Presence Of Squalene Research Articles

Bioactive compounds from GC-MS analysis in methanolic extract of Gongronema latifolium inhibits aluminium chloride induced oxidative stress in rat spermatozoa in vitro

This study investigated the effect of Gongronema latifolium extract on aluminum chloride-induced sperm oxidative stress. Spermatozoa were obtained from the epidydimis and dispersed in a buffer consisting of tris-(hydroxymethyl)-aminomethane, citric acid anhydrous and D (−) fructose (TCF buffer) and incubated with Gongronema latifolium alone (0.8 mg/ml), AlCl3 alone (50 mM) and Gongronema latifolium (0.2 – 0.8 mg/ml) + AlCl3 (50 mM) repectively, for 3 h at 32 0C. We found that exposure to aluminum chloride alone led to significant increase in lipid peroxidation (LP) 14.41±1.18µM and protein carbonyl content 19.21±0.9nM, but significant decrease on glutathione content 5.32±3.11mM. When Gongronema latifolium was added in the incubation medium, it improved and protected spermatozoa against the harsh effect of AlCl3 on sperm cells. Bioactive compounds detected from GC-MS analysis also revealed the presence of squalene, ascorbic acid, gamma tocopherol, phthalic acid,carbazic acid amongst others. This study showed that Gongronema latifolium could protect spermatozoa against AlCl3 induced sperm damage.

Chimeric virus-like particles presenting tumour-associated MUC1 epitope result in high titers of specific IgG antibodies in the presence of squalene oil-in-water adjuvant: towards safe cancer immunotherapy

BackgroundImmunotherapy is emerging as a powerful treatment approach for several types of cancers. Modulating the immune system to specifically target cancer cells while sparing healthy cells, is a very promising approach for safer therapies and increased survival of cancer patients. Tumour-associated antigens are favorable targets for cancer immunotherapy, as they are exclusively expressed by the cancer cells, minimizing the risk of an autoimmune reaction. The ability to initiate the activation of the immune system can be achieved by virus-like particles (VLPs) which are safe and potent delivery tools. VLP‐based vaccines have evolved dramatically over the last few decades and showed great potential in preventing infectious diseases. Immunogenic potency of engineered VLPs as a platform for the development of effective therapeutic cancer vaccines has been studied extensively. This study involves recombinant VLPs presenting multiple copies of tumour-specific mucin 1 (MUC1) epitope as a potentially powerful tool for future immunotherapy.ResultsIn this report VLPs based on the structural protein of Norovirus (NoV VP1) were genetically modified to present multiple copies of tumour-specific MUC1 epitope on their surface. Chimeric MUC1 particles were produced in the eukaryotic Leishmania tarentolae expression system and used in combination with squalene oil-in-water emulsion MF59 adjuvant to immunize BALB/c mice. Sera from vaccinated mice demonstrated high titers of IgG and IgM antibodies which were specifically recognizing MUC1 antigen.ConclusionsThe obtained results show that immunization with recombinant chimeric NoV VP1- MUC1 VLPs result in high titers of MUC1 specific IgG antibodies and show great therapeutic potential as a platform to present tumour-associated antigens.Graphical

Evaluation of the Protective Efficacy of UV Absorbers on Stratum Corneum Carbonylation Induced by UV Irradiation in the Presence of Squalene

Evaluation of the Protective Efficacy of UV Absorbers on Stratum Corneum Carbonylation Induced by UV Irradiation in the Presence of Squalene

CDNA cloning, prokaryotic expression, and functional analysis of squalene synthase (SQS) in Camellia vietnamensis Huang

