脂多糖对猪组织和IPEC-J2细胞P-糖蛋白表达及其对恩诺沙星药动学的影响

发布时间：2018-06-16 19:24

本文选题：LPS + P-gp　；参考：《南京农业大学》2015年硕士论文

【摘要】：P-gp是由Abcb1基因编码的分子量为170kD的药物外排泵,其在机体肝脏、肾脏、肠道等组织中广泛分布,故推测其对药物的吸收和排泄过程也会产生影响。有报道称LPS诱导的炎症会引起啮齿类动物和人P-gp表达和活性的改变,进而使得口服底物药物的生物利用度发生改变,影响药物疗效的发挥。有关P-gp的研究大多集中在肿瘤细胞和人类以及啮齿动物上,而在猪上的研究非常有限,尤其LPS处理对猪组织中P-gp表达和活性的影响更为少见。本文以此为切入点,研究了LPS对猪肝脏、肾脏、空肠、回肠和IPEC-J2细胞系中P-gp表达的影响及此变化对口服恩诺沙星药动学的影响,为炎症状态下猪的合理用药提供理论支持。本试验首先采用荧光定量及免疫组化的方法检测了LPS处理不同时间(3 h和6 h)对健康60日龄三元仔猪肝脏、肾脏、空肠和回肠中P-gp定位和表达的影响。荧光定量的结果表明,在LPS处理3 h后,各组织内Abcb1 mRNA的表达均下降,且差异显著或极显著;6 h后空肠内Abcb1 mRNA与对照相比差异不显著,其余组织与对照比显著或极显著降低。免疫组化的结果表明P-gp在猪肝脏中主要分布于肝细胞间的胆小管细胞膜上,用Image-Pro Plus软件对其阳性反应面积进行半定量分析发现LPS处理3h后胆小管细胞膜上P-gp表达量显著上升(P0.05),6h后降至正常水平。猪肾脏中P-gp主要分布于近端小管和远端小管的细胞膜上,LPS处理6 h后其在膜上的表达量显著升高(P0.05)。猪空肠和回肠中P-gp主要分布于小肠绒毛上皮细胞顶端和小肠腺靠近腔面细胞的顶端,LPS处理3 h后P-gp表达在空肠中有上升趋势,在6 h后降低至正常水平,但是LPS对猪回肠中P-gp的表达未产生显著影响(P0.05)。该试验表明经LPS处理后,P-gp在细胞膜上的表达升高,但各组织中Abcb1的总mRNA水平呈下降趋势。其次,为进一步探究LPS对猪组织细胞内P-gp表达的影响,本试验检测了LPS处理对IPEC-J2细胞膜表面P-gp定位、总mRNA和总蛋白水平的影响。免疫荧光的结果表明,10μg/mLLPS处理后细胞膜上P-gp定位并没有发生改变,但1 h荧光亮度轻微减弱,3 h-24 h增强。荧光定量结果表明,LPS处理1 h后Abcb1 mRNA表达略微下降,且50μg/mL浓度下差异极显著;处理3 h后,Abcb1 mRNA表达和对照相比均明显增加;6 h后又显著降低,但和对照相比差异不显著;12 h后其表达逐渐上升接近正常水平;24 h后mRNA的表达回复至对照水平。Western blot的结果显示,LPS处理1 h后细胞内P-gp总体表达水平呈浓度依赖性的降低;但3 h后细胞内P-gp总体表达水平呈浓度依赖性的增加;而6 h后又呈现抑制状态;12 h后1μg/mL组P-gp水平与对照比略有增加,10μg/mL和50μg/mL LPS仍抑制P-gp的表达,但抑制程度比6 h低;24 h后1μg/mL组P-gp表达量和对照相比又极显著降低,而另两个浓度下P-gp表达量则逐渐回复接近对照水平。试验结果表明LPS处理后P-gp在IPEC-J2细胞膜上的表达量变化与猪组织细胞膜上的表达变化一致,均呈增加趋势,但其总RNA和总体蛋白表达水平与体内试验结果相似,均呈下降趋势。基于之前LPS对猪组织及细胞P-gp表达影响的研究结果,本试验最后采用高效液相色谱的方法揭示LPS通过影响P-gp功能而对恩诺沙星在猪体内药代动力学过程产生影响。结果表明,口服恩诺沙星组中,维拉帕米处理组与对照组相比Tpeak延长(1.81vs 3.18 h),Ke极显著降低(0.24 vs 0.12 h~(-1),P0.01),T1/2ke显著延长(3.02 vs 6.33 h,P0.05),AUC0-24h极显著增加(4.30 vs 8.95μg·mL~(-1)·h,P0.01),CL/F极显著降低(1.23 vs0.57mg-kg~(-1)·h~(-1)/μg·mL~(-1),P0.01),生物利用度增加(42%vs78%),其他药动学参数与对照比没有显著差异;LPS组恩诺沙星在体内的吸收相没有明显变化,Ke极显著降低为0.03 h~(-1)(P0.01),达峰时间明显延后,AUC0-24h增加为7.31μg·mL~(-1)·h(00.05),CL/F降低至0.27 mg·kg~(-1)·h~(-1)/μg·mL~(-1)(P0.01),V/F为11.10 mg·kg~(-1)/μg·mL~(-1)(P0.05),生物利用度升高至72%。静脉注射恩诺沙星组药动学参数之间均无显著差异。结果表明,维拉帕米和LPS处理均使得恩诺沙星药时曲线下面积增加,消除减慢,增加了恩诺沙星的口服生物利用度,推测与P-gp的表达减少或活性降低有关。综上所述,LPS处理使得猪组织及IPEC-J2细胞P-gp的总体表达量下降,但是在细胞膜上的表达量升高,同时恩诺沙星的口服生物利用度增加。该实验结果提示炎症状态可增加P-gp底物药物的口服吸收,对药效的发挥有一定好处,但同时也应注意由此引起的药物蓄积和药物间相互作用,因此要在综合考虑后制定合理的给药方案。
[Abstract]:P-gp is a drug efflux pump, encoded by the Abcb1 gene, 170kD, which is widely distributed in the body's liver, kidney, intestine and other tissues. Therefore, it is speculated that its absorption and excretion process can also affect the process of absorption and excretion of drugs. It is reported that the inflammation induced by LPS can cause changes in the expression and activity of P-gp in rodents and human P-gp, and then the oral base The bioavailability of drugs has changed to affect the efficacy of the drug. Most of the studies on P-gp are focused on tumor cells and human and rodents, but the study on pigs is very limited, especially