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

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.