Fish oil fatty acids as cardiovascular drugs

Starting in the 1970s the hypothesis that the low mortality from coronary heart disease among the Greenland Eskimos was due to their high consumption of n-3 fish oil fatty acids, initiated many studies to find if the n-3 polyunsaturated fatty acids in fish oils (PUFAs) could prevent cardiac atherosclerosis. To date this possibility has not achieved clinical recognition. The recent literature shows an increase of intervention studies to learn if the fish oil fatty acids can reduce mortality from sudden cardiac death, and the mechanism(s) of such a protective effect. Indeed the most definite beneficial cardiac action of these n-3 PUFAs seems now to be their ability in the short term to prevent sudden cardiac death. It is apparent that over long periods of time the n-3 fish oil fatty acids also prevent atherosclerosis. Definition of the fatty acids to which I will be referring in the text: n-6 (omega-6) polyunsaturated fatty acids; linoleic acid (18:2n-6, LA); arachidonic acid (C20:4n-6, AA). n-3 (omega-3) fatty acids; alpha-linolenic acid (18:3n-3, ALA); eicosapentaenoic acid (20:5n-3, EPA); docosahexaenoic acid (C22:6n-3, DHA). The bold, underlined abbreviation will appear in the text to identify the fatty acid being discussed.     

Omega-3 fatty acids induce Ca2+ mobilization responses in human colon epithelial cell lines endogenously expressing FFA4

Aim:

Free fatty acid receptor 4 (FFA4; formerly known as GPR120) is the G protein-coupled receptor (GPCR) for omega-3 polyunsaturated fatty acids. FFA4 has been found to express in the small intestines and colons of mice and humans. In this study we investigate the effects of omega-3 polyunsaturated fatty acids on FFA4 in human colon epithelial cells in vitro.

Methods:

HCT116 and HT-29 human colon epithelial cell lines endogenously expressing FFA4 were used. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in fura 2-AM-loaded cells with fluorescence spectrophotometry. RT-PCR and immunohistochemistry were used to detect FFA4.

Results:

Ten to 100 μmol/L of omega-3 polyunsaturated fatty acids α-linolenic acid (αLA) or eicosapentaenoic acid (EPA) induced dose-dependent [Ca²⁺]_i increase in HCT116 and HT-29 cells, whereas docosahexaenoic acid (DHA) had no effect. In addition, the omega-6 fatty acids linoleic acid and γ-linoleic acid also dose-dependently increase [Ca²⁺]_i, but the mono-unsaturated fatty acid oleic acid and saturated fatty acids such as stearic acid and palmitic acid had no effect. In HCT116 and HT-29 cells, the αLA-induced [Ca²⁺]_i increase was partially inhibited by pretreatment with EGTA, phospholipase C inhibitor edelfosine, cADPR inhibitors 8-bro-cADPR or DAB, and abolished by pretreatment with Ca²⁺ATPase inhibitor thapsigargin, but was not affected by G_i/o protein inhibitor PTX or IP₃R inhibitor 2-APB.

Conclusion:

Omega-3 and omega-6 long-chain polyunsaturated fatty acids (C18-20) induce Ca²⁺ mobilization responses in human colonic epithelial cells in vitro through activation of FFA4 and PTX-insensitive G_i/o protein, followed by Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores and Ca²⁺ influx across the plasma membrane.     

Formation of cyclic deoxyguanosine adducts from omega-3 and omega-6 polyunsaturated fatty acids under oxidative conditions

