Study On The Mechanism Of Intestinal Barrier Disorder Induced By Epoxy Triglyceride In Deep-Frying Oil

Recently,deep-frying oil has been found to be associated with the risk of chronic inflammation,atherosclerosis,diabetes,hypertension,cardiovascular disease and even cancer.The total polar components(TPC)produced by deep-frying oil will lead to programmed cell death.However,due to the variety of lipid oxidation products,complex structure,difficulty in separation and purification,and unclear cytotoxicity mechanism,it is still unable to effectively target to inhibit or improve a series of diseases caused by deep-frying oil.Intestine is the main absorption site of nutrients needed for human health,and also an important barrier to protect the body from external harmful substances.Recently,the effects of diet on intestinal homeostasis and intestinal barrier has been the focus.The mechanism of dietary effects on intestinal barrier may be the direct contact between digestive products of diet and intestinal epithelium.Dozens of studies have shown that the intake of deep-frying oil can induce intestinal homeostasis imbalance,but the mechanism is still unknown.In this study,the composition of deep-frying oil was identified using high-performance liquid chromatography-mass spectrometry(HPLC-MS);the main cytotoxic oxidized triglyceride(ox-TG)was determined by cell viability;moreover,the animal model was applied to identify the molecular mechanism of intestinal barrier disorder induced by main cytotoxic ox-TG in deep-frying oil;finally,the mechanism was verified through in vitro experiments.The main results are as follows:(1)Caco-2 cells were treated with either the total polarizing component(TPC)or one of its separated components(total polymerized triglyceride(TPG),ox-TG,and total triglyceride degradation products(TDG))from deep-frying oil.Cytotoxicity assays and flow cytometry analysis showed that TPC and its components induce apoptosis,where ox-TG was the main toxic component.Subsequently,the components of ox-TG in deep-frying oil were analyzed by HPLC-MS.Sixteen ox-TGs were identified,and the epoxy group was identified as the main oxidation component.(2)In order to explore the biological toxicity of epoxide triglycerides-the main toxic component of deep-frying oil,this study investigated the digestive characteristics of epoxide triglycerides in the intestine using an in vitro digestion model.First,the high-purity epoxy triglycerides,1-epoxy stearic acid,2,3-dioleoleic glyceride(EGT),were synthesized by chemical method.Compared with glycerol trioleate(GT),the digestion rate analysis showed that the free fatty acid release rate of EGT(1007.34±29.13 μmol)was significantly lower than GT(1452.69±21.56 μmol).This phenomenon was then analyzed from three aspects: the binding ability of the compound to salt ions in the digestive juice,the molecular size and the lipase activity.The particle size analysis showed that the particle size of EGT(3.51 nm)was significantly larger than that of GT(3.10 nm),which would reduce the contact area between EGT and lipase.The interface behavior analysis showed that the binding ability of EGT to salt ions in digestive juice(1986.84)was significantly weaker than that of GT(2049.5).Spectral analysis showed that EGT caused the red shift of lipase peak,indicating that EGT changed the structure of lipase.Molecular dynamics simulation showed that EGT resulted in the looseness of lipase structure.These results suggest that EGT can stay longer in the intestine,thus increasing the chance of EGT contacting intestinal epithelial cells.(3)In order to further-explore the biological toxicity of EGT,this study investigated the lipid metabolism of EGT in multiple organs(heart,liver,duodenum,brain,white adipose tissue and brown adipose tissue)using non-targeted lipidomics.long-term animal chronic experiments were conducted to simulate the long-term consumption of fried food by humans.A hematogenomic analysis showed that the high consumption of epoxy groups significantly increases the levels of lipopolysaccharide(LPS),diamine oxidase(DAO),and fatty acid binding protein(i FABP)in the body,suggesting that it can promote the permeability of the intestinal barrier.Lipidomics results also revealed that the ingestion of epoxy groups causes significant changes in the white and brown adipose tissue,the duodenum,the liver,the brain,and the heart’s lipidic groups.Furthermore,these differences in lipidic metabolites in various tissues analyzed were significantly correlated to the LPS,DAO,and i FABP levels,suggesting that the damage caused by the epoxy groups might occur through an intestinal barrier disorder.(4)In order to elucidate the molecular mechanism of the intestinal barrier disorder induced by epoxy triglycerides,the short-term acute toxicity experiment and long-term chronic experiment were designed in this study.The physical characteristics of C57BL/6J mice showed that EGT could significantly decrease the body weight,and increased the disease activity index in C57BL/6J.At the end of the experiment,the mice with long-term chronic uptake presented hematochezia,indicating that the EGT induced significant intestinal inflammation.H&E staining revealed that the EGT led to diffused duodenal villi and an increased infiltration of inflammatory cells.Immunohistochemical staining demonstrated that the epoxy group also induced the disintegration of tight junction protein(TJ)between intestinal epithelial cells,proving that the chronic ingestion of epoxy group-enriched deep-fried oils causes intestinal barrier disorders.To clarify the molecular mechanism associated with intestinal barrier disorders,52 healthy and 116 intestinal biopsy samples from intestinal barrier disorder patients were analyzed through bioinformatics.The results showed that pyroptosis is the main molecular mechanism of intestinal barrier disorder,indicating EGT may lead to intestinal barrier disorder by inducing intestinal epithelial pyroptosis.(5)Based on the above results,this study further clarified the molecular mechanism of intestinal barrier disorder induced by EGT through model animal experiment and cell experiment.The results showed that EGT could induce Caspase-1 to perceive danger through the presence of the inflammasomes-NLRP3.Recruitment and activation of Caspase-1 could activate IL-1β and other inflammatory factors.Caspase-1 could also activate the GSDMD protein by cutting the N-terminal sequence of GSDMD,binding it to the cellular membrane,and generating holes.This leaded to cellular swelling and cytoplasm increase,and finally to the rupture of the cell membrane.The inflammatory cytokine IL-1β and TNF-α,which released after cell membrane rupture,could disintegrate TJ proteins,enhancing intestinal permeability and promoting intestinal barrier disorder.Meanwhile,EGT could also activate the c GASSTING-NF-κB signal pathway,further inducing intestinal inflammation TJ protein disintegration,enhancing intestinal permeability,and aggravating intestinal barrier disorder.To further explore the main functional substances of EGT and their actions in the gut,the result showed that its digestive product,epoxy stearic acid(ESA),is a compound that mainly induces intestinal barrier disorder.Therefore,the EGT present in deep-frying oils may induce intestinal epithelial cell scorch and inflammation through a combination of gastrointestinal digestion of ESA and activation of the TLR4 signaling pathway present on the epithelial cellular membrane.Altogether this action should further enhance intestinal permeability and intestinal barrier disorder.In conclusion,this study investigated how avidly consuming deep-frying oils induces intestinal barrier disorder.Meanwhile,this study provides knowledge on epoxy groups present in deep-frying oil and their effect on the body health.Furthermore,this study may help clarify the biological safety of deep-frying oil,reducing its biological hazard by targeting the inhibition and/or formation of EGT.