Mechanism of CREG regulating PINK1/Parkin to promote mitophagy in sepsis-induced acute lung injury
CAO Liang
ZOU Fang
ZHANG Changhong
XU Kailun
ZHAO Jianqing
LI Jingqi
LIU Jianhua
TANG Zhanhong
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 quanti-tative 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 deter-mine 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 expres-sion,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 expres-sion,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 Over-expression of CREG enhances lysosomal activity and exerts anti-inflammatory and antioxidant effects.These find-ings suggest that CREG alleviates LPS-induced sepsis-induced lung injury by activating the PINK1/Parkin pathway to promote mitophagy.
Keywords：sepsisacute lung injurylipopolysaccharidecellular repressor of e1a-stimulated genesautophagy
Publication Date：2026-03-10
Online Publishing Date：2026-03-20(First online date of this platform, not the publication date of the document)
Pages：8( 861-868 )