Effects Of PM_2.5 Exposure On Human Small Intestinal Epithelial Cells HIEC-6 And Its Regulatory Mechanism Of Non-coding RNA

PM_2.5 is one of the major air pollutants in China.The toxic effects of PM_2.5exposure on multiple organs have been widely reported,while the toxic effects of PM_2.5on the digestive system are relatively less discussed.According to the existing epidemiological investigation and corresponding experimental studies,PM_2.5 exposure can increase the risk of intestinal health,but the existing studies pay little attention to the damage of intestinal barrier function caused by PM_2.5,and there is a conclusion bias(PM_2.5 exposure has adverse effects on intestinal health,some experiments have confirmed that PM_2.5 exposure induces inflammation,while no inflammatory effect was observed in other experiments).Therefore,the impact of PM_2.5 on intestinal health still needs to be supported by experimental data.This study foused on the effects of PM_2.5exposure on oxidative damage,inflammation,apoptosis and intestinal barrier function of normal intestinal epithelial cells HIEC-6,and initially explored its mechanism from the perspective of non-coding RNA.The research methods and results of this study are as follows:1.Effects of PM_2.5 exposure on oxidative damage,inflammation,apoptosis and intestinal barrier function of HIEC-6 cells.（1）Use TH1000C large flow particle sampler to collect PM_2.5 samples and prepare PM_2.5 suspension.（2）Normal intestinal epithelial cells HIEC-6 were selected as experimental subjects.The cells were exposed to graded PM_2.5 for 24 h,and cell viability was measured after exposure by CCK8method.The results showed that PM_2.5 exposure concentration was negatively correlated with cell viability,and PM_2.5 concentrations of 0,25,50 and 100μg/m L were selected as the experimental concentrations in subsequent experiments.（3）The total ROS content of cells which exposed 24 h in PM_2.5 was determined by DCFH-DA probe method.The intracellular ROS content showed an increasing trend with the increase of exposure concentration,and ROS content was significantly increased at 100μg/m L high concentration compared with the control group,indicating that the increase of PM_2.5 concentration led to the increase of oxidative stress level of HIEC-6 cells.（4）m RNA levels and protein expression levels of inflammatory markers IL-6,TNF-αand ICAM-1 were measured 24 h after exposure by RT-q PCR and ELISA,and the results showed that the m RNA levels of other indicators except IL-6 had no statistical difference.Proteins levels of the three indicators were significantly higher as a result of exposure to PM_2.5,suggesting that increased PM_2.5 concentrations lead to increased levels of cellular inflammation.（5）The transcription and protein expression levels of apoptosis-related markers Bcl2 and Caspase-3 were determined by RT-q PCR and western blot,respectively,24 h after exposure.The results showed that the m RNA expression level of Caspase-3 was significantly increased in the 100μg/m L exposure group.The expression level of Bcl2 protein was significantly decreased,and the level of Cle-Caspase-3 protein was significantly increased,indicating that PM_2.5 exposure promoted the apoptosis process of cells.（6）m RNA and protein expression levels of cell barrier related markers after 24 h of PM_2.5 exposure were detected by RT-q PCR and western blot,respectively.Compared with the control group,the expression levels of CFTR and NKCC1 proteins were significantly decreased,while ZO-1 protein expression showed a downward trend but no statistical difference,the m RNA changes of the three indicators were not significant,indicating that PM_2.5 caused damage to intestinal barrier function.2.A preliminary study on the regulatory mechanism of long non-coding RNAs on intestinal toxic effects of PM_2.5.After 24 h of PM_2.5 exposure,the cells’RNA were analyzed by full transcriptional sequencing,differentially expressed lnc RNAs were screened and then verified by RT-q PCR,and the subcellular localization was performed on lnc RNAs whose verified expression trends were consistent with the sequencing results.Twelve lnc RNAs that might be related to the above effects and successfully performed subcellular localization were obtained.In conclusion,PM_2.5 exposure reduced the survival rate of normal intestinal epithelial cells HIEC-6,increased cellular oxidative stress and inflammatory effect,promoted apoptosis,and impaired intestinal barrier function.Through full transcriptome sequencing analysis,gene screening and RT-q PCR verification,12lnc RNAs that may be related to the above effects were obtained and successfully located.lnc RNAs that may be involved in the regulation of enterotoxic effects of PM_2.5were preliminarily identified for further study.