乌司他丁通过miR-21／p38MAPK通路对脓毒症保护作用的研究  

The protective effect of Ulinastatin on sepsis through miR-21/p38MAPK pathway

作　　者：陶杨[1] 肖玲 黄健[1] 马渝[1] Tao Yang, Xiao Ling, Huang Jian, Ma Yu(Intensive Care Unit, Chongqing Emergency Medical Center, Chongqing 400014, China)

机构地区：[1]重庆市急救医疗中心重症医学科,重庆400014

出　　处：《中华急诊医学杂志》2018年第11期1242-1247,共6页Chinese Journal of Emergency Medicine

基　　金：重庆市卫生计生委科研资助项目（2016ZDXM030）

摘　　要：[目的 探讨乌司他丁对脓毒症中miR-21/p38 丝裂原活化蛋白激酶（p38MAPK）信号通路影响。方法 将SPF 级BALB/c 小鼠（30 只）随机（随机数字法）分为3 组（每组n=10）：脓毒症组（LPS 组，LPS 10 mg/kg 一次性腹腔内注射），乌司他丁预处理组（UTI 组，乌司他丁105 U/kg 腹腔注射预处理2 h 后给予LPS 10 mg/kg 腹腔内注射），正常组（相同时间点注射等体积生理盐水）。24 h 后处死小鼠观测肺组织病理学结果，采用荧光实时定量PCR 法测定肺组织中miR-21 表达，免疫组化法检测各组肺部组织中PTEN，p-p38MAPK 的表达。另外，小鼠巨噬细胞RAW264.7 细胞采用LipofectamineTM 2000 转染试剂以及miR-21模拟剂（100 nmol/L）、抑制剂（100nmol/L） 处理 6 h 后，弃掉上清液，加入乌司他丁（1 000 U/mL） 处理2 h 后加入LPS（500 ng/ mL） 刺激，培养24 h 后收集细胞，应用PCR 法测定肺组织中miR-21 表达，Western-blot 法分别检测PTEN、p-p38MAPK 蛋白表达情况。结果 动物实验中，与正常组（1.00±0.00） 比较，LPS 组中miR-21（2.73±0.17） 及p-p38MAPK 表达上调，miR-21 下游靶基因PTEN 蛋白表达下调, 而与LPS组比较，UTI 组miR-21（2.14±0.13） 及p-p38MAPK 表达水平下降，PTEN 蛋白表达上调（P〈0.05）。细胞实验中, 与LPS 组（1.93±0.21）比较，LPS＋miR-21 抑制剂组p-p38MAPK 蛋白表达量（1.51±0.06）下降，而LPS＋miR-21 模拟剂组p-p38MAPK 蛋白表达量（2.52±0.07）升高（P〈0.05）。而加入UTI 干预后，结果显示LPS＋UTI 组p-p38MAPK 蛋白表达量（0.61±0.04）比LPS 组低；而与LPS＋UTI 组（0.61±0.04）比较，LPS＋UTI＋miR-21 抑制剂组p-p38MAPK 蛋白表达（2.36±0.15） 升高，LPS＋UTI＋miR-21 模拟剂组p-p38MAPK 蛋白表达（0.22±0.05） 下降（P〈0.05）。结论 乌司他丁在脓毒症中发挥保护作用，其机制与调控miR-21/p38MAPK 通路有关。Objective To investigate the effect ofulinastatin on miR-21/p38 mitogen-activated protein kinase （p38MAPK） signaling pathway in sepsis. Methods SPF BALB/c mice （n=30） were randomly divided into 3 groups （n=10, each group）： LPS group （LPS 10 mg/kg disposable intraperitoneal injection）, UTI group （Ulinastatin 105 U/kg was pretreated by intraperitoneal injection for 2 hours, and LPS was administered intraperitoneally 10 mg/kg）, and the normal group （same volume of normal saline was injected at the same time point）. After 24 h, the mice were sacrificed and the pathological results of lung tissue were observed. The expression of miR-21 in lung tissue was detected by real-time quantitative PCR. The expression of PTEN and p-p38MAPK in lung tissues of each group were detected by immunohistochemistry. In addition, mouse macrophage RAW264.7 cells were treated with Lipofectamine^TM 2000 transfection reagent, miR-21 mimic agent （100 nmol/L） and inhibitor （100 nmol/L） for 6 h, then the supernatant was discarded and Ulinastatin （1 000 U/mL） was added for 2 h treatment, and finally 500 ng/mL LPS were added. After 24 h, the cells were collected. The expression of miR-21 in lung tissue was determined by PCR, and the expression of PTEN and p-p38MAPK protein were detected by Western blot. Results In animal experiments, the expression of miR-21 （2.73±0.17） and p-p38MAPK was up-regulated in the LPS group, and the expression of PTEN protein was down-regulated in the downstream target gene of miR-21. Whereas the expression levels of miR-21 （2.14±0.13） and p-p38MAPK in the UTI group were decreased, and the expression of PTEN protein was up-regulated. In the cell experiment, compared with the LPS group（1.93±0.21）, the expression of p-p38MAPK protein in the LPS＋miR-21 inhibitor group（1.51±0.06） decreased, while the expression of p-p38MAPK protein in the LPS＋miR-21 mimic group increased（2.52±0.07）. After UTI intervention, the expression of p-p38MAPK protein in the LPS＋UTI

关 键 词：乌司他丁 脓毒症 MIR-21 PTEN P38MAPK 

分 类 号：R459.7[医药卫生—急诊医学]