Effects Of Berberine On Expression And Function Of P-gp In Broilers

Oral administration was one of the most important routes in veterinary treatment, and the efficacy of orally administered drug could be displayed because they are mostly absorbed into systemic circulation through the membranes of gastrointestinal tract. The studies indicated that P-glycoprotein(P-gp) located in intestinal epithelial cell membrane would limit the absorption of oral administration and depress the bioavailability of oral medicine. Therefore, screening of P-gp inhibitor was safe and effective to improve the oral bioavailability of drug had become a hot research topic in the field of pharmaceutical preparation and their application in veterinary clinically practices. Berberine had a good antibacterial activity and had been used for anti-infection drug in clinic. Recently several studies showed that berberine could affect the expression and function of P-gp in rats. However, effects of berberine on mRNA expression and activity of P-gp in the intestine of broilers had not been explored. Therefore, the aims of the study were to figure out the effect of berberine on the expression and function of P-gp, as well as whether nuclear receptors (CR,CAR and PXR) regulated Abcbl mRNA expression or not.First, the CDS sequences of Abcbl gene from poultry would be cloned, as well as transmembrane topology, putaive N-glycosylation site and phylogenetic of Abcbl was also forecasted. After being identified by enzymes digestion and sequencing, results showed that the CDS of Abcbl gene had 3864 base pairs and 1288 amino acids. Sequences blasting indicated similarities of Abcbl with human, pig,cattle, sheep, duck and turkey were 72%, 72%, 68%, 72%, 74% and 95%, respectively. Furthermore Bioinformatics analysis indicated that P-gp for Homo sapiens and chicken has analogous topology: two transmembrane domains, two cytoplasm domains and 12 transmembrane helices. Besides them, seven putaive N-glycosylation sites were identified and located in amino acid sequences, which were less 3 putaive N-glycosylation sites as compared to Homo sapiens. Phylogenetic tree of chP-gp indicated a more close genetic relationship with turkey than the mammal species P-gp.Then we constructed MDCK-chAbcbl cell lines with high expression of chicken P-gp by liposome transfection method and the function and characteristic of P-gp were also studied as well. In this experiment, the pcDNA3.1-chAbcbl was transfected into MDCK cell by LipofectamineTM2000 reagent,and Hygromycin B was used to select single stable cell clones. The mRNA and protein expression of P-gp was detected by RT-PCR, immunohistochemical and immunofluorescence in MDCK-chAbcbl cell.Finally, the function of chicken P-gp in stable cell strain was identified through translocation and accumulation of Rho 123. The result of RT-PCR showed mRNA of gallus sapiens Abcbl was expressed in MDCK-chAbcbl. Immunological detection indicated that the expression of protein encoded by gallus Abcbl gene was three times more than controls using Image-Pro Plus5.2 software. The results of Rho 123 accumulation displayed that uptake of Rho 123 in MDCK-chAbcbl was significantly (P<0.01)decreased at 10, 20 and 50 minutes comparing with MDCK-pcDNA3.1; but the uptake of Rho 123 was slightly more than content in Caco-2. Inhibition test was performed through verapamil, the accumulation of Rho123 was significantly (P<0.05) increased in Caco-2 and MDCK at 20 and 50 minutes; but the obvious trend of recovery was not presence in MDCK-pcDNA3.1. Further analysis showed kinetic parameters for the high- and low-affinity components of Rho 123 uptakes in MDCK-chAbcbl cells were 0.73 and 51.75 ?M (Km) and 0.87 and 10.14 nmol/mg protein/min (Vmax),respectively. But, there was only one binding component for Rho123 in Caco-2 with a Km value of 2.38 mM and Vmax of 22.8 pmol/mg protein/min. Significant values of efflux experiments displayed Pappba of Rho 123 were founded as 72.33 and 54.42 cm·min-1 as comparison with control 11.35 cm·min-1. The efflux ratios of Caco-2,MDCK-chAbcb1 and MDCK-pcDNA3.1 (control) were 7.49, 6.24 and 1.64, respectively. It was concluded from the present experiment, the chicken P-gp was over expressed