Schisandra lignans ameliorate nonalcoholic steatohepatitis by regulating aberrant metabolism of phosphatidylethanolamines

Nonalcoholic steatohepatitis (NASH) is a spectrum of chronic liver disease characterized by hepatic lipid metabolism disorder. Recent reports emphasized the contribution of triglyceride and diglyceride accumulation to NASH, while the other lipids associated with the NASH pathogenesis remained unexplored. The specific purpose of our study was to explore a novel pathogenesis and treatment strategy of NASH via profiling the metabolic characteristics of lipids. Herein, multi-omics techniques based on LC-Q-TOF/MS, LC-MS/MS and MS imaging were developed and used to screen the action targets related to NASH progress and treatment. A methionine and choline deficient (MCD) diet-induced mouse model of NASH was then constructed, and Schisandra lignans extract (SLE) was applied to alleviate hepatic damage by regulating the lipid metabolism-related enzymes CES2A and CYP4A14. Hepatic lipidomics indicated that MCD-diet led to aberrant accumulation of phosphatidylethanolamines (PEs), and SLE could significantly reduce the accumulation of intrahepatic PEs. Notably, exogenous PE (18:0/18:1) was proved to significantly aggravate the mitochondrial damage and hepatocyte apoptosis. Supplementing PE (18:0/18:1) also deteriorated the NASH progress by up regulating intrahepatic proinflammatory and fibrotic factors, while PE synthase inhibitor exerted a prominent hepatoprotective role. The current work provides new insights into the relationship between PE metabolism and the pathogenesis of NASH.

Phosphatidylethanolamine functionalized biomimetic monolith for immobilized artificial membrane chromatography / 药物分析学报

In this research,a new phospholipid based monolith was fabricated by in situ co-polymerization of 1-dodecanoyl-2-(11-methacrylamidoundecanoyl)-sn-glycero-3-phosphoethanolamine and ethylene dimethacrylate to mimick bio-membrane environment.Excellent physicochemical properties of this novel monolith that were achieved included column efficiency,stability,and permeability.Moreover,the biomimetic monolith showed outstanding separation capability for a series of intact proteins and small molecules.In particular,it exhibited good potential as an alternative to the commercial immobilized artificial membrane(IAM)column(IAM.PC.DD2)for studying drug-membrane interactions.This study not only enriched the types of IAM stationary phases,but also provided a simple model for the prediction of phosphatidylethanolamine related properties of drug candidates.

MiR-4443 promotes migration and invasion of breast cancer cells by inhibiting PEBP1 expression / 南方医科大学学报

OBJECTIVE@#To investigate the effect of miR-4443 expression on migration and invasion of breast cancer.@*METHODS@#We examined the expression of miR-4443 in breast carcinoma in situ and paired adjacent tissues from 3 breast cancer patients with high-throughput sequencing and verified the results using TCGA database. We also detected miR-4443 expressions using real-time quantitative PCR (RT-qPCR) in low invasive and highly invasive breast cancer cells (MCF-7 and MDA-MB-231 cells, respectively). The changes in apoptosis, migration and invasion of MCF-7 and MDA-MB-231 cells after transfection with miR-4443 mimics, mimics-NC, miR-4443 inhibitor or inhibitor-NC were analyzed using flow cytometry, wound healing assay and Transwell invasion assay. The target gene of miR-4443 was predicted by bioinformatics software and validated by a dual luciferase reporter gene system. RT-qPCR and Western blotting were performed to detect the expression of recombinant human phosphatidyl ethanolamine binding protein 1 (PEBP1) in the transfected cells.@*RESULTS@#The expression of miR-4443 was significantly higher in the breast cancer tissues than in the adjacent tissues (@*CONCLUSIONS@#MiR-4443 promotes the migration and invasion of breast cancer cells by inhibiting the expression of PEBP1, suggesting the possibility of suppressing miR-4443 expression as a potential therapeutic strategy for breast cancer.