The discovery of the cyclic 1,N(2)-propanodeoxyguanosine adducts of acrolein (Acr), crotonaldehyde (Cro), and t-4-hydroxy-2-nonenal (HNE) as endogenous DNA lesions from lipid peroxidation has raised questions regarding the role of different types of fatty acids as sources for their formation. In this study, we carried out reactions at pH 7 and 37 degrees C with deoxyguanosine 5'-monophosphate and omega-3 polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA), linolenic acid (LNA), and eicosapentaenoic acid (EPA); or omega-6 PUFAs, including linoleic acid (LA) and arachidonic acid (AA), each in the presence of ferrous sulfate. The formation of Acr, Cro, and HNE-derived 1,N(2)-propanodeoxyguanosine adducts (Acr-, Cro-, and HNE-dG) in the incubation mixture was determined by reversed-phase HPLC analysis. The results showed that Acr and Cro adducts are primarily derived from omega-3 PUFAs, although Acr adducts are also formed, to a lesser extent, from oxidized AA and LA. HNE-dG adducts were detected exclusively in incubations with AA. The kinetics of the formation of these adducts was determined during incubations for 2 weeks and 5 days. The rate of Acr adduct formation was about 5-10-fold that of Cro adducts, depending on the type of PUFAs, and the rate of formation of HNE adducts from AA was also considerably slower than that of Acr adducts. Unlike other cyclic adducts, the formation of Acr adducts was independent of types of PUFAs, but its yield was proportional to the number of double bonds in the fatty acid. Only one of the isomeric Acr adducts was detected, and its stereoselective formation is consistent with that observed previously in vivo. Two previously unknown cyclic adducts, one derived from pentenal and the other from heptenal, were also detected as products from omega-3 and omega-6 fatty acids, respectively. This study demonstrated the specificity for the formation of the cyclic adducts of Acr, Cro, and HNE and other related enals by oxidation of omega-3 and omega-6 PUFAs. These results may be important for the understanding of the specific roles of different types of fatty acids in tumorigenesis.     

Omega-6 polyunsaturated fatty acid-stimulated cellular internalization of phosphorothioate oligodeoxynucleotides: evidence for protein kinase C-zeta dependency.

The rate of cellular internalization of phosphorothioate oligodeoxynucleotides is determined predominantly by adsorptive plus fluid-phase endocytosis. Internalization of a 5'-fluoresceinated phosphorothioate 15mer homopolymer of thymidine (FSdT15) in K562 cells in medium containing lipid-depleted albumin was reduced consistently versus nondepleted albumin. Treatment of K562 and several other cell lines with omega-6 polyunsaturated fatty acids (omega-6 PUFAs; e.g. arachidonic and linoleic acids) but not saturated fatty acids dramatically increased FSdT15 internalization in a concentration-dependent manner and over a wide albumin concentration range. The rate of efflux of FSdT15 from K562 cells was not affected by the omega-6 PUFA, implying that an increase of cellular fluorescence was due to an increase in the in-rate. These data were consistent with the observation that the binding of FSdT15 to the cell surface was also increased in the presence of omega-6 PUFAs. Omega-6 PUFAs are stimulators of protein kinase C (PKC) activity. Inhibition of PKC activity in K562 cells by Go6976, an inhibitor of the classical PKC isoforms, did not block the linoleic acid-induced stimulation of FSdT15 internalization. On the other hand, treatment of cells with Ro318220, which has considerably less isoform specificity, almost totally blocked the effect of linoleic acid on FSdT15 internalization, implying the involvement of a nonclassical PKC isoform in the process. Finally, since the only PKC isoform expressed in K562 cells that also is activated by omega-PUFAs is PKC-zeta, we obtained NIH 3T3 cells expressing a doxycycline-repressible dominant negative PKC-zeta mutant. Expression of the mutant blocked the stimulation of FSdT15 internalization by linoleic acid. Stimulated internalization also was blocked by wortmannin and LY 294002, which are relatively specific inhibitors of phosphatidylinositol 3-kinase (PI 3-K). Taken together, our data suggest that omega-6 PUFA stimulation of fluoresceinated phosphorothioate oligomers may be PKC-zeta dependent, and perhaps PI-3K dependent as well.     

