The Effect Of High Altitude Hypoxia On The Function And Expression Of Drug Transporters And The Regulatory Mechanism Of MiRNA-873-5p

Objective:To investigate the effects of high altitude hypoxia environment on the function and expression of drug transporters,through screening out mi RNAs that significantly change under hypoxic conditions as well as can act on MDR1 and PXR,and explore the regulatory role of MDR1and PXR by mi RNA-873-5p mediated by hypoxia.Methods:For in vivo experiments,male SD rats were randomly divided into a total of 5 groups:plain group,acute moderate altitude hypoxia group,chronic moderate altitude hypoxia group,acute high altitude hypoxia group,chronic high altitude hypoxia group.Each group was divided into 5sub-groups—MDR1,MRP2,BCRP,PEPT1 and OATP2B1.After the model was established,the rat in vivo intestinal perfusion experiment was performed.The concentrations of Rhodamine 123,Vincristine,Prazosin,Ubenimex,and Glibenclamide in the perfusate were determined by HPLC,and the effective permeability coefficient and absorption rate P_effabsorption rate K_awere calculated.The liver tissues of rats were taken after the intestinal perfusion.Western-blot and qPCR were used to determine the expression of MDR1,MRP2,BCRP,PEPT1 and OATP2B1 on protein and mRNA level,respectively.Caco-2 cells were used for in vitro experiment,with 3 hypoxic oxygen concentrations of 21%（normal oxygen）,2%,5%,and 10%,all of which were treated with hypoxia condition for 24 hours.In addition,there are a total of 9groups with 5 hypoxic treatment times of 2,6,12,24,and 48 hours,and with an oxygen concentration of 5%.After completion of the model,Western-blot and qPCR were used to determine the protein and mRNA expression of MDR1,MRP2,BCRP,PEPT1 and OATP2B1 in the cells.Caco-2 cells were cultured continuously for 14 days and then received hypoxia treatment.Rhodamine 123,Vincristine,Prazosin,Ubenimex,and Glibenclamide were added to the cells and incubated together for 30,60,90,120 min,and the cells were disrupted by ultrasound.The concentration of the drug in the cells was measured by LC-MS.The Caco-2 monolayer cell model was constructed,and the cell transport experiment was performed after hypoxia treatment.LC-MS was used to determine the drug concentration,and the apparent permeability coefficient(P_app)and rate of efflux（R_E）were calculated.The expression profile of mi RNAs in Caco-2 cells were screened through high-throughput sequencing under hypoxic conditions,and mi RNAs that can regulate PXR and MDR1 were analyzed through bioinformatics.mi RNA-873-5p mimics and inhibitors were synthesized to transfect Caco-2cells for the construction of mi RNA-873-5p over-expression and low-expression models.Western-blot and qPCR were conducted to determine the protein and mRNA expression of PXR and MDR1 in mi RNA-873-5p over-expression and low-expression models,confirming the regulatory effect of mi RNA-873-5p on PXR and MDR1.Results:In vivo experiment:High altitude hypoxia can significantly reduce the protein expression of MDR1 in rats（P<0.05）,significantly increase the protein expression of PEPT1（P<0.05）,and has no significant role on the protein expression of OATP2B1（P>0.05）;compared with the plain group,the protein expression of MRP2 in the acute moderate altitude hypoxia group was significantly reduced（P<0.05）,and MRP2 expression in the chronic high altitude hypoxia group was significantly increased（P<0.05）;The protein expression of BCRP in the chronic moderate altitude hypoxia group and the acute and chronic high altitude hypoxia group was significantly increased（P<0.05）.High altitude hypoxia can significantly increase the mRNA expression of MRP2 and BCRP（P<0.05）,but has no significant effect on the mRNA expression of OATP2B1（P>0.05）;The mRNA expression of MDR1 in the acute moderate and high altitude hypoxia group was significantly increased,and the chronic high altitude hypoxia group was significantly decreased compared with that in the plain group（P<0.05）;PEPT1 mRNA expression was significantly increased in the acute moderate altitude,acute and chronic high altitude hypoxia group（P<0.05）,and significantly decreased in the chronic moderate altitude hypoxia group（P<0.05）;The activity of MDR1,OATP2B1,PEPT1 increased significantly（P<0.05）,and the activity of BCRP decreased significantly（P<0.05）under high altitude hypoxic environment;the activity of MRP2 decreased significantly in the acute moderate altitude hypoxia group,and significantly increased in chronic high altitude hypoxia group（P<0.05）.In vitro experiment:Under hypoxic conditions,the protein expression of MDR1,BCRP,PEPT1,OATP2B1 in Caco-2 cells increased significantly（P<0.05）,and the protein expression of PXR and MRP2 decreased significantly（P<0.05）.The mRNA expression of MDR1,BCRP and PEPT1increased significantly（P<0.05）;the mRNA expression of OATP2B1 and PXR decreased significantly（P<0.05）,and there was no significant difference in MRP2（P>0.05）.In the drug delivery experiment,the activities of MDR1,BCRP,and PEPT1 were significantly increased（P<0.05）;the activity of MRP2 was significantly decreased（P<0.05）.OATP2B1 expression was significantly reduced after 30min of drug incubation（P<0.05）,while significantly increased after 60 or 90 min of drug incubation（P<0.05）.Expression of 83 mi RNAs was changed significantly in Caco-2 cells,of which 27 were up-regulated and 56 were down-regulated under hypoxic conditions,mi RNA-873-5p may act on PXR and MDR1 with the use of mi RNA target prediction and screening.In the Caco-2 cell model with overexpression and underexpression of mi RNA-873-5p,the expressions of PXR and MDR1 were significantly decreased and increased respectively in protein and mRNA level（P<0.05）.Conclusion:High altitude hypoxia can significantly affect the expression and function of MDR1,MRP2,BCRP,PEPT1 and OATP2B1 in rat liver.High altitude hypoxia can significantly affect the expression and transport function of MDR1,MRP2,BCRP,PEPT1 and OATP2B1 in Caco-2 cells.mi RNA-873-5p mediates the regulation of high altitude hypoxia on the expression of MDR1 and PXR,which in turn affects the physiological disposition of the drug.