肝素对急性肝损伤小鼠保护作用及对细胞外组蛋白水平影响  

Protective effect of heparin on mice with acute liver injury and its effect to extracellular histone level

作　　者：温韬[1] 朱钧[2] 李惠玲[2] 李安[2] 

机构地区：[1]首都医科大学附属北京朝阳医院医学研究中心,北京100020 [2]首都医科大学附属北京朝阳医院职业病与中毒医学科,100020

出　　处：《中国职业医学》2015年第6期620-625,共6页China Occupational Medicine

基　　金：国家自然科学基金(81372094);北京市卫生系统高技术人才培养项目-学科骨干(2013-3-077);北京市自然科学基金资助项目(7142064)

摘　　要：目的研究肝素对D-氨基半乳糖联合脂多糖(Gal N/LPS)染毒所致小鼠急性肝损伤的保护效应,探讨其作用机制是否通过作用于细胞外组蛋白而实现。方法 1肝素保护作用实验(体内实验)。无特定病原体级健康雄性C57BL/6小鼠随机分为正常对照组(6只)、肝素对照组(6只)、肝损伤组(8只)和肝素干预组(8只)。肝损伤组和肝素干预组小鼠均一次性腹腔注射Gal N 600 mg/kg体质量和LPS 20μg/kg体质量进行急性肝损伤造模,肝素干预组小鼠同时经皮下注射肝素300 U/kg体质量;正常对照组和肝素对照组小鼠分别予一次性腹腔注射等体积质量分数为0.9%的氯化钠溶液或肝素300 U/kg体质量。染毒后12 h时间点,采集4组小鼠静脉血分离血浆检测肝损伤指标、细胞外组蛋白和相关细胞因子的水平,并进行肝组织病理学观察。另设急性肝损伤组和肝素干预组小鼠,每组10只,观察染毒后24 h的生存率。2肝素-细胞外组蛋白结合实验(离体实验)。取肝损伤组小鼠血浆,按照1∶1比例与分别加入肝素琼脂糖凝胶CL-6B(肝素组)和二乙氨基乙醇琼脂糖凝胶(对照组),孵育1 h后分离上清液,检测细胞外组蛋白和细胞因子水平化。结果 1体内实验。染毒处理后12 h时间点,肝损伤组小鼠出现大量肝细胞变性和坏死的急性肝损伤组织病理学改变,而肝素干预组小鼠肝细胞损伤程度及范围均较肝损伤组明显减轻。与正常对照组比较,肝损伤组小鼠血浆丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、总胆红素(T-BIL)、细胞外组蛋白、白细胞介素(IL)-1β、IL-6、IL-10和肿瘤坏死因子-α(TNF-α)水平均升高(P<0.01)。与肝损伤组比较,肝素干预组小鼠血浆ALT、AST、T-BIL、细胞外组蛋白、IL-6和TNF-α水平均下降(P<0.01)。肝损伤组小鼠24 h生存率低于肝素干预组(10%vs 80%,P<0.01)。2离体实验。肝素组血浆细胞外组蛋白水平低于对照组(P<0.05),但2�Objective To investigate the protective effects of heparin against acute liver injury in mice caused by D-galactosamine plus lipopolysaccharide（ Gal N / LPS） and to explore whether its mechanism is conducted by affecting the extracellular histone. Methods i） In vivo assay for the protective effect of heparin. Specific pathogen free healthy male C57 BL /6 wild type mice were randomly divided into four groups： normal control group（ n = 6）,which received saline（ 0. 9% mass fraction of sodiumchloride solution） once by subcutaneous injection; heparin-treated control group（ n = 6）,which received heparin once at a dose of 300 U / kg body weigh（ bw） by subcutaneous injection; acute liver injury group（ n = 8）,which were treated with an intraperitoneal injection of Gal N（ 600 mg/kg bw） plus lipopolysaccharide（ LPS）（ 20 μg/kg bw） once to establish acute liver injury model; heparin-intervention group（ n = 8）,which were treated with heparin together with Gal N / LPS once. Separated plasma hepatic damage indicators,extracellular histones,the relevant cytokines were measured and the liver tissue histopathologic changes were observed 12 hours after Gal N / LPS treatment. In another set of experiments,the survival rate was compared between liver-injury group（ n = 10） and heparin-intervention group（ n = 10） within 24 hours period. ii） In vitro assay for the binding of heparin to extracellular histones. The plasma from acute liver injury group mice were collected and incubated with heparin-sepharose CL-6B solution（ heparin group）and diethylaminoethyl-sepharose solution（ control group） at a ratio of 1∶ 1 at room temperature for 1 hour. Then the levels of extracellular histone and the relevant cytokines were measured in the supernatant of the mixture. Results i） In vivo experiments. Histopathological analysis at the time point of 12 hours following Gal N / LPS administration,the mice in acute liver injury group showed changes of acute liver injury such as massive hepa

关 键 词：肝损伤 急性损伤 肝素 细胞外组蛋白 细胞因子 小鼠 

分 类 号：R114[医药卫生—卫生毒理学] R575[医药卫生—公共卫生与预防医学]