Omega 3-fatty acids: health benefits and cellular mechanisms of action

Epidemiological evidence has established that ingestion of long-chain polyunsaturated omega-3 fatty acids (omega-3 PUFAs), abundant in fish oils, have profound effects on many human disorders and diseases, including cardiovascular disease and cancer. Here we briefly review the dietary recommendations and the food sources that are naturally enriched by these fatty acids. There are also a number of products including eggs, bread, and cereals available to supplement omega-3 fatty acid dietary intake. Some of these supplements are proposed to aid different pathological conditions. While the beneficial effects of omega-3 fatty acids can no longer be doubted, their molecular mechanism of action remains elusive. Without question, the action of omega-3 fatty acids is complex and involves a number of integrated signaling pathways. This review focuses on one of the possible cellular mechanisms by which the omega-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), may function. Studies with cancer cells suggest that DHA induces cell cycle arrest and apoptosis by activating protein phosphatases, leading to dephosphorylation of retinoblastoma protein (pRB). Protein phosphatases are also involved with the protein Bcl2, which regulates the release of cytochrome c from mitochondria, and eventually, activation of the apoptotic enzyme caspase 3.     

Effects of the cannabimimetic fatty acid derivatives 2-arachidonoylglycerol, anandamide, palmitoylethanolamide and methanandamide upon IgE-dependent antigen-induced beta-hexosaminidase, serotonin and TNF alpha release from rat RBL-2H3 basophilic leukaemic cells

There are conflicting reports in the literature as to whether palmitoylethanolamide affects the function of mast cell-related cell lines in vitro, in contrast to the well-documented effects of this compound upon mast cell function in vivo. In the present study, we have reinvestigated the effects of palmitoylethanolamide upon antigen-induced release of [3H]serotonin and beta-hexosaminidase from rat basophilic leukemia RBL-2H3 cells and compared these effects with those of 2-arachidonoylglycerol, anandamide and R1-methanandamide. RBL-2H3 cells were sensitized with a monoclonal anti-DNP IgE, after which they were stimulated with antigen (DNP-HSA). Palmitoylethanolamide produced a small, but significant reduction in antigen-stimulated [3H]serotonin release at high concentrations, whereas anandamide was without effect. In contrast, 2-arachidonoylglycerol and methanandamide increased the antigen-stimulated release of both [3H]serotonin and beta-hexosaminidase. It is concluded that in RBL-2H3 cells, these cannabimimetic fatty acid derivatives do not have potent stabilizing effects upon antigen-induced degranulation.     

Effects of omega-3, omega-6 and omega-9 fatty acids on vascular smooth muscle tone.

The comparative effects of omega-3, omega-6 and omega-9 fatty acids on vascular smooth muscle tone were investigated. Docosahexaenoic acid (1-255 microM) and eicosapentaenoic acid (31-255 microM) inhibited phenylephrine-induced contractions, (8-63%) and (20-65%), respectively, which were not altered by indomethacin, NDGA, or by removal of the endothelium. Linoleic acid (18:2n6) and arachidonic acid (20:4n6) also induced significant relaxation. Therefore, fatty acid-induced relaxation of the rat aorta is specific to polyunsaturated fatty acids, 20:5n3, 22:6n3, 18:2n6 and 20:4n6.     

Gamma linolenic acid: an antiinflammatory omega-6 fatty acid

Inflammation plays an important role in health and disease. Most of the chronic diseases of modern society, including cancer, diabetes, heart disease, arthritis, Alzheimer's disease, etc. have inflammatory component. At the same time, the link between diet and disease is also being recognized. Amongst dietary constituents, fat has gained most recognition in affecting health. Saturated and trans fatty acids have been implicated in obesity, heart disease, diabetes and cancer while polyunsaturated fatty acids (PUFAs) generally have a positive effect on health. The PUFAs of omega-3 and omega-6 series play a significant role in health and disease by generating potent modulatory molecules for inflammatory responses, including eicosanoids (prostaglandins, and leukotrienes), and cytokines (interleukins) and affecting the gene expression of various bioactive molecules. Gamma linolenic acid (GLA, all cis 6, 9, 12-Octadecatrienoic acid, C18:3, n-6), is produced in the body from linoleic acid (all cis 6,9-octadecadienoic acid), an essential fatty acid of omega-6 series by the enzyme delta-6-desaturase. Preformed GLA is present in trace amounts in green leafy vegetables and in nuts. The most significant source of GLA for infants is breast milk. GLA is further metabolized to dihomogamma linlenic acid (DGLA) which undergoes oxidative metabolism by cyclooxygenases and lipoxygenases to produce anti-inflammatory eicosanoids (prostaglandins of series 1 and leukotrienes of series 3). GLA and its metabolites also affect expression of various genes where by regulating the levels of gene products including matrix proteins. These gene products play a significant role in immune functions and also in cell death (apoptosis). The present review will emphasize the role of GLA in modulating inflammatory response, and hence its potential applications as an anti-inflammatory nutrient or adjuvant.     

