| P-gp encoded by the Abcbl gene is a drug efflux pump with molecular weight of 170 kD.P-gp widely distributes in liver,kidney and intestinals which therefore affects the disposion of xenobiotics including oral drugs.It is reported that inflammation induced by LPS can cause changes of expression and activity of P-gp in rodents and human,thus affecting the oral bioavailability and efficacy of the drug.Currently,related studies have been widely undertaken in human and rodent,however rare have been reported in the fields of farm animals involving pigs.For this reason,this article focused on the effect of LPS on P-gp expression in liver,kidney,jejunum,ileum,IPEC-J2 cells and its effects on the pharmacokinetics of enrofloxacin orally administrated in pigs which may provide theoretical support for rational use of drugs under inflammatory status.First of all,real time RT-PCR and immunohistochemistry method were used to study the influence of LPS on expression and localization of P-gp in tissues of 60-day-old piglets and Image-Pro Plus software was used for semi-quantitative analysis of specific staining of P-gp.The results showed that after LPS treatment for 3h,Abcbl mRNA expression decreased in liver,kidney and ileum with significant difference（P<0.05）,and in jejunum with extremely significant difference（P<0.01）.After 6h,Abcbl mRNA expression in jejunum changed little compared with control,but Abcbl mRNA expression in kidney decreased significantly（P<0.05）,and decreased extremely different（P<0.01）in liver and ileum.Immunohistochemical results showed that P-gp was mainly distributed on bile canalicular membrane between hepatocytes in liver after LPS treatment or not,and the expression of P-gp was significantly increased（P<0.05）after LPS treatment for 3 h,and declined to normal levels after 6h.In kidney,P-gp distributed on the membrane of proximal tubule and distal tubule in both healthy and LPS treated pigs,and P-gp expression levels were significantly increased（P<0.05）after LPS treatment for 6 h.In intestinal,P-gp was mainly distributed on apical surface of the small intestine villi and glandular epithelium.And the intensity was enhanced following LPS treated 3 h（P<0.05）but decreased to normal leval after 6 h in the jejunum.No significantly difference was found in ileum（P>0.05）.In conclusion,P-gp located on cell membrane was increased after LPS treatment,though the total expression of Abcb1 mRNA changed with a downward trend.Secondly,to further explore the effect of LPS on the expression of P-gp in pigs,IPEC-J2 cells were used to detect changes of localization,mRNA and protein expression levels of P-gp after LPS treatment.Immunofluorescence results indicated that localization of P-gp on the cell membrane had not changed after LPS（10 μg/mL）treatment,but the apical staining of P-gp decreased slightly after lh,and increased from 3h to 24h.Real time RT-PCR results showed that Abcbl mRNA expression decreased after LPS treatment for 1 h and the difference was significant at 50 μg/mL.While after 3h,Abcbl mRNA expression was significantly increased compared with control,then decreased constantly until 12 h.After LPS treatment for 24 h,the mRNA expression returned to control levels.Western blot results showed that intracellular P-gp expression levels were concentration-dependent lower than control after LPS treatment for 1h.After 3h,P-gp expression levels were concentration-dependent increased compared with control and decreased after 6h.A slight increase of P-gp expression level occurred at 1 μg/mL after 12h,but LPS at 10 μg/mL and 50 μg/mL still inhibited P-gp expression,though the extent of inhibition was lower than 6h.After 24h,P-gp expression level was significantly reduced with the concentration of 1μg/mL,and the other two concentrations induced P-gp expression near to control level.The results show that P-gp on cell surface increased as the P-gp distributed in tissues of pigs,which is inconsistency with the total Abcbl and P-gp expression.As we know the effect of LPS on expression of P-gp in tissues of pigs and IPEC-J2 cells,the paper finally aimed to explore the impact of LPS on pharmacokinetics of enrofloxacin in pigs by HPLC to reveal the function change of P-gp after LPS treatment.The results showed that verapamil group exhibited longer Tpeak（1.81 vs 3.18 h）,lower Ke(0.24 vs 0.12 h-1,P<0.01),longer T1/2ke（3.02 vs 6.33 h,P<0.05）,higher AUC（0-24h）(4.30 vs 8.95μg·mL-1·h,P<0.01)and lower CL/F(1.23vs 0.57 mg·kg-1·h-1/μg·mL-1（P<0.01）than control group.After LPS treatment,the absorption of enrofloxacin haven’^ changed significantly,and Ke reduced to 0.03 h-1（P<0.01）,the Tpeak was significantly delayed,AUC（0-24h）increased to 7.31 μg·mL-1·h（P=0.05）,CL/F decreased to 0.27 mg·kg-1·h-1/μg·mL-1（P<0.01）and V/F increased to 11.10 mg·kg-1/μg·mL-1（P<0.05）compared with the control group.The bioavailability of enrofloxacin in verapamil group（78%）and LPS group（72%）were higher than control group（42%）.However,there were no significant differences between intravenous injection groups.The results preliminary indicated that verapamil and LPS treatment could increase the AUC and bioavailability,decrease the elimination of enrofloxacin possibly by reduced function of P-gp.In summary,LPS decreased the expression of P-gp in tissues of pigs and IPEC-J2 cells and increased the oral bioavailability of enrofloxacin,indicating that inflammation is good for the efficacy of certain drugs.At the same time,it should be noted that dosage regimen has to be formulated reasonably considering the cumulative toxicity and withdrawal period of the drug. |