Camellia vietnamensis Huang, which belongs to Camellia oleifera, is a traditional Chinese medicinal plant widely planted on Hainan Island. Tea saponin is an important functional component of C. vietnamensis, and squalene is the precursor substance that controls its formation. Squalene synthase (SQS: EC 2.5.1.21) synthesizes squalene from 2 molecules of farnesyl pyrophosphate (FPP). In this study, 1683 bp of the C. vietnamensis SQS gene, designated as CvSQS, was cloned and encoded 414 amino acids. Bioinformatics and phylogenetic tree analysis revealed the high homology of CvSQS with squalene synthases from other plants. For soluble proteins, the carboxy-terminal deleted CvSQS was obtained for expression in Escherichia coli Transetta (DE3), and the recombinant protein with a weight of 42.5 kDa was detected using SDS-PAGE and Western blot. After an enzymatic reaction, the presence of squalene in the product was analyzed using GC-MS detection, which indicated that CvSQS had catalytic activity. The tissue specificity of CvSQS and its presence in seeds at various ripening stages was detected by q-RT PCR. CvSQS had the highest transcriptional level in leaves, followed by seeds, roots, and flowers; the amount of CvSQS in the seeds was highest in September. The identification and functional characterization of CvSQS is essential for further studies on the regulation mechanism of tea saponin in C. vietnamensis.

Antioxidative and Hepatoprotective Effects of Rubus canescens DC. Growing Wild in Lebanon

Background: Plants of the genus Rubus (family Rosaceae) have been used for diverse medicinal purposes for centuries. We hypothesized that the scarcely investigated R. canescens DC., like other species of the genus Rubus, exhibit prominent antioxidant activity. Objective: The present study investigates the in vitro and in vivo antioxidant activity of fruit juice as well as aqueous and methanolic extracts of the areal parts of Rubus canescens DC., and deciphers the phytochemical profile of the methanolic extracts by GC-MS. Methods: The in vitro antioxidant activity was assessed by DPPH radical scavenging assay and β- carotene bleaching assays. The in vivo antioxidant activity of the extracts was evaluated by measuring ALT, AST, CAT, and SOD levels in CCl4-challenged mice in two experimental models (chronic exposure and preventive). Histological analysis was conducted on H&E stained liver sections, and the phytochemical profile of methanolic extracts was investigated by GC-MS. Results: DPPH radical scavenging assay revealed that the methanolic leaves extract exhibited the highest activity, while the juice was the most active in terms of the β-carotene bleaching assay. The in vivo experiments suggested that the extracts have promising antioxidant potential and hepatoprotective effects capable of promoting liver functions. Histological analysis of liver sections revealed that administrating juice extract regenerated hepatocytes while reducing inflammation. GC-MS analysis indicated the presence of squalene, β-amyrin, and γ-sitosterol that may have contributed to the observed activity. Conclusion: The current study provided the first in vivo evidence supporting the antioxidative and hepatoprotective effects of R. canescens DC. growing wild in Lebanon.

Improving Vesicular Integrity and Antioxidant Activity of Novel Mixed Soy Lecithin-Based Liposomes Containing Squalene and Their Stability against UV Light.

In order to improve the membrane lipophilicity and the affinity towards the environment of lipid bilayers, squalene (SQ) could be conjugated to phospholipids in the formation of liposomes. The effect of membrane composition and concentrations on the degradation of liposomes prepared via the extrusion method was investigated. Liposomes were prepared using a mixture of SQ, cholesterol (CH) and Tween80 (TW80). Based on the optimal conditions, liposome batches were prepared in the absence and presence of SQ. Their physicochemical and stability behavior were evaluated as a function of liposome constituent. From the optimization study, the liposomal formulation containing 5% (w/w) mixed soy lecithin (ML), 0.5% (w/w) SQ, 0.3% (w/w) CH and 0.75% (w/w) TW80 had optimal physicochemical properties and displayed a unilamellar structure. Liposome prepared using the optimal formulation had a low particle size (158.31 ± 2.96 nm) and acceptable %increase in the particle size (15.09% ± 3.76%) and %trolox equivalent antioxidant capacity (%TEAC) loss (35.69% ± 0.72%) against UV light treatment (280–320 nm) for 6 h. The interesting outcome of this research was the association of naturally occurring substance SQ for size reduction without the extra input of energy or mechanical procedures, and improvement of vesicle stability and antioxidant activity of ML-based liposome. This study also demonstrated that the presence of SQ in the membrane might increase the acyl chain dynamics and decrease the viscosity of the dispersion, thereby limiting long-term stability of the liposome.

