Effects Of Hypoxia On Expression Of MRP2?PEPT1 Transporters And Its Substrates Pharmacokinetics

With the upsurge of the plateau tourism,the implementation and rapid development of the "the Belt and Road" national strategy and many other reasons,the population from the plain into the plateau region were gradually increased,especially the crowd who acutely exposed to high altitude.However,the usage and dosage of drugs under high altitude are still the same as the plains,it will affect the using rationality,safety and effectiveness of drugs at high altitude.Pharmacokinetics is an important theoretical basis and foundation for rational use of drugs,there are few researches about the changes of pharmacokinetics in the plateau environment.There are many factors affecting the pharmacokinetics of drugs and the previous results were currently concentrated in CYP450 enzymes.Moreover,transport is an important factor affecting the pharmacokinetic characteristics and membrane transporters are key elements of drugs from cells in the active transport process.Drug transports express on a number of important organs,such as the small intestine,liver and kidney,etc.This localization determines its importance in the drug absorption,distribution,metabolism and excretion processes.Nevertheless,there is little information available in literature about drug transporters how to change and influence drug pharmacokinetics at high altitude.Therefore,it is necessary to systematically study the effects of hypoxia on drug transporters and regulatory mechanism,which will provide more resourceful data support and theoretical basis for reasonable medication at high altitude.In this study,we selected the most important and representative efflux transporter MRP2 and uptake transporter PEPT1,to investigate the expressions,the pharmacokinetics parameter changes of norfloxacin and amoxicillin,and the related regulatory mechanism after hypoxia.In this study,the differences of blood gas,biochemistry and pathological indexes were firstly compared between the high altitude group and the plain area group,and the degree of hypoxia and injury were evaluated in rats.Blood gas results showed that the levels of pH,PaCO2,PaO2,SaO2,HCO3-,BB and BE were significantly decreased after acute exposed to plateau,indicating that the rats appeared hypoxia symptoms.The reduction of oxygen partial pressure in the environment causes the imbalance of oxygen supply in the body tissue,leading to metabolic disorder of compensatory respiratory alkalosis and metabolic acidosis in rats.Biochemical results showed that the levels of ALB,AST,ALT,TBIL,BUN and UA in rats were significantly higher than those in the control group,illustrating that acute hypoxia could affect the function of liver and kidney in rats.Pathological results showed that the small intestine,liver and kidney tissues of rats had different degrees of injury after hypoxia compared with the plain group.Secondly,the expressions of multiple drug transporters in the small intestine,liver and kidney of rats after acute exposure to high altitude were explored by RT2 Profiler PCR Arrays technology,and in order to screen the differential transporters susceptible to hypoxia.The results showed that compared with the plain group,the rats in the acute plateau group showed a significant changes in numerous drug transporters.In the small intestine,liver and kidney,there were 12,3 and 8 categories of drug transporters exceed tow times respectively and 5,18 and 4 species decreased more than two times respectively.Then,we selected the important drug transporters Mrp2 and Pept1,which have significant differences,common changes and the wide application and verified the expression changes in different tissues under high altitude by Real-time PCR and Western blot methods.At the same time,the effects of hypoxia on the pharmacokinetics of norfloxacin and amoxicillin were studied.The results showed that the expressions of Mrp2 and Pept1 in the hypoxic group were different from that in the control group.In the small intestine and kidney,the mRNA and protein expressions of Mrp2 and Pept1 at high altitude group were significantly increased respectively,and however,the expressions of Mrp2 and Pept1 were decreased in liver.The pharmacokinetic parameters of norfloxacin showed a significant decrease of AUC,Cmax and t1/2.The MRT,CL and Vd were significantly enhanced in the plateau group.Compared with the pharmacokinetic parameters of amoxicillin,it revealed that the AUC,Cmax,MRT,Tmax and t1/2 of amoxicillin in the plateau group were significantly heightened respectively,while the CL and Vd were distinctly reduced.Finally,the expressions of H19,MRP2,PEPT1,related transcription factors and nuclear receptors were investigated in the hypoxic surroundings at Caco-2 cell level.And then based on the laboratory preliminary experiments about screening for hypoxia differences in lncRNAs by lncRNA high-throughput gene chip,we used lentiviral vector-mediated RNAi technology to silence lncRNA-H19.The influences on the expressions of MRP2,PEPT1,related transcription factors and nuclear receptors in Caco-2 cells after disturbed lncRNA-H19 were investigated.All these researches are in order to preliminarily explore the regulatory mechanism of MRP2 and PEPT1 under hypoxia conditions.The results showed that the expression levels of MRP2 and PEPT1 were positively correlated with H19 under hypoxia environment.After the LV-H19-RNAi lentivirus stably transfecting into Caco-2 cells,the gene expressions of MRP2 and PEPT1 were decreased by half and the mRNA levels of PXR and CAR were significantly reduced,while HIF1 a and NF-?B weren't changed.At the level of protein expression,MRP2,PEPT1,related transcription factors NF-?B,HIF1 a,and nuclear receptor PXR and CAR were significantly inhibited.In addition,the degree of inhibition of MRP2,PEPT1,HIF1 a,NF-?B,PXR and CAR expressions was aggravated after hypoxia treatment with LV-H19-RNAi in Caco-2 cells.In this study,we can draw the following conclusion: the expressions of drug transporters,especially Mrp2 and Pept1 were distinctly changed in the small intestine,liver and kidney tissues,which affected the pharmacokinetic parameters of norfloxacin and amoxicillin after acute exposure to high altitude.After silencing lncRNA-H19 in Caco-2 cells,the expressions of MRP2,PEPT1,nuclear receptors and related transcription factors were significantly inhibited.These results preliminarily manifested that lncRNA-H19 is a pivotal molecule regulating MRP2 and PEPT1 expressions in hypoxic condition,which may be through the regulation of HIF1 a,NF-?B,PXR and CAR expressions at the transcription level and post-transcriptional level,and finally forms a regulatory network for MRP2 and PEPT1 expressions.The study will provide a richer data base and theoretical support for rational medication at high altitude.