Sphingomyelin synthase activity measurement by the fluorescent product in cell culture medium or animal plasma

Sphingolipids, a new class of lipid mediators, are involved in a variety of important physiological and pathological processes. Sphingomyelin synthase （SMS） is an enzyme to convert the ceramide （Cer.） and phosphatidylcholine into sphingomyelin （SM） and diacylglycerol, which plays a key role in sphingolipid biosynthesis. Two SMS isoforms, SMS1 and SMS2, have been identified with different subceUular localizations and expression level in tissues. Previous studies have shown that SMS may serve as a potential therapeutic target for the treatment of various diseases, such as cardiovascular and metabolic diseases. Thus, there is an urgent need for a rapid and sensitive method for SMS activity analysis. In our study, we developed a novel method for SMS activity by monitoring the appearance of the product, NBD-SM, in the tissue culture medium or blood and applied this method in cells and mice. In Huh7 cells, the interassay coefficient of variation of the SMS activity assay was （3.60±0.07）% . In wild type （WT） mice, we observed accumulation of NBD-SM in blood in a time dependent fashion. In SMS2 KO mice, NBD-SM in plasma collected at 5- （0%, P〈0.01）, 30- （16%, P〈0.01）, and 60 min （21%, P〈0.01） after injection of fluorescence liposome solution was significantly decreased compared with WT mice. However, in SMS1 KO mice, NBD-SM in plasma collected 5- and 30 min is similar to that in WT mice. Our results suggest that this method could be used for SMS activity measurement in vitro and in vivo.

Design, Synthesis, and Biological Evaluation of Novel Dual Inhibitors of Secretory Phospholipase A2 and Sphingomyelin Synthase

A novel series of eight SMS and sPLA2 dual inhibitors containing indole and a-amino cyanide fragments of different length and substitution position was synthesized and evaluated by three different in vitro assays. Biological evaluation showed that all compounds provided inhibitory effects against SMS （about 50% inhibition at 100 μmol/L） and sPLA2 （14-32 μmol/L）. All the compounds had the SMS activity better than the positive control compound D609 in SMS2 homogenate, with compounds 5b and fie ideal for liver homogenate and SMS2 high expression cell homogenate, respectively.

Molecular Modeling of the Three-Dimensional Structure of Human Sphingomyelin Synthase

Sphingomyelin synthase （SMS） produces sphingomyelin and diacylglycerol from ceramide and phosphatidyl- choline. It plays an important role in cell survival and apoptosis, inflammation, and lipid homeostasis, and therefore has been noticed in recent years as a novel potential drug target. In this study, we combined homology modeling, molecular docking, molecular dynamics simulation, and normal mode analysis to derive a three-dimensional struc- ture of human sphingomyelin synthase （hSMS 1） in complex with sphingomyelin. Our model provides a reasonable explanation on the catalytic mechanism of hSMS 1. It can also explain the high selectivity of hSMS 1 towards phos- phocholine and sphingomyelin as well as some other known experimental results about hSMS1. Moreover, we also derived a complex model of D609, the only known small-molecule inhibitor of hSMS 1 so far. Our hSMS 1 model may serve as a reasonable structural basis for the discovery of more effective small-molecule inhibitors of hSMS 1.

The relationship among amyloid-βdeposition,sphingomyelin level,and the expression and function of P-glycoprotein in Alzheimer’s disease pathological process

