Adenosine dialdehyde regulates methylation metabolism and H3K4me3 modification to inhibit the NF-κB pathway and attenuate sepsis-induced inflammation and tissue injury

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.

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.

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.

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.