过氧化物酶增殖体激活受体α、氧固醇7α羟化酶及雌激素受体问调控关系及其与孕鼠肝内胆汁淤积发生的相关性

Interaction among peroxisome proliferators-activated receptor alpha, cytochrome P450 oxysterol 7α-hydroxylase and estrogen receptor and its association with intrahepatic cholestasis in pregnant rats

目的 探讨过氧化物酶增殖体激活受体α（PPARα）、氧固醇7α羟化酶（CYP7B1）、雌激素受体（ER）α及ERβ之间的调控关系与孕鼠肝内胆汁淤积发生的相关性.方法 选择清洁级SD孕鼠80只,随机分为4组,每组20只,自孕第13天起：对照组孕鼠皮下注射精制植物油2.0ml·kg-1·d-1;低剂量组孕鼠皮下注射17-α-乙炔雌二醇（1.0 mg·kg-1·d-1）;中剂量组孕鼠皮下注射17-α-乙炔雌二醇（1.25 mg·lg-1·d-1）;高剂量组孕鼠皮下注射17-α-乙炔雌二醇（1.5 mg·kg-1·d-1）.4组孕鼠于妊娠第21天处死后提取肝脏组织.应用酶联免疫吸附试验检测各组孕鼠血清中丙氨酸转氨酶（ALT）、门冬氨酸转氨酶（AST）、总胆酸（TBA）及胆红素（BIL）水平;应用实时定量PCR技术检测各组孕鼠肝脏组织中PPARα mRNA、CYP7B1 mRNA、ERα mRNA及ERβ mRNA的表达水平.结果 （1）生化指标：对照组孕鼠ALT、AST、TBA及BIL水平分别为（41.1±2.8）U/L、（44.4±3.6）U/L、（26.4±5.6）μmol/L、（2.8±0.2）U/L,低剂量组孕鼠分别为（48.2±3.4）U/L、（47.9±3.7）U/L、（36.4±4.2）μmol/L、（4.2±0.2）U/L,中剂量组孕鼠分别为（70.4±5.3）U/L、（68.4±5.6）U/L、（64.3±3.8）μmol/L、（6.2±1.2）U/L,高剂量组孕鼠分别为（72.4±7.6）U/L、（70.2±3.8）U/L、（72.4±7.8）μmol/L、（8.2±2.2）U/L.低剂量组、中剂量组、高剂量组孕鼠ALT、AST、TBA、BIL水平明显高于对照组（P〈0.05）;中剂量组、高剂量组孕鼠各生化指标水平明显高于低剂量组（P〈0.05）.（2）ERαmRNA及ERβmRNA表达水平：ERαmRNA的表达水平在低剂量组（0.76±0.02）、中剂量组（0.99±0.04）和高剂量组（1.21±0.01）孕鼠肝脏组织中呈逐渐升高趋势（P〈0.05）,并明显高于对照组（0.65±0.01）,分别与对照组比较,差异均有统计学意义（P〈0.05）;ERβ表达水平在4组孕鼠间分别比较,差异均无统计学意义（P〉0.05）.（3）CYP7B1 mRNA及PPARα mRNA表达水平：CYP7B1 mRNA的表达水平在低剂量组（0.93±0.01）、中剂量组（0.99±0.06）和高剂量组（1.22±0.04）孕鼠肝脏组织中呈逐渐升高趋势（P〈0.05）,并明显高于对照组（0.75±0.02）,分别与对照组比较,差异均有统计学意义（P〈0.05）;PPARα mRNA表达水平在低剂量组（0.83±0.05）、中剂量组（0.71±0.02）和高剂量组（0.64±0.03）孕鼠肝脏组织中呈逐渐降低趋势（P〈0.05）,并明显低于对照组（1.35±0.05）,分别与对照组比较,差异均有统计学意义（P〈0.05）.结论 随着雌激素剂量的增加,PPARα表达水平降低,对CYP7B1表达的抑制作用解除,而导致CYP7B1表达水平升高;而CYP7B1有促进ERα高表达的作用,最终由ERα介导了雌激素诱导的肝内胆汁淤积的发生.提示PPARα、CYP7B1及ER的异常表达,是调控雌激素诱导孕鼠肝内胆汁淤积的发生机制之一.

