Abstract:

Objective To investigate the effects of translocator protein （TSPO） ligand XBD173 on the pulmonary inflammatory response and M1 polarization of macrophages of the mice induced by cigarette smoke extraction （CSE）， and to clarify their realted molecular mechanisms. Methods A total of 18 adult male C57BL/6 mice were randomly divided into control group， CSE group，and CSE+XBD173 group.The mice in CSE group were intranasally given 20 μL CSE，and the mice in CSE+XBD173 group were given the same dose of CSE and intraperitoneally injectied with 10 mg·kg^－1 XBD173. The pathomorphology of lung tissue of the mice in various groups was observed by HE staining and PAS staining. The RAW264.7 macrophage were cultured and divided into control group， CSE group， and CSE + XBD173 group. The CSE administration concentration was 1%， the XBD173 administration concentration was 4 mg·L^－1， and the cells in control group were given the same volume of dimethyl sulfoxide；after 18 h of stimulation， the expression levels of CD80， CD86， inducible nitric oxide synthase （iNOS）， reactive oxygen species （ROS） in the RAW264.7 cells，and the apoptotic rates of the RAW264.7 cells in various groups were detected by flow cytometry； Western blotting method was used to detect the expression levels of nuclear factor-κB/p65 （NF-κB/p65） and phosphorylated NF-κB/p65 （p-NF-κB/p65）proteins in the cells in various groups. The siRNA was used to knock down the TSPO expression in the RAW264.7 cells in various groups， and the expression levels of interleukin-6 （IL-6） and tumor necrosis factor-α（TNF-α） mRNA in the cells in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） method. Results The animal experiment results showed that compared with control group， the number of inflammatory cells in lung tissue of the mice in CSE group was increased significantly and the bronchial wall was thicker；compared with CSE group， the number of inflammatory cells in lung tissue of the mice in XBD173+CSE group was decreased and the bronchial wall was thinner. The cell experiment results showed that compared with control group， the expression levels of CD80， CD86， iNOS， and ROS in the RAW264.7 cells in CSE group were increased（P<0.05）， the apoptotic rate was increased（P<0.05）， the expression levels of IL-6 and TNF-α mRNA were increased（P<0.05），and the expression level of p-NF-κB/p65 protein was significantly increased（P<0.05）， while the expression level of NF-κB/p65 had no significant difference （P>0.05）； compared with CSE group， the expression levels of CD80， CD86， iNOS， and ROS in the RAW264.7 cells in CSE+XBD173 group were decreased（P<0.05）， the apoptotic rate was decreased（P<0.05）， the expression levels of IL-6 and TNF-α mRNA were decreased（P<0.05）， and the expression level of p-NF-κB/p65 protein was decreased significantly（P<0.05）. After the expression of TSPO protein in the RAW264.7 cells was knocked down by siRNA， the expression levels of IL-6 and TNF-α mRNA in the RAW264.7 cells among various groups had no significant differences（P>0.05）. Conclusion XBD173 can alleviate the inflammatory response of lung tissue of the mice and inhibit the M1 polarization of the macrophage induced by CSE， and its mechanism may be related to the NF-κB protein.

Key words: Translocator protein, XBD173, Cigarette smoke extraction, Macrophage, Nuclear factor-κB