The relation between degranulation and rapid metabolic responses in RBL-2H3 cells

The relation between degranulation and rapid metabolic responses (acidification rate changes) in RBL-2H3 cells was studied using a cytosensor microphysiometer, a silicon-based biosensor system. The metabolic responses in RBL-2H3 cells by antigen stimulation were compared with those by inhibitors of Ca2+ -ATPase. The former resulted in a rapid transient increase in the acidification rate of RBL-2H3 cells while the latter resulted in gradual decreases. When RBL-2H3 cells were costimulated with the inhibitors of Ca2+ -ATPase and an activator (PMA, phorbol-12-myristoyl-13-acetate) of protein kinase C, the metabolic responses increased again in RBL-2H3 cells. This seems to indicate that degranulation in RBL-2H3 cells was accelerated by costimulation with the inhibitors and PMA. However, costimulation was not able to completely mimic the way antigen stimulated RBL-2H3 cells in degranulation and in rapid metabolic responses.     

Mast cell degranulation induced by chlorogenic acid

Aim:

To investigate the mechanism of chlorogenic acid (CA)-induced anaphylactoid reactions.

Methods:

Degranulation of peritoneal mast cells was assayed by using alcian blue staining in guinea pigs, and the degranulation index (DI) was calculated. CA-induced degranulation of RBL-2H3 cells was also observed and assayed using light microscopy, transmission electron microscopy, flow cytometry, and β-hexosaminidase release.

Results:

CA 0.2, 1.0, and 5.0 mmol/L was able to promote degranulation of peritoneal mast cells in guinea pigs in vitro, but it did not increase the degranulation of peritoneal mast cells in CA-sensitized guinea pigs compared with control (P>0.05). Treatment with CA 0.2, 1.0, and 5.0 mmol/L for 30, 60, and 120 min induced degranulation in RBL-2H3 cells in a dose- and time-dependent manner (P<0.01). Under transmission electron microscope typical characteristics of degranulation, including migration of granular vesicles toward the plasma membrane and integration combined with exocytosis, were observed, after CA or C48/80 treatment. Fluorescent microscopy and flow cytometric analysis showed that CA induced concentration-dependent translocation of phosphatidylserine in RBL-2H3 cells. β-hexosaminidase release in RBL-2H3 cells was significantly increased after incubation with 1 mmol/L CA for 60 min and 5 mmol/L CA for 30 min (P<0.01).

Conclusion:

CA induces degranulation of peritoneal mast cells and RBL-2H3 cells in guinea pigs, which might be one of the mechanisms of the generation of anaphylactoid reactions induced by CA.     

Omega-3 fatty acids in major depressive disorder. A preliminary double-blind, placebo-controlled trial.

Patients with depression have been extensively reported to be associated with the abnormality of omega-3 polyunsaturated fatty acids (PUFAs), including significantly low eicosapentaenoic acid and docosahexaenoic acid in cell tissue contents (red blood cell membrane, plasma, etc.) and dietary intake. However, more evidence is needed to support its relation. In this study, we conducted an 8-week, double-blind, placebo-controlled trial, comparing omega-3 PUFAs (6.6 g/day) [corrected] with placebo, on the top of the usual treatment, in 28 patients with major depressive disorder. Patients in the omega-3 PUFA group had a significantly decreased score on the 21-item Hamilton Rating Scale for Depression than those in the placebo group (P < 0.001). From the preliminary findings in this study, omega-3 PUFAs could improve the short-term course of illness and were well tolerated in patients with major depressive disorder.     