Study on Preparation ，Characterization and Cytotoxicity of Baicalin PEG-PE Nanomicelles / 中国药房

OBJECTIVE： To prepare Baicalin-loaded Polyethylene glycol-derivatized phosphatidylethanolamine （BAI@PEG-PE） nanomicelles， and to characterize it and study its cytotoxicity. METHODS： BAI@PEG-PE nanomicelles were prepared by film hydration method and their appearance characteristics were observed. The particle size， polydispersity index， Zeta potential， drug-loading amount and encapsulation efficiency of the nanomicelles were detected. Drug release of BAI raw material and BAI@PEG-PE nanomicelles in pH 7.4 phosphate buffer were compared within 1-84 h. Using coumarin 6 as fluorescent probe， the distribution of PEG-PE nanomicelles in H9c2 cardiomyocytes were observed. H9c2 cardiomyocytes were divided into model group， BAI raw material group and BAI@PEG-PE nanomicelles group. After treated with serum-free DMEM medium containing no or corresponding drugs for 0.5 h， isoproterenol was used to induce cardiomyocyte apoptosis. Nuclear morphology， cell apoptosis rate and protein expression of Bcl-2 and Bax were compared with among 3 groups. RESULTS： Prepared BAI@PEG-PE nanomicelles were uniform globular shape. The particle size was （16.7±0.8） nm， PDI was 0.11±0.01 and Zeta-potential was （－18.4±0.6） mV； drug-loading amount was （7.84±0.65）％， encapsulation efficiency was （85.7±4.9）％ （n＝3）. Accumulative release rate was 76.5％ within 84 h. BAI raw material was released completely within 24 h. PEG-PE nanomicelles could strengthen the intake of coumarin 6 in H9c2 cardiomyocytes， mainly gathering around mitochondria. Compared with model group， the apoptosis morphology of cardiomyocytes were improved significantly in BAI raw material group and BAI@PEG-PE nanomicelles group； apoptosis rate was decreased significantly； protein expression of Bcl-2 was increased significantly； protein expression of Bax was decreased significantly with statistical significance （P＜0.05 or P＜0.01）. Above effects of BAI@PEG-PE nanomicelles group were more significant （P＜0.05 or P＜0.01）. CONCLUSIONS： BAI@PEG-PE nanomicelles are prepared successfully， and show significant sustained-release effect and myocardial targeting， and can prevent cardiomyocyte apoptosis.

Preparation of Norcantharidin Nano-micelle and Study on Its Antitumor Effect / 中国药房

OBJECTIVE:To prepare the norcantharidin (NCTD) nano-micelle and study its antitumor effect. METHODS:NCTD nano-micelle was self-formed in water using Triblock copolymers distearyl phosphatidylethanolamine-polyethylene glycol-ma-leimide;its shape was observed,the drug-loading rate,entrapment efficiency,particle size,Zeta potential were investigated. MTT was used to investigate the cell survival rate of human lung cancer A549 cells in negative control group (Phosphate buffer solu-tion),carrier group (blank nano-micelle),positive control group (NCTD APIs,5-320 μg/mL) and NCTD nano-micelle group (NCTD,5-320 μg/mL) after acting different time (24,48,72 h). Tumor nude mice were randomly divided into blank control group,NCTD injection group(1 mg/kg),NCTD low-dose,high-dose groups(0.5,1 mg/kg),6 in each group. All mice were in-travenously injected relevant medicines in tail,once a day,for 8 weeks. Tumor size was measured every week,and tumor quality was detected after the second day of finishing administration. RESULTS:NCTD nano-micelle was round,drug-loading rate was (2.82±0.05)%,entrapment efficiency was(83.67±1.78)%,particle size was(138.6±45.8)nm,Zeta potential was -(12.75± 0.34)mV(n=6). Cell survival rate of A549 cells in carrier group had no obvious changes,and was obviously decreased in posi-tive control group and NCTD nano-micelle group,which was positively correlated with concentration and time. And the decrease degree of cell survival rate in NCTD nano-micelle group was stronger than positive control group(P<0.01). Compared with blank control group,the tumor quality of mice in 3 administration groups was reduced (P<0.05),the reduction degree in NCTD na-no-micelle high-dose group was stronger than NCTD nano-micelle injection group (P<0.05). CONCLUSIONS:NCTD nano-mi-celle is successfully prepared,which has good in vitro and in vitro anti-tumor effect on A549 cells.