PECULIARITIES OF THE PROCESS OF PURIFICATION OF AMRANT GRAIN FROM IMPURITIES

Proper and balanced nutrition is the key to a person's physical health, especially in the face of deteriorating environmental conditions and the effects of daily stress caused by the accelerated pace of life in recent years. In this regard, the attention of many researchers and entrepreneurs is focused on the development and implementation in mass production of foods that would fully meet human needs for protein, vitamins, micro-and micronutrients, etc. Recently, ingredients made from non-traditional vegetable raw materials have been used for the production of functional foods. Amaranth belongs to such vegetable raw materials. Amaranth culture has been known since ancient times, used by the ancient Aztecs. The main feature of this culture is the unique chemical composition, which causes an extremely wide range of its use, which is not limited to the food industry. In terms of amino acid composition, amaranth grain protein is close to the composition of an ideal protein. The presence of squalene in amaranth oil makes it possible to use it for the manufacture of medicines and cosmetics. However, despite the prospects for the use of amaranth, producers face difficulties with the characteristics of the grain of this crop. Small grain sizes require careful selection of mode parameters of technological equipment for post-harvest processing. To substantiate the cleaning regimes, a study of the particle size distribution, physical-mechanical and aerodynamic properties of amaranth grain mass has been carried out. Based on the research, it is established that despite the fact that some amaranth species like shchyrytsya” have almost the same grain size and shape, the transgression coefficients calculated according to certain divisibility values can predict the possibility of separating a mixture of freshly harvested amaranth grain and its impurities, in particular from difficult to separate . It is proposed to use a set of sieves with round holes of the following sizes (mm) to separate this mixture: B1 – 1.0…1.1; B2 – 1.0…1.1; B2 – 1.0…1.2; G1 – 0.7…0.9; and elongated holes with dimensions (mm): B1 – (0.8…1.0)×20, B2 – (0.5…0.7)×20, B1 – (0.4…0.6)×20 .

Enzymatic Synthesis of Ether Lipids Rich in Docosahexaenoic Acid with Squalene as Reaction Medium

AbstractUsing squalene as reaction medium was tried for the enzymatic synthesis of ether lipids rich in docosahexaenoic acid (DHA) via transesterification of alkylglycerols (AKG) obtained from shark liver oil and DHA‐enriched ethyl esters (DHA‐EE) from Schizochytrium sp. The effects of reaction time, temperature, molar ratio (AKG /DHA‐EE), and enzyme dosage were investigated. DHA conversion of 74.47% was achieved for 48 hours of reaction at 60 °C using a molar ratio of 1:2 and an enzyme dosage of 30% based on AKG in the presence of squalene with Lipozyme® 435 (Novozymes, Tianjin, China) as the catalyst. With a high boiling point, squalene could act as a solvent without being vaporized from the reaction system under vacuum and improve the operational stability of immobilized lipase, which may be useful for enzymatic reactions under vacuum for lipid modification with byproducts of low boiling points.

Profiling of the bioactive components of safflower seeds and seed oil: cultivated (Carthamus tinctorius L.) vs. wild (Carthamus oxyacantha M. Bieb.)