In Alzheimer’s disease,the transporter P-glycoprotein is responsible for the clearance of amyloid-βin the brain.Amyloid-βcorrelates with the sphingomyelin metabolism,and sphingomyelin participates in the regulation of P-glycoprotein.The amyloid cascade hypothesis describes amyloid-βas the central cause of Alzheimer’s disease neuropathology.Better understanding of the change of P-glycoprotein and sphingomyelin along with amyloid-βand their potential association in the pathological process of Alzheimer’s disease is critical.Herein,we found that the expression of P-glycoprotein in APP/PS1 mice tended to increase with age and was significantly higher at 9 and 12 months of age than that in wild-type mice at comparable age.The functionality of P-glycoprotein of APP/PS1 mice did not change with age but was significantly lower than that of wild-type mice at 12 months of age.Decreased sphingomyelin levels,increased ceramide levels,and the increased expression and activity of neutral sphingomyelinase 1 were observed in APP/PS1 mice at 9 and 12 months of age compared with the levels in wild-type mice.Similar results were observed in the Alzheimer’s disease mouse model induced by intracerebroventricular injection of amyloid-β1-42 and human cerebral microvascular endothelial cells treated with amyloid-β1-42.In human cerebral microvascular endothelial cells,neutral sphingomyelinase 1 inhibitor interfered with the changes of sphingomyelin metabolism and P-glycoprotein expression and functionality caused by amyloid-β1-42 treatment.Neutral sphingomyelinase 1 regulated the expression and functionality of P-glycoprotein and the levels of sphingomyelin and ceramide.Together,these findings indicate that neutral sphingomyelinase 1 regulates the expression and function of P-glycoprotein via the sphingomyelin/ceramide pathway.These studies may serve as new pursuits for the development of anti-Alzheimer’s disease drugs.

Ceramide is involved in alcohol-induced neural proliferation

Prenatal alcohol exposure, especially during early pregnancy, can lead to fetal alcohol syndrome. The pharmacological and toxicological mechanisms of ethanol are related to the effects of ceramide In this study, we established an alcohol exposure model in wild-type mice and in knockout mice for the key enzyme involved in ceramide metabolism, sphingomyelin synthase 2. This model received daily intragastric administration of 25% ethanol, and pups were used at postnatal days 0, 7, 14, 30 for experiments. Serology and immunofluorescence staining found that ethanol exposure dose-dependently reduced blood sphingomyelin levels in two genotypes of pups, and increased neural cell proliferation and the number of new neurons in the hippocampal dentate gyrus. Western blot analysis showed that the relative expression level of protein kinase C e increased in two genotypes of pups after ethanol exposure. Compared with witd-type pups, the expression level of the important activator protein of the ceramide/ceramide-l-phosphate pathway, protein kinase C a, was reduced in the hippocampus of sphingomyelin synthase 2 knockouts. Our findings illustrate that ceramide is involved in alcohol-induced neural proliferation in the hippocampal dentate gyrus of pups after prenatal ethanol exposure, and the mechanism may be associated with increased ex- pression of protein kinase C a activating the ceramide/ceramide-l-phosphate pathway.

Substance P promotes epidural fibrosis via induction of type 2 macrophages

In response to spinal surgery,neurons secrete a large amount of substance P into the epidural area.Substance P is involved in macrophage differentiation and fibrotic disease.However,the specific roles and mechanisms of substance P in epidural fibrosis remain unclear.In this study,we established a mouse model of L1–L3 laminectomy and found that dorsal root ganglion neurons and the macrophages infiltrating into the wound area released sphingolipids.In vitro experiments revealed that type 1 macrophages secreted substance P,which promoted differentiation of type 1 macrophages towards a type 2 phenotype.High-throughput mRNA-seq analysis revealed that the sphingolipid metabolic pathway may be involved in the regulation of type 2 macrophages by substance P.Specifically,sphingomyelin synthase 2,a component of the sphingolipid metabolic pathway,promoted M2 differentiation in substance P-treated macrophages,while treating the macrophages with LY93,a sphingomyelin synthase 2 inhibitor,suppressed M2 differentiation.In addition,substance P promoted the formation of neutrophil extracellular traps,which further boosted M2 differentiation.Blocking substance P with the neurokinin receptor 1 inhibitor RP67580 decreased the number of M2 macrophages in the wound area after spinal surgery and alleviated epidural fibrosis,as evidenced by decreased fibronectin,α-smooth muscle actin,and collagen I in the scar tissue.These results demonstrated that substance P promotes M2 macrophage differentiation in epidural fibrosis via sphingomyelin synthase 2 and neutrophil extracellular traps.These findings provide a novel strategy for the treatment of epidural fibrosis.