| Part1 the effects of isoflurane pretreatment on mice macrophage efferocytosis in vivo and in vitroObjective: To investigate the effects of isoflurane pretreatment on mice macrophage efferocytosis in vivo and in vitro.Methods: Mouse bone marrow-derived macrophage, alveolar macrophages and neutrophils are isolated and cultured in vitro. After pretreatment by different concentrations of 0.5MAC, 1MAC and 2MAC isoflurane for one hour, the ability of efferocytosis of mice bone marrowderived macrophages and alveolar macrophages were detected by immunofluorescence in vitro.In vivo two models were used in the study:Model one:There were three groups of mice: the control group, the BMDM group and the ISO group. Among the three groups of mice alveolar macrophages were depleted by clodronate liposomes treatment. Two days later, LPS(3.5mg/kg) was administrated to mice via intratracheal instillation. Five days later, mice were given to the same volume of saline at in CON group, BMDM cells(2×106) in BMDM group or BMDM cells(2×106) pretreated with isoflurane in BMDM group, respectfully, through intratracheal instillation. Two hours afterward, the bronchoalveolar lavage fluids of the mice were collected; macrophage phagocytosis was monitored with the methods of cytospin and Diff-quick staining.Model two:There were three groups of mice: the control group, the LPS group and the LPS+ISO group. The mice were given to the saline at the same volue in CON group, LPS(3.5mg/kg) in LPS group and in LPS+ISO group via intratracheal instillation. Three days later, the mice were exposed to isoflurane(1MAC) or air for one hour; two hours later macrophage phagocytosis was monitored.Results: In vitro, compare to the control group, treatment with 1MAC and 2MAC isoflurane significantly enhanced mouse bone marrow-derived macrophage efferocytosis(P<0.05). However, pretreatment of 0.5MAC isoflurane did not enhance mouse bone marrow-derived macrophage efferocytosis. Treatment with 1MAC isoflurane enhanced mouse alveolar macrophages efferocytosis(P<0.05). Same pretreatment of mouse bone marrow-derived macrophage also enhanced their efferocytic ability when transplanted into the lungs of the mice. There is significant difference between control group and treated group(P<0.05). Treatment of 1MAC isoflurane in vivo also increased the efferocytic ability of the aveolar macrophages(P<0.05).Conclusion: 1MAC isoflurane pretreatment enhanced the efferocytic ability of mouse bone marrow-derived macrophage and mouse alveolar macrophages in vivo and in vitro.Part 2 the study of mechanism of isoflurane pretreatment on mouse macrophages efferocytosisObjective: To investigate the effect of isoflurane inhalation anesthetics on Mer protein expression, the change of AMPK phosphorylation and ADAM17 protein translocation in BMDM; to explore the mechanism of isoflurane pretreatment on mice macrophage efferocytosis.Methods: BMDM cells were pretreated by isoflurane at different concentrations of 0.5MAC、1MAC and 2MAC, and total protein, membrane and cytoplasm protein were isolated with membrane cytoplasmic protein isolation kit. Then the change of Mer protein level was detected by western blotting; total protein, membrane and cytoplasmic protein of BMDM cells pretreated by 1MAC isoflurane were extracted at zero, 0.5, one, 1.5 and two hours, and the changes of Mer protein and ADAM17 were detected by western blotting. Total AMPK and phospho-AMPK in total lysates of BMDM cells pretreated by 1MAC isoflurane were observed. BMDM cells pretreated with AMPK kinase inhibitor Compound C(25μM) or si RNA were treated with 1MAC of isoflurane and cell lysates were collected, and change in Mer TK, ADAM17, AMPK and phospo-AMPK in the total lysates were detected again. Mer protein, membrane bound or cytoplasmic ADAM17 protein of BMDM cells was ralso detected. AMPK gene in BMDM cells was knockdown with RNA interference, the change of BMDM efferocytosis was observed.Results: The expression of Mer in total protein increased in BMDM cells pretreated with 1MAC or 2MAC isoflurane but there was no significant difference between cells untreated and those pretreated by 0.5MAC isoflurane. When compared with cells untreated, the expression of either total or membrane Mer protein in BMDM cells pretreated by 1MAC isoflurane increased at one, 1.5 and two hours. Although there was no significant difference in the expression of Mer in the cytoplasmic portion between cells untreated and pretreated, there was a large decrease of Mer protein in the cell culture media after isoflurane treatment. The AMPK phosphorylation increased in BMDM cells pretreated with 1MAC isoflurane for zero, 0.5, one, 1.5 and two hours. The expression of