The composition of lipophilic and hydrophilic components in cultivated (C. tinctorius) and wild (C. oxyacantha) safflower seed oils was studied. By LC–HRMS/MS2, a total of seven highly abundant bioactive compounds with hydrophilic nature, a lignan glycoside (tracheloside), two flavonoids (acacetin–glucuronide pentoside and acacetin-7-O-D-glucuronide), and four alkaloids (N-coumaroylserotonin glucoside, N-feruloylserotonin glucoside, N-coumaroylserotonin, and N-feruloylserotonin), in seeds of both species, were identified. Only a minor part of the hydrophilic compounds (≤ 0.05%) present in the seeds was transferred into the seed oil during the extraction. The linoleic (~ 78%), oleic (~ 15%), palmitic (~ 5%), and stearic (~ 2%) acids—constituted 99% of all detected fatty acids in both species. α-Tocopherol was a main form of tocochromanols (over 94%) in both safflower seed oils. β-Sitosterol was the predominate form (over 36%) of phytosterols, while high levels were also recorded for gramisterol (17.1%) and avenasterol (19.6%) in C. oxyacantha and C. tinctorius seed oils, respectively. Zeaxanthin was a predominated form of carotenoids (over 37%), while high levels were recorded for lutein and β-carotene 15 and 25%, mainly in C. oxyacantha. The total amount of minor lipophilic compounds such as tocochromanols, carotenoids and sterols in C. oxyacantha vs. C. tinctorius seed oil was 57.9 vs. 58.2, 0.76 vs. 0.5, and 185.5 vs. 274 mg/100 g oil, respectively. The presence of squalene was detected only in C. oxyacantha (10.4 mg/100 g oil). Despite the similar composition and levels of fatty acids and tocochromanols, species differed by the phytosterols, carotenoids, and bioactive compounds with hydrophilic nature.

NMR characterization and evaluation of antibacterial and antiobiofilm activity of organic extracts from stationary phase batch cultures of five marine microalgae (Dunaliella sp., D. salina, Chaetoceros calcitrans, C. gracilis and Tisochrysis lutea)

The chemical composition of five marine microalgae (Dunaliella sp., Dunaliella salina, Chaetoceros calcitrans, Chaetoceros gracilis and Tisochrysis lutea) was investigated through nuclear magnetic resonance (NMR) spectroscopic study of the soluble material obtained by sequential extraction with hexane, ethyl acetate (AcOEt) and methanol of biomass from stationary phase cultures. Hexane extracted the major lipids present in the microalgae during the stationary phase of growth, which correspond to storage lipids. Triacylglycerols (TGs) were the only storage lipids produced by Dunaliella and Chaetoceros. In contrast, T. lutea predominantly stored polyunsaturated long-chain alkenones, with sterols also detected as minor components of the hexane extract. The molecular structure of brassicasterol was determined in T. lutea and the presence of squalene in this sample was also unequivocally detected. Monogalactosyldiacylglycerols (MGDGs) and pigments were concentrated in the AcOEt extracts. C. calcitrans and D. salina constituted an exception due to the high amount of TGs and glycerol produced, respectively, by these two strains. Chlorophylls a and b and β-carotene were the major pigments synthesized by Dunaliella and chlorophyll a and fucoxanthin were the only pigments detected in Chaetoceros and T. lutea. Information concerning the acyl chains present in TGs and MGDGs as well as the positional distribution of acyl chains on the glycerol moiety was obtained by NMR analysis of hexane and AcOEt extracts, with results consistent with those expected for the genera studied. Fatty acid composition of TGs in the two Dunaliella strains was different, with polyunsaturated acyl chains almost absent in the storage lipids produced by D. salina. Except in C. calcitrans, the polar nature of soluble compounds was inferred through the relative extraction yield using methanol as the extraction solvent. Glycerol was the major component of this fraction for the Dunaliella strains. In T. lutea 1,4/2,5-cyclohexanetetrol (CHT) and dimethylsulfoniopropionate (DMSP) preponderated. CHT was also the major polyol present in the Chaetoceros strains in which DMSP was not detected, but prominent signals of 2,3-dihydroxypropane-1-sulfonate (DHSP) were observed in the 1H NMR spectra of methanolic extracts. The presence of DHSP confirms the production of this metabolite by diatoms. In addition, several other minor compounds (digalactosyldiacyglycerols (DGDGs), sulphoquinovosyldiacylglycerols (SQDGs), amino acids, carbohydrates, scyllo-inositol, mannitol, lactic acid and homarine) were also identified in the methanolic extracts. The antibacterial and antibiofilm activities of the extracts were tested. The AcOEt extract from C. gracilis showed a moderate antibiofilm activity.

