Objective: To establish ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of 22 phospholipids in serum. Methods: In September 2022, Using synthetic non endogenous phospholipids as internal standard, phospholipids in serum were extracted by methanol-dichloromethane (2∶1, V/V) protein precipitation method. Chromatographic separation was achieved on an ACQUITY UPLC BEH shield RP18 column, and the mobile phase was methanol/water (5∶95, V/V) containing 10 mM ammonium formate and methanol. Detection was performed in multiple reaction monitoring mode with ion mode switching. And the method was applied by analyzing phospholipids in the serum of coal workers' pneumoconiosis patients. Results: The 22 phospholipids showed good linear relationships in their respective concentration ranges and the correlation coefficients were higher than 0.990. The spiked recoveries of the 22 phospholipids were 81.03%-121.63% at the three spiked levels. The intra-assay were less than 14.52%, and the inter-assay were less than 15.00%. Conclusion: The method with the advantages of simplicity, stability and high sensitivity, and it can be used for the analysis of phospholipids in serum.
Humans ; Chromatography, Liquid ; Chromatography, High Pressure Liquid/methods* ; Tandem Mass Spectrometry/methods* ; Phospholipids ; Methanol

Long-chain acyl-coenzyme A (CoA) synthase 4 (ACSL4) is an enzyme that esterifies CoA into specific polyunsaturated fatty acids, such as arachidonic acid and adrenic acid. Based on accumulated evidence, the ACSL4-catalyzed biosynthesis of arachidonoyl-CoA contributes to the execution of ferroptosis by triggering phospholipid peroxidation. Ferroptosis is a type of programmed cell death caused by iron-dependent peroxidation of lipids; ACSL4 and glutathione peroxidase 4 positively and negatively regulate ferroptosis, respectively. In addition, ACSL4 is an essential regulator of fatty acid (FA) metabolism. ACSL4 remodels the phospholipid composition of cell membranes, regulates steroidogenesis, and balances eicosanoid biosynthesis. In addition, ACSL4-mediated metabolic reprogramming and antitumor immunity have attracted much attention in cancer biology. Because it facilitates the cross-talk between ferroptosis and FA metabolism, ACSL4 is also a research hotspot in metabolic diseases and ischemia/reperfusion injuries. In this review, we focus on the structure, biological function, and unique role of ASCL4 in various human diseases. Finally, we propose that ACSL4 might be a potential therapeutic target.
Humans ; Ferroptosis ; Apoptosis ; Phospholipids/metabolism* ; Nitric Oxide Synthase

Exosomes are phospholipid bilayer membrane-enclosed vesicles released from cells with diameters of 30-150 nm, exosomes can directly reflect the physiological and functional state of secretory cells, participate in material transport and information communication between cells, which are of great significance as biomarkers for early tumor diagnosis and treatment evaluation. There are many detection methods for exosomes, among which aptasensor technology with the properties of low price and easy operation, fast response, high sensitivity, remarkable specificity helps tumor patients to find, diagnose and treat early, improve the survival rate, and provide important basis for the evaluation of the prognosis. There are seven types of common aptasensors: fluorescent, electrochemical, colorimetric, luminescence, lateral flow strips, surface-enhanced Raman scattering and surface plasmon resonance sensors. Different aptasensors have different characteristics, this article focuses on the research progress of several common aptasensor for tumor exosomes detection.
Humans ; Exosomes/metabolism* ; Biomarkers/analysis* ; Neoplasms/diagnosis* ; Phospholipids/metabolism* ; Biosensing Techniques/methods*

Phospholipids are important components of biomembrane and lipoproteins. Phospholipids can be oxidized by free radicals/nonradicals and enzymes to form oxidized phospholipids (OxPLs), which can lead to further generation of oxidation products with different biological activities. Clinical evidence shows that OxPLs are constantly generated and transformed during the pathogenesis of atherosclerosis and accumulated at the lesion sites. OxPLs are highly heterogeneous mixtures that can influence the progress of atherosclerosis through a variety of related receptors or signaling pathways. This review summarizes the process of phospholipid oxidation, the related products, the interaction of OxPLs with endothelial cells, monocytes/macrophages, smooth muscle cells, platelets and lipoproteins involved in the pathological process of atherosclerosis, and the progress of the researches using OxPLs as a target to inhibit atherosclerosis in recent years.
Atherosclerosis ; Endothelial Cells ; Humans ; Myocytes, Smooth Muscle ; Oxidation-Reduction ; Phospholipids

