羧酸酯酶在免疫性肝损伤大鼠体内的活性研究

发布时间：2018-04-23 17:53

本文选题：免疫性肝损伤 + 脂多糖　；参考：《华中科技大学》2013年硕士论文

【摘要】：免疫性肝损伤与自身机体免疫应答有关。由卡介苗(BCG)+脂多糖(LPS)诱导的急性免疫性肝损伤模型更能模拟由病毒或内毒素引发的急性肝炎模型。在肝脏疾病状态下，药物代谢酶的活性可能会发生改变，，从而会对药物的药物代谢动力学及药物疗效产生影响。因此，本研究首先采用BCG+LPS法建立免疫性肝损伤大鼠模型，将SD大鼠随机分为3组，低剂量LPS组，高剂量LPS组和正常对照组，先腹腔注射BCG (8mg)，连续7d后，分别腹腔注射给予三组大鼠5mg·kg~(-1)LPS,10mg·kg~(-1)LPS和等体积的生理盐水。12h后，检测血清中谷丙转氨酶（ALT）和谷草转氨酶（AST）含量变化，将肝脏经HE染色，在光镜下观察病理学变化。结果发现，与正常对照组相比，低剂量LPS组和高剂量LPS组大鼠血清ALT和AST活性显著升高（P 0.05），且随剂量的增加而升高；普通光镜下观察肝脏病理学变化，LPS组的大鼠肝脏出现炎症细胞浸润和肝细胞坏死，且损伤程度随LPS剂量的升高而加重。采用BCG+LPS（5mg·kg~(-1)）可以成功的建立免疫性肝损伤大鼠模型。在模型建立成功的基础上，我们考察了CES在免疫性肝损伤大鼠体内的代谢活性，选取咪达普利和伊立替康(CPT-11)分别作为CES两种同工酶CES1和CES2的特异性底物。我们首先建立了LC-MS/MS法和HPLC法测定血浆中咪达普利的活性代谢产物咪达普利拉和CPT-11的活性代谢产物SN-38的浓度，两种方法均简便，快捷，专属性好，在线性范围内灵敏，准确，精密度高，为研究药物体内药动学打下了基础。接着我们考察了咪达普利和CPT-11在免疫性肝损伤大鼠体内的药动学特征。给模型组大鼠分别灌胃给予咪达普利(10mg·kg~(-1))和尾静脉注射CPT-11(20mg·kg~(-1))，正常对照组大鼠给予等体积生理盐水，于给药后不同时间点采血，测定血浆中咪达普利拉和SN-38的浓度。结果显示与正常对照组大鼠相比，模型组大鼠体内咪达普利拉的AUC0-12h从1759.93±386.57mg·h-1·L-1降至620.93±197.16mg·h-1·L-1， Cmax从234.66±68.85mg·L-1下降至113.1±19.69mg·L-1(P0.05)；SN-38的AUC0-24h从646.1±110.9ng·h·mL-1降至275.9±52.5ng·h·mL-1，Cmax从145.6±15.9ng·mL-1下降至57.8±9.5ng·mL-1(P0.05)。以上结果表明咪达普利和CPT-11在免疫性肝损伤大鼠体内的代谢均受到了抑制，药动学特征发生了改变。最后，为进一步考察CES1和CES2在免疫性肝损伤大鼠体内表达特征的变化，我们采用荧光定量PCR (RT-PCR)和Western blot技术对肝脏中CES1和CES2的mRNA和蛋白表达水平进行分析。RT-PCR结果显示，CES1mRNA及CES2的两种主要同工酶AB010635和AY034877的mRNA含量均比正常对照组含量低(P0.05)。Western blot结果显示，CES1和CES2的蛋白表达水平均下降(P0.05)。因此，本研究发现免疫性肝损伤状态下，CES1和CES2的表达和代谢活性显著降低。本研究成果对临床免疫性肝损伤患者服用经CES代谢的药物剂量调整具有一定的参考意义。
[Abstract]:Immune liver injury is associated with autoimmune response. The model of acute immune liver injury induced by BCG lipopolysaccharide (LPS) can simulate the acute hepatitis induced by virus or endotoxin. Under the condition of liver disease, the activity of drug metabolizing enzymes may change, which will affect the pharmacokinetics and efficacy of drugs. In this study, the immunological liver injury model was established by BCG LPS method. SD rats were randomly divided into three groups: low dose LPS group, high dose LPS group and normal control group. The rats were injected intraperitoneally with BCG 8 mg / g for 7 days. Three groups of rats were injected intraperitoneally with 10 mg kg~(-1)LPS and 10 mg kg~(-1)LPS of 5mg, and normal saline for 12 h. The changes of serum alanine aminotransferase (alt) and aspartate aminotransferase (AST) levels were detected. The liver was stained with HE and the pathological changes were observed under light microscope. The results showed that the activities of serum ALT and AST in the low dose LPS group and the high dose LPS group were significantly higher than those in the normal control group (P 0.05), and increased with the increase of the dose. The pathological changes of lipopolysaccharide group were observed under light microscope. Inflammatory cell infiltration and hepatocyte necrosis occurred in lipopolysaccharide group, and the degree of injury increased with the increase of LPS dose. The immunological liver injury model could be established successfully by BCG LPS(5mg. On the basis of successful establishment of the model, we investigated the metabolic activity of CES in immunological liver injury rats, and selected midazapril and Ilitecan CPT-11 as specific substrates of CES isozyme CES1 and CES2, respectively. We first established a LC-MS/MS and HPLC method for the determination of the concentrations of the active metabolites of midazapril and CPT-11 in plasma. The two methods were simple, rapid and specific, and were sensitive and accurate in the linear range. The high precision laid the foundation for the study of pharmacokinetics in vivo. Then we investigated the pharmacokinetic characteristics of imidapril and CPT-11 in immune liver injury rats. Rats in the model group were given intragastric administration of imidapril (10 mg / kg) and caudal vein injection of CPT-11(20mg (KGG) and normal control group (control group). Blood samples were collected at different time points after administration, and the concentrations of midapriline and SN-38 in plasma were determined. The results showed that compared with the normal control group, the AUC0-12h of the model group was decreased from 1759.93 卤386.57mg h -1 to 620.93 卤197.16mg h -1 L -1, and the AUC0-24h of Cmax from 234.66 卤68.85mg L -1 to 113.1 卤19.69mg L -1 P0.05N 38 decreased from 646.1 卤110.9ng h mL-1 to 275.9 卤15.9ng h ml -1 C max from 145.6 卤15.9ng mL-1 to 57.8 卤9.5ng ml -1 P 0.05N. These results indicated that both imidapril and CPT-11 were inhibited in vivo and pharmacokinetic characteristics were changed in immune liver injury rats. Finally, in order to investigate the expression characteristics of CES1 and CES2 in immunological liver injury rats, The expression levels of mRNA and protein of CES1 and CES2 in liver were analyzed by fluorescence quantitative PCR RT-PCR and Western blot. The results of RT-PCR showed that the contents of AB010635 and AY034877, the two main isozymes of CES2, were lower than those of normal control group (P 0.05). Western blot The results showed that the protein expression levels of CES1 and CES2 were both decreased (P 0.05). Therefore, we found that the expression and metabolic activity of CES1 and CES2 decreased significantly in immune liver injury. The results of this study have some reference significance for the dosage adjustment of CES metabolism in patients with immune liver injury.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.8

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