腺花素对急性肺损伤模型小鼠的保护作用及其机制

Effects and Mechanism of Adenanthin on Acute Lung Injury Model in Mice

目的探讨腺花素对急性肺损伤(ALI)的治疗作用及其机制。方法C57BL/6雄性小鼠30只,随机分为6组:正常对照组、模型对照组(10 mg·kg^(-1)脂多糖)、地塞米松组(5 mg·kg^(-1)地塞米松)和腺花素小、中、大剂量组(5,10和20 mg·kg^(-1)腺花素),每组5只。造模6 h后分别腹腔注射相应药物。18 h后,记录体质量变化,麻醉处死,取肺部样本。酶联免疫吸附测定(ELISA)检测肺组织匀浆中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-6的含量;二喹啉甲酸(BCA)比色法和瑞氏-吉姆萨染色法分别检测肺泡灌洗液中总蛋白的浓度、巨噬细胞和中性粒细胞数目;苏木精-伊红(HE)染色观察肺组织病理学改变。采用噻唑蓝(MTT)法检测不同浓度腺花素对RAW264.7细胞生长的影响。AnnexinV-FITC/PI双染法检测腺花素对RAW 264.7细胞凋亡的影响。免疫印迹法(Western blotting)检测RAW 264.7细胞和肺组织中Toll样受体4(TLR4)、MyD88、p-NF-κB、NF-кB、Bcl-2、Bax及β-Tubulin的表达。结果腺花素显著降低TNF-α、IL-1β、IL-6和肺泡灌洗液中总蛋白浓度及巨噬细胞和中性粒细胞的百分比,显著改善肺组织病理损伤;0~12μmol·L^(-1)腺花素对巨噬细胞RAW264.7生长无明显抑制作用;腺花素显著抑制脂多糖诱导的RAW 264.7细胞凋亡以及TLR4、MyD88、p-NF-кB、Bax的表达,促进Bcl-2的表达;显著抑制肺组织中TLR4、MyD88、p-NF-кB、Bax的表达,促进Bcl-2的表达。结论腺花素可改善ALI,其机制可能是抑制TLR4/MyD88/NF-κB炎症通路并减少细胞凋亡。

Objective To investigate the therapeutic effect and mechanism of adenanthin(Ade)on acute lung injury(ALI).Methods Thirty male C57BL/6 mice were randomly divided into 6 groups:normal control group(n=5 in each group),model control group(10 mg·kg^(-1) lipopolysaccharide),dexamethasone group(5 mg·kg^(-1) dexamethasone),and 3 different dose groups of Ade(5,10 and 20 mg·kg^(-1) Ade).After 6 hours of modeling,therapeutic drugs were given.After 18 hours,mass changes were recorded.The contents of TNF-α,IL-1βand IL-6 in lung tissue homogenate were determined by ELISA.The concentration of total protein,the numbers of macrophages and neutrophils were determined by BCA colorimetry and Ray-Giamsa staining in alveolar lavage fluid,respectively.The histopathological changes of lung were observed by H&E staining in lung tissue.MTT assay was used to detect the effect of different concentrations of Ade on the viability of RAW264.7 cells.AnnexinV-FITC/PI double staining was used to detect the effect of Ade on the apoptosis of RAW 264.7 cells.The expressions of TLR4,MyD88,p-NF-кB,NF-κB,Bcl-2,Bax andβ-tubulin were detected by Western blotting in RAW 264.7 cells and lung tissues.Results Ade significantly inhibited the TNF-α,IL-1βand IL-6 generation,and decreased the concentration of total protein as well as the percentage of macrophages and neutrophils in alveolar lavage fluid,and significantly improved the LPS-induced lung injury.Ade had no effect on the viability of RAW264.7 cells in the concentration range from 0 to 12μmol·L^(-1).Ade significantly inhibited LPS-induced apoptosis of RAW 264.7 cells and the expression of TLR4,MyD88,p-NF-κB and Bax along with increased expression of Bcl-2.Ade significantly inhibited the expressions of TLR4,MyD88,p-NF-кB,Bax and promoted the expression of Bcl-2 in lung tissues of ALI mice.Conclusion Ade alleviates macrophage apoptosis and improves ALI by inhibiting TLR4/MyD88/p-NF-кB inflammatory pathway.