Protective Effcets And Mechanism Of PQ401 On Acute Peritonitic Mice Induced By LPS

Objective:PQ401 is a selective inhibitor of insulin-like growth factor-1 receptor (IGF-1R). The research was executed to investigate the anti-inflammatory effects of PQ401 on lipopolysaccharide (LPS)-induced peritonitis and explore the potential mechanism, which provide new strategy for peritonitis clinical treatment.Methods:Established acute peritonitis model of C57BL/6 mice induced by LPS. The mice were randomly divided into control group, model group, PQ401 group (25mg/kg,50mg/kg, 100mg/kg).12 hours later all mice were broken the neck to death.The blood and hepatic tissue collected from mice were separately extracted and frozen stock as serum and protein. The observation of mice physical condition was used to determinate the effects of PQ401 on the mice mental status and activity. The concentration of tumor necrosis factor-a (TNF-a) and interleukin (IL-1(3 and IL-6) in serum and hepatic tissue were examined by enzyme linked immunoabsorbent assay (ELISA), which evaluate the effects of PQ401 on the cytokines of acute peritonitis mice. The activity of alanine transaminase (ALT) and aspartate transaminase (AST) as well as the concentration of total bilirubin (TBIL) in serum measured by automatic biochemistry analyzer were used to investigate the effects of PQ401 on the liver of peritonitis mice. The expression of nuclear transcription-κB (NF-κB) was examined by Western blot for evaluating the anti-inflammatory mechanism of PQ401.In vitro experiments, the inflammatory model was established by LPS-induced RAW264.7 cells. After the treatment with different concentration of PQ401, the culture supernatant and cell protein were collected at different time. The concentration of TNF-a, IL-1β and IL-6 in supernatant were mesured by ELISA for evaluating the anti-inflammatory action of PQ401 on macrophage. The phosphorylation level of IGF-1R was determinated by Western bot for investigating the relationship between PQ401 and IGF-1R phosphorylation. Through two patterns of IGF-1 addition and transfection by IGF-IRa/p shRNA lentiviral particles, the relationship of IGF-1 R signal pathway and the anti-inflammatory effects of PQ401 was evaluated. The NF-κB signal pathway examined by Western blot were for studying the anti-inflammatory effects of PQ401 on NF-κB signal pathway.Results:In the LPS-induced acute peritonitis mice model, the model mice became lethargic and quiet, but the mice with the treatment of PQ401 (25 mg/kg,50 mg/kg,100 mg/kg) were found to greatly improve the general status of animals compared with the model group. The data show that PQ401 could dramatically reduce the concentration of TNF-α, IL-1β and IL-6 in serum and the concentration of TNF-α and IL-1β in hepatic tissue of acute peritonitic mice induced by LPS. PQ401 had no significant effects on the activity of ALT and AST as well as the concentration of TBIL in serum of mice. PQ401 had no significant effects on the expression of NF-κB in hepatocyte.In vitro experiments, the treatment of PQ401 (0.1 μM,1 μM,10 μM) could effectively decrease the concentration of TNF-α, IL-1β and IL-6 produced by LPS-induced inflammatory RAW264.7 cells at different time (6 h,12 h,24 h). And PQ401 could inhibit the phophorylation of IGF-1R in inflammatory cells. The activation of IGF-1R by IGF-1 addition or decreasing the expression of IGF-1R by IGF-IRα/β shRNA lentiviral particles transfection, had no effects on the anti-inflammatory action of PQ401. PQ401 had no effects on the NF-κB signal pathway induced by LPS.Conclusion:These research indicate that PQ401 has significant anti-inflammatory effects in vivo and vitro. However, the anti-inflammatory action of PQ401 has no relevance with the phosphorylation of IGF-1R, which imply that IGF-1R is not the efficacious target of anti-inflammatory action of PQ401. PQ401 has no effects on the NF-κB signal pathway induced by LPS, suggesting that PQ401 may play the anti-inflammatory role through other signal pathways.