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Abstract

Therapeutic options for the treatment of sepsis/septic shock are still limited and mortality remains unacceptably high. Besides autonomic, endocrine, hematological, and immunological alteration in the septic patient, many metabolic changes occur as a result of inflammation, which lead to the release of reactive compounds such as reactive carbonyl species. The reactive carbonyl metabolite and advanced glycation end-product precursor methylglyoxal was identified as an early biomarker for the diagnosis and outcome prediction in sepsis. The extent to which increases in methylglyoxal play a role in the course of the disease in sepsis and septic shock is not known yet. In order to test a potential adjuvant therapy that has already been shown to reduce advanced glycation end-products, oxidative stress and glucose homeostasis-regulating properties associated with diabetic damage, the histidine-rich dipeptide anserine was applied in the septic context. Within the here presented approach, methylglyoxal-derived carbonyl stress was identified as a major contributor to the disruption respectively reconstruction of the endothelial barrier forming proteins claudin 5 and zonula occludens-1 in a cell culture model, comparable to inflammatory insults following lipopolysaccharide and tumor necrosis factor administration. Methylglyoxal leads to a disturbed cell integrity as well as to an increased paracellular leakage of small and big molecules. These effects were not dependent on matrix metalloproteinase cleavage of the extracellular matrix. Anserine was able to prevent and to restore methylglyoxal-induced damage in vitro and showed a protective effect on the level of barrier forming proteins claudin 5 and zonula occludens-1. In an in vivo mouse model of sepsis, anserine accumulated stably for 36 h in several organs and showed protective effects via reducing methylglyoxal - advanced glycation end-product formation in lungs as well as kidneys and reduced capillary leakage and mortality. Furthermore, anserine improved survival probability to 60 % compared to vehicle controls (36.6 %) and improved the clinical outcome of the animals. On the immunological base, methylglyoxal leads to non-apoptotic and non-pyroptotic nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 inflammasome assembly, as well as an increase of the interleukin-1beta precursor and proinflammatory cytokines like interleukin-1beta, interleukin-6 and interleukin-8 in vitro. Methylglyoxal did not activate nuclear factor k-light-chain-enhancer of activated B cells subunit p65 assembly or the protein kinase B pathway, but rather activated mitogen-activated protein kinases c-Jun and p38 via different pathways. C-Jun-N-terminal kinase activation by methylglyoxal could be identified as receptor of advanced glycation end-product dependent, whereas p38 activation by methylglyoxal could persist even when the receptor of advanced glycation end-products was inhibited. The results propose a pathway in addition to direct toxic effects of methylglyoxal, by which methylglyoxal might causally contribute to the development as well as the severity of septic shock via an methylglyoxal - advanced glycation end-product/ receptor of advanced glycation end-product/ mitogen-activated protein kinase pathway functionally resulting in the activation of the nucleotide- 88 binding oligomerization domain-like receptor containing pyrin domain 3 inflammasome with a subsequent release of proinflammatory cytokines as well as a loss of tight junctions alongside. In summary, using a translational approach, sepsis was shown to be associated with methylglyoxal-derived carbonyl stress causally impacting outcome. Moreover, anserine was able to prevent and restore methylglyoxal-induced damages of the endothelial barrier and improved outcome in experimental sepsis. Therefore, anserine might be an innovative therapeutic option for the treatment of sepsis and septic shock.