CAJ | 학술논문

目的：课题组之前的研究已证实在 OVA 诱导的哮喘小鼠模型中，乌司他丁通过抑制 ROS 的产生、诱导抗氧化酶的表达，起到治疗哮喘的作用，但具体机制尚不清晰；此研究将以 Nrf2／HO-1这一氧化应激的关键通路为切入点，深入探讨乌司他丁抗氧化作用的潜在机制。方法建立 OVA 诱导的小鼠哮喘模型，腹腔注射乌司他丁（100 kU·kg －1·d －1）进行治疗，对照组用 PBS（pH 7.4）代替；检测气道高反应性；ELISA 法检测小鼠 BALF 中 IL-4、IFN-γ、总 IgE 及特异性 IgE的表达水平；双氢罗丹明（DHR）-123方法检测小鼠支气管肺泡灌洗液（BALF）细胞中 ROS 含量；检测小鼠肺组织中蛋白羰基含量（protein carbonyl）和丙二醛（MDA）水平；抗氧化酶试剂盒检测小鼠 BALF 中的抗氧化酶；Western blot 及 Re-al-time PCR 检测小鼠肺组织中 HO-1在蛋白和基因的表达水平；Western blot、IF 及 EMSA 检测 Nrf2核转移及结合活性。结果乌司他丁治疗组明显降低气道高反应；降低BALF 中 IL-4、特异性 IgE 及 ROS 的表达水平，降低小鼠肺组织 Protein carbonyl 含量和 MDA 水平，提高 IFN-γ、SOD、GSH和 TAOC 等抗氧化酶的表达水平，其治疗作用可被 HO-1抑制剂 Znpp 逆转；乌司他丁诱导小鼠肺组织中的 HO-1在蛋白水平的表达呈明显剂量和时间依赖性；乌司他丁诱导 Nrf2的核转移，增强 Nrf2的结合活性并提高 HO-1基因水平表达。结论乌司他丁在 OVA 诱导的哮喘小鼠模型发挥抗氧化治疗作用，其作用机制可能是通过 Nrf2／ARE 途径诱导HO-1的表达实现。
목적：과제조지전적연구이증실재 OVA 유도적효천소서모형중，오사타정통과억제 ROS 적산생、유도항양화매적표체，기도치료효천적작용，단구체궤제상불청석；차연구장이 Nrf2／HO-1저일양화응격적관건통로위절입점，심입탐토오사타정항양화작용적잠재궤제。방법건립 OVA 유도적소서효천모형，복강주사오사타정（100 kU·kg －1·d －1）진행치료，대조조용 PBS（pH 7.4）대체；검측기도고반응성；ELISA 법검측소서 BALF 중 IL-4、IFN-γ、총 IgE 급특이성 IgE적표체수평；쌍경라단명（DHR）-123방법검측소서지기관폐포관세액（BALF）세포중 ROS 함량；검측소서폐조직중단백탄기함량（protein carbonyl）화병이철（MDA）수평；항양화매시제합검측소서 BALF 중적항양화매；Western blot 급 Re-al-time PCR 검측소서폐조직중 HO-1재단백화기인적표체수평；Western blot、IF 급 EMSA 검측 Nrf2핵전이급결합활성。결과오사타정치료조명현강저기도고반응；강저BALF 중 IL-4、특이성 IgE 급 ROS 적표체수평，강저소서폐조직 Protein carbonyl 함량화 MDA 수평，제고 IFN-γ、SOD、GSH화 TAOC 등항양화매적표체수평，기치료작용가피 HO-1억제제 Znpp 역전；오사타정유도소서폐조직중적 HO-1재단백수평적표체정명현제량화시간의뢰성；오사타정유도 Nrf2적핵전이，증강 Nrf2적결합활성병제고 HO-1기인수평표체。결론오사타정재 OVA 유도적효천소서모형발휘항양화치료작용，기작용궤제가능시통과 Nrf2／ARE 도경유도HO-1적표체실현。
Aim To explore the potential mechanism of ulinastatin’s antioxidant effect by examining the Nrf2 /HO-1 pathway.Methods OVA-induced asthma of mice was cured by intraperitoneal injection of ulinas-tatin (1 00 kU·kg -1 ·d -1 ).Control mice were given the same volume of PBS (pH 7.4).To investigate the effect of ulinastatin on airway hyperresponsiveness, levels of interleukin IL-4,IFN-γand OVA specific IgE in bronchoalveolar lavage fluid (BALF)were measured using enzyme-linked immunosorbent assays (ELISAs). The content of ROS from BALF of mice was tested in double hydrogen rhodamine (DHR)-1 23 method.The level of protein carbonyl and MDA from lung tissue of mice was detected with Protein carbonyl content assay kit and MDA kit.And antioxidative enzyme in mice BALF was tested by antioxidant enzyme kit.The levels of HO-1 in lung tissue from mice were detected by Western blot and Real-time PCR.Nuclear transfer and binding activity of Nrf2 were tested respectively by Western blot,IF and EMSA.Results Ulinastatin could alleviate the airway hyperresponsiveness,dis-tinctly reduce the content of IL-4,OVA specific IgE, ROS,protein carbonyl and MDA,but upraise the ex-pression of IFN-γand antioxidative enzyme such as SOD,GSH and TAOC. Moreover, the antioxidant effect of ulinastatin could be reversed by Znpp,which was the inhibitor of HO-1 .Ulinastatin could obviously induce the expression of HO-1 in protein level in a dose-and time-dependent manner.Ulinastatin could also induce the nuclear transfer of Nrf2 and increase the binding activity of Nrf2 as well as the expression of HO-1 in gene level;Conclusion Ulinastatin could induce the activation of Nrf2 /HO-1 pathway,which may contribute to the protective effects of ulinastatin a-gainst OVA-induced oxidative stress.