Protective Effects And Mechanisms Of Lipoxin On Endotoxin-induced Acute Lung Injury

Partâ… The effects of lipoxin on calcium signal transduction and production of reactive oxygen species in macrophages induced by LPSObjective To investigate the effects of lipoxin on calcium signal transduction and production of reactive oxygen species in macrophages induced by lipopolysaccharide (LPS). Methods Macrophages were randomly assigned to one of the following six groups:â‘ control group;â‘¡LPS group;â‘¢Thapsigargin group;â‘£Lipoxin + LPS group;⑤Lipoxin + Thapsigargin group;â‘¥2-Aminoethoxydiphenylborate + Thapsigargin group. The intracellular [Ca²⁺]_i was analyzed by confocal laser microscopy. The production of ROS was assayed by flow cytometry. Results Both LPS and Thapsigargin induced an increase in intracellular [Ca²⁺]_i, either in the presence or absence of extracellular Ca²⁺ in murine macrophages. The Ca²⁺ signal was sustained in the presence of external Ca²⁺ and only initiated a mild and transient rise in the absence of external Ca²⁺. SOC channel inhibitor 2-APB completely suppressed the Ca²⁺ entry signal evoked by thapsigargin and suppressed approximately 93% of the Ca²⁺ entry signal evoked by LPS. The increase in intracellular [Ca²⁺]_i was associated with increased ROS production which was completely abolished in the absence of extracellular Ca²⁺ or in the presence of SOC channel inhibitors 2-APB or lipoxin. Conclusions These findings indicate that the LPS-induced intracellular [Ca²⁺]_i increase depends on the Ca²⁺ entry through SOC channels, and lipoxin inhibits Ca²⁺ influx and ROS production through SOC channels in murine macrophages induced by LPSPartâ…¡The effects of lipoxin on expression of TNF-αconverting enzyme in RAW264.7 macrophages induced by LPSObjective To observe the effects of lipoxin on expression of TNF-αconverting enzyme in RAW264.7 macrophages induced by LPS. Methods RAW264.7 macrophages were cultured in vitro with 1-2ug/ml lipopolysaccharide in the absence or presence of lipoxin at different concentrations for 6 and 24 hours, then the supernatant was collected for measuring TNF-αby ELISA kit and the expressions of TNF-αand TNF-αconverting enzyme were measured by semi-quantitative RT-PCR. Western blotting was applied to detect the expression of TNF-αconverting enzyme. The expressions of membrane-bound TNF-αprotein were assessed by flow cytrometry. Results Lipoxin inhibited the gene and protein expression of TNF-α, lipoxin inhibited up-regulation of TACE protein expression but did not inhibit TNF-αconverting enzyme mRNA expression. Conclusions Lipoxin inhibits the release of TNF-αthrough inhibition the protein expression of TNF-αconverting enzyme in RAW264.7 macrophages. Partâ…¢Protective effect of lipoxin on LPS-induced acute lung injury in miceObjective To investigate whether lipoxin could attenuate LPS induced acute lung injury in mice. Methods All of the animals were randomly assigned to one of six groups (n = 6 per group). In the sham-vehicle group, mice were treated with 0.9% saline 60 mins after they were challenged with saline. The sham-ATL group was identical to the sham-vehicle group except that ATL (0.7 mg/kg, intravenously) was administered instead of vehicle. The sham-ZnPP group was identical to the sham-vehicle group except that Zinc protoporphyrin IX (ZnPP, 25 mg/kg intravenously) was administered instead of vehicle. In the LPS-vehicle group, mice were treated with vehicle 60 mins after they were challenged with LPS. The LPS-ATL group was identical to the LPS-vehicle group but received ATL (0.7 mg/kg, intravenously) instead of vehicle. The ZnPP-ATL-LPS group was identical to the LPS-ATL group, but ZnPP (25 mg/kg intravenously) was administered 30 mins before ATL. Results Inhalation of LPS increased inflammatory cell counts, TNF (Tumor Necrosis Factor)-αand protein concentration in BALF and also induced lung histological injury and edema. Post-treatment with ATL inhibited TNF-α, nitric oxide and malondialdehyde production, with the outcome of decreased pulmonary edema, lipid peroxidation and the infiltration of neutrophils in lung tissues. In addition, Western blot and immunohistochemical analysis revealed that ATL promoted the formation of HO-1 in the lung tissues. Heme oxygenase-1(HO-1) activity was also increased in the lung tissues after ATL stimulation. The beneficial effects of ATL were abolished by ZnPP. Conclusions This study demonstrates that post-treatment with ATL significantly reduces LPS-induced acute lung injury in mice. HO-1 plays an essential role in the anti-inflammatory and pro-resolution bioactions of lipoxin.