ADAM17 on membrane of BMDM cells pretreated with 1MAC isoflurane increased at one, 1.5 and two hours, but there was no significant difference in the expression of total ADAM17 between cells untreated and treated. AMPK kinase inhibitor inhibited the increase of Mer protein, the change of ADAM17 protein and AMPK phosphoryation in BMDM cells induced by isoflurane. Meanwhile, the same results were acquired by silencing the AMPK gene in BMDM cells with RNA interference. Silencing the AMPK gene in primary BMDM inhibited the increase of macrophage efferocytosis enhanced by isoflurane.Conclusion: isoflurane could induce AMPK phosphorylation, inhibit ADAM17 translocation, and lead to the increase of mer protein. Then macrophage efferocytosis was enhanced.Part 3 Effect of isoflurane on resolution of LPS-induced lung inflammation in miceObjective: To investigate the effect of 1MAC isoflurane pretreatment on resolution of LPS-induced lung inflammation in mice.Methods: The LPS-induced mice lung injury model was established as follows: Experiment design 1: Mice were divided into five groups: the CON group, the LPS group, the CLOD+LPS group, the CLOD+ LPS+BMDM-CON group and the CLOD+ LPS+BMDM-ISO group. Two days prior to LPS treatment, the mice were administered with or without CLOD via intratracheal instillation in order to deplete aveolar macrophages. On the day of lung injury induction, the mice were challenged with LPS(3.5mg/kg)or the same volume of saline through intratracheal instillation. Three days later, the mice were transplanted with or without BMDM cells(2×106cells/40 micro liter)that were pretreated with or without 1MAC of isoflurane for 1 hour or 40 microliter of saline(in the control group) via intratracheal instillation. Experiment design 2: There were three groups of mice: the CON group, the LPS group and the LPS +ISO group. The mice in the LPS group and the LPS+ISO group were challenged with LPS(3.5mg/kg) through intratracheal instillation to induce acute lung injury. Three days later, the mice of the LPS+ISO group were administered with isoflurane for one hour, and the LPS and control group were put under the same procedure with no isoflurane. Bronchoaveolar lavage fluids and lung tissues were collected on day one, and days three, five, seven, and nine post-LPS challenge. The numbers of neutrophils, protein level, and cytokines(TNF-α、IL-6、TGF-β and IL-10) in the bronchoaveolar lavage fluids, lung tissue MPO and wet to dry ratio of lung tissues were detected. HE stained lung tissue sections were observed 5 day after LPS stimulation.Results: The number neutrophils, protein level and cytokines(TNF-αand IL-6) in the broncoaveolar lavage fluids, lung tissue MPO, wet to dry ratio of the lung tissues and lung injury scores in the CLOD+ LPS+BMDM-CON group and in the CLOD+ LPS+BMDM-ISO group were lower than those in the CLOD+ LPS group on days five, seven, and nine post-LPS challenge. Compared to those in the CLOD+ LPS+BMDM-CON group, the number of neutrophils, protein level and cytokines(TNF-αand IL-6) in the bronchoaveolar lavage fluid, lung tissue MPO, wet to dry ratio of the lung tissues and lung injury scores in the CLOD+ LPS+BMDM-ISO group decreased significantly. TGF-β and IL-10 in the lavage fluids of the CLOD+ LPS+BMDM-CON group and the CLOD+ LPS+BMDM-ISO group were lower than those in the CLOD+ LPS group on days five, seven and nine post LPS challenge. TGF-β and IL-10 in the lavage fluids of the CLOD+ LPS+BMDM-ISO group decreased significantly when compared with the CLOD+ LPS+BMDM-CON group. In the second experiment model, the number of neutrophils, protein level and cytokines(TNF-αand IL-6) in the bronchoaveolar lavage fluids, lung tissue MPO, wet to dry ratio of the lung tissues and lung injury scores in LPS+ ISO group were lower than those in LPS group on days five, seven and nine post LPS challenge. TGF-β and IL-10 in the broncotracheal lavage fluids of the LPS+ ISO group increased significantly comparing to those in the LPS group. The decrease of TNF-αand IL-6 and increase of TGF-β and IL-10 in the bronchotracheal lavage fluid were diminished in the CLOD+ LPS+BMDM(AMPKsi RNA)-ISO group.Conclusion: BMDM pretreated by 1MAC isoflurane decreased the number of neutrophils, protein level and cytokines(TNF-αand IL-6) level in the bronchotracheal lavage fluids, lung tissue MPO, and wet to dry ratio of lung tissues in the LPS induced lung inflammation mice model. It also lowered the lung injury scores and increased the level of TGF-β and IL-10 in the bronchotracheal lavage fluids in LPS induced mice. Thus, isoflurane can promote the resolution of LPS induced mice lung inflammation. |
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