The mechanism of roflumilast in treating sepsis-induced inflammation

XU Man; SHEN Yue; MI Yanyan

doi:10.3969/j.issn.1002-7386.2025.11.007

Abstract：Objective To explore the mechanism of roflumilast in treating sepsis-induced inflammation by negatively regulating Vesicular overexpressed in cancer prosurvival protein 1(VOPP1)via the intervention of miRNA-218 by the Janus kinase/signal transducer and activator of the transcription(JAK/STAT)signaling pathway.Methods A total of 54 mice were randomly divided into the normal group,model group and roflumilast group,with 18 mice in each group.Mice in the normal group were fed with a normal diet and treated with blank control.Sepsis model was built in mice of the model group and roflumilast group by the cecal ligation and puncture(CLP).They were intervened by daily intraperitoneal injection of 0.9％sodium chloride injection,and roflumilast group,respectively.After 7 days of intervention,mice were sampled.The biochemical detector counted the number of inflammatory cells in the lavage solution.The protein expressions of JAK and STAT-3 were detected by western blot.Aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels were detected by the enzyme-linked immunosorbent assay(ELISA).The mRNA expressions of IL-6 and TNF-α were detected by quantitative polymerase chain reaction(qPCR).The binding conditions of miRNA-218 and VOPP1 were determined by dual-luciferase reporter assay and RNA immunoprecipitation(RIP)assay.Results Compared with the normal group,the symptom score of sepsis mice after CLP significantly increased(P＜0.05).Compared with the model group,symptoms were delayed and improved in mice treated with roflumilast at 1.0 mg/kg(P＜0.05).Compared with normal group,the numbers of lymphocytes,neutrophils and monocytes in the model group and roflumilast group significantly increased(P＜0.05),which were significantly less in the roflumilast group than the model group(P＜0.05).The expression of miRNA-218 in sepsis mice was significantly lower than that in the normal group(P＜0.05),and pretreatment with roflumilast could upregulate miRNA-218.The protein expressions of JAK and STAT-3 were significantly lower in the normal group than the model group(P＜0.05).Their protein expressions were significantly higher in the model group and roflumilast group than the normal group(P＜0.05).The protein expressions of JAK and STAT-3 in the roflumilast group were significantly lower than the model group(P＜0.05).AST and ALT levels were significantly higher in the model group and the roflumilast group than the normal group(P＜0.05),which were significantly lower in the roflumilast group than the model group(P＜0.05).Peritoneal and plasma IL-6 and TNF-α levels were significantly higher in sepsis mice than the controls(P＜0.05).Roflumilast significantly reduced their levels than the model group(P＜0.05).Significantly higher mRNA levels of NF-κB and p38 MAPK,and lower mRNA level of IKB-α were detected in the model group and roflumilast group than the normal group(P＜0.05).Compared to the model group,the mRNA levels of NF-κB and p38 MAPK were significantly lower,while the mRNA level of IκB-α was significantly higher in the roflumilast group(P＜0.05).Transfection of miRNA-218 mimic significantly upregulated miRNA-218,and transfection of miRNA-218 inhibitor significantly downregulated miRNA-218(P＜0.05).Co-transfection of miRNA-218 mimic significantly decreased the luciferase activity in the VOPP1-wt,while co-transfection of miRNA-218 inhibitor significantly increased the luciferase activity in the VOPP1-wt(P＜0.05).Conclusion Roflumilast can alleviate sepsis-induced inflammation and liver injury,and improve the survival rate of multiple episodes of sepsis.