Xestospongin C, a novel blocker of IP3 receptor, attenuates the increase in cytosolic calcium level and degranulation that is induced by antigen in RBL-2H3 mast cells

1. We evaluated the role of the cross-linking of Fc epsilon RI-mediated inositol 1,4,5-triphosphate (IP(3)) in the increase in cytosolic Ca(2+) level ([Ca(2+)](i)) using xestospongin C, a selective membrane permeable blocker of IP(3) receptor, in RBL-2H3 mast cells. 2. In the cells sensitized with anti-dinitrophenol (DNP) IgE, DNP-human serum albumin (DNP-HSA) and thapsigargin induced degranulation of beta-hexosaminidase and a sustained increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited both of these changes that were induced by DNP-HSA without changing those induced by thapsigargin. 3. In the absence of external Ca(2+), DNP-HSA induced a transient increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited this increase in [Ca(2+)](i). 4. In the cells permeabilized with beta-escin, the application of IP(3) decreased Ca(2+) in the endoplasmic reticulum (ER) as evaluated by mag-fura-2. Xestospongin C (3 - 10 microM) inhibited the effect of IP(3). 5. After the depletion of Ca(2+) stores due to stimulation with DNP-HSA or thapsigargin, the addition of Ca(2+) induced capacitative calcium entry (CCE). Xestospongin C (3 - 10 microM) inhibited the DNP-HSA-induced CCE, whereas it did not affect the thapsigargin-induced CCE. 6. These results suggest that Fc epsilon RI-mediated generation of IP(3) contributes to Ca(2+) release not only in the initial phase but also in the sustained phase of the increase in [Ca(2+)](i), resulting in prolonged Ca(2+) depletion in the ER. The ER Ca(2+) depletion may subsequently activate CCE to achieve a continuous [Ca(2+)](i) increase, which is necessary for degranulation in the RBL-2H3 mast cells. Xestospongin C may inhibit Ca(2+) release and consequently may attenuate degranulation.     

UDP-glucose acting at P2Y14 receptors is a mediator of mast cell degranulation

UDP-glucose (UDPG), a glycosyl donor in the biosynthesis of carbohydrates, is an endogenous agonist of the G protein-coupled P2Y₁₄ receptor. RBL-2H3 mast cells endogenously express a P2Y₁₄ receptor at which UDPG mediates degranulation as indicated by β-hexosaminidase (HEX) release. Both UDPG and a more potent, selective 2-thio-modified UDPG analog, MRS2690 (diphosphoric acid 1-α-d-glucopyranosyl ester 2-[(2-thio)uridin-5″-yl] ester), caused a substantial calcium transient in RBL-2H3 cells, which was blocked by pertussis toxin, indicating the presence of the G_i-coupled P2Y₁₄ receptor, supported also by quantitative detection of abundant mRNA. Expression of the closely related P2Y₆ receptor was over 100 times lower than the P2Y₁₄ receptor, and the P2Y₆ agonist 3-phenacyl-UDP was inactive in RBL-2H3 cells. P2Y₁₄ receptor agonists also induced [³⁵S]GTPγS binding to RBL-2H3 cell membranes, and phosphorylation of ERK1/2, P38 and JNK. UDPG and MRS2690 concentration-dependently enhanced HEX release with EC₅₀ values of 1150 ± 320 and 103 ± 18 nM, respectively. The enhancement was completely blocked by pertussis toxin and significantly diminished by P2Y₁₄ receptor-specific siRNA. Thus, mast cells express an endogenous P2Y₁₄ receptor, which mediates G_i-dependent degranulation and is therefore a potential novel therapeutic target for allergic conditions.     