Polygonum multiflorum is a traditional Chinese herbal medicine and has many biological activities such as hair-blacking, anti-atherosclerosis, anti-inflammatory and anti-aging. However, the liver injury induced by P. multiflorum has aroused wide attention in recent years. 2,3,5,4'-tetrahydroxystibane-2-O-β-D-glucoside(TSG) is a main component of P. multiflorum, but the role of TSG in inducing liver injury is unclear. The aim of present study was to evaluate TSG's potential liver injury and effects on bile acid homeostasis and phospholipids efflux. C57 BL/6 J mice received intraperitoneal administration of 400 mg·kg~(-1) of TSG daily for 15 days, and then biochemical indexes of liver injury and changes of phospholipid content were detected. The changes of bile acid compositions were detected by LC-MS/MS. The results showed TSG 400 mg·kg~(-1) significantly increased the content of serum total bile acid(TBA) and alkaline phosphatase(ALP). Elevated free bile acid levels were observed in TSG-treated groups, including β-muricholic acid(β-MCA), ursodeoxycholic acid(UDCA), hyodeoxycholic acid(HDCA), chenodeoxycholic acid(CDCA), deoxcholic acid(DCA) in serum and β-MCA, CDCA in liver. TSG inhibited the protein expression of farnesoid X receptor(FXR) and down stream bile salt export pump(BSEP), which may result in the accumulation of bile acid. TSG also inhibited the expression of 25-hydroxycholesterol-7 alpha-hydroxylase(CYP7 B1), which may disturb the alternative pathway for bile acid synthesis. In addition, intraperitoneal injection of TSG 400 mg·kg~(-1) significantly decreased the content of phospholipids in bile. The research showed that TSG significantly inhibited the expression of multidrug resistance protein 2(MDR2) and destroyed the regular distribution of MDR2 on the bile duct membrane of liver. In vitro results showed that the IC_(50) of TSG on HepG2 cells was about 1 500 μmol·L~(-1) and TSG at 500 μmol·L~(-1)(for 24 h) could destroy the distribution of MDR2 on the bile duct membrane of liver. In conclusion, TSG induced liver injury by disrupting bile acid homeostasis and phospholipids efflux.
Animals ; Bile Acids and Salts ; Chromatography, Liquid ; Glucosides ; Homeostasis ; Liver ; Mice ; Phospholipids ; Tandem Mass Spectrometry

Human secreted phospholipase A2 GIIE (hGIIE) is involved in inflammation and lipid metabolism due to its ability of hydrolyzing phospholipids. To reveal the mechanism of substrate head-group selectivity, we analyzed the effect of mutation of hGIIE on its activity and selectivity. hGIIE structural analysis showed that E54 might be related to its substrate head-group selectivity. According to the sequence alignment, E54 was mutated to alanine, phenylalanine, and lysine. Mutated genes were cloned and expressed in Pichia pastoris X33, and the enzymes with mutations were purified with 90% purity by ion exchange and molecular size exclusion chromatography. The enzymatic activities were determined by isothermal microthermal titration method. The Km of mutant E54K towards 1,2-dihexyl phosphate glycerol decreased by 0.39-fold compared with that of wild type hGIIE (WT), and the Km of E54F towards 1,2-dihexanoyl-sn-glycero-3-phosphocholine increased by 1.93-fold than that of WT. The affinity of mutant proteins with phospholipid substrate was significantly changed, indicating that E54 plays an important role in the substrate head-group selectivity of hGIIE.
Humans ; Kinetics ; Mutation ; Phospholipases A2, Secretory ; Phospholipids ; Saccharomycetales ; Substrate Specificity

In the present study, liquiritigenin-phospholipid complex (LPC) was developed and evaluated to increase the oral bioavailability of liquiritigenin. A single-factor test methodology was applied to optimize the formulation and process for preparing LPC. The effects of solvent, drug concentration, reaction time, temperature and drug-to-phospholipid ratio on encapsulation efficiency were investigated. LPCs were characterized by UV-visible spectroscopy, differential scanning calorimetry (DSC), fourier transform infrared spectroscopy (FTIR), and powder X-ray diffractometry (PXRD). The apparent solubility and n-octanol/water partition coefficient were tested. The pharmacokinetic characteristics and bioavailability of the LPC were investigated after oral administration in rats in comparison with liquiritigenin alone. An LPC was successfully prepared. The optimum level of various parameters for liquiritigenin-phospholipid complex was obtained at the drug concentration of 8 mg·mL
Administration, Oral ; Animals ; Biological Availability ; Flavanones/pharmacokinetics* ; Phospholipids/pharmacokinetics* ; Rats ; Solvents

