| Background and objectivesThe amounts of oxidant and anti-oxidantare kept precisely balanced in healthy individual.When free radicals accumulate,a wide range of symptoms can be caused by elevated levels of oxidative stress.Superoxide anion,as a free radical produced in the process of organism metabolism,can attack nucleic acid,protein,lipid and other biological macromolecules.Superoxide can cause the destruction of cell structure and function,which is closely related to the tissue damage of organism.As the representation of chronic oxidative stress is not easy to be detected,it is extremely harmful to health.The gastrointestinal tract is the main organ of nutrient abosorption and metabolism,imbalanced of the oxidative state can easily induce gastrointestinal diseases and disorders of digestion.With the introduction of intestinal oxidative stress in recent years,the oxygen free radicals have gradually become a key factor for intestinal barrier damage.Thus,our study applied the in vitrointestinal barrier model to explore the mechanism of extracellular superoxide disrupting the intestinal barrier.MethodsExperiment 1:Intestinal barrier model was constructed in vitro using human colorectal cancer cell line Caco-2.The experimental treatment group was divided into control group,stimulation group with superoxide anion treatment and recovery group with SOD.After the barrier construction was succeed,the control group was given complete medium,the superoxide anion stimulation group was given superoxide anion-producing bacteria and xanthine/xanthine oxidase reaction to co-culture with cells,and the SOD group was added SOD on the basis of superoxide anion stimulation.The effect of superoxide anions on barrier permeability was observed by detecting TEER of monolayer cells and the permeability of fluorescein sulfonic acid,and the expression and distribution of intercellular tight junction proteins were observed after the experiment.Experiment 2:Caco-2 cells were stimulated by extracellular superoxide anion and administered SOD intervention.Transcriptome sequencing was used to observe the changes of gene level in cells under superoxide anion stimulation,and subsequent experiments were conducted to verify the changes.Results1.The model of in vitro intestinal barrier was successfully constructed,and the barrier permeability was significantly increased under the stimulation of superoxide anion.Meanwhile,the intercellular tight junction protein ZO-1,Occludin expression was decreased,and the Claudin-4,Occludin distribution changes from compact to disordered.In addition,SOD could effectively improve the damage of superoxide anion to the intestinal barrier.2.Transcriptome sequencing analysis showed that the expression of 550 genes increased and 351 genes decreased after superoxide anion stimulation.Moreover,the genes related to oxidation and apoptosis increased,while the antioxidant genes decreased.MitoSOX probe detection and apoptosis experiment showed that extracellular oxidative stress stimulated the production of mitochondrial superoxide anion and cytochrome C was released from mitochondria into the cytoplasm.Furthermore,PCR showed apoptosis-related genes were significantly up-regulated.ConclusionIt was found that superoxide stimulation can cause structural and functional damage to the in vitro intestinal barrier model.During the imbalance of extracellular oxidative stress,mitochondria produce a large number of superoxide and induce apoptosis.SOD intervention can reduce the superoxide of mitochondria and inhibit the process of apoptosis,ultimately maintaining the integrity of the intestinal barrier.Cells could be recovered to nomal state by the disproportionation reaction of SOD. |
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