Effects Of Porcine P-glycoprotein On Drug Transmembrane Transport And Its Transcription Regulatory Mechanism

P-glycoprotein(P-gp)was first found in drug-resistant tumor cells.Later it was also found in different tissues of healthy animals and human,especially in absorptive and excretive tissues and plays important roles in drug absorption,distribution and excretion.It is gradually drawing the interests of the researchers and become the focus in the fields of pharmaceutics.Nowadays,the studies of P-gp mainly involved in rodents and human.The P-gp related studies on livestock and poultry were still scarce.Therefore,we will focus on studying the porcine P-gp in this paper.The porcine Abcbl gene was first analyzed by cloning and bioinformatics method.Additionally,the expression profile of P-gp in different tissues of healthy piglets was explored.Then,the porcine Abcb1 gene was transfected into MDCK cells to establish a stable cell line stably expressing porcine P-gp and used to screen the porcine P-gp substrates.Based on the above study,the regulatory mechanism of P-gp was finally studied.Therefore,it has a far-reaching significance.The main contents and results are involved as follows.1.Cloning and tissue expression profile of porcine Abcbl geneThe full-length porcine P-gp cDNA was first cloned and its secondary and tertiary structure was analyzed by bioinformatics method.The P-gp expression levels in different tissues were determined using qRT-PCR and Western blot technique.The results showed that the coding region of pig Abcb1 gene was 3,861 bp,encoding 1,286 amino acid residues(Mw = 141,966).Sequence comparison analysis showed that pig Abcbl gene presented high conservativeness with other species both in nucleotide sequence and amino acid sequence.It contains Walker A-,WalkerB-and Walker C-motif,which are the characteristics of the ABC transporter.Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep.Protein transmembrane structure analysis found that porcine P-gp had a similar structure with human P-gp,suggesting that they may have a similar biological function,however,I-TASSER predicted that 14 amino acids sites participating in substrate binding were located at TMD1 sites 5 and 6 and TMD2 sites 7,11 and 12 of porcine P-gp,whereas previous studies have shown that human P-gp substrate binding sites were located in TMD1 sites 5 and 6 and TMD2 sites 11 and 12,this may lead to different substrate types.The expression level of P-gp or its encoding gene Abcbl in different tissues was detected by Western blot and qRT-PCR.The expression level of P-gp in healthy tissues was not the same.It was highly expressed in the brain capillaries,jejunum,ileum and liver;lowly expressed in the duodenum,cecum,colon and rectum;and in the cerebral cortex,midbrain,hypothalamus,hippocampus,cerebellum,kidney were almost undetectable.The high expression of P-gp in healthy porcine absorption-and excretive-associated tissues and brain suggests that it may affect the absorption,excretion and distribution of some oral drugs or poisons.2.Establishment of MDCK-pAbcbl and its effect on transmembrane transport of enrofloxacin and ivermectinIn order to further study the function of porcine P-gp,we established MDCK-pAbcbl cell line stably expressing pig Abcbl gene and used this cell line to study its effect on transmembrane transport of enrofloxacin and ivermectin.To achieve this,pcDNA3.1(+)vector containing G418 resistance gene was used to insert the porcine Abcb1 gene into the two restriction endonuclease sites of Xho I and Xba I,and thus construct the pcDNA3.1-pAbcbl plasmid.By plotting the G418 tolerance to cell growth curve,500 ?g/mL of G418 was determined as the optimal resistance for screening.Finally,pcDNA3.1-pAbcbl was transfected into MDCK cells by Lipofectamine 2000,and monoclonals were screening with 500 ?g/mL G418.Successfully,one monoclonal cell,named as MDCK-pAbcb1,was screened and proved by detecting the expression level of P-gp with RT-PCR and western blot method.Accumulation of Rho123 in MDCK cells and constructed MDCK-pAbcbl was detected by flow cytometry.The results showed that Rho123 accumulated in MDCK-pAbcb1 cells was significantly lower than that in control MDCK cells at 15,30,60 and 120 min,respectively(P<0.05).However,the accumulated amount of Rhol23 in MDCK-pAbcb1 cells was significantly increased(P<0.05)when P-gp inhibitor verapamil was added.Therefore,P-gp overexpressed in MDCK-pAbcbl cells has biological activity and could be used to screen the P-gp substrates.Drug transport assay showed that the efflux rate of enrofloxacin in MDCK-pAbcbl cells was 2.58,which was significantly higher than that in MDCK cells(P<0.01).The addition of P-gp inhibitor