| With the improvement of people’s living standards and the popularity of fried foods,the safety of fried foods has also attracted more and more attention.Vegetable oil undergoes a series of chemical reactions such as oxidation,polymerization,and hydrolysis during high-temperature frying,resulting in a class of compounds with greater polarity than triglycerides.Polar compounds are the main substances that reduce the quality of frying oil and endanger human health,include oxidized triglycerides（ox-TGM）,triglyceride polymers（TGP）,and triglyceride degradation products.At present,the research on the specific harmful components in polar compounds and its mechanism of action are unclear.Based on this,this study separated the polar compounds from frying oil,analyzed the effects of polar compounds on the metabolic pathways,compared the effects of different components of polar compounds on liver cells,and clarified the mechanism of cytotoxicity caused by polar compounds.This research is aimed to provide scientific basis and theoretical reference for the safety of frying oil and related metabolic syndrome.Firstly,a frying experiment of palm oil was carried out.After frying for 40 h,the content of total polar compounds（TPC）reached the upper limit of 27%.At this time,the contents of TGP,ox-TGM,and triglyceride degradations were 14.9%,9.2%,and 2.6%,respectively.The TPC in the frying oil were separated by traditional silica gel column chromatography.After concentrating and collecting,TPC were further separated by dextran gel column chromatography,using methanol and dichloromethane（2:3,v/v）to obtain TGP,ox-TGM and triglyceride degradations.Secondly,the effects of polar compounds on the metabolic pathways of mice were studied by means of untargeted metabolomics,and it was found that polar compounds disturbedβ-oxidation and tricarboxylic acid cycle,leading to disorders of lipid metabolism and energy metabolism.Then the polar compounds were applied to liver cells,it was found that ox-TGM inhibited the growth of THLE-2 cell in a concentration-dependent manner,with an IC50 of 150μg/m L,while other components of TPC have little effect on the survival rate of THLE-2 cells.Further lipidomics analysis was performed on THLE-2 cells treated with ox-TGM.In the experimental group,the levels of triglyceride,phosphatidylethanolamine,ceramide,lysophosphatidylethanolamine,and fatty acids increased significantly,while the levels of phosphatidylcholine,sphingomyelin,and lysophosphatidylcholine decreased.It indicated that ox-TGM destroyed phospholipid metabolism and fatty acid metabolism in liver cells,and affected the normal physiological functions of cells.Subsequently,the effect of ox-TGM on the biological function of liver cells were studied.At the concentration of 100μg/m L,ox-TGM can induce lipid deposition in THLE-2 cells,and the intracellular triglyceride content increased from 0.09 to 0.87 mmol/gprot.The ox-TGM increased reactive oxygen species（ROS）and malondialdehyde（MDA）level,and reduces superoxide dismutase（SOD）activity in a dose-dependent manner.After 24 h of treatment,the number of autophagosomes increases,and the expression levels of autophagy-related genes LC3B,Beclin1,Atg5,Atg7,and Atg12 were up-regulated.As the treatment time increases,ox-TGM induced an increased apoptosis rate of THLE-2 cells.The path of ox-TGM-induced cytotoxicity was analyzed by adding antioxidant NAC,autophagy inhibitor chloroquine,and autophagy agonist rapamycin,ox-TGM first caused lipid deposition in THLE-2 cells,thus inducing lipid peroxidation and disrupting the intracellular redox balance,then the level of autophagy were enhanced,abnormal level of autophagy further aggravated oxidative damage,and leading to apoptosis.Finally,the pathway of ox-TGM damage on cells was analyzed from a molecular perspective.After treatment with ox-TGM,the concentration of the second messenger Ca2+in THLE-2 cell increased significantly.It was found that ox-TGM activated the intracellular Ca MKKβ/AMPK/m TOR pathway.The intracellular lipid deposition,oxidative stress,autophagy and apoptosis levels were measured after adding pathway inhibitors.It was found that the inhibition of the Ca MKKβ/AMPK/m TOR pathway can significantly alleviate the cytotoxicity caused by ox-TGM,which further verified the activation of the Ca MKKβ/AMPK/m TOR pathway induced by ox-TGM. |