Objective To investigate the relationship between interaction of peroxisome proliferators-activated receptor alpha （PPARα）, cytochrome P450 oxysterol 7α-hydroxylase （CYP7B1） and estrogen receptor （ER） and intrahepatic cholestasis in pregnant rats. Methods Eighty clean SD pregnant rats were selected and divided into four groups randomly with 20 in each. Since the 13th day of pregnancy,rats in the control group was injected subcutaneously with refined vegetable oil 2.0 ml · kg-1 · d -1 , those in the low-dose, moderate-dose and high-dose groups received 17-α-ethynylestradiol （EE） 1.0 mg · kg-1 · d-1,1.25 mg · kg-1 · d-1 and 1.5 mg · kg-1 · d-1, respectively. All rats were sacrificed at the 21at day of pregnancy and maternal hepatic tissues were collected. The serum levels of alanine aminotransferase（ALT）, aspartate transaminase （AST）, total bile acid （TBA） and bilirubin （BIL） were determined by enzyme linked immunosorbent assay （ELISA）. The mRNA expressions of PPARα, CYP7B1, Erα and Erβ in maternal rat livers were examined by real-time PCR. Results （1） Biochemical indicators： the serum levels of ALT,AST, TBA and BIL were significantly lower in the control group than in the rest 3 groups,respectively [ control group： （41.1 ± 2.8 ） U/L, （44.4 ± 3.6） U/L, （26.4 ± 5.6 ） μmol/L and（ 2.8 ± 0.2）U/L;low-dose group： （48.2 ±3.4） U/L,（47.9 ±3.7） U/L,（36.4 ±4.2） μmol/L and （4.2 ±0.2） U/L;moderate-dose group： （70.4 ± 5.3 ） U/L, （68.4 ± 5.6） U/L, （64.3 ± 3.8 ） μmol/L and （ 6.2 ± 1.2）U/L; high-dose group： （72.4 ±7.6） U/L, （70.2 ±3.8） U/L, （72.4 ±7.8） μmol/L and （8.2 ±2.2）U/L, P〈0.05], and those in the moderate or high-dose groups were higher than in the low-dose group （P〈0.05）. （2） mRNA expression of Erα and Erβ： the mRNA expression of Erα in pregnant rat livers increased in a dose-dependent manner, which were all significantly higher than that in the control group,respectively （ low-dose group： 0.76 ± 0.02 ）; moderate -dose group： （ 0.99 ± 0.04; high-dose group：1.21 ±0.01 ;control group：0.65 ±0.01, P 〈0.05）, but no difference was found among the 4 groups in the mRNA expression of Erβ （ P 〉 0.05 ）. （3） mRNA expression of CYP7B1 and PPARα： the mRNA expression of CYP7B1 in pregnant rat livers increased from the low-dose group to the high-dose group, and were all higher than that of the control group （ low-dose group： 0.93 ± 0.01; moderate-dose group： 0.99 ±0.06; high-dose group： 1.22 ± 0.04; control group： 0.75 ± 0.02, P 〈 0.05 ）. However, the mRNA expression of PPARα decreased from the low-dose group to the high-dose group, and were all lower than that of the control group （low-dose group： 0.83 ± 0.05; moderate-dose group： 0.71 ± 0.02; high-dose group：0.64 ± 0.03; control group： 1.35 ± 0. 05; P 〈 0.05 ） . Conclusions The down regulated mRNA expression of PPARα, caused by higher dose of estrogen, may increase the expression of CYP7B1 due to the ineffectiveness of the inhibition of PPARα on CYP7B1, which may further stimulate the Erα activity and then induce intrahepatic cholestasis. Abnormal expression of PPARα, CYP7B1 and ER may play a role in the pathogenesis of estrogen-induced intrahepatic cholestasis.