Beclin-1、P62、LC3B在肺炎链球菌感染小鼠肺部中的表达变化

Changes in the expression of Beclin-1, P62，and LC3B in the lungs of mice infected with Streptococcus pneumoniae

摘要:

目的：探讨自噬相关蛋白Beclin-1、P62及微管相关蛋白轻链3B（microtubule-associated protein light chain 3B，LC3B）在肺炎链球菌感染小鼠肺部中的表达及意义。方法：生长至对数生长期的肺炎链球菌标准株D39用无菌PBS调节浓度为1.5×109 CFU/mL，采用鼻滴的方法对50只SPF级雌性Balb/c鼠（6~8 周，16~20 g）进行处理，分别在感染后0、6、12、24、48 h收集Balb/c小鼠支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）及肺组织，瑞氏-吉姆萨染色进行BALF细胞计数，HE染色观察肺组织病理学改变，Western blot 检测自噬相关蛋白Beclin-1、P62、LC3B在肺组织中的表达水平，免疫荧光观察LC3B在肺组织中的表达情况。结果：HE染色显示，小鼠鼻饲肺炎链球菌后12 h及24 h后肺组织内出现大量炎症细胞浸润。BALF中总细胞数量及中性粒细胞数量于感染后6 h开始增高[（1.280±0.506） vs. （6.272±1.312），P=0.000；（0.000±0.000） vs. （3.456±1.308），P=0.000]，在12 h时达到高峰[（1.280±0.506） vs. （19.456±0.997），P=0.000；（0.000±0.000） vs. （15.040±1.012），P=0.000]，48 h后基本恢复正常水平[（1.280±0.506） vs. （3.520±0.933），P=0.005；（0.000±0.000） vs. （0.128±0.175），P=0.835]。Western blot结果显示：自噬相关蛋白Beclin-1于感染后6 h开始增加[（0.626±0.078） vs. （0.921±0.093），P=0.002]，48 h时达到最高水平[（0.626±0.078） vs. （1.884±0.158），P=0.000]；自噬相关蛋白P62于感染后6 h开始减少[（2.915±0.094） vs. （2.275±0.269），P=0.000]，48 h时降低至最低水平[（2.915±0.094） vs. （1.178±0.142），P=0.000]；自噬相关蛋白LC3B于感染后12 h开始升高[（0.747±0.197） vs. （1.429±0.318），P=0.006]，24 h时达到高峰[（0.747±0.197） vs. （2.002±0.451），P=0.000]，48 h后降低[（0.747±0.197） vs. （1.563±0.501），P=0.002]。免疫荧光显示：小鼠感染后12 h与24 h，肺组织中LC3B蛋白表达水平增加。结论：肺炎链球菌感染小鼠肺部后激活自噬的发生，且自噬在免疫调节过程中发挥着重要作用。

Abstract:

Objective：To investigate the expression of autophagy-related proteins，Beclin-1，P62，and microtubule-associated protein light chain 3B（LC3B），in the lungs of mice infected with Streptococcus pneumoniae. Methods：A standard strain of Streptococcus pneumoniae（D39） in the logarithmic growth phase was adjusted to a concentration of 1.5×109 CFU/mL by sterilized phosphate-buffered saline；then the resulting solution was administered to 50 specific pathogen-free female Balb/c mice aged 6-8 weeks and with a weight of 16-20 g by intranasal instillation. The bronchoalveolar lavage fluid（BALF） and lung tissues of the Balb/c mice were col－lected at 0 h，6 h，12 h，24 h，and 48 h after infection. Cells in the BALF were counted after Wright-Giemsa staining；pathological changes in the lung tissues were evaluated after hematoxylin-eosin（HE） staining. Western blot was used to determine the expression levels of autophagy related proteins Beclin-1，P62，and LC3B in the lung tissues. Immunofluorescence assay was employed to observe the expression of LC3B in the lung tissues. Results：HE staining showed massive inflammatory cell infiltration in the lung tissues of the mice at 12 h and 24 h after intranasal instillation of Streptococcus pneumoniae. The total cell count and neutrophil count in the BALF increased significantly at 6 h after infection（6.272±1.312 and 3.456±1.308，respectively，both P=0.000），reached a peak at 12 h（19.456±0.997 and 15.040±1.012，respectively，both P=0.000），and decreased to a normal level at 48 h（3.520±0.933 and 0.128±0.175，P=0.005 and 0.835，respectively），as compared with the baseline levels of 1.280±0.506 and 0.000±0.000，respectively. Western blot results showed that the autophagy-related protein Beclin-1 increased from 6 h after infection（0.921±0.093，P=0.002） and reached a peak at 48 h（1.884±0.158，P=0.000） compared with the baseline level of 0.626±0.078；the autophagy-related protein P62 decreased from 6 h after infection（2.275±0.269，P=0.000） and decreased to a trough level at 48 h（1.178±0.142，P=0.000） compared with the baseline level of 2.915±0.094；the autophagy-related protein LC3B increased from 12 h after infection（1.429±0.318，P=0.006），reached a peak at 24 h（2.002±0.451，P=0.000），and decreased after 48 h（1.563±0.501，P=0.002），as compared with the baseline level of 0.747±0.197. Immunofluorescence assay showed that the expression of LC3B protein increased at 12 h and 24 h after infection. Conclusion：After infecting the lungs of mice，Streptococcus pneumoniae activates autophagy，which plays an important role in the immune modulation in mice.