Objective: To evaluate the effects of glutamine (Gln) on macrophages cytokines release and heat shock protein 72 (HSP72) expression in vivo and in vitro, and explore the anti-inflammation mechanisms of Gln during sepsis.
Methods: In experiment one, mouse peritoneal macrophage cell line RAW264.7 cells were divided into 0, 0.5 and 8 mmol/L Gln groups, and cells and supernatants were harvested at 0, 1, 4, 12 and 24 hours after lipopolysaccharide (LPS) challenge. In experiment two, forty-five Kunming mice were randomized into sham-operation group (Sham), sepsis model group, and Gln group. Sepsis was induced by cecal ligation and puncture (CLP). Either Gln 0.75 g/kg (Gln group) or saline (Sham and model group) was administered immediately after CLP via tail-vein injection. Blood sample was collected at 6 hours, and macrophages were harvested by peritoneal lavage. Tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-10 contents in supernatant, serum and cell lysate were analyzed with enzyme-linked immunosorbent assay (ELISA), macrophages HSP72 expression was assessed with Western blotting.
Results: Gln promoted RAW264.7 cells to release TNF-alpha, IL-6 and IL-10 in a dose-dependent and time-dependent manner in vitro (P<0.05 or P<0.01), and significantly increased HSP72 expression in 8 mmol/L Gln group at 4 hours after LPS stimulation (both P<0.01). In vivo, in animals given Gln intracellular TNF-alpha and IL-6 levels were significantly lower than sepsis animals (P<0.01 and P<0.05), but there was no statistically significant difference in intracellular IL-10 levels among three groups. The serum levels of TNF-alpha in Gln group were significantly lower than in model group (P<0.05), while serum IL-6 and IL-10 levels were similar between two groups. Gln treatment led to significant HSP72 expression compared to model and Sham groups (both P<0.01).
Conclusion: Gln can promote inflammatory cytokines release from macrophages in vitro, which cannot be attenuated by HSP72 expression induced by Gln in LPS challenged RAW264.7 macrophages. Gln treatment significantly decreases intracellular TNF-alpha and IL-6 levels in vivo during sepsis. HSP72 expression increases after Gln treatment both in vivo and in vitro. These data implicate that HSP72 may not play a major role in attenuating the inflammatory response after Gln administration in sepsis.