Analysis of brain PEBP and ERK in morphine-induced CPP model in rats / 中国药理学通报

Aim To analyze the expression of phosphatidylethanolamine-binding protein(PEBP) and ERK in critical brain regions of psychological dependence rats.Methods Morphine-induced rats conditioned place preference(CPP) model was established to mimic different stages of morphine psychological dependence, during which PEBP expression and ERK activity were assayed in different brain regions.Results PEBP expression in hippocampus, prefrontal cortex, striatum and nucleus accumbens showed no change at three stages of psychological dependence.However, ERK activity increased notably in prefrontal cortex on CPP formation, and decreased remarkably in hippocampus on CPP reinstatement.Conclusions The formation and retrieval of associated memory between morphine effects and environment involve different neural circuits, in which ERK activity is critical, and PEBP might not be involved in such a memory-related ERK regulation.

Study on the phospholipid composition of human milk at different lactation stages / 上海交通大学学报（医学版）

Objective · To obtain the latest data on phospholipid composition of human milk in Shanghai and compare the differences in phospholipid composition at different lactation stages. Methods · Healthy postpartum women who delivered full-term infants in the Obstetrical Department of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine between April and July, 2016 were enrolled. The colostrum, transitional milk, and mature milk were collected at Day 3, 10, and 45 after delivering babies, respectively. Human milk fat was extracted with Folch's method and phospholipids were separated with solid phase extraction (SPE). The phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were quantitatively analyzed with HPLC/VWD. The differences in phospholipid composition at different lactation stages were compared with univariate analysis of variance and Games-Homell test. Results · One hundred women who provided at least one breast milk sample were enrolled. A total of 70 colostrum samples, 96 transitional milk samples, and 82 mature milk samples were collected. The total phospholipid content of mature milk [(281.93±118.54) μg/g] was significantly lower than that of colostrum [(381.99±205.90) μg/g]. At all lactation stages, the relative content of phosphatidylcholine was the highest (53.74%-59.36%), followed by sphingomyelin (28.12%-32.74%). The relative content of phosphatidylethanolamine was constant (P=0.617), the relative content of phosphatidylcholine gradually decreased (P=0.000), and that of sphingomyelin gradually increased (P=0.000) during the lactation. Conclusion · Sphingomyelin and phosphatidylcholine are major components of human milk phospholipids. The amount of phospholipids varies during the lactation. The total amount of phospholipids is lower in mature milk than in colostrum and transitional milk. The relative content of phosphatidylethanolamine is consistent at all lactation stages, the relative content of phosphatidylcholine gradually decreases, and that of sphingomyelin gradually increases.

Rapid analysis of glycerophospholipids in RAW264.7 macrophage with UHPLC-QTOF/MS / 药学学报

An ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometric method was developed for rapid analysis of glycerophospholipids in RAW264.7 macrophage. The modified Bligh-Dyer was applied to extract glycerophospholipids from RAW264.7 macrophage. The target compounds, detected by mass spectrometry in ESI+ and ESI- mode, were separated by gradient elution with mobile phase (A) water (containing 10mmol·L-1 ammonium acetate and 0.25% acetic acid) and (B) acetonitrile/isopropanol (1:1) (containing 10mmol·L-1 ammonium acetate and 0.25% acetic acid). A total of 82 glycerophospholipids including 57 phosphatidylcholines (PCs), 21 phosphatidylethanolamines (PEs), three phosphatidylglycerols (PGs) and one phosphatidylinositol (PI) were deduced. The UHPLC-QTOF/MS method is rapid, simple and credible for targeting analysis of glycerophospholipids of RAW264.7 macrophage.

Mechanism of Raf kinase inhibitor protein down-regulation in cancer / 国际肿瘤学杂志

Raf kinase inhibiting protein (RKIP)plays a vital role in various physiological processes and participates in multiple signaling pathways,with a close releationgship with tumor.The mechanism of RKIP down-regulation in neoplasms includes transcriptional regulation,methylation,acetylation,histone modifica-tion,microRNA regulation and NF-κB/Snail/RKIP loop regulation.