Biotransfromation of Dibenzothiophene by Resting Cells of a Newly Isolated Serratia marscens Sp. Strain Originated from Industrial Wastewater

Five isolates able to use dibenzothiophene (DBT) as a sole sulfur source with high rates were selected to investigate their potentialities as biocatalysts of biodesulfurization reactions. The desulfurizing activities of selected strains were investigated in growing and resting cell state. The biodegradation yields were considerably higher in resting cell reaction especially for two strains tentatively named S1 (98.8%) and S27 (97.5%). These results insinuated that biodegradation activity was mainly related to secondary metabolism on these strains. Their biotransformation potentialities were also evaluated under various conditions in order to evaluate their stability in both aqueous and organic media; and their sensitivity to the presence of squalene, used in this study as a representative of hydrocarbons in petroleum. The results showed that the 5 selected strains were still active in the presence of 95% of squalene but no transformation observed at 99% of squalene. The sulfur substrate selectivity was studied in presence of other organosulfur compounds such us dimethylsulfoxide DMSO and benzothiazole BTH. The presence of these substrates inhibited the DBT uptake by the bacteria and consequently decreased its degradation rate. Moreover, conventional analysis of 16S ribosomal DNA sequencing showed that the strain-with highest bioconversion rates-belonged to Serratia marcescens species. To far of our knowledge, Serratia sp. was rarely reported as DBT degrder strain. Thus, the rate and the extent of the biodesulfurization reaction, exhibited by the strain Serratia marcescens S27, suggested that it could be used in practical scale.

Does squalene alter the antioxidant potential of astaxanthin and fucoxanthinol? In vitro evidence in RAW 264.7 cells, a murine macrophage.

Astaxanthin (Ax) and fucoxanthin/fucoxanthinol (FuOH) are marine xanthophylls exhibiting anti-oxidant effects. Squalene (SQ) is a triterpenoid and is a precursor of sterols. This study aimed to determine if SQ can improve the effect of Ax/FuOH on lipid peroxidation. RAW 264.7 cells were treated with different concentrations of Ax, FuOH and SQ and corresponding rate of cell survival was noted. In addition,combination groups - Ax + SQ and FuOH + SQ- were also run. Cells treated with Ax, FuOH, SQ, Ax+SQ and FuOH+SQ were stimulated with lipopolysaccharide and lipid hydroperoxides were estimated. Results showed that 5μM Ax, 2μM FuOH and 10μM SQ supported cell survival. In presence of SQ, cell viability improved for higher concentrations of FuOH (5, 10μM). Lipid hydroperoxides were supressed by Ax, FuOH, Ax+SQ and FUOH +SQ and were significantly lower in Ax+SQ, indicating the synergistic effect of Ax and SQ. To conclude, combination of Ax with SQ enhances its ability to supress lipid peroxidation while with FuOH, SQ attenuates the toxic effect at higher doses. Moreover, this is the first time that the combined effect of SQ and carotenoids has been studied and reported.

Lipid Profile and Antioxidant Activity of Macadamia Nuts (<i>Macadamia integrifolia</i>) Cultivated in Venezuela

Macadamia nuts (Macadamia integrifolia) grown in Venezuela have showed an average total fat content of 70%. Oleic acid (18:1) was the main monounsaturated fatty acid (MUFA) (51.3%), followed by palmitoleic acid (16:1, 22.6%). The content of polyunsaturated fatty acids (PUFAs), C18:2 and C18:3 represented 5.4%. Thus, MUFAs and PUFAs together constituted more than 80% of the total fatty acids present. Trans-vaccenic acid was also present (3%). As regards to other phytochemical compounds, tocopherols and tocotrienols were not found in the sample, but the presence of squalene was detected. The antioxidant activity (44.2%) of the extract was produced by the phytochemicals present.