Regulation of M2-type pyruvate kinase mediated by the high-affinity IgE receptors is required for mast cell degranulation

BACKGROUND AND PURPOSE: M2-type pyruvate kinase (M2PK) was found to interact directly with the 'ITAM' region of the gamma chain of the high-affinity IgE receptor (FcvarepsilonRI). Our hypothesis was that mast cell degranulation might require the FcvarepsilonRI-mediated inhibition of M2PK activity. EXPERIMENTAL APPROACH: In rat basophilic leukaemia (RBL-2H3) cells, the effects of directly inhibiting M2PK or preventing the FcvarepsilonRI-mediated inhibition of M2PK (disinhibition) on degranulation was measured by hexosaminidase release. Effects of blocking the FcvarepsilonRI-mediated inhibition of M2PK was also assessed in vivo in a mouse model of allergen-induced airway hyper-responsiveness. KEY RESULTS: Activation of FcvarepsilonRI in RBL-2H3 cells caused the rapid phosphorylation of tyrosine residues in M2PK, associated with a decrease in M2PK enzymatic activity. There was an inverse correlation between M2PK activity and mast cell degranulation. FcvarepsilonRI-mediated inhibition of M2PK involved Src kinase, phosphatidylinositol 3-kinase, PKC and calcium. Direct inhibition of M2PK potentiated FcvarepsilonRI-mediated degranulation and prevention of the FcvarepsilonRI-mediated inhibition of M2PK attenuated mast cell degranulation. Transfection of RBL-2H3 cells with M1PK which prevents FcvarepsilonRI-induced inhibition of M2PK, markedly reduced their degranulation and exogenous M1PK (i.p.) inhibited ovalbumin-induced airway hyper-responsiveness in vivo. CONCLUSIONS AND IMPLICATIONS: We have identified a new control point and a novel biochemical pathway in the process of mast cell degranulation. Our study suggests that the FcvarepsilonRI-mediated inhibition of M2PK is a crucial step in responses to allergens. Moreover, the manipulation of glycolytic processes and intermediates could provide novel strategies for the treatment of allergic diseases.     

Inhibitory effects of saturated and polyunsaturated fatty acids on the cytochrome P450 3A activity in rat liver microsomes

This study was conducted to investigate the inhibitory effects of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) on cytochrome P450 3A (CYP3A). The inhibition of 6beta-hydroxy testosterone formation from testosterone in rat liver microsomes was used as an index of CYP3A activity. In the present study, among the three types of fatty acids, the rank order of inhibitory effects of fatty acids was SFAs相似文献   

Inhibitory effect of juniperonic acid (Delta-5c,11c,14c,17c-20:4, omega-3) on bombesin-induced proliferation of Swiss 3T3 cells

Juniperonic acid (Delta-5c,11c,14c,17c-20:4, JA) is a polymethylene-interrupted (PMI) fatty acid that occurs in Biota orientalis. In this study, we found that JA has an antiproliferative activity. Swiss 3T3 cells were preloaded with fatty acids before stimulation with bombesin, a mitogenic neuropeptide, and proliferation of the cells was assessed by [(3)H]thymidine incorporation. Preloading of linoleic acid (Delta-9c,12c-18:2) significantly enhanced bombesin-induced proliferation. In contrast, preloading of eicosapentaenoic acid (Delta-5c,8c,11c,14c,17c-20:5, EPA) suppressed proliferation. Likewise, cells preloaded with JA showed a significantly curtailed response to bombesin. The antiproliferative potency of JA was equivalent to that of EPA. Sciadonic acid (Delta-5c,11c,14c-20:3), an omega-6 analogue of JA did not show antiproliferative activity, suggesting the importance of the omega-3 double bond rather than the PMI structure. The EPA-like activity of JA may be involved in the pharmaceutical activity of biota seeds, a psychoactive Chinese traditional medicine.     

Omega-3 fatty acid preparations--a comparative study

Fifteen food supplements and 1 medicine, formulated as soft capsules and containing omega-3 fatty acids, were evaluated with different tests, including desegregation, determination of the anisidine and peroxide values and assay of the omega-3 acids according to the European Pharmacopoeia. All the products contained purified fish oil rich in omega-3 fatty acids, mainly eicosapentaenoic acid (C20:5; EPA) and docosahexaenoic acid (C22:6; DHA), and available as triglycerides, ethyl esters or free fatty acids. The medicinal product complied with the fixed requirements whereas 7 of the 15 food supplements deviated from 1 or more of the criteria with regard to the peroxide value and the content of 1 or more of the fattty acids.     