the effect of LPS treatment on the expression and activity of P-gp in pig tissues is more rare. This is the breakthrough point to study LPS to pig liver. The effects of P-gp expression in the kidney, jejunum, ileum and IPEC-J2 cell lines and the effect of this change on oral enrofloxacin pharmacokinetics were provided to provide theoretical support for the rational use of drugs in the inflammatory state. First, the fluorescence quantitative and immunohistochemical methods were used to detect the dissimilar time of LPS treatment (3 h and 6 h) on healthy 60 days old three yuan pig liver. The effect of P-gp localization and expression in dirty, kidney, jejunum and ileum. The fluorescence quantitative results showed that the expression of Abcb1 mRNA in each tissue decreased significantly after LPS treatment 3 h, and the difference was significant or very significant. The difference of Abcb1 mRNA in jejunum after 6 h was not significant, and the other groups were significantly or significantly lower than the control. The results showed that P-gp was mainly distributed on the membrane of bile duct cells between liver cells in pig liver. Semi quantitative analysis of the positive reaction area was carried out by Image-Pro Plus software. It was found that P-gp expression on the membrane of bile duct cells increased significantly after LPS treatment (P0.05), and 6h decreased to normal level. The main distribution of P-gp in pig kidney was in proximal tubule and distal end. On the cell membrane of the tubule, the expression of LPS on the membrane increased significantly after 6 h treatment (P0.05). The P-gp in the jejunum and ileum was mainly distributed at the top of the small intestinal villous epithelial cells and the small intestinal gland near the top of the cavity surface cells. After LPS treatment, the expression of P-gp in the jejunum was rising, and decreased to the normal level after 6 h, but LPS was in the ileum of the pig. The expression of P-gp was not significantly affected (P0.05). The test showed that after LPS treatment, the expression of P-gp increased on the cell membrane, but the total mRNA level of Abcb1 in each tissue decreased. Secondly, to further explore the effect of LPS on the expression of P-gp in the pig tissue cells. This test examined the P-gp localization of LPS treatment on the membrane surface of IPEC-J2 cells and the total mRNA. The results of immunofluorescence showed that the location of P-gp on the membrane of the cell was not changed after 10 g/mLLPS treatment, but the luminance of 1 h was slightly weakened and 3 h-24 h enhanced. The fluorescence quantitative results showed that the mRNA expression of Abcb1 was slightly decreased after LPS treatment 1 h, and the difference was very significant at 50 mu g/ mL concentration. After processing 3 h There was a significant increase in the ratio of the 6 h to the camera, but the difference was not