清瘟败毒饮抑制TLR4/NF-κB通路调控小鼠肺泡巨噬细胞自噬减轻脓毒症肺损伤的实验研究

Qingwen Baidu Decoction Regulated Mouse Alveolar Macrophages Autophagy to Reduce Sepsis Induced Lung Injury via Inhibiting TLR4/NF-κB Signaling Pathway： an Experimental Study

免费下载全文查看/发表评论下载PDF阅读器

DOI：10.7661/j.cjim.20220815.320

中文关键词: 清瘟败毒饮脓毒症肺损伤肺泡巨噬细胞 Toll样受体4 核因子-κB 自噬

英文关键词:Qingwen Baidu Decoction sepsis induced lung injury alveolar macrophage Toll-like receptor 4 nuclear factor-κB autophagy

基金项目:湖南省中医药科研计划项目青年基金（No.2021175）；湖南省卫健委一般项目（No.202117011236）；湖南中医药大学校级科研联合基金一般项目（No.2020XJJJ041）

作者	单位
易琼,戴飞跃,王建湘,丁灿,廖杨,张彬彬,蔡鹏	1.湖南中医药大学第一附属医院重症医学科（长沙 410007）；2.湖南中医药大学中西医结合学院（长沙 410208）

摘要点击次数: 281

全文下载次数: 228

中文摘要:

目的探讨清瘟败毒饮调控小鼠肺泡巨噬细胞自噬减轻脓毒症肺损伤的机制。方法将60只 C57BL/6小鼠按随机数字表法分为空白组、模型组、清瘟败毒饮低、中、高剂量组及地塞米松组，每组10只。采用腹腔注射脂多糖（LPS）建立脓毒症肺损伤动物模型。清瘟败毒饮各剂量组每天分别予607.75、1 215.5、2 431 mg/kg灌胃，每日1次；地塞米松组予尾静脉注射地塞米松1.3 mg/kg，每日1次；空白组予以等量生理盐水腹腔注射，模型组予以腹腔注射生理盐水，干预时间均为1周。实验结束时，用HE染色观察小鼠肺组织的病理学改变，计算肺组织病理学评分，取肺组织称重测定湿重/干重比，蛋白免疫印迹法（Western Blot）检测小鼠肺组织中Toll样受体4（TLR4）、核因子-κB（NF-κB）、核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）、微管相关蛋白1轻链3-Ⅱ（LC3-Ⅱ）、自噬相关蛋白beclin-1的蛋白表达水平，电子显微镜观察肺泡巨噬细胞超微结构。结果光镜示空白组小鼠肺组织肺泡壁光滑，结构清晰完整，肺泡腔无渗出。模型组小鼠肺泡腔内大量红细胞和炎性细胞浸润，肺泡间隔及肺泡壁增厚，部分肺泡腔塌陷，肺组织病理学评分和肺组织湿重/干重比较空白组明显升高（P<0.01）。与模型组比较，清瘟败毒饮各剂量组及地塞米松组小鼠肺组织水肿和炎性浸润均减轻，肺组织结构破坏改善，肺组织病理学评分和肺组织湿重/干重比明显降低（P<0.05，P<0.01）。与清瘟败毒饮低剂量组比较，清瘟败毒饮中、高剂量组和地塞米松组的小鼠肺组织水肿和炎性浸润进一步减轻，肺组织结构破坏改善，肺组织病理学评分和肺组织湿重/干重比明显降低（P<0.05，P<0.01）。透射电镜示空白组肺泡巨噬细胞超微结构正常，模型组肺泡巨噬细胞皱缩、胞质饱满度降低，游离面微绒毛变短、稀疏、排列不整齐，胞质内很少有自噬小体形成，线粒体嵴消失，板层小体破溃，可见细胞核固缩、核碎裂及核溶解。与模型组比较，清瘟败毒饮各剂量组和地塞米松组的肺泡巨噬细胞形态及细胞内超微结构均明显改善。与空白组比较，模型组TLR4、NF-κB、NLRP3蛋白表达显著增加（P<0.01），LC3-Ⅱ、beclin-1蛋白表达显著降低（P<0.01）。与模型组比较，清瘟败毒饮各剂量组及地塞米松组TLR4、NF-κB、NLRP3蛋白表达显著降低（P<0.05，P<0.01），LC3-Ⅱ、beclin-1蛋白表达显著增加（P<0.05，P<0.01）。与清瘟败毒饮低剂量组比较，清瘟败毒饮中、高剂量组及地塞米松组TLR4、NF-κB、NLRP3的蛋白表达显著降低（P<0.05，P<0.01），LC3-Ⅱ、beclin-1的蛋白表达显著增加（P<0.05，P<0.01）。结论清瘟败毒饮对脓毒症小鼠肺损伤有保护作用，其机制可能是通过抑制TLR4/NF-κB信号通路，增加小鼠肺泡巨噬细胞自噬来实现的。