Development and characterization of a digestion model based on olive oil microemulsions

AbstractOlive oil microemulsions were used as biomimetic model to simulate aspects of the digestion process in the small intestine. Hydrolytic activity of trypsin and alkaline phosphatase in microemulsions in the presence and in the absence of squalene and phenolic acids at pH 8.5 and 37°C was examined. In the case of trypsin kinetic constants were calculated and found Km = 0.080 ± 0.002 mM and Vmax = 3.8 × 10−5 ± 0.06 × 10−5 mM/s. When gallic acid and squalene were added, hydrolytic activity of trypsin was reduced to 77 ± 2% and 69 ± 3%, respectively. In the presence of o‐, m‐, and p‐coumaric acid, protocatechouic acid, and caffeic acid, enzymatic activity was increased up to 142 ± 6%. Hydrolytic activity of alkaline phosphatase was inhibited in the presence of squalene and phenolic acids. To evaluate the size of the dispersed aqueous droplets in the presence and in the absence of enzymes, substrates, and antioxidants, dynamic light scattering (DLS) measurements were carried out and diameters in the range of 8.9–13.4 nm were observed. Membrane flexibility and micropolarity were studied by electron paramagnetic resonance (EPR) spectroscopy. Rotational correlation time (τR), order parameter (S), and hyperfine splitting constant (A0) of doxyl stearic acid in the microemulsions were calculated and found 2.06 ns, 0.17, and 14.30 G, respectively. These values were slightly affected in the presence of additives.Practical applications: The main aim of the present study is to enhance our understanding on the digestibility of proteins and lipids in the presence of food antioxidants suggesting an in vitro digestion model for hydrolases based on virgin olive oil microemulsions. The information generated in this study could potentially facilitate the design and development of novel functional food formulations based on food‐grade colloidal delivery systems. Since modification or fortification of different food products focusing on different areas of health concern are of growing interest among the various sectors of food industry, the proposed research results could be of particular interest.

Identification of Volatile Compounds in Coconut Toddy by GC-MS - Assisted With Different Solvent System

Thirty one Volatile compounds from fermented coconut toddy were isolated by extracting with three different organic solvents viz., Diethyl ether, Dichloromethane and Chloroform simultaneously and analyzed by GC-MS. Eleven Distinct flavor compounds were extracted using diethyl ether in which greater quantity constitutes hydroquinone, ethyl hydrogen succinate, 2,4,6,8 – tetraazabicyclo [3.3.0] octon-3-one, 7-nitroimino, Phenylethyl alcohol is followed by oleic acid, hexanoic acid, squalene and n-Hexadecanoic acid in moderate level. 1, 2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester, Dibutyl phthalate and n-Decanoic acids were found to be very trace amount. Dichloromethane was able to find out another set of twelve compounds including ethyl hydrogen succinate, Di-n-octyl phthalate, Pentanoic acid (10-undecenyl ester) and nonanoic acid as a major compounds followed by 3-Pentanol, 2, 3-dimethyl, 1,2-Benzenedicarboxylic acid (butyl octyl ester and diheptyl ester) in moderate level and lower levels of dichloroacetic acid, 3,4-Hexanediol and 2-Buten-1-ol propanoate. Another set of extraction using chloroform repeatedly showed the presence of squalene as studied in diethyl ether extraction. It also shows the lupeol in higher level along with lower concentrations of 1,2-Benzenedicarboxylic acid, diisooctyl ester, 2,3-Epoxyhexanol, Propanedioic acid, and Aminocyanoacetic acid.