functionally in MDCK-chAbcbl and transport kinetics of Rho 123 by chicken P-gp was different with human P-gp in Caco-2 cell line, but transport activity was corresponded in both cells.Based on above results, effects of berberine and verapamil on transport of P-gp in MDCK-chAbcb1 were compared in Caco-2 cell. The results showed that transport of Rho123 from BL to AP was significantly inhibited along with the increasing of verapamil concentration from 0.01 to5 mM, and transport of Rho 123 from AP to BL was only slightly increased in both cells. When incubation concentration of verapamil was 0.1, 1 and 5 mM in MDCK-chAbcb1, inhibition of ER was 43.8%,68.9% and 78%, respectively. After the same treatment in Caco-2 cells, inhibition of ER was 45.7%,61.1% and 71.4%, respectively. Transport of Rho123 from BL to AP was also significantly inhibited at 5,20 and 40?M berberine for 2 and 8 hours in MDCK-chAbcbl cell, and transport of Rho123 from AP to BL was only slightly increased. Finally the results indicated that the activity of P-gp was also reversed by verapamil in MDCK-chAbcb1; transmembrane transport of Rho 123 was depressed by berberine which was inferred as possible inhibitor.For further confirming the inhibitory effect of berberine on P-gp activity in chicken intestine, the in-situ perfusing method was performed in chicken ileum. In the experiment six weeks old broilers were divided into several groups. PBS was given as control. Berberine with different dosage regimes of 40 and 80 mg/kg body weight (b.w.) were given per orally for 1 day and for 3 days, respectively. The results demonstrated that Ka of 40 mg/kg.bw and 80 mg/kg.bw for 1 day were 1.87 min-1 and 2.25 min-1,comparing with control (1.12 min-1), respectively. Papp in each group were 0.0074, 0.0132 and 0.0076 cm-min-1 (as control), respectively. However, induction of berberine for 3 days, there were no significant difference about the values of Ka and Papp in each group. It was showed that Berberine could significantly depress activity of P-gp in chicken ileum.Finally we aimed to analyse the correlation of Abcbl mRNA expression change with nuclear receptor CXR, CAR and PXR gene expression patterns. The mRNA expression levels of Abcbl, PXR,CAR and CXR in cell models and chicken ileum were measured using real-time RT- PCR and the expression levels of Abcbl encoding protein P-gp in Caco-2 and MDCK-chAbcbl cells were detected by immunofluorescence at different time with different concentrations. 5?M of berberine had little effect on mRNA expression of Abcb1 and CAR in Caco-2 cells, however, when its concentrations increased to 20 and 40 ?M, the mRNA expression of Abcbl and CAR could be inhibited significantly within 8 hours.But mRNA expression level of Abcbl and CAR could be recovered to control levels after 12 or 24 hours.P-gp expression change style was consistent with mRNA expression of Abcb1 by the same treatment of berberine. In MDCK-chAbcbl cell,mRNA expression of Abcbl and CAR showed a dose and time dependent decreasing (P<0.05) when treated with berberine. Positive fluorescence intensity of P-gp was also decreased when treated with different concentrations of berberine. In chicken ileum tissue, mRNA expression of Abcbl and CXR were inhibited obviously after orally administrating berberine with dose of 40 and 80mg/kg body weight for 1 day (P<0.05). However, mRNA expression Abcbl and CXR came back to control level when broilers treated by high concentration of berberine for three days. The results reveal that berberine inhibited the expression and functions of P-gp within special dose and treatment time to some degree and the changes induced by berberine was mediated by nuclear receptor CXR and CAR.In summary, we established MDCK-chAbcbl with functional overexpression P-gp from gallus species, and berberine decreased the expression and activity of P-gp in stable cells lines.Down-regulation of expression and activity of P-gp in broilers caused by berberine was closely related with CXR and CAR signal pathway, but its regulatory mechanism remained to be further studied. The results of this study had enriched fundamental theory about gallus spcies P-gp,and provided a platform for researching the interaction of veterinary drugs.