| Palmitoleic acid is a representative trans-fatty acid in ruminants.It mainly comes from ruminant fat and dairy products.It can not be synthesized in human body,but can be produced by endogenous trans-isooleic acid.In recent years,epidemiological studies have reported that trans-palmitoleic acid has the function of reducing blood lipids,and also reported that trans-palmitoleic acid is inversely proportional to the incidence of cardiovascular disease.As one of the most harmful diseases in the world,it is not clear whether trans-palmitoleic acid will play a positive role in preventing cardiovascular diseases.Epidemiological analysis alone does not determine its role.Therefore,it is of great significance to study the relationship betweentrans-palmitoleic acidacid and lipid through specific experiments.In this study,hepatocyte LO2 cells were used as experimental objects to study the effect of trans-palmitoleic acid on the fatty acid composition of cells by adding different concentrations of trans-palmitoleic acid.To study the effect of trans-palmitoleic acid on triglyceride and cholesterol content in hyperlipidemic LO2 cells induced by oleic acid,and to observe its effect on the related indicators of lipid oxidation.In addition,the effect of trans-palmitoleic acid on the lipid group of hyperlipidemic LO2 cells was studied by using lipidomics technology,and differential lipids were found.The mechanism of a trans-palmitoleic acid acting on lipids was further elucidated by the determination of related lipid metabolic proteins in AMPK pathway.The experimental results are as follows:1.The effects of trans palmitoleic acid on lipid metabolism were studied by measuring cell survival rate,intracellular fatty acid composition,lipid content and cell oxidation.The results showed that there was no significant toxicity of trans palmitoleic acid below 100 uM.The results showed that the saturated fatty acids decreased by 34.89%,the monounsaturated fatty acids increased by 16.44% and the polyunsaturated fatty acids increased by 30.50% when human hepatocytes were treated with 9t16:1 of 100 uM.9t16:1 was partly transformed into 11t18:1 and CLA in hepatocytes,and the conversion rates were 17.50% and 16.42% respectively.9t16:1significantly reduced the triglyceride and cholesterol contents in steatotic hepatocytes,with the reduction rates of 61.76% and 37.80%,respectively.In addition,9t16:1 significantly increased the MDA level(increased by 45.36%)and decreased SOD activity(decreased by 74.42%)in hepatocytes to promote intracellular lipid oxidation.2.The total lipids in cells were extracted and identified by high-efficiency liquid.Then the differences between the two groups were compared.The results showed that there were significant differences in lipid metabolites between oleic acid group and control group,oleic acid and trans palmitoleic acid mixed group and oleic acid group.The effect of adding oleic acid and trans palmitoleic acid was obvious through PCA total score map and PLS-DA remote statistical analysis.According to the ionic peak strength,100 different lipids were screened out,of which glycerol phospholipids were the majority.Through volcanic map and hierarchical cluster analysis,it can be clearly seen that lipid is up-regulated or down-regulated.For example,compared with the control group,the up-regulated lipids in the OA group included diol phosphatidylethanolamine,1,2-diol-sn-glycerol-3-phosphate-N,N-dimethylethanolami ne,1,2-diol-sn-glycerol-3-phosphate-l-serine,1,2-diol-sn-glycerol,etc.However,in the OA group,the down-regulated lipids were ceramide(d18:1/16:0),1-(1Z hexaenol)-2-(4Z,7Z,10 Z,13Z,16 Z,19Z docosahexaenol)-sn-glycerol-3-phosphate ethanolamine,1-palmitoyl-2-palmitoyl-sn-glycerol,1-stearic-2-palmitoyl-sn-glycerol,etc.Compared with the oleic acid group,the up-regulated lipids in the mixed group of trans palmitoleicacid and oleic acid were as follows:1-hexadecacyl-2-(9Z,12 Z octadecenyl)-sn-glycerol-3-phosphoethanoamine,(2R)-3-[(2-amino ethoxy(hydroxy)phosphoric acid] oxo-2-[(9Z)-9-hexadecanoxy] propyl(9Z)-9-hexadecadecadecadecadecadecanoate,(19R)-(19R)-25-amino-22-hydroxy-22-hydro xhydroxy-22-22-oxoxo-16-oxy-16-17,17,17,21,21,23-trioxheterheterhetero-22-lamla mp-5-phosphophosphophosphorus-5-5-phosphophosphophosphophosphorus-19-penta methyl-19-19-19-phosphophosphophosphoZ,15Z)-9,12,15-octadecanoic triesters,1-palmitoyl-2-oleoyl-sn-glycyrrhiza-3-octanoate ethanolamine,etc.The down-regulated lipids were 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine,n-hexadecanoimide,1,2-dipalmitoyl-sn-glycerol-3-phosphateethanolamine,(2R)-3-[(2-aminoethoxy)(hydroxy)phosphoric acid]oxy-2-(tetraacyloxy)palmitate propyl ester,etc.The possible metabolic pathway is AMPK signaling pathway through the analysis of differential lipids on KEGG website.3.The effect of trans palmitoleic acid on AMPK signaling pathway was studied by Western Blot experiment.The results showed that trans palmitoleic acid could increase the expression of AMPK alpha and activate various lipid metabolism related proteins downstream of AMPK alpha.The activities of lipid synthesis related proteins ACC and FAS were inhibited,while the activities of PGC1,CPT1 A and PPAR alpha were increased.These results suggest that trans palmitoleic acid can activate AMPK pathway and regulate lipid metabolism in hyperlipidemicLO2 cells. |
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