非诺贝特改善脂多糖诱导的急性肺损伤及其机制研究

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、80 mg/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（5 μmol/L）、LPS+Fen（10 μmol/L）、LPS+Fen（20 μmol/L）组。A549细胞经LPS（10 mg/L）处理后分别加入5、10、20 μmol/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（20 μmol/L）处理后添加H2O2（100 μmol/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（20 μmol/L）处理后添加JNK激动剂Anisomycin（3 μmol/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。

Fenofibrate improves acute lung injury induced by lipopolysaccharide

Abstract: Objective　To investigate the effect and mechanism of fenofibrate (Fen) on acute lung injury (ALI). Methods　(1) In vivo experiment:thirty male C57BL/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 blotassay 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, H2O2 (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-JNK and Bax expression were significantly increased in the model group, and the expression of Bcl-2 was significantly decreased. Compared with the model group, the changes of above indexes were significantly reversed in the Fen low, medium and high dose groups (20, 40, 80 mg/kg). (2) Fenofibrate significantly reduced ROS content, decreased apoptosis rate, inhibited p-JNK and Bax expression and promoted Bcl-2 expression in LPS-induced A549 cells, which were reversed by H2O2 treatment. Up-regulation of Bcl-2 and down-regulation of Bax expression induced by Fen in LPS-induced A549 cells were reversed by anisomycin treatment. Conclusion　Fenofibrate inhibits apoptosis through ROS/JNK signaling, so as to reduce ALI.