significant after the 12 h, but the expression gradually increased close to the normal level after 12 h, and the expression of the expression back to the control level.Western blot after 24 h showed that the level of the P-gp overall table in the cells was reduced in a concentration dependent manner after the LPS treatment 1 h; but after 3 h, the cells were within the cells. The overall expression level of P-gp increased in a concentration dependent manner, while the 6 h showed an inhibitory state, and the P-gp level of the 1 mu g/mL group increased slightly after 12 h, while 10 and 50 mu g/mL LPS still inhibited the expression of P-gp, but the degree of inhibition was lower than that of the 6 h; and the expression of 1 mu g/mL group was significantly lower than that of the control after 24 h, while the other two concentrations were expressed. The test results showed that the expression of P-gp on the IPEC-J2 cell membrane after LPS treatment was in accordance with the changes of the expression on the membrane of the porcine tissue cells, and all showed an increasing trend, but the total RNA and total protein expression levels were similar to that in the test results in the body. Based on the previous LPS to the pig tissues and cell P The results of the effect of -gp expression were found. In the end, the effect of LPS on the pharmacokinetics of enrofloxacin in pigs was revealed by high performance liquid chromatography (HPLC). The results showed that in the oral enrofloxacin group, the Vera Pammy treatment group was longer than the control group (1.81vs 3.18 h), and the Ke was significantly reduced (0.2). 4 vs 0.12 h~ (-1), P0.01), T1/2ke significantly prolonged (3.02 vs 6.33 h, P0.05), AUC0-24h significantly increased (4.30 vs 8.95 mu g mL~). There was no obvious change in the absorption phase in the body, and the Ke was significantly reduced to 0.03 h~ (-1) (P0.01), and the peak time was significantly delayed, and the AUC0-24h increased to 7.31 g mL~ (-1) H (0.05), CL/F decreased to 0.27 mg. The results showed that both Vera Pammy and LPS treatments increased the area under the curve of enrofloxacin, eliminated slowing down, increased the oral bioavailability of enrofloxacin, presumed to be related to the decrease in the expression of P-gp or the decrease of activity. The LPS treatment made P-gp of pig tissues and IPEC-J2 cells P-gp The overall expression decreased, but the amount of expression on the cell membrane increased and the oral bioavailability of enrofloxacin increased. The results suggested that the inflammatory state could increase the oral absorption of P-gp substrates and benefit the efficacy of the drug, but at the same time, attention should be paid to the accumulation of drugs and the interaction between drugs. A rational drug delivery scheme should be formulated after comprehensive consideration.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.7

【参考文献】