Effects of bile duct ligation on function and expression of OCT1/2 on the blood-brain barrier in rats and their mechanisms
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摘要:
探讨胆管结扎（BDL）诱导的肝损伤对血脑屏障（BBB）上有机阳离子转运体1/2（OCT1/2）功能和表达的影响及其可能机制。构建BDL大鼠模型，通过试剂盒、Western blot和LC-MS考察BDL大鼠生理生化指标、BBB完整性、皮层OCT1/2蛋白表达和功能以及血浆鹅去氧胆酸（CDCA）浓度。连续14 d灌胃CDCA后测定大鼠生理生化指标、血浆各胆汁酸浓度和皮层OCT1/2蛋白表达。结果显示，BDL大鼠血清天冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）和碱性磷酸酶（ALP）等浓度升高，血浆CDCA浓度升高，金刚烷胺脑血浓度比值（Kp）降低，皮层Claudin-5和Occludin无明显变化，OCT1表达下调，OCT2无明显变化。大鼠灌胃CDCA后，血清AST、ALT和ALP无明显变化，血浆CDCA浓度升高，皮层OCT1表达下调，OCT2无明显变化。本研究表明，BDL大鼠BBB上OCT1功能与表达下调与血液中升高的CDCA有关。
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关键词:
- 胆管结扎 /
- 肝损伤 /
- 有机阳离子转运体1/2 /
- 鹅去氧胆酸
Abstract:This study investigated the effects of bile duct ligation (BDL)-induced liver injury on the function and expression of organic cation transporter 1/2 (OCT1/2) at blood brain barrier (BBB) and their potential mechanisms. BDL rat model was constructed, and physiological and biochemical parameters, BBB integrity, cortical OCT1/2 protein expression and function, and plasma chenodeoxycholic acid (CDCA) level were then examined by kits, Western blot, and LC-MS. Physiological and biochemical parameters, plasma bile acid levels, and cortical OCT1/2 protein expression were determined in rats after ig administration of CDCA for 14 d. The results showed that serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels increased, plasma CDCA level increased, the brain-to-blood concentration ratio (Kp) of amantadine decreased, while cortical Claudin-5 and Occludin did not significantly change, OCT1 expression was downregulated, while OCT2 did not significantly change in BDL rats. Serum AST, ALT, and ALP levels did not significantly change, plasma CDCA level increased, cortical OCT1 expression was downregulated, and OCT2 did not significantly change in rats after ig administration of CDCA. This study suggests that downregulation of OCT1 function and expression at BBB of BDL rats is related to elevated CDCA in plasma.
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Figure 1. Effect of BDL on distributions of amantadine in rats at 30 min following tail vein administration of amantadine (0.5 mg/kg) ($ \bar{x}\pm s,n=6 $)
A: Amantadine concentration in the plasma; B: Amantadine concentration in the brain; C: Ratio of brain to plasma of amantadine*P<0.05, **P<0.01 vs sham group
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Figure 2. Effect of BDL on protein levels of Claudin-5, Occludin, OCT1, and OCT2 in the cortex of rats ($ \bar{x}\pm s $)
A: Expression of Claudin-5 in the cortex of sham and BDL rats (n=8); B: Expression of Occludin in the cortex of sham and BDL rats (n=8); C, D: Expression of organic cation transporter 1/2 (OCT1/2) in the cortex of sham and BDL rats (n=6)**P<0.01 vss sham group
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Figure 3. Effect of ig administration of CDCA (180 mg/(kg·d)) for 14 d on concentrations of bile acids in plasma of rats ($ \bar{x}\pm s,n=6 $)
DCA:Deoxycholic acid; HDCA: Hyodeoxycholic acid; CA: Cholic acid; GCA: Glycocholic acid; LCA: Lithocholic acid;CON:Control**P<0.01 vs CON group
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Figure 4. Effect of ig administration of CDCA (180 mg/(kg·d)) for 14 d on the expression of OCT1 and OCT2 in the cortex of rats ($ \bar{x}\pm s,n=8 $)
**P＜0.01 vs CON group
Table 1 Physiological and biochemical parameters in serum and chenodeoxycholic acid(CDCA) concentration in plasma of sham and BDL rats ($ \bar{x}\pm s,n=5 $)
Parameter Sham BDL Body weight (BW)/g 223.80±8.73 229.40±12.20 Liver index /(% BW) 2.70±0.10 6.32±0.73** Spleen index/(% BW) 0.24±0.03 0.40±0.05** Kidney index/(% BW) 0.72±0.05 0.85±0.05** AST/(IU/L) 36.52±1.85 112.73±37.12** ALT/(IU/L) 16.65±2.30 42.89±15.35** ALP/(IU/L) 15.79±2.15 32.30±6.70** Total bilirubin/(μmol/L) 1.26±0.13 160.28±23.73** Conjugated bilirubin/(μmol/L) 0.56±0.02 135.41±12.10** Ammonia/(μmol/L) 181.95±23.24 225.32±29.19* Total bile acids/(μmol/L) 15.33±3.52 195.25±42.78** CDCA/(μg/mL) 1.06±1.04 4.83±1.86** BDL:Bile duct ligation; AST:Aspartate aminotransferase; ALT:Alanine aminotransferase; ALP:Alkaline phosphatase
*P＜0.05, **P＜0.01 vs sham group
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Table 2 Physiological and biochemical parameters of CON and CDCA-treated (180 mg/(kg·d)) for 14 d rats ($ \bar{x}\pm s,n=6 $)
Parameter Control CDCA Body weight/(BW)/ g 248.33±9.83 251.67±38.17 Liver index/(% BW) 2.88±0.13 2.78±0.23 Spleen index/(% BW) 0.24±0.03 0.24±0.09 Kidney index/(% BW) 0.77±0.04 0.81±0.06 AST/(IU/L) 42.56±3.86 45.75±9.79 ALT/(IU/L) 26.81±2.74 35.58±11.29 ALP /(IU/L) 21.93±3.56 18.17±5.47
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