Anvances in research on Raf kinase inhibitory protein in common digestive system malignant tumors / 肿瘤

RKIP (Raf kinase inhibitor protein) is a member of PEBP (phosphatidylethanolamine-binding protein) family, which plays a pivotal modulatory role in MAPK (mitogen-activated protein kinase), CPCR (G-protein-coupled receptor) and NF-KB (nuclear factor-kappa B) signaling transduction pathways. In recent years, many studies reported that RKIP expression is weakened or lost in a series of malignant tumors such as prostate cancer, breast cancer and melanoma. RKIP may also play an important role in inhibition of tumor metastasis via inhibition of cell invasion and tumor angiogenesis. Recently, many experiments revealed that RKIP which is a metastasis-suppressor gene participates in the suppression of metastasis in a variety of digestive system malignant tumors and it will become a new target for prevention and control of digestive system cancer. In this article, the advances of research on RKIP in common digestive system malignant tumors were reviewed. Copyright © 2013 by TUMOR.

Apoptosis of human cervical cancer HeLa cells induced by phosphatidylethanolamine / 西安交通大学学报(医学版)

Objective Phosphatidylethanolamine (PE) is an important phospholipid component in the cell membrane and is involved in the formation of membrane asymmetry. PE is exposed on the cell surface with phosphatidylserine during apoptosis. However, the effects of PE on cell apoptosis are not clear. In this study, we investigated effects of PE on apoptosis in human cervical cancer HeLa cells. Methods HeLa cells were used as the experiment material, and were divided into five groups: blank PE, respectively. The cell growth was tested by MTT assay; the cell cycle and apoptosis were analyzed using flow cytometry. Results Compared with the control group, PE inhibited the growth of HeLa cells in all the treatment groups in dose- and time-dependent manners, and induced the apoptosis, but did not change the cell cycle. Conclusion PE inhibits the growth of HeLa cells by inducing the apoptosis.

Relationship between raf kinase inhibitor protein and metastasis of ovarian cardnoma / 中华妇产科杂志

Objective To investigate the relationship between raf kinase inhibitor protein (SKIP), a novel metastasis suppressor gene, and metastasis of ovarian carcinoma. Methods Immunohistochemistry, RT-PCR, and western blot analysis were performed to examine the expression of SKIP in clinical samples of ovarian tumors and five human ovarian carcinoma cell lines. Stable cell lines over-expressed or deleted of SKIP were cloned to investigate the function of SKIP in ovarian cancer cells. The recombinant plasmids expressing sense (ss) or antisense (as) SKIP cDNA or empty vector was transfected into ovarian cancer cell line SKOV3 by lipofectamine. The expression level of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in ovarian cancer cells were detected by western blot analysis. Assays of cell proliferation, soft-agar colony formation, cell adhesion, and cell invasion in vitro were used to examine the malignant phenotypes of the transfected cells. Flow cytometric analysis was performed to observe the effect of SKIP on cell cycle distribution before and after transfection. Results (1 ) The expression levels of SKIP protein in ovarian carcinoma tissues from patients were found to be reduced than those in ovarian benign tumor and borderline tumor. SKOV3 clones stably expressing full-length recombinant ssRKIP, asRKIP, and their respective empty vector were obtained. (2)RKIP was able to block basal levels of MEK and ERK in ovarian cancer cells. The expression level of phosphorylation MEK in ssRKIP#1 and ssRKIP#4 cells were 0. 35, 0. 34; while the expression level of phosphorylation ERK in ssRKIP#1 and ssRKIP#4 cells were 0.48 and 0.46. (3) Abilities of cell proliferation in the ssRKIP vector-transfected cells were decreased compared with that in the non-transfected cells (P ＜0. 01 ). (4)Anchorage-independent growth in the ssRKIP#1 and ssRKIP#4 cells (83.7 ± 5.7, 106. 0±9. 2) were decreased compared with that in the empty vector-transfected cells (158.3 ± 14. 6, P＜ 0. 01). (5)Cell adhesion in the ssRKIP#1 and ssRKIP#4 cells [(68.3±0. 8)%, (64. 1±0. 9)%] were decreased compared with that in the non-transfected cells [(100. 0 ± 1.1 )%, P ＜ 0. 01]. (6) Cell invasion in the ssRKIP#1 and ssRKIP#4 cells (24 ± 5, 25±4) were decreased compared with that in the non-transfected cells (68 ± 5, P ＜ 0. 01 ). (7) ssRKIP cells had a significant increase in the G1 phase and decrease in the G2 + S phase. Conclusion RKIP could inhibits the metastasis, but also the growth of ovarian cancer cells.

