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

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1.5×109CFU/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。

Objective:To investigate the expression of autophagy-related proteins,Beclin-1,P62,and microtubule-associated protein light chain 3 B(LC3 B),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/m L 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 collected 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 LC3 B in the lung tissues.Immunofluorescence assay was employed to observe the expression of LC3 B 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 LC3 B 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 LC3 B 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.