Investigation of the contribution of RİPK1/RİPK3/MLKL necrosome to inflammatory hyperalgesia induced by lipopolysaccharide in mice

In this thesis, by using the selective receptor-interacting serine/threonine-protein kinase (RIPK) 1 inhibitor necrostatin (Nec)-1s, the contribution of RIPK1/RIPK3/mixed lineage kinase domain like pseudokinase; MLKL) necrosome to inflammatory hyperalgesia induced by lipopolysaccharide (LPS) was investigated. Mice were divided into 8 groups: (1) saline, (2) LPS, (3) saline and dimethyl sulfoxide, (4) saline and Nec-1s (0.01 mg/kg), (5) LPS and Nec-1s (0.001 mg/kg), (6) LPS and Nec-1s (0.01 mg/kg), (7) LPS and Nec-1s (0.1 mg/kg), and (8) LPS and Nec- 1s (1 mg/kg). Nec-1s (10 ml/kg) was administered simultaneously with saline (10 ml/kg) or LPS (10 mg/kg) intraperitoneally. Reaction time to thermal stimuli within 60 seconds was evaluated in mice that received either saline or LPS after 6 hours. At the end of the experiments, the brains and spinal cords of the mice were removed after they were killed. In tissue homogenate supernatants prepared from brains and spinal cords, expression of β-tubulin, toll- like receptor (TLR4), RIPK1, phosphorylated RIPK1, RIPK3, phosphorylated RIPK3, MLKL, phosphorylated MLKL, high-mobility-group-box (HMGB1), caspase-11 p20, p30-gasdermin D (GSDMD), interleukin (IL)-1β, semaphorin (SEMA) 3A, and myelin proteolipid protein (PLP) proteins was measured by using the immunoblotting method. It was observed that LPS-induce decrease in the response time of mice to thermal stimulation was prevented with Nec-1s only at a dose of 0.01 mg/kg. When Nec-1s was injected at the lowest effective dose (0.01 mg/kg), it prevented the increase induced by LPS in expression of TLR4, phosphorylated RIPK1, phosphorylated RIPK3, phosphorylated MLKL, HMGB1, caspase-11 p20, p30-GSDMD, IL-1β, and SEMA3A as well as decreased expression of myelin PLP. These results suggested that suppression of the RIPK1/RIPK3/MLKL necrosome-mediated necroptosis, caspase-11/GSDMD- mediated pyroptosis, and SEMA3A expression, in addition to increased expression of myelin PLP in the central nervous system contributes to the preventive effect of Nec-1s on inflammatory hyperalgesia mediated by the LPS/TLR4 pathway.