In vitro cytoprotective activity of squalene on a bone marrow versus neuroblastoma model of cisplatin-induced toxicity : implications in cancer chemotherapy

The development of a non-toxic selective cytoprotective agent that preferentially protects normal tissues from chemotherapy toxicity, without protecting malignant tissues, is a major challenge in cancer chemotherapy research. The available cytoprotective agents are either toxic or lack selective cytoprotective activity. Here, we report the in vitro selective cytoprotective activity of squalene, an isoprenoid molecule with antioxidant properties. Normal human bone marrow (BM) derived colony-forming unit (CFU) growth was increased by squalene in a dose-dependent manner. Squalene (12.5–25 μM) treatment significantly protected the CFUs from cisplatin-induced toxicity; the protective effect was equivalent to reduced glutathione (GSH), a known cytoprotective agent. Squalene also increased the long-term survival of cisplatin-treated 4-week-old CFUs. Cisplatin-induced apoptosis of CFUs as measured by the TUNEL assay was reduced by squalene. To examine the squalene-induced protection of tumours, several neuroblastoma cell lines, including five MYCN-amplified cell lines, were grown in monolayers, as well as in anchorage-independent cultures, in the presence of squalene and cisplatin. Squalene did not protect the neuroblastoma (NBL) cell lines from cisplatin-induced toxicity. In addition, squalene did not protect the NBL cells from carboplatin, cyclophosphamide, etoposide and doxorubicin-induced toxicity. In conclusion, our results suggest that squalene has a selective in vitro cytoprotective effect on BM-derived haematopoietic stem cells that is equipotent to GSH.

Fluvastatin induces apoptosis in rat neonatal cardiac myocytes: a possible mechanism of statin-attenuated cardiac hypertrophy.

Hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) have been shown to reduce atherosclerotic cardiovascular mortality and morbidity. Recent evidence indicates that statins may also exert direct effects on vascular wall cells (including endothelial cells and smooth muscle cells) independently of their hypocholesterolemic properties. However, little is known about whether statins have direct effects on myocardium. The effect of lipophilic and hydrophilic statins (fluvastatin and pravastatin) on apoptosis and protein synthesis in rat neonatal cardiac myocytes was investigated. The presence of apoptosis was evaluated by morphologic criteria, electrophoresis of DNA fragments, 4",6"-diamidine-2"-phenylindole (DAPI) staining, and TUNEL assay. Protein synthesis was measured by H-leucine incorporation into the cells. Fluvastatin, but not pravastatin, induced apoptosis in cardiac myocytes in a time- and dose-dependent manner. The pro-apoptotic effect of fluvastatin was reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate (GGPP), but not in the presence of squalene. The addition of protein prenylation inhibitor perillic acid and Rho-kinase inhibitor Y27632 significantly increased apoptosis. Fluvastatin decreased RhoA protein in the membrane fraction, whereas there were no significant changes of the RhoA protein in the cytosol fraction. Interleukin-1beta-stimulated H-leucine incorporation was completely inhibited by fluvastatin, but not by pravastatin. The findings suggest that fluvastatin induces apoptosis in cardiac myocytes via protein prenylation and the subsequent inhibition of Rho, and may play a role in the pathogenesis of cardiac hypertrophy and remodeling.

Regulation of interleukin-8 expression by HMG-CoA reductase inhibitors in human vascular smooth muscle cells

Interleukin-8 (IL-8) is a potent chemotactic factor that has been implicated in atherogenesis. HMG-CoA reductase inhibitors (statins) may reduce the cardiovascular risk and vulnerability of atherosclerotic plaque through nonlipid mechanisms such as inhibition of cytokine expression. In this study, we investigated the effects of statins on IL-8 synthesis in human vascular smooth muscle cells (VSMCs). Addition of angiotensin II (Ang II) increased IL-8 production in VSMCs in a time (0–24 h)- and dose (10 −8–10 −6 mol/l)-dependent manner with increased IL-8 mRNA accumulation. The Ang II type 1 receptor (AT1R) antagonist candesartan, but not the Ang II type 2 receptor (AT2R) antagonist PD123319, significantly blocked Ang II-induced IL-8 production. Addition of fluvastatin decreased the basal and Ang II-induced IL-8 production in VSMCs in a dose (10 −8–10 −5 mol/l)-dependent manner with a decrease in IL-8 mRNA accumulation. The effect of fluvastatin on IL-8 production was completely reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate, but not in the presence of squalene or farnesyl-pyrophosphate. Lipophilic cerivastatin also significantly decreased IL-8 production, while hydrophilic pravastatin showed no effect on IL-8 levels. In conclusion, we demonstrated for the first time that Ang II increased IL-8 production and fluvastatin decreased the basal and Ang II-induced IL-8 production in human VSMCs. These findings suggested that Ang II may exacerbate atherosclerosis through induction of IL-8 in VSMCs, while statins may exert therapeutic effects by modulating IL-8 synthesis in patients with atherosclerotic disease.

