RESUMO
Objective To evaluate the regulatory role of acetylcholine receptor in muramyl dipeptide (MDP)-induced activation of Nod-like receptor 2/receptor-interacting protein 2 (2NLR2/RIP2) pathway in macrophages of mice.Methods RAW264.7 cells at the logarithmic growth phase were seeded in 12-well plates (density 1 × 106 cells/ml,2 ml/well),a total of 108 wells.The cells were randomly divided into 3 groups (n =36 each) using a random number table:control group (group C),MDP group (group M),and GTS-21 (a7nAChR specific agonist) group (group G).The cells were routinely cultured in group C.MDP with the final concentration of 10 μg/ml was added to the culture medium in group M.MDP with the final concentration of 10μg/ml and GTS21 with the final concentration of 50 μg/ml were added to the culture medium in group G.The cells were incubated for 24 h.At 1,6 and 24 h of incubation with MDP,12 wells were chosen and the cell suspension was obtained for measurement of NLR2 mRNA expression (by real-time fluorescent quantitative PCR),RIP2 expression (by Western blot),and concentrations of tumor necrosis factor-alpha (TNF-α) and high mobility group box-1 (HMGB1) in the culture media (by ELISA).Results Compared with group C,the levels of NLR2 mRNA,RIP2,TNFα and HMGB1 were significantly increased at each time point in group M (P ＜ 0.05).Compared with group M,the levels of NLR2 mRNA,RIP2,TNF-α and HMGB1 were significantly decreased at each time point in group G (P ＜ 0.05).Conclusion Acetylcholine receptor can suppress MDP-induced transduction of NLR2/RIP2 pathway in macrophages of mice.
RESUMO
Objective To establish a rat model of sepsis induced by muramyl dipeptide (MDP) after scald burn.Methods Fifty SPF male Sprague-Dawley rats,aged 2-3 months,weighing 200-250 g,were randomly divided into 3 groups:control group (group C,n =10),scald group (group S,n =10) and MDP group (n =30).The rats were subjected to a third-degree scald burn covering 20% of total body surface area in groups S and MDP.The rats were only exposed to 20 ℃ water in group C.MDP 5 mg/kg was injected via the femoral vein at 24 h after scald bum in group MDP.Arterial blood samples were collected at 1,6 and 24 h after MDP injection in group MDP,at 24 h after scald burn in group S,or at 24 h after exposure to 20 ℃ water in group C for blood gas analysis and for measurement of white blood cell (WBC) and platelet (Plt) counts,serum aminotransferase (ALT),aspartate transferase (AST),total bilirubin (TB),creatinine (Cr) and blood urea nitrogen (BUN) levels,creatine kinase isoenzyme-MB (CK-MB) activity,and plasma tumor necrosis factor-α (TNF-α),interferon-γ(IFN-γ),interleukin-6 (IL-6),IL-10 and high mobility group box 1 protein (HMGB-1) levels.The rats were sacrificed after collecting blood samples,and heart,liver,lung,and kidney specimens were obtained for microscopic examination of pathologic changes.The activity of myeloperoxidase (MPO) in lung tissues was measured.Another 90 male Sprague-Dawley rats were randomly divided into 3 groups and treated as the method previously described for record of the survival rate within 72 h.Results Compared with C group,the plasma IL-6,IL-10,IFN-γ and HMGB1 levels,WBC count,serum ALT,AST,and BUN levels and MPO activity were significantly increased,and the survival rate within 72 h was decreased in S group,and the plasma TNF-α,IL-6,IL-10,IFN-γand HMGB-1 levels,serum ALT,AST,TB,BUN,Cr and CK-MB levels,MPO activity,PaCO2 and BE value were significantly increased,and WBC and PLT counts,pH value,PaO2 and survival rate within 72 h were decreased in MDP group (P ＜ 0.05).Compared with S group,the plasma TNF-α,IL-6,IFN-γ and HMGB-1 levels,serum ALT,AST,TB,BUN,Cr and CK-MB levels,MPO activity,PaCO2 and BE value were significantly increased,and WBC and Plt counts,pH value,PaO2 and survival rate within 72 h were decreased in MDP group (P ＜ 0.05).The pathologic changes of heart,liver,lung and kidney were obvious in S and MDP groups and severer in MDP group.Conclusion After a third-degree 20% total body surface area scald burn,MDP induces excessive production of inflammatory cytokines accompanying with multiple organ damage ; thus the model of sepsis is successfully established after scald burn in rats.