verapamil reduced the efflux rate of enrofloxacin to 1.24,significantly lower than that of the non-inhibitor group(P<0.01).The efflux rate of ivermectin was 3.79,and its efflux rate decreased to 1.29 after the addition of verapamil.The above results indicated that enrofloxacin and ivermectin may be substrates of porcine P-gp.Thus,under different physiological and pathological conditions we should adjust the dosing program to reduce the incidence of drug poisoning.3.Screening core promoter of porcine Abcbl gene and the effect of Spl on its transcriptionIn different physiological and pathological conditions,porcine P-gp expressed differently due to the difference in transcription level.However,the mechanism of transcriptional regulation of porcine Abcb1 gene has not been reported till now.In this study,the transcription initiation site(TSS)of porcine Abcb1 gene was identified by 5'-RACE for the first time.A 1905 bp in 5'-untranslated region was obtained using porcine genomic DNA as template and then the transcription factor binding sites and CpG islands were predicted.It was found that there were several transcription factors binding sites in the 5' untranslated region of porcine Abcb1 gene,such as API,CEBP?,CEBP ?,Spl and two CpG islands.The region in Abcbl promoter which is critical for promoter activity was investigated via progressive deletions,by which we proved that-195?+ 25 bp determine the basal transcription level of the gene.Multiple Spl transcription factor binding sites exist in the core promoter of porcine Abcbl gene.Through site-specific mutations of-61/-51 bp and-43/-31 bp significantly reduced the activity of porcine Abcbl promoter.Interference of the intracellular Sp1 transcription factor by siRNA reduced the promoter activity of porcine Abcbl gene,and then affected the P-gp mRNA and protein expression levels.Finally,it was proved that the intracellular Spl transcription factor can binds the porcine Abcb1 gene promoter directly detected by CHIP.Hence,the current work will contribute valuable information to understanding the molecular regulatory mechanisms of pig Abcbl.4.The action mechanism of LPS on porcine P-gp expressionPathogen infection and inflammation can affect the expression and activity of drug metabolite enzymes and transporters,which may lead to adverse drug interactions.In this study,the effect of LPS-induced inflammation on porcine P-gp expression and its mechanism of action were studied.Firstly,the safe concentrations of LPS on IPEC-J2 cell viability were determined by MTT method.The safe concentrations were within 0.1,1?10?g/mL and these concentrations could be used for subsequent studies.The expression of IL-1? and IL-6 was not significantly affected by LPS,but the release of IL-8 was significantly increased(P<0.01)after LPS treated.Detected by qRT-PCR and Western blot,it was found that the expression levels of P-gp were significantly decreased in a concentration-dependent manner after different concentrations of LPS treated the cells.Further study by western blot and immunofluorescence assay showed that LPS could increase the phosphorylation of p65 in IPEC-J2 cells and detect the entry of p65 into nucleus,which indicated that LPS could activate NF-?B pathway in IPEC-J2 cells.Pharmacological inhibition of nuclear factor ?B(NF-?B)transcriptional signaling pathway significantly reversed the down-regulation of porcine Abcbl expression in LPS-treated cells,which proved that LPS decreased the expression of Abcbl expression through NF-?B pathway.In addition,PXR agonist rifampicin and overexpression of PXR demonstrated that PXR can promote the expression of porcine Abcb1 gene.Previous studies have shown that NF-?B can bind to RXR?,destruction of PXR-RXR? heterodimer,thus reducing the expression of drug-metabolizing enzymes.Considering pig Abcb1 gene,we can use Co-IP to explore whether there is an interaction between porcine RXR? and NF-?B to further elucidate the regulation mechanism of LPS on porcine Abcb1.In summary,this study first cloned pig Abcb1 gene and analyzed its sequence and structure characteristics.Then the MDCK-pAbcb1 cell line was established by transfecting the pig Abcb1 gene into MDCK cells and used to screen whether enrofloxacin and ivermectin were the substrates of porcine P-gp.The most important in this study is that we screened the core promoter of the pig Abcbl gene and confirmed that the Sp1 transcription factor binding site in the core promoter positively regulates the expression of pig Abcb1,LPS can reduce the expression of porcine Abcbl through NF-?B pathway.The results of the study will provide a comprehensive understanding on porcine P-gp and guide the rational use of drugs in porcine industry.