BACKGROUND/OBJECTIVES: Consumption of cholesterol-rich foods, such as eggs, has a minimal effect on circulating cholesterol levels in healthy humans. To gain insight, we investigated whether phospholipids rich in eggs (EPL) interfere with intestinal cholesterol absorption in vivo. MATERIALS/METHODS: To investigate the acute effect of EPL on intestinal cholesterol absorption, male C57BL/6J mice were orally administered with 6, 11, or 19 mg of EPL for three days. We also tested the effect of chronic EPL consumption on cholesterol metabolism in the small intestine and the liver in mice with diet-induced hypercholesterolemia. Male C57BL/6J mice were fed a high fat/high cholesterol (HF/HC; 35% fat, 0.25% cholesterol, w/w) diet for 4 weeks to induce hypercholesterolemia, and subsequently the mice were either fed 0, 0.4 or 0.8% (w/w) of EPL for 6 weeks. RESULTS: Intestinal cholesterol absorption was significantly decreased by the highest dose of acute EPL administration compared to control. Chronic EPL supplementation did not significantly alter intestinal cholesterol absorption nor plasma levels of total cholesterol and low-density lipoprotein cholesterol. In the small intestine and the liver, EPL supplementation minimally altered the expression of genes which regulate cellular cholesterol levels. CONCLUSION: Although chronic EPL consumption was not able to counteract hypercholesterolemia in HF/HC-fed mice, acute EPL administration decreased intestinal cholesterol absorption. This study provides in vivo evidence that acute administration of PLs in eggs prevent cholesterol absorption in the intestine, suggesting a mechanism for a minimal effect of egg consumption on circulating cholesterol levels.
Absorption ; Animals ; Cholesterol ; Diet ; Eggs ; Humans ; Hypercholesterolemia ; Intestinal Absorption ; Intestine, Small ; Intestines ; Lipoproteins ; Liver ; Male ; Metabolism ; Mice ; Ovum ; Phosphatidylcholines ; Phospholipids ; Plasma

Background: Signal transducer and activator of transcription 3 (STAT3) was strongly expressed and activated in psoriatic keratinocytes (KCs) and correlated with the severity of psoriasis. The study aimed to investigate the effects of STAT3 small interfering RNA (siRNA) combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis in KCs of psoriatic lesions and the relative mechanisms. Methods: Psoriatic KCs were transfected under four experimental conditions: (1) STAT3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group); (2) STAT3 siRNA only carried by Lipofectamine 3000 (L group); (3) the negative control of siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (siRNA-NC); (4) not treated as Blank. Cell Counting Kit-8 assay was used to evaluate the cell proliferation. Cell cycle analysis was detected with cycle test Plus DNA reagent kit associated with flow cytometer. FITC Annexin V apoptosis detection kit associated with flow cytometer was applied for apoptosis analysis. Fluo calcium indicator associated with flow cytometer was used to analyze intracellular free calcium concentration ([Ca]). The expressions of cyclin D1 and Bcl-xL were detected both at the mRNA level by real-time reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. The obtained data were statistically evaluated by two-way analysis of variance. Results: STAT3 siRNA inhibited the growth of KCs in a time-dependent manner showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72h (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced an altered cell cycle distribution of KCs showing the highest increases in G2/M-phase population up to 18.06% ± 0.36% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced late apoptosis of KCs with the highest late apoptosis percentage of 22.87% ± 1.28% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the elevation of [Ca]of KCs with the highest calcium fluorescence intensity mean of 1213.67 ± 60.51 in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the downregulation of cyclin D1 and Bcl-xL expressions of KCs at mRNA and protein levels with the lowest expressions in LUS group with cyclin D1 expression of 51.81% ± 9.58% and 70.17% ± 4.22% at mRNA level and at protein level, respectively, and with Bcl-xL expression of 37.58% ± 4.92% and 64.06% ± 7.78% at mRNA level and at protein level, respectively (P < 0.05 vs. L group, siRNA-NC, or Blank). Conclusions STAT3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca], and downregulating cyclin D1 and Bcl-xL expressions. Silencing the target gene STAT3 in psoriatic KCs with siRNA combined with ultrasonic irradiation and microbubbles would contribute to a significant innovation as a new clinical therapy for psoriasis.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Keratinocytes ; Microbubbles ; Phospholipids ; Psoriasis ; therapy ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor ; metabolism ; Sulfur Hexafluoride ; Ultrasonics

The aim of this study was to provide a basis for the molecular mechanism underlying the pharmacological action of ethanol. We studied the effects of 1-propanol on the location of n-(9-anthroyloxy)palmitic acid or stearic acid (n-AS) within the phospholipids of synaptosomal plasma membrane vesicles (SPMV). The SPMV were isolated from the bovine cerebral cortex and liposomes of total lipids (SPMVTL) and phospholipids (SPMVPL). 1-Propanol increased the rotational mobility of inner hydrocarbons, while decreasing the mobility of membrane interface, in native and model membranes. The degree of rotational mobility varied with the number of carbon atoms at positions 16, 12, 9, 6 and 2 in the aliphatic chain of phospholipids in the neuronal and model membranes. The sensitivity of increasing or decreasing rotational mobility of hydrocarbon interior or surface by 1-propanol varied with the neuronal and model membranes in the following order: SPMV, SPMVPL and SPMVTL.
1-Propanol* ; Carbon ; Cell Membrane ; Cerebral Cortex ; Ethanol ; Hydrocarbons ; Liposomes ; Membranes* ; Neurons* ; Phospholipids