| Triazole pesticides play a bactericidal role through inhibiting lanosterol-14alpha-demethylase of cytochrome P450 proteins（CYP）enzyme.For eukaryotes,CYP enzyme participates in the biotransformation of many drugs,endogenous and exogenous substances.The non-specific effect of triazole pesticides on CYP enzyme will affect the activities of many CYP enzyme.Bile acids（BAs）,as a natural surfactant to promote the absorption of intestinal lipids,are mainly synthesized by cholesterol 7α-hydroxylase（CYP7A1）and cholesterol 27-hydroxylase（CYP27A1）.Previous study showed that tebuconazole exposure affected the content and composition of BAs pool,and changed the statins oral absorption.Drug absorption is closely depended on the permeability of intestinal barrier.So,the mechanism of intestinal barrier affected by BAs disturbance remains to be explored.Objective:Cell models were established to evaluate the absorption of statins and the function of intestinal barrier,and the mechanism of intestinal barrier dysfunction caused by BAs pool disturbance was analyzed to explain the changes in drug absorption.Methods:（1）Prepared the BAs solution according to the research data from normal and tebuconazole long-/short-term oral exposure model rats,detected the change of the equilibrium solubility of liposoluble drugs,and explored the effect of BAs disturbance on its solubilization ability.（2）Caco-2 monolayer model and Caco-2/HT29 co-culture model were eatablished as intestinal epithelial cell models in vitro.The apparent permeability coefficient（Papp）of fluorescein was used to detect the integrity of the models,the morphology of the cell model and the production of mucus were observed by microscope to determine whether the models were successfully established.（3）MTT method was used to screen the concentration of drugs and BAs with cell survival rate greater than 80%and the absorption behavior of statins-BAs complex solution in in vitro intestinal epithelial models was detected by liquid chromatography-mass spectrometry（LC-MS/MS）.（4）The expression of tight junction proteins（Occludin and ZO-1）in Caco-2 cells was detected by immunofluorescence and western bloting.The oxidative stress level of Caco-2 cells were detected by reactive oxygen species（ROS）,malondialdehyde（MDA）and superoxide dismutase（SOD）kit.Immunohistochemical method was used to detect the production of HT29 cell mucus to explore the mechanism of BAs disturbance on intestinal barrier function.Results:（1）The BAs disturbance will affect its solubilization ability to liposoluble drugs.（2）Papp of fluorescein in intestinal epithelial cell model was less than 5×10-7cm/s.The cells were closely arranged and have differentiated into orderly and compact microvilli under microscope.Caco-2/HT29 co-culture model produced significantly more mucus than Caco-2 monolayer model.The intestinal epithelial models were successfully established.（3）The BAs pool of tebuconazole long-/short-term oral exposure rats model reduced the penetration promoting effect of normal rats BAs pool on simvastatin and pravastatin in the Caco-2 monolayer model.The BAs pool of tebuconazole long-/short-term oral exposure rats model will reduce the distribution of simvastatin and penetration of fluvastatin and pravastatin in Caco-2/HT29 co-culture model.BAs disturbance significantly affected the drug absorption in in vitro intestinal epithelial models.（4）The BAs pool of tebuconazole long-/short-term oral exposure rats model significantly reduced the oxidative stress level of Caco-2 cells,increased the tight junction expression level of Caco-2 cells,and reduced the mucus production of HT29 cells.Conclusion:The BAs disturbance of tebuconazole long-/short-term oral exposure rats model can change the drug absorption behavior.It can increase the expression of tight junction,reduce the oxidative stress level and reduce the production of intestinal mucus to change the permeability of intestinal barrier. |
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