腺苷二醛调控甲基化代谢及H3K4me3修饰抑制NF-κB通路减轻脓毒症炎症与组织损伤OA

Objective To investigate the effects of adenosine dialdehyde(Adox)on sepsis-induced inflammatory response and tissue injury,and to analyze its regulatory roles in methylation metabolic homeostasis,H3K4me3 modification,and the NF-κB signaling pathway.Methods In vitro experiments:① Mouse mononuclear macrophage leukemia cells(RAW264.7)and human monocytic leukemia cells(THP-1)were subjected.CCK-8 assay was performed to determine the effects of Adox(2.5-40 µmol/L)on cell viability after 24 h of treatment in RAW264.7 and THP-1 cells to screen the safe dose range of Adox.② ELISA was conducted to assess the impact of various Adox concentrations(2.5,5,10,20,40 µmol/L)on lipopolysaccharide(LPS)-induced inflammatory cytokine release,and to examine the effect of 10 µmol/L Adox on the release of inflammatory cytokines at different time points following 100 ng/mL LPS stimulation.③ RAW264.7 and THP-1 cells were divided into a Medium group,a LPS100 group(stimulated with 100 ng/mL LPS for 4 h),and a LPS100+Adox group(co-treated with 10 µmol/L Adox and LPS for 4 h).ELISA was employed to measure TNF-α,IL-6,SAM and SAH levels in cell supernatants;Western blot was utilized to detect the protein expression of H3K4me3,SAHH and NF-κB/IκBα signaling pathway-related molecules;RT-qPCR was carried out to measure the mRNA expression of TNF-α and IL-6.Animal experiments:Male C57BL/6J mice(6 to 8 weeks old,weighing 18 to 22 g)were randomly assigned to a Sham group,a cecal ligation and puncture group(CLP,as a sepsis model),and a CLP+Adox group(intraperitoneal injection of 2 mg/kg Adox administered 4 h prior to CLP surgery).Samples were collected 6 h after modeling.ELISA was used to detect serum TNF-α and IL-6 levels in mice;HE staining and pathological scoring(0-12)were utilized to evaluate lung and spleen tissue injury;Western blot was performed to detect the expression of H3K4me3,SAHH,and NF-κB/IκBα signaling pathway-related proteins in lung and spleen tissues.Results In vitro experiments:CCK-8 results demonstrated that Adox had no obvious toxicity to RAW264.7 and THP-1 cells within the range of 2.5 to 40 µmol/L.ELISA revealed that Adox significantly reduced the released levels of TNF-α and IL-6 in a concentration-and time-dependent manner(P<0.01).Notably,10 µmol/L Adox significantly suppressed inflammatory cytokines without affecting cell viability,and was therefore selected for subsequent experiments.Furthermore,Adox treatment decreased SAM levels,increased SAH levels,and reduced the SAM/SAH ratio in RAW264.7 cell supernatants(P<0.01).Western blot analysis demonstrated that Adox significantly downregulated the protein expression of SAHH and H3K4me3 in RAW264.7 and THP-1 cells under inflammatory conditions(P<0.01),and inhibited the activation of the NF-κB signaling pathway.In vivo experiments:ELISA results showed that Adox significantly decreased serum TNF-α and IL-6 levels in CLP-induced septic mice(P<0.01).HE staining and pathological scoring demonstrated that Adox ameliorated lung and spleen injuries in septic mice.Western blot analysis indicated that Adox significantly reduced H3K4me3 levels and suppressed NF-κB/IκBα signaling pathway activation in the lung and spleen tissues of CLP mice.Conclusion Adox alleviates sepsis-related inflammatory response and tissue injury,and its mechanism may be related to the regulation of SAHH-mediated SAM/SAH metabolism,reduction of H3K4me3 modification levels,and inhibition of NF-κB signaling pathway activation.These findings suggest that targeting methylation metabolism and histone methylation modifications may provide a novel therapeutic strategy for intervening in sepsis-associated inflammation.