Effect Of Ruminant Trans Fatty Acid TAG On Human Umbilical Vein Endothelial Cells Phospholipids Profile

The health effects of trans-fatty acids have been the focus of public attention for a long time and it’s the point of scientific research.A large number of epidemiological and clinical studies have found that industrial hydrogenated trans fatty acids(ITFAs)have adverse effects on human health,including increase blood lipids level,lead to atherosclerosis and cardiovascular disease(CHD).But the result of study of another trans-fatty acid,the ruminant trans fatty acid(RTFA)is inconclusive,whether it has a similar health impact with ITFA,and whether there is a safe intake threshold have been still widely studied.Studies have shown that RTFA and ITFA have different effects on CHD at a certain concentration.The difference is mainly due to the structure of the isomers.The main isomer of RTFA is 11 t 18:1,the vaccenic acid(VA).The major isomer of ITFA is 9t 18:1,the elaidic acid(EA).However,there is no clear biological explanation for such differences currently.Most studies researched in the health effects of trans fatty acids have focused on free fatty acids and diets containing trans fatty acids.There is no study comparing the effects of VA and EA directly on pure triglyceride structure currently.The study have found that the positional distribution of the two trans fatty acids in triglycerides is different.So,whether the distribution of the location affects the health effects of the two trans fatty acids or not is very important for guiding a reasonable diet.Phospholipids are the main component of cell membranes,and their structural abnormalities will directly alter the structure of cell membranes,causing irregular cell functions.After triglycerides acts on cells,it will affect the phospholipid fatty acid composition of the cell membrane,causing irregular cell and vascular function.So,what is the difference between the effects of trans fatty acid triglycerides OOV and OEO on endothelial cell membrane phospholipids,and whether it influence endothelial cell function via cellular phospholipids or not? That is point of our research.Therefore,this study synthesizes a trans fatty acid triglyceride OOV containing 11 t 18:1 by mimicking the configuration of ruminant trans fatty acids and industrial hydrogenated oil trans fatty acids in triglycerides.The effects of triglyceride OOV and OEO on endothelial function were evaluated by MTT method,LDH exudation rate,NOS-NO system and expression of inflammatory factors;and the effect of triglyceride on endothelial cell membrane phospholipids were investigated based on phospholipidomics techniques and determined by multivariate statistical analysis methods such as PCA and OPLS-DA to identify significant differential phospholipid metabolites,thereby performing KEGG pathway analysis to identify metabolic pathways associated with differential metabolites.And the relationship between phospholipase and inflammatory factor prostaglandins was analyzed by Western Blot.The research results obtained are as follows:1.The synthesis method of asymmetric structural TAG(OOV)was studied by using free fatty acid and acetone glycerol as substrates.First,the high-purity concentration of VA and solketal was used as a substrate to synthesize 1-VA solketal under the catalysis of Nozym435 lipase.Then,the monoglyceride was separated and purified by a solvent crystallization method to obtain a monoglyceride with purity of 91%.The purified monoglyceride and oleic acid were catalysted to synthesize OOV.The effects of reaction temperature,reaction time,substrate ratio and additive ratio DCC/DMAP on the content of TAGs in the product were investigated.The results of single factor experiments showed that the optimal conditions for the synthesis of TAGs by this chemical reaction were as follows: the molar ratio of VA and solketal was 1:2.2,DCC/DMAP =(1: 0.08),reacted in 5 ml of chloroform at 30 °C for 3 h.Under this condition,the purity of the TAGs was 97%.The fatty acid composition of OOV was determined to be OOV by gas chromatography,which verified the rationality of the synthesis method.2.The effects of triglyceride OOV and OEO containing two different sources of trans fatty acids on endothelial cell function were evaluated by MMT method,LDH exudation rate,NOS-NO system and expression of inflammatory factors.The results showed that both OOV and OEO can damage cell viability,increase cell membrane permeability,and inhibit NO production by reducing NOS enzyme activity,thereby promoting inflammation.At the same time,the expression levels of inflammatory factors ICAM-1,VCAM-1 and IL-6 are increased,but the damage of OEO to cells is more obvious than that of OOV.Part of the reason may due to the higher absorption rate of OEO than OOV.And another is that a small amount of 11t18:1 was converted to CLA,and CLA has anti-inflammatory and anti-atherosclerotic effects.They are the reason for the differences in cell damage between OOV and OEO.3.Based on the UPLC/MS/MS(QE)Plus ? liquid chromatography mass spectrometry system of non-targeted phospholipidomics analysis platform,the lipid extract was analyzed after different trans triglycerides act on endothelial cells.It can be seen from the PCA results,the composition of the phospholipids were changed in cell membranes by different triglycerides.Through the analysis of OPLS-DA and volcano map,it was found that there were 8 kinds of significantly different phospholipid metabolites.PEGPEtn(21:0/22:6(4Z,7Z,10 Z,13Z,16 Z,19Z))、 PE(22:0/24:1)、GPEtn(21:0/24:0)[U]、GPEtn(18:1(9E)/18:1(9E))[U]、PE(16:0/18:1)、PE(18:0/22:6)、PE(18:0/20:4)、GPEtn(15:0/16:0)[U].The content of these eight PE in OEO group was more than 2 times higher in OOV(P<0.05).Based on these differential phospholipid metabolites,KEGG pathway analysis revealed 8 differential metabolic pathways.Among them,we paid more attention to glycerophospholipid metabolism(P<0.01),linoleic acid metabolism(P<0.05),alpha-linolenic acid metabolism(P<0.05)and arachidonic acid metabolism(P<0.05).4.The effects of trans fatty acid triglycerides on human umbilical vein endothelial cells phospholipase were determined by Western Blot,and the relationship between phospholipase and prostaglandin was discussed in this part.The results showed that the expression of HUVEC phospholipase A2 was increased by OEO,OOV and OOO.And OEO had a more significant effect on sPLA2 expression,while OOV had a more significant effect on iPLA2 expression(P<0.05).In addition,OOV is more likely to activate the phosphorylation level of cPLA2.Phospholipase A2 regulates trans fatty acid triglycerides to induce inflammatory responses in HUVEC cells.The secretion of intracellular inflammatory cytokines was significantly reduced after pretreatment with three phospholipase A2 inhibitors,PYR(cPLA2 inhibitor),BEL(iPLA2 inhibitor)and DTT(sPLA2 inhibitor),compared with that induced by OEO and OOV alone.