英文摘要:

Objective To study the mechanism of Qingwen Baidu Decoction （QWBDD） in regulating autophagy of mouse alveolar macrophages and alleviating sepsis induced lung injury. Methods A total of 60 C57BL/6 mice were divided into blank group，model group，low， medium， high dose QWBDD groups， and dexamethasone group （DXM） by random digit table， 10 mice in each group. The animal model of sepsis induced lung injury was established by intraperitoneal injection of lipopolysaccharide （LPS）. QWBDD at 607.75， 1215.5， 2431.0 mg/（kg·d） was respectively administered to mice in corresponding groups by gastrogavage， once per day. DXM at 1.3 mg/（kg·d） was injected to mice in DXM group from the tail vein， once per day. Mice in the blank group were administered with the same amount of normal saline by gastrogavage. Mice in the model group were intraperitoneally injected with normal saline. All intervention course was 1 week. By the end of the experiment， HE staining was used to observe the pathological changes of lung tissues. The pathological scores of lung tissues were calculated. The wet weight/dry weight （W/D） ratio of the lung tissue was weighed. Western Blot was used to detect protein expressions of Toll - like receptor 4（TLR4）， nuclear factor-κB （NF-κB）， nucleotide binding oligomerization domain like receptor protein 3 （NLRP3）， microtubule-associated protein 1 light chain3 -Ⅱ （LC3-Ⅱ），and beclin-1 in mouse lung tissues. The ultrastructure of alveolar macrophage was observed under electron microscope. Results The alveolar wall of the lung tissue in the blank group was smooth， the structure was clear and complete，with no exudation in the alveolar cavity. In the model group， numerous erythrocytes and inflammatory cells infiltrated in the alveolar cavity， the alveolar septum and alveolar wall were thickened， parts of the alveolar cavity were collapsed， and the lung pathological score and W/D significantly increased than those in blank group （P<0.01）. Compared with the model group， the alveolar tissue edema and inflammatory infiltration were attenuated， the destruction of alveolar tissue structure was alleviated， and the lung pathological score and W/D significantly decreased in each dose QWBDD group and DXM group（P<0.05， P<0.01）. Compared with low dose QWBDD group， edema and inflammatory infiltration of the alveolar tissue were further attenuated in medium and high dose QWBDD groups， the destruction of alveolar tissue structure was alleviated，and the lung pathological score and W/D were significantly lowered （P<0.05， P<0.01）. The ultrastructure of alveolar macrophage in the blank group was normal. The alveolar macrophage in the model group shrank，the plumpness of cytoplasm decreased，the microvilli on the free surface became shorter and sparser， and were irregularly arranged. There were few autophagy bodies in the cytoplasm， the mitochondrial cristae disappeared， the lamellar bodies collapsed. Cell nuclear pyknosis， nuclear fragmentation， and nucleolysis were observed. Compared with the model group， the morphology and intracellular ultrastructure of alveolar macrophages in each dose QWBDD group and DXM group were significantly improved. Compared with the blank group， the protein expressions of TLR4，NF-κB，NLRP3 significantly increased （P<0.01）， protein expressions of LC3-Ⅱ and beclin-1 significantly decreased （P<0.01） in the model group. Compared with the model group， protein expressions of TLR4，NF-κB，NLRP3 decreased significantly in each dose QWBDD group and DXM group （P<0.05，P<0.01）， protein expressions of LC3 -Ⅱ and beclin-1 significantly increased （P<0.05，P<0.01）. Compared with low dose QWBDD group，the protein expressions of TLR4，NF-κB，NLRP3 significantly decreased （P<0.05，P<0.01）， and protein expressions of LC3 -Ⅱ and beclin-1 significantly increased （P<0.05，P<0.01） in medium/high dose QWBDD and DXM group. Conclusion QWBDD had protective effect on sepsis induced lung injury mice， and its mechanism might increase autophagy of mouse alveolar macrophages via inhibiting TLR4/NF-κB signaling pathway.

关闭