Fluvastatin inhibits matrix metalloproteinase-1 expression in human vascular endothelial cells.

Matrix metalloproteinase-1 (MMP-1), also called interstitial collagenase, may play an important role in the pathogenesis of atherosclerosis and atherosclerotic plaque rupture. We investigated the effects of fluvastatin on MMP-1 expression in human vascular endothelial cells (ECs). The addition of fluvastatin decreased the basal MMP-1 levels in the culture media of ECs in a time-dependent (0 to 48 hours) and dose-dependent (10(-)(8) to 10(-)(5) mol/L) manner. On the other hand, fluvastatin did not affect tissue inhibitor of metalloproteinase-1 levels. Collagenolytic activity in conditioned media of ECs was also dose-dependently reduced by fluvastatin. The effect of fluvastatin on MMP-1 expression was completely reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate, but not in the presence of squalene. Inhibition of Rho by C3 exoenzyme also significantly decreased MMP-1 expression in ECs. Our findings revealed that fluvastatin decreases MMP-1 expression in human vascular ECs through inhibition of Rho.

Isoprenoid-mediated changes in the glycerophospholipid molecular species of the sterol auxotrophic fungus Lagenidium giganteum.

The mosquito pathogenic fungus Lagenidium giganteum (Oomycetes: Lagenidiales) is a sterol auxotroph that can grow vegetatively in the absence of these compounds, but requires an exogenous source of sterols to enter its sexual and asexual reproductive cycles. Electrospray mass spectrometry (MS) and electrospray MS/MS were used to examine three major glycerophospholipid molecular species--glycerophosphocholine (GPC), glycerophosphoethanolamine (GPE) and glycerophosphoinositol (GPI)--from fungal mycelium and nuclei grown in defined medium with and without isoprenoids which induce (cholesterol and ergosterol) or do not induce (squalene, cholestane) reproduction. Testosterone supplementation of defined media inhibited growth of L. giganteum, so the effect of this steroid on phospholipid metabolism could not be assessed. Mycelium grown in defined media supplemented with these isoprenoids produced significantly different quantities of total phospholipid relative to unsupplemented media and to each other, ranging from a mean of 292 micrograms phosphate per g wet weight for cholesterol-supplemented media to 56 micrograms phosphate per g wet weight for mycelium grown in the presence of squalene. A very large percentage of the GPC (69-80 mol%) and GPI (74-79 mol%) molecular species from mycelia and nuclei contained ether linkages. GPE molecular species had 13-20 mol% ether-containing moieties. The elevated levels of ether lipids may be related to the sterol auxotrophic nature of the fungus. Isoprenoid supplementation of defined growth media resulted in many significant changes in molecular species for all three lipid classes. Significant differences (P < 0.05) in the percentage of total cell ether lipids in GPC and GPE were generated by isoprenoid supplements to culture media. Mycelium grown in the presence of the two sterols which induce asexual and sexual reproduction in L. giganteum, cholesterol and ergosterol, had a significantly greater percentage of ether-containing GPE moieties. The glycerolipid species from nuclei isolated from cultures grown with cholesterol and ergosterol were similar to the composition of nuclei isolated from fungus cultured in defined medium without any supplement or supplemented with squalene. The nuclear membrane from mycelia grown in cholestane-supplemented media, however, had a very different glycerophospholipid composition relative to either whole cells or nuclei from cells grown on other media. It appears that one of the reasons that cyclic isoprenoids such as cholestane do not induce fungal reproduction is that they drastically alter the nuclear membrane glycerophospholipid composition.