The effect of high dose endotoxin on CYP3A2 expression in the rat
The effect of high dose endotoxin on CYP3A2 expression in the rat
复制标题
DOI：
10.1023/a:1011915402914
复制
发表时间：
1998-10-01
影响因子：
3.7
通讯作者：
Blouin, RA
中科院分区：
文献类型：
--
作者：
Roe, AL;Warren, G;Blouin, RA
目的。我们研究的目的有两个方面：1)进一步表征急性期反应（APR）期间CYP3A2 mRNA、蛋白和活性的下调；2)最重要的是，将核蛋白的时间依赖性激活与CYP3A2 5'侧区假定的DNA结合序列，以及CYP3A2表达的缺失联系起来。给大鼠注射LPS (2.0 mg/只，i.p.)，于1、2、4、6、8、24、48和72小时处死。分离肝核蛋白，分析其与AP-1、NF κ B和NF- il6的结合活性。采用溶液杂交法测定肝脏CYP3A2 mRNA水平，测定肝脏微粒体中CYP3A2蛋白水平、CYP3A2活性及总P450。计算机分析CYP3A2的5'侧区域显示存在5个NF-IL6和4个AP-1推测的DNA结合位点。AP-1结合活性最强的增加发生在6 - 24小时之间，结合复合物对NF-IL6寡核苷酸的改变发生在4 - 24小时之间。CYP3A2 mRNA的最大损失发生在lps注射后8小时，并在24小时时保持降低。CYP3A2蛋白在lps处理后24,48和72小时显著降低，CYP3A2活性和总p450相应降低。LPS处理后NF-IL6和AP-1结合的变化似乎与CYP3A2 mRNA的变化相关，再加上位于CYP3A2 5'侧区域的假定NF-IL6和AP-1位点的存在，可能表明NF-IL6和AP-1在APR期间CYP3A2下调中的潜在作用。
Purpose. The purpose of our research was two-fold: 1) to further characterize the downregulation of CYP3A2 mRNA, protein, and activity during an acute phase response (APR); 2) most importantly, to relate the time-dependent activation of nuclear proteins to putative DNA binding sequences within the CYP3A2 5'-flanking region, with the loss in CYP3A2 expression.Methods. Rats were injected (2.0 mg/animal, i.p.) with LPS and sacrificed at 1, 2, 4, 6, 8, 24, 48, and 72 hours. Hepatic nuclear protein was isolated and analyzed for binding activity to AP-1, NF kappa B, and NF-IL6 consensus sequences. Hepatic CYP3A2 mRNA levels were determined by solution hybridization and CYP3A2 protein, CYP3A2 activity, and total P450 were measured in hepatic microsomes.Results. Computer analysis of the 5'-flanking region of CYP3A2 revealed the presence of 5 NF-IL6 and 4 AP-1 putative DNA binding sites. The strongest increase in AP-1 binding activity occurred between 6 and 24 hr, and the alteration in binding complexes to an NF-IL6 oligonucleotide occurred between 4 and 24 hr. Maximum loss in CYP3A2 mRNA occurred at 8 hr post-LPS injection and remained lowered at the 24 hr timepoint. CYP3A2 protein was significantly decreased at 24, 48, and 72 hours post-LPS treatment with corresponding decreases in CYP3A2 activity and total P450.Conclusions. The changes in NF-IL6 and AP-1 binding after LPS treatment, which appears to correlate with the changes in CYP3A2 mRNA, combined with the presence of putative NF-IL6 and AP-1 sites located in the CYP3A2 5'-flanking region, may indicate a potential role for NF-IL6 and AP-I in CYP3A2 downregulation during an APR.