非诺贝特改善脂多糖诱导的急性肺损伤及其机制研究  

Fenofibrate improves acute lung injury induced by lipopolysaccharide

作　　者：董超 李华宇 区豪杰 孙嘉 张陆勇[2] 刘冰 DONG Chao;LI Huayu;OU Haojie;SUN Jia;ZHANG Luyong;LIU Bing(School of Pharmacy,Guangdong Pharmaceutical University,Guangzhou 510006,China;New Drug Research and Development Center,Guangdong Pharmaceutical University,Guangzhou 510006,China)

机构地区：[1]广东药科大学药学院,510006 [2]广东药科大学新药研发中心

出　　处：《天津医药》2022年第1期59-66,共8页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81973562);广东省普通高校药物早期毒性评价创新团队项目(2018KCXTD016)。

摘　　要：目的探究非诺贝特(Fen)对急性肺损伤(ALI)的作用及机制。方法(1)体内实验。30只C57BL/6雄性小鼠采用随机数字表法分成6组:正常组,模型组(LPS组),阳性药地塞米松(DXMS)组,Fen低、中、高剂量(20、40、80mg/kg)组。分组给药12 h后,收集肺泡灌洗液(BALF),处死小鼠,获取肺组织,记录肺组织湿/干质量比;酶联免疫吸附试验(ELISA)检测肺组织肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6和IL-1β含量;BCA比色法和瑞氏-吉姆萨染色检测BALF中总蛋白含量和免疫细胞数目;苏木精-伊红(HE)染色观察肺组织病变并进行病理评分。Western blot检测肺组织B淋巴细胞瘤-2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)及c-Jun氨基末端激酶(JNK)、磷酸化JNK(p-JNK)表达。(2)体外实验。实验设Control、LPS(10 mg/L)、LPS+Fen(5μmol/L)、LPS+Fen(10μmol/L)、LPS+Fen(20μmol/L)组。A549细胞经LPS(10 mg/L)处理后分别加入5、10、20μmol/L的Fen处理12 h,分别用DCFH-DA、Annexin V-FITC/PI法和Western blot检测Fen对A549细胞ROS、凋亡和凋亡相关蛋白Bcl-2、Bax及p-JNK影响。另选取LPS(10 mg/L)+Fen(20μmol/L)处理后添加H2O2(100μmol/L),观察细胞ROS、凋亡率和凋亡相关蛋白Bcl-2、Bax表达变化。此外,选取LPS(10 mg/L)+Fen(20μmol/L)处理后添加JNK激动剂Anisomycin(3μmol/L),观察Bcl-2、Bax表达变化。结果(1)与正常组相比,模型组小鼠肺组织湿/干质量比,肺组织损伤评分,TNF-α、IL-6、IL-1β含量,肺泡灌洗液总蛋白含量,免疫细胞数目,p-JNK和Bax表达均出现显著升高,Bcl-2表达显著降低。与模型组相比,Fen低、中、高剂量组上述指标变化均被逆转。(2)Fen能显著减少LPS诱导的A549细胞ROS含量,降低凋亡率,抑制p-JNK和Bax表达并促进Bcl-2表达。H2O2可逆转Fen引起的上述效应。Anisomycin也能抑制Fen引起的Bcl-2上调和Bax蛋白下调。结论Fen可通过ROS/JNK信号抑制细胞凋亡,从而减轻ALI。Objective To investigate the effect and mechanism of fenofibrate(Fen) on acute lung injury(ALI).Methods(1) In vivo experiment:thirty male C57 BL/6 mice were divided into 6 groups by random number table method including the normal group,the model group(LPS group),the positive drug(dexamethasone) group,and the fenofibrate(Fen)low,medium and high dose(20,40 and 80 mg/kg) groups.After 12 h of administration,the rats were sacrificed,wet/dry ratio of lung tissue was recorded.The contents of TNF-α,IL-1β and IL-6 in lung tissue were detected by enzyme linked immunosorbent assay(ELISA).The total protein content and immune cell number in the bronchoalveolar larage fluid(BALF)were determined by BCA colorimetry and Wright-Giemsa staining.Hematoxylin-eosin(HE) staining was used to observe lung lesions and pathological scores.Western blotting was used to detect the expressions of B-cell lymphoma-2(Bcl-2),bcl-2-Associated X(Bax),c-Jun N-terminal kinase(JNK) and phosphorylated c-Jun N-terminal kinase(p-JNK) in lung tissues.(2) In vitro experiment:the experiment consisted of 7 groups including the control group,the LPS(10 mg/L) group,the LPS+Fen(5 μmol/L) group,the LPS+Fen(10 μmol/L) group,the LPS+Fen(20 μmol/L) group.A549 cells were treated with LPS(10 mg/L) and Fen(5,10 and 20 μmol/L) for 12 h,respectively.DCFH-DA,Annexin V-FITC/PI and Western blot assay were used to detect effects of Fen on ROS levels,apoptosis and expression of apoptosis-related proteins Bcl-2,Bax and p-JNK in A549 cells.After LPS(10 mg/L)+Fen(20 μmol/L) treatment,H_(2) O_(2)(100 μmol/L) was added to observe the changes of ROS,apoptosis rate and expression of apoptosis-related proteins Bcl-2 and Bax.In addition,LPS(10 mg/L) and Fen(20 μmol/L) were treated and JNK agonist anisomycin(3 μmol/L) was added to observe the expression changes of Bcl-2 and Bax.Results(1) Compared with the normal group,the lung wet-dry ratio,lung histopathological score,the contents of TNF-α,IL-1β,IL-6,the number of total proteins and immune cells in alveolar lavage fluid,p

关 键 词：急性肺损伤 非诺贝特 脂多糖类 活性氧 JNK丝裂原活化蛋白激酶类 细胞凋亡 小鼠 近交C57BL A549细胞 

分 类 号：R563[医药卫生—呼吸系统]