TLR9-MyD88-TRAF6 signaling mediates mitochondrial DNA-induced systemic inflammatory response after radiofrequency ablation of hepatic hemangioma, with Merlin as a downstream participant.

OBJECTIVE

Radiofrequency ablation (RFA) is an effective minimally invasive treatment for hepatic hemangioma, but systemic inflammatory response (SIR) syndrome-associated complications should be considered. This study aimed to explore the mechanism and potential targets in SIR caused by RFA of hepatic hemangioma.

METHODS

Murine orthotopic hepatic hemangioma model with RFA treatment and heme-stimulated human lung microvascular endothelial cells (HLMVECs) were utilized for investigations. Colocalization staining was conducted to observe mitochondrial DNA (mtDNA) oxidation damage and the colocalization of TLR9, MyD88 and TRAF6. Co-IP was conducted to test the interactions among TLR9, TRAF6 and MyD88. Inflammatory cytokines were detected by ELISA. VBIT-4 was utilized to inhibit the release of mtDNA.

RESULTS

Along with the SIR caused by RFA, there are mitochondrial swelling and damage, increased release of mtDNA and increased TLR9 expression. Inhibiting mtDNA release using VBIT-4, generation of inflammatory cytokines IL-1β, IL-18, IL-6 and TNF-α significantly reduced and TLR9 expression was reduced. Further rescue experiment by overexpressing TLR9 counteracted the inhibition role of VBIT-4 pretreatment on inflammatory cytokines generation. In hepatic hemangioma mice with RFA treatment, injection of sh-TLR9 could alleviate the development of SIR, manifested as markedly reduced inflammatory cells (lymphocyte, monocyte and neutrophil) and inflammatory cytokines. Mechanistically, TLR9 interacted with MyD88 and TRAF6, and TLR9 silencing attenuated the SIR in association with the changes of Merlin phosphorylation.

CONCLUSION

This study demonstrated the involvements of mtDNA/TLR9 mediated inflammatory response in SIR of hepatic hemangioma following RFA. Targeting TLR9 might be an available strategy to prevent SIR in RFA of hepatic hemangioma.