| PurposeCornea is one of the tissues that are in direct contact with the external environment,and is very sensitive to pollutants in the air.Epidemiological investigations have shown that air pollution can cause damage to ocular surface tissues such as the corneal epithelium and tear film.As modern people spend most of their daily time indoors,the impact of indoor pollution on ocular surface is getting more and more attention.Cooking oil fumes is one of the main sources of indoor pollution,of which trans,trans-2,4-decadienal(tt-DDE)is the most abundant mutagenic aldehyde,which can be released into indoor air through daily cooking activities and widely present in heated fat foods and food additives.Previous studies have confirmed that exposure to tt-DDE has adverse effects on human health,but most of these studies have focused on the digestive and respiratory systems,and there has no record on biotoxicity of tt-DDE in human cornea.Based on this background,the present study aims to investigate the effects of tt-DDE on human corneal epithelial cells(HCECs)for the first time.MethodsFirstly,cell viability of HCECs treated with tt-DDE at different concentrations or durations were examined by the CCK-8 assay,and the changes in cell morphology were observed and recorded under a microscope.Terminal deoxynucleotidyl transferase-mediated d UTP Nick End Labeling(TUNEL)assay was used to detect the proportion of apoptotic cells.2,7-Dichlorodi-hydrofluorescein diacetate(DCFH-DA)probe was used to detect the production of reactive oxygen species(ROS).JC-1 fluorescent probe and luciferase were used to detect mitochondrial membrane potential(MMP)and intracellular adenosine triphosphate(ATP)level,respectively.Immunofluorescence staining was used to observe the localization and expression of ER stress-related proteins(Bip,p IRE1,XBP-1s,p PERK,pe IF2α,ATF4,and CHOP)which were further quantified by western blot analysis.Secondly,HCECs were pre-treated by two ER stress inhibitors(integrated stress response inhibitor(ISRIB)and sodium 4-phenylbutyrate(4-PBA))for 6 h before exposed to 20 μM tt-DDE for 12 h.Methods as above were used to detect cell viability,apoptotic cell ratio,ROS,MMP,ATP level and ER stress-related protein expression.Finally,32 C57BL/6 mice were divided into 4 groups.The first group was left untreated.The second,third,and fourth groups were treated with 20 μM tt-DDE 3 times a day.Additionally,from the day before tt-DDE treatment,the third or fourth group was injected intraperitoneally with ISRIB(2.5 mg/kg)or 4-PBA(20 mg/kg)once a day.On the 15 th and 30 th days,ocular surface fluorescein staining was performed,slit lamp microscope was used to observe and record the ocular surface conditions,tear break up time(TBUT)was also conducted,and phenol red cotton silk was used to assess tear secretion.After 30 days of treatment,the eyes were taken for paraffin sections and HE staining to observe the morphological changes of the corneal epithelium.ResultsThe present study found that tt-DDE exposure can cause dose-and time-dependent decrease in cell viability,morphological changes(cytoplasmic vacuolation and cell shrinkage),and a significantly higher proportion of apoptotic cells in HCECs.tt-DDE can also lead to increased ROS production,decreased MMP,and decreased intracellular ATP level.In addition,expression of ER stress-related proteins(Bip,p IRE1,XBP-1s,p PERK,pe IF2α,ATF4,CHOP)all increased after tt-DDE treatment,and the localization to the nucleus of some of the proteins(p IRE1,pe IF2α)was increased.Compared with the group exposed to tt-DDE alone,pretreatment with ER stress inhibitor can significantly alleviate the cell viability decrease,the increase in ratio of apoptotic cells,the excessive ROS production and mitochondrial dysfunction induced by tt-DDE exposure.At 15 th and 30 th days,the tt-DDE treated mice had higher ocular surface staining scores,TBUT was significantly shortened,tear secretion was reduced,and the corneal epithelium was thinner in HE staining.In mice pre-treated with ISRIB and 4-PBA,the above indicators were significantly improved compared with the group treated with tt-DDE alone.ConclusionsThis study revealed for the first time the toxic effects of tt-DDE,the most mutagenic chemical component of cooking oil fumes,on HCECs.tt-DDE could cause apoptosis of HCECs by triggering ER stress and ROS-mediated mitochondrial dysfunction.The regulation of ER stress can be considered as a potential protective method for tt-DDE-induced ocular surface disorders.Moreover,tt-DDE can cause damage to the tear film and HCECs of mice,which can be further developed as a new way to construct dry eye model. |
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