Study on the protective effect of reduced glutathione on pristane-induced diffuse alveolar hemorrhage in mice

LIAN Shaopan; ZHOU Ying; LU Conghua; SHI Chenjun; LIN Caiyu; LI Li; DAI Huanzi

doi:10.3969/j.issn.1674-3806.2026.02.11

Abstract：Objective To explore the protective effect and mechanism of reduced glutathione(GSH)on a mouse model of pristane-induced systemic lupus erythematosus-associated diffuse alveolar hemorrhage(SLE-DAH).Methods In vivo experiments:Female C57BL/6J mice aged 6 to 10 weeks were selected and used to construct a diffuse alveolar hemorrhage(DAH)model by intraperitoneal injection of 0.5 mL pristane.Subsequently,these mice were intraperitoneally injected with GSH on the third day,the 6th day,the 9th day and the 12th day after constructing the model to observe the gross changes in the lungs,pathological changes in lung tissues,and changes in body weight of the mice.The levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β)in the bronchoalveolar lavage fluid(BALF)of the mice were detected.In vitro experiments:Mouse lung epithelial(MLE)-12 cells were treated with β-CD-pristane and the morphology and density of the cells were observed,and the viability of the cells was assessed.The changes in gene expressions were analyzed by using miRNA-sequencing(mRNA-seq),and the cell proliferation and the level of reactive oxygen species(ROS)were detected after GSH intervention.Results After the treatment with pristane,the lungs of the mice showed the changes in different degrees of hemorrhage on the whole,and the percentage of gross lung hemorrhage in the lung tissues and DAH scores were significantly increased(P＜0.05),with disordered lung tissue structure,significant infiltration of inflammatory cells and significantly decreased body weight(P＜0.05),and the levels of TNF-α,IL-6,IL-1β in BALF were significantly increased(P＜0.05).After the treatment with β-CD-pristane,the morphology of MLE-12 cells changed,and their viability was significantly decreased(P＜0.05).The combination of RNA sequencing(RNA-seq)with Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis and Gene Ontology(GO)functional enrichment analysis suggested that β-CD-pristane might cause cell damage through glutathione metabolism pathways and inflammatory response.After the intervention with GSH,the proliferation of MLE-12 cells was restored(P＜0.05),and the intracellular ROS levels were significantly decreased(P＜0.05),and the percentage of gross lung hemorrhage in pristane-induced DAH mice and DAH scores were significantly decreased(P＜0.05),and the body weight was significantly increased(P＜0.05),and the levels of TNF-α,IL-6 and IL-1β in BALF were significantly decreased(P＜0.05).Conclusion GSH intervention can alleviate pristane-induced DAH and inflammatory response in mice.The mechanism of action may be associated with the inhibition of oxidative stress in alveolar epithelial cells and the promotion of proliferation of alveolar epithelial cells.