紫花地丁止痒复方对RBL-2H3细胞脱颗粒的影响

目的考察紫花地丁止痒复方(Viola yedoensis makino antiitching compound,VYAC)对RBL-2H3细胞脱颗粒的影响及机制。方法CCK-8检测VYAC对RBL-2H3细胞的毒性;C48/80诱导RBL-2H3细胞发生脱颗粒,台盼蓝染色、β-氨基己糖胺酶释放、组胺释放、细胞内Ca2+浓度,评价VYAC对C48/80诱导的RBL-2H3细胞脱颗粒情况;Western blot法检测相关蛋白(Syk、p-Syk、PI3K、Akt、p-Akt)的表达。结果100 mg·L^-1的C48/80能够明显刺激RBL-2H3细胞发生脱颗粒,脱颗粒率达74%(P<0.05);VYAC呈剂量依赖性抑制β-氨基己糖胺酶和组胺的释放(P<0.05),且剂量在800 mg·L^-1以下时不影响RBL-2H3细胞存活;VYAC能够明显减弱细胞内荧光强度,降低细胞内Ca2+浓度;VYAC(25、100 mg·L^-1)明显抑制PI3K蛋白表达、抑制Syk、Akt蛋白的磷酸化(P<0.05)。结论VYAC能够抑制过敏性皮炎中肥大细胞脱颗粒,抑制Ca2+内流,其机制可能是抑制Syk/PI3K/Akt活化。     

非免疫性过敏反应细胞模型的建立

目的:通过非免疫性刺激物C48/80诱导大鼠嗜碱性粒细胞白血病细胞株RBL-2H3细胞脱颗粒反应正交试验,优化非免疫性过敏反应细胞模型建立条件.方法:在不同的孵育时间下,C48/80与不同浓度的RBL-2H3细胞共孵育,通过测定β-氨基己糖苷酶的释放率确定建立非免疫型过敏反应细胞模型的最优实验条件,并且分析不同检测方法间的差异.结果:不同浓度、不同孵育时间下的RBL-2H3细胞均可受C48/80诱导发生典型的脱颗粒反应,但不同条件下的脱颗粒程度和β-氨基己糖苷酶释放率都有显著差异.结论:当RBL-2H3细胞浓度为2×105 mL、孵育时间60 min时,细胞的感受性较好,其脱颗粒程度与药物浓度呈正相关.     

PMX-53 as a dual CD88 antagonist and an agonist for Mas-related gene 2 (MrgX2) in human mast cells

Human mast cells express the G protein coupled receptor (GPCR) for C5a (CD88). Previous studies indicated that C5a could cause mast cell degranulation, at least in part, via a mechanism similar to that proposed for basic neuropeptides such as substance P, possibly involving Mas-related gene 2 (MrgX2). We therefore sought to more clearly define the receptor specificity for C5a-induced mast cell degranulation. We found that LAD2, a human mast cell line, and CD34(+) cell-derived primary mast cells express functional MrgX1 and MrgX2 but the immature human mast cell line HMC-1 does not. A potent CD88 antagonist, PMX-53 (10 nM) inhibited C5a-induced Ca(2+) mobilization in HMC-1 cells, but at higher concentrations (≥30 nM) it caused degranulation in LAD2 mast cells, CD34(+) cell-derived mast cells, and RBL-2H3 cells stably expressing MrgX2. PMX-53 did not, however, activate RBL-2H3 cells expressing MrgX1. Although C5a induced degranulation in LAD2 and CD34(+) cell-derived mast cells, it did not activate RBL-2H3 cells expressing MrgX1 or MrgX2. Replacement of Trp with Ala and Arg with dArg abolished the ability of PMX-53 to inhibit C5a-induced Ca(2+) mobilization in HMC-1 cells and to cause degranulation in RBL-2H3 cells expressing MrgX2. These findings demonstrate that C5a does not use MrgX1 or MrgX2 for mast cell degranulation. Moreover, it reveals the novel finding that PMX-53 functions as a potent CD88 antagonist and a low-affinity agonist for MrgX2. Furthermore, Trp and Arg residues are required for the ability of PMX53 to act as both a CD88 antagonist and a MrgX2 agonist.