Mannose Prevents Lipopolysaccharide-induced Acute Lung Injury In Rats

Background:Acute lung injury(ALI)is characterized by an acute inflammatory process in the airspaces and lung parenchyma,and lipopolysaccharide(LPS)has been recognized as a principal component causing ALI.In typical ALI,the alveolar spaces are filled with protein-rich edema fluid and inflammatory cells,which are mostly polymorphonuclear neutrophils(PMNs).PMNs are known to be important contributors to the pathogenesis of ALI.Thus,compounds which inhibit the marked infiltration of leukocytes,especially PMNs,into the lung are likely to attenuate LPS-induced ALI.Mannose is a simple hexose sugar with a molecular weight of 180.2,and it has been found to have anti-inflammatory effects.Mannose treatment inhibits the inflammatory reaction in wound healing and decreases leukocytes, especially neutrophils,in wound fluid.Mannose also inhibits the neutrophil oxidative burst,which plays an important role in inflammation.Mannose-6-phosphate,the mannose derivative,has been shown to inhibit adjuvant arthritis and have anti-inflammatory activity in wound healing.Aims:The aim of this study was to evaluate the preventive effects of mannose on the rat model of endotoxin induced ALI.The effects of the other three hexose sugars (glucose,fructose and galactose),which have the same molecular weight as mannose, were compared with the effects of mannose.Materials and methods:Intratracheal administration of LPS induced acute lung injury.Ten groups of Sprague-Dawley rats were used:1)the control group received an intratracheal instillation of saline,2)the LPS group received an intratracheal instillation of LPS(3 mg/kg),3-6)the mannose groups were injected i.v.with 15,45,135,and 405 mg/kg mannose,7-9)the glucose,galactose,and fructose groups were injected with different hexoses(135 mg/kg),and 10)the dexamethasone(DXM)group was injected with DXM(2 mg/kg).In groups 3-8,drugs were administered by intravenous injection in the tail vein 5 min before and 3 h after intratracheal instillation of LPS.Lung wet/dry weight ratio,permeability index(PPI),total leukocytes and polymorphonuclear neutrophils(PMNs)counts in bronchoalveolar lavage fluid(BALF),myeloperoxidase (MPO)and superoxide dismutase(SOD)activity,tumor necrosis factor(TNF)-αand interleukin(IL)-10 in lung and BALF were determined.Results:1.Mannose inhibited pulmonary edema and protein exudation induced by LPS.Intratracheal instillation of LPS into the rats increased the lung wet/dry weight ratio from 4.30±0.18 to 4.56±0.16(P＜0.01).This LPS-induced pulmonary edema was significantly inhibited by DXM(2 mg/kg)(P＜0.05)as well as by mannose at the doses of 135 and 405 mg/kg.The potency of mannose at a dose of 405 mg/kg was similar to that of DXM at a dose of 2 mg/kg.And galactose,glucose and fructose had no significant effect on wet/dry weight ratio.Mannose significantly inhibited the increase of PPI as compared with the LPS group.The inhibitory effect of mannose at the doses of 135(4.32±0.45)and 405 mg/kg(4.04±0.86)was greater than that of DXM at a dose of 2 mg/kg(4.37±0.88).In contrast,there was no detectable effect in glucose,galactose or fructose groups.2.Mannose inhibited pulmonary inflammatory cell infiltration induced by LPS.The inflammatory cells infiltration induced by LPS was significantly attenuated by 45 mg/kg or higher dosage of mannose in a dose-dependent manner,whereas glucose,galactose,or fructose at a dose of 135 mg/kg had no significant protective effect.There were significant differences between 135 mg/kg mannose group and the other three saccharides(135mg/kg)groups(p＜0.05).3.Mannose attenuated changes in MPO and SOD activity.MPO activity was increased by 26.3%in the lung tissue homogenate samples and 9.5%in BALF in the LPS group compared with the control group.Mannose inhibited LPS-induced MPO activity in a dose-dependent manner in both the lung and BALF,and the inhibitory effect of mannose at 405 mg/kg(1.09±0.21 u/g in lung, 75.43±4.46 u/L in BALF)is similar to the effect of DXM at a dose of 2 mg/kg(1.14±0.15 u/g in lung,74.24±7.23 u/L in BALF).LPS decreased SOD activity,whereas intravenously administered mannose dose-dependently attenuated the effect of LPS.4.Mannose inhibited production of TNF-αand IL-10.LPS induced a dramatic increase in the concentration of TNF-αin both lung homogenate and BAL fluid.Treatment with mannose or DXM attenuated this response,whereas glucose,galactose and fructose did not significantly attenuate the effect of LPS.The IL-10 level changed in line with TNF-αin the BALF.5.Mannose improved histological changes induced by LPS.An obvious hemorrhage,edema,thickened alveolar septum,formation of hyaline membranes and the infiltration of inflammatory cells in alveolar spaces were yielded in the LPS group.Mannose at a dose of 45 mg/kg or higher and DXM at a dose of 2 mg/kg markedly attenuated LPS-induced inflammation in the lung.Similarly,the mean lung injury score was increased from 0.2±0.1 in the control group to 4.8±0.4 in the LPS group.Mannose at the doses of 15,45,135,405 mg/kg significantly abrogated lung injury in a dose-dependent manner,as the mean lung injury scores were 4.6±0.3,3.8±0.4,2.5±0.4,and 1.9±0.3.However,the mean lung injury scores in the glucose,galactose,and fructose groups remained high.Conelution:Pre-treatment with an intravenous injection of mannose attenuated the extent of LPS-induced ALI in rats,as evaluated by the wet/dry weight ratio,leukocytes in BALF,PPI,cytokines and histological changes.Furthermore,the other three hexose sugars,glucose,galactose and fructose,had no significant effects on ALI,suggesting that the mechanisms by which mannose attenuates ALI may be due to specific sugar-binding properties. Background:Mannose is a simple hexose sugar with a molecular weight of 180.2.In our previous studies,it had been found to have anti-inflammatory effects on lipopolysaccharide(LPS)-induced acute lung injury in rats.Mannose pretreatment inhibited pulmonary edema and protein exudation induced by LPS,inhibited LPS