Expression Of P-GP In Tissues Of Healthy And E.Coli Infected Broilers And Its Role In Pharmacokinetics Of Oral Enrofloxacin In Broilers

P-gp encoded by the mdrl gene is a transmembrane transporter protein with the relative molecular mass of 170 kda. P-gp is an ATP-dependent drug efflux pump. It was found that P-gp is not only expressed in cancer cells, but also distributed in the normal tissues of humans and animals, such as bile duct, intestinal epithelium, and blood-brain barrier tubular. It limits the absorption of drugs and exogenous substances in vivo, leading to a reduced drug bioavailability. The expression of P-gp may be modified by a variety of factors, including chemicals, age, sex, physical condition, disease and hormones. Colibacillosis is an important disease affecting broiler breeding efficiency. Birds infected with E. coli present the clinical manifestations of severe site lesions in small intestines that affect the growth of broilers.Till now studies about the effects of E. coli infection on P-gp expression and function are still rare. Therefore, the aims of the study were to figure out the effect of E. coli infection on the expression and function of P-gp as well as its regulatory mechanism on Abcbl mRNA expression.First, the study designed to prove that enrofloxacin is substrate of P-gp. Thirty broilers were chosen to randomly divide into six groups (n=5). The first group received a single dose of 10 mg/kg body weight (b.w.) of enrofloxacin orally through crop tube gavage. The second group was first orally administrated with verapamil (15 mg/kg b.w.) and then enrofloxacin (10 mg/kg b.w.). The third group was first orally administrated with rifampicin (500 mg for each bird) and then enrofloxacin (10 mg/kg b.w.). The fourth group received a single dose of 10 mg/kg b.w. of enrofloxacin intravenously (i.v.) through the left brachialis vein.The fifth was first orally administrated with verapamil (15 mg/kg b.w.) and then enrofloxacin (10 mg/kg b.w.). The sixth group was first orally administrated with rifampicin (500 mg for each bird) and then enrofloxacin (10 mg/kg b.w.). The serum concentrations of enrofloxacin were detected by HPLC method. The results showed that broilers treated with verapamil exhibited significantly higher AUC0-∞ (P=0.001), Ka (P=0.008), as well as lower Tmax (P=0.026) than that of the control broilers. The bioavailability of enrofloxacin in verapamil treated broilers was increased by 30.7%, compared with that in control birds. Broilers treated with rifampicin exhibited significantly lower Cmax (P=0.002), AUC0-∞ (P=0.036), Ka (P=0.029) than that of the control broilers. The bioavailability of enrofloxacin in rifampicin treated broilers was decreased by 19.3%, compared with that in control birds. However, when enrofloxacin was i.v. administered, there were no significant differences of the main parameters between the each two groups of broilers. The results indicated that P-gp inhibitor and inducer could change enrofloxacin concentrations in broliers which proved enrofloxacin is a substrate of P-gp.Aimed at exploring effect of infection on expression and function of P-gp, ten birds aging four weeks were chosen to divide into control and infected group. Expression of P-gp mRNA was measured by real time RT-PCR with β-actin as house-keeping gene. The results indicated that E. coli infection up-regulated expression of Abcb1 mRNA levels significantly in the kidney (P=0.032), jejunum (P=0.017) and ileum (P=0.046), but not significantly in the liver and duodenum (P>0.05). Then immunohistochemical method was used to identify the localization of P-gp in healthy and infected broilers. It was showed that the total amount of P-gp positioned within the liver and kidney cells increased, but the localization in the membrane decreased after infection. There was almost no change of P-gp immunostaining in the duodenum. In healthy birds, immunoreactivity of P-gp was both visualized on the apical surface of the enterocytes of ileum and jejunum, while remaining the same position in infected broilers, but the intensity was significantly increased. Meanwhile, pharmacokinetics of orally administered enrofloxacin was also investigated in healthy and infected broilers by HPLC. The infection reduced absorption of orally administered enrofloxacin, significantly decreased Cmax (P=0.000) and AUC0-12h (P=0.042, absorptive phase) as well as obviously longer Tmax (P=0.040) and T1/2a (P=0.045). Treatment with verapamil, an inhibitor of P-gp, significantly improved the absorption of enrofloxacin in infected broilers. The results strongly suggested that E. coli infection could affect the pharmacokinetics of enrofloxacin in broilers through up-regulating the expression of P-gp, which was different with mammals.Finally we aimed to analyse the correlation of Abcbl mRNA expression change with proinflammatory cytokines IL-6, IL-1(3 and TNFa which were induced by E. coli infection as well as nuclear receptor CXR and nuclear transcription factor NF-kB gene expression patterns. The significant inductions of IL-1(3 mRNA levels were observed in the liver (P=0.018), kidney (P=0.022), duodenum (P=0.000), jejunum (P=0.001) and ileum (P=0.002), while IL-6 significantly increased only in the liver (P=0.023) of broilers after challenged with E. coli. Similar to the modification of P-gp expression, the expression level of TNFa were significantly increased in the kidney (P=0.010), jejunum (P=0.000) and ileum (P=0.001). The result of CXR and NF-κB showed that both of them were significantly up-regulated in jejunum and ileum (P<0.05). Meanwhile, a significant increasing trend of CXR was also observed in the duodenum (P<0.05). TNFα expression level showed significantly consistent with Abcbl expression level in kidney (r=0.653, P=0.016), jejunum (r=0.806, P=0.000)and ileum(r=0.613, P=0.026). NF-κB expression level showed close correlation with Abcb1 expression level in jejunum (r=0.659, P=0.01) and ileum (r=0.563, P=0.043. CXR expression level is significantly consistent with Abcb1 expression level in kidney (r=0.711, P=0.006), jejunum (r=0.685, P=0.007) and ileum (r=0.657, P=0.02).IL-1β expression level is significantly consistent with Abcbl expression level in jejunum (r=0.813, P=0.000) and ileum (r=0.634, P=0.015).IL-6 expression level did not present significant correlation with Abcbl expression level in all tissues. It indicated that the modulation mechanism of Abcbl mRNA is tissues-dependent and the most important regulators were CXR and TNF0/NF-κB siginal factors.In summary, E. coli infection modulates the pharmacokinetics of orally administered enrofloxacin by increasing intestinal P-gp expression. Up-regulation of expression and activity of P-gp in broilers caused by E. coli infection is closely related with CXR, TNFα/ NF-κB signal pathway, but its regulatory mechanism remains to be further studied.