arachidonyl trifluoromethyl ketone, prevented the accumulation of 7KC esters and inhibited 7KC-induced apoptosis in P388D1 cells. The decrease in 7KC ester accumulation produced by the inhibition of cPLA2 was reversed by supplementing with either oleic or arachidonic acid (AA); however, only AA supplementation restored the induction of apoptosis by 7KC. These results suggest that 7KC not only initiates the apoptosis pathway by activating cPLA2, as we have reported previously, but also participates in the downstream signaling pathway when esterified by ACAT to form 7KC-arachidonate.

In vivo and in vitro anti-inflammatory activity of Mangifera indica L. extract (VIMANG).

A standard aqueous extract of Mangifera indica L., used in Cuba as an antioxidant under the brand name of VIMANG, was tested in vivo for its anti-inflammatory activity using commonly accepted assays. M. indica extract, administered topically (0.5-2 mg per ear), reduced ear edema induced by arachidonic acid (AA) and phorbol myristate acetate (PMA, ED50 = 1.1 mg per ear) in mice. In the PMA model, M. indica extract also reduced myeloperoxidase (MPO) activity. This extract p.o. administered also inhibited tumor necrosis factor alpha (TNFalpha) serum levels in both models of inflammation (AA, ED50 = 106.1 mg kg(-1) and PMA, ED50 = 58.2 mg kg(-1)). In vitro studies were performed using the macrophage cell line RAW264.7 stimulated with pro-inflammatory stimuli (LPS-IFNgamma or the calcium ionophore A23187) to determine PGE2 or LTB4 release, respectively. The extract inhibited the induction of PGE2 with IC50 = 64.1 microg ml(-1) and LTB4 IC50 = 22.9 microg ml(-1). M. indica extract also inhibited human synovial secretory phospholipase (PL)A2 with IC 50 = 0.7 microg ml(-1). These results represent an important contribution to the elucidation of the mechanism involved in the anti-inflammatory and anti-nociceptive effects reported by the standard M. indica extract VIMANG.