Abstract
Baicalein has strong anti-inflammatory activity, but its efficacy and mechanism in pneumonia of mice is still unclear. Herein, RAW264.7 cells were infected with M. tuberculosis H37Rv (MOI 1:10) after pre-incubation with 50 μM baicalein, and the result found that baicalein incubation ameliorated the increase of bacterial load, and reduced the levels of ROS, IL-12p40, TLR2 protein, phosphorylated JNK protein and M1 polarization markers (iNOS and CD11c), and increased the levels of IL-10, M2 polarization markers (Arg1, Mrc1 and CD206) and NEDD4L protein in H37Rv infected-RAW264.7 cells. And lentivirus-mediated NEDD4L overexpression vector (LV-NEDD4L) transfection had the same effect as baicalein, while small interfering RNA target NEDD4L (si-NEDD4L) transfection or JNK pathway inhibitor IQ 3 treatment reversed the effects of baicalein, and ubiquitin inhibitor MG132 treatment reversed the effects of LV-NEDD4L. Co-immunoprecipitation (Co-IP) and TLR2 ubiquitination assays found that the binding of NEDD4L and TLR2 promoted the ubiquitination of TLR2. In vivo, C57BL/6 mice were nasal infected with H37Rv (1000 ± 150 CFU/mouse) to establish a mouse pneumonia model, and then administered with baicalein. The result found that baicalein improved H37Rv-induced pneumonia in mice. In conclusion, baicalein increased NEDD4L-mediated ubiquitination of TLR2, promoting macrophage M2 polarization and alleviating H37Rv-induced pneumonia.
Highlights
Mycobacterium tuberculosis (M. tb) does not develop immediately after infection, but manifests itself in a clinically asymptomatic state called the incubation period of tuberculosis (TB) infection
We found that compared with the control group, the pro-inflammatory cytokine IL-12p40 level, the anti-inflammatory cytokine IL-10 level, Reactive oxygen species (ROS) level and the mRNA levels of M1 polarization markers iNOS and CD11c were significantly increased (Figure 2A-D), and the mRNA levels of M2 polarization markers Arg1, Mrc1 and CD206 were significantly decreased (Figure 2E) in RAW264.7 cells in the M. tb H37Rv infection group
We found that compared with the control group, the expression level of NEDD4L protein was significantly reduced, and the expression levels of toll-like receptor 2 (TLR2) and phosphorylated Jun N-terminal kinase (JNK) proteins were significantly increased of RAW264.7 cells in H37Rv infection group (Figure 6A, B)
Summary
Mycobacterium tuberculosis (M. tb) does not develop immediately after infection, but manifests itself in a clinically asymptomatic state called the incubation period of tuberculosis (TB) infection. About 22 to 32% of the world’s population is in this state. It is reported that Chinese medicine can significantly alleviate the decline in lung function and the production of alveolar inflammatory factors caused by various stimuli (Huang et al, 2021). Baicalein is one of the flavonoids with the highest content in the traditional Chinese medicine Scutellaria, and it has strong anti-inflammatory activity (Dinda et al, 2017). It is reported that baicalin can relieve acute respiratory distress syndrome in rats by reducing the secretion of alveolar inflammatory factors and improving lung function (Bao et al, 2021). A report claimed that baicalein can protect mice from death caused by Streptococcus pneumoniae by reducing alveolar capillary membrane permeability, lung infiltration of inflammatory cells, inflammatory cytokine production and lung cell apoptosis. The effect of baicalein on M. tb has not been reported yet
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