Effects Of PEPT1 On The Transport Of Cinnabar In Caco-2 Cell Model

Objective:The effect of PEPT1 on the transmembrane absorption of cinnabar was studied based on the Caco-2 cell model,and further clarify the absorption mechanism of cinnabar.Methods:1.A UPLC-ICP/MS method was established to detect the inorganic mercury content in the apical?AP side?,cellular and basolateral side?BL side?in the Caco-2 cell model after administration.2.Caco-2 cell model was established and the integrity and function of Caco-2 cell monolayers was assessed by cell morphology,transmembrane resistance?TEER?and fluorescein permeability.3.After the model established,the experiment was conducted with four groups,namely,blank group,cinnabar group,mercury sulfide group?HgS?and mercury chloride group?HgCl₂?for the following experiments.?1?To investigate the transport levels of cinnabar,HgS and HgCl₂ in Caco-2 cells:0.2?M cinnabar,HgS or HgCl₂ was added into the AP side,respectively.The added medium for AP side was collected and replaced with fresh medium containing with cinnabar,HgS or HgCl₂ at 6,12,18 and24 h during the incubation.After 24 h,the medium in AP and BL sides and cells were recovered,and their inorganic mercury contents were examined by UPLC-ICP/MS.?2?Gene and protein expression levels of PEPT1:After pretreatment of the above cell samples,PEPT1 mRNA and protein expression levels were assayed by RT-PCR and Western-blot method,respectively.?3?Inhibition of PEPT1 on the transport of cinnabar,HgS and HgCl₂:siRNA silencing and chemical inhibition were used to inhibit the activity of PEPT1 in Caco-2 cells,respectively.Then the transport of cinnabar,HgS and HgCl₂ in Caco-2 cells were investigated by UPLC-ICP/MS.Results:1.A rapid and selective ultra-high liquid chromatography-inductively coupled plasma mass spectrometer?UPLC-ICP/MS?method has been developed to determine inorganic mercury in AP,cellular and BL sides.2.Caco-2 cell monolayer membrane has good tightness and integrity,TEER value is greater than 500?.cm² and fluorescein permeability is less than 1×10^-6 cm/s,indicating Caco-2 cells established in this experiment and the Caco-2 cell model can be used for drug transport studies.3.After exposure of cinnabar,the amount of inorganic mercury transported in AP,cellular and BL sides were 97.71%,1.95%and 0.34%of the total added respectively.For HgS,the amount of inorganic mercury transported in AP,cellular and BL sides were97.72%,1.81%and 0.47%of the total added respectively.For HgCl₂,the amount of inorganic mercury transported in AP,cellular and BL sides were 97.56%,1.77%and0.68%of the total added respectively.The contents of inorganic mercury in the BL side of cinnabar and HgS groups were 49.39%and 30.41%of that in HgCl₂ group,respectively.4.Compared with the control group,cinnabar and HgS were significantly decreased the mRNA expression of PEPT1.Furthermore,cinnabar also significantly decreased the PEPT1 protein expression as compared with the control group,and HgS slightly down-regulate the levels of PEPT1 protein expression.5.In PEPT1-siRNA cells,the mercury content was reduced 30.03%in cellular accumulation and 57.85%in transport to the BL side,compared with control group.No changes were seen in HgS incubation.The mercury content increased 34.48%in the BL side of HgCl₂ of control after intervention by PEPT1-siRNA,compared with control group.6.Transport of cinnabar was decreased by 33.32%in BL side with the treatment of ibuprofen to control.There were no observed changes in HgS and HgCl₂ group.Conclusion:?1?The intestinal transport levels of cinnabar and HgS are lower than that of HgCl₂;?2?The transport of cinnabar be mediated by PEPT1 transporter in Caco-2 cell model.