Abstract:

Objective To investigate the role of cellular repressor of E1A-stimulated genes （CREG） in alleviating lipopolysaccharide （LPS）-induced sepsis-induced acute lung injury（S-ALI） by regulating the PTEN-induced kinase 1 （PINK1）/Parkin pathway to promote mitophagy. Methods Alveolar macrophage cell line MH-S was treated with LPS at concentrations of 1， 5， 10， and 15 μg/mL. Cell viability was detected using the Cell Counting Kit-8 （CCK-8） assay， while CREG protein and mRNA expressions were measured via Western blotting and quantitative reverse transcription-polymerase chain reaction （qRT-PCR）. The 5 μg/ml LPS concentration was selected for subsequent experiments due to its ability to induce the highest cell viability. MH-S cells were divided into four groups： Normal group， LPS group， LPS + pLNCX2-CREG group， and LPS + pSM2-siCREG group. Except for the Normal group， all other groups were exposed to 5 μg/mL LPS. The LPS + pLNCX2-CREG group and LPS + pSM2-siCREG group were transfected with pLNCX2-CREG plasmid and pSM2-siCREG plasmid， respectively， after LPS treatment， while the Normal and LPS groups received no transfection. Lysosomal activity was assessed using the Lyso-Tracker Red fluorescent probe. The expressions of cluster of differentiation （CD） 86 and CD206 were detected by flow cytometry. Enzyme-linked immunosorbent assay （ELISA） kits were used to measure the levels of interleukin （IL）-1β， IL-6， IL-10， C-reactive protein （CRP）， reactive oxygen species （ROS）， malondialdehyde （MDA）， and superoxide dismutase （SOD） in each group. Western blot and qRT-PCR were performed to determine the protein and mRNA expressions of CREG， PINK1， and Parkin. Results Compared with the 1 μg/mL LPS group， the MH-S cell viability in the 5 μg/mL LPS group was significantly increased （P < 0.05）. In contrast， cell viability was remarkably decreased in the 10 μg/mL and 15 μg/mL LPS groups compared with the 5 μg/mL LPS group， with the lowest viability observed in the 15 μg/mL LPS group （P < 0.05）. The protein and mRNA expressions of CREG were significantly higher in the 5 μg/mL LPS group than in the 1 μg/mL LPS group （P< 0.05）， but were notably reduced in the 10 μg/mL and 15 μg/mL LPS groups compared with the 5 μg/mL LPS group （P < 0.05）. Compared with the Normal group， the LPS group exhibited decreased lysosomal quantity， CD206 expression， SOD activity， and the protein/mRNA expressions of CREG， PINK1， and Parkin， along with increased CD86 expression， levels of IL-1β， IL-6， IL-10， CRP， ROS， and MDA （all P < 0.05）. Compared with the LPS group， the LPS + pLNCX2-CREG group showed significantly elevated lysosomal quantity， CD206 expression， IL-10 level， SOD activity， and the protein/mRNA expressions of CREG， PINK1， and Parkin， as well as reduced CD86 expression and levels of IL-1β， IL-6， CRP， ROS， and MDA （all P<0.05）. Conversely， compared with the LPS + pLNCX2-CREG group， the LPS + pSM2-siCREG group displayed decreased lysosomal quantity， CD206 expression， IL-10 level， SOD activity， and the protein/mRNA expressions of CREG， PINK1， and Parkin， along with increased CD86 expression and levels of IL-1β， IL-6， CRP， ROS， and MDA （all P < 0.05）. Conclusion Overexpression of CREG enhances lysosomal activity and exerts anti-inflammatory and antioxidant effects. These findings suggest that CREG alleviates LPS-induced sepsis-induced lung injury by activating the PINK1/Parkin pathway to promote mitophagy.

Key words: sepsis, acute lung injury, lipopolysaccharide, cellular repressor of e1a-stimulated genes, autophagy