Progress of the phosphatidylethanolamine binding protein / 中国药理学通报

Proteins of the phosphatidylethanolamine binding protein(PEBP) family are highly conserved throughout nature and have multiple biological functions.These small,cytosolic proteins have a typical large central sheet and a putative ligand-binding site which shares an affinity for a variety of ligands such as phospholipids,opioids,nucleotides,hydrophobic odorant molecules.PEBP plays a pivotal modulatory role in Raf-1/MEK/ERK、I?B/NF-?B、GPCR signaling cascades.As the precursor of the hippocampal cholinergic neurostimulating peptide(HCNP),PEBP may play an important role in the septal cholinergic development of the hippocampal.In addition,PEBP has the potential to contribute to neural protection,biogenesis and refinement of the membrane,Alzheimers disease(AD),opioid dependence,diabetic nephropathy,cancer and other physiological or pathophysiological processes.

Increased activity of large conductance Ca2+-activated K+ channels in negatively-charged lipid membranes

The effects of membrane surface charge originated from lipid head groups on ion channels were tested by analyzing the activity of single large conductance Ca2+-activated K+ (maxi K) channel from rat skeletal muscle. The conductances and open-state probability (Po) of single maxi K channels were compared in three types of planar lipid bilayers formed from a neutral phosphatidyledianolamine (PE) or two negatively-charged phospholipids, phosphatidylserine (PS) and phosphatidylinositol (PI). Under symmetrical KCl concentrations (3 apprx 1,000 mM), single channel conductances of maxi K channels in charged membranes were 1.1 apprx 1.7 times larger than those in PE membranes, and the differences were more pronounced at the lower ionic strength. The average slope conductances at 100 mM KCl were 251 +/- 9.9, 360 +/- 8.7 and 356 +/- 12.4 (mean +/- SEM) pS in PE, PS and PI membranes respectively. The potentials at which Po was 1/2, appeared to have shifted left by 40 mV along voltage axis in the membranes formed with PS or PI. Such shift was consistently seen at pCa 5, 4.5, 4 and 3.5. Estimation of the effect of surface charge from these data indicated that maxi K channels sensed the surface potentials at a distance of 8 apprx 9 ANG from the membrane surface. In addition, similar insulation distance (7 apprx 9 ANG) of channel mouth from the bilayer surface charge was predicted by a 3-barrier-2-site model of energy profile for the permeation of K+ ions. In conclusion, despite the differences in structure and fluidity of phospholipids in bilayers, the activities of maxi K channels in two charged membranes composed of PS or PI were strikingly similar and larger than those in bilayers of PE. These results suggest that the enhancement of conductance and Po of maxi channels is mostly due to negative charges in the phospholipid head groups.

Effect of phospholipase-D on rat kidney mitochondria.

Incubation of purified rat kidney mitochondrial fraction with phospholipase- D resulted in the accumulation of phosphatidic acid in the membrane due to the degradation of membrane-bound phosphatidylcholine, -serine and -ethanolamine Simultaneously with the hydrolysis of the phospholipids, cholesterol and protein were released from the mitochondrial membrane into the medium, and binding of Ca2+ by mitochondrial membranes increased. Phospholipase Dtreated mitochondrial fraction exhibited increased swelling in vitro in the early stages of incubation (15 min) after which the mitochondria were ruptured. Membrane- bound adenosine triphosphatase was partially inactivated and the enzyme activity was not significantly restored by incubation with sonicated dispersions of phosphatidylcholine, -serine and cholesterol. These results indicate that removal of choline, serine and ethanolamine from membrane-bound phospholipids disrupt phospholipid-cholesterol and phospholipid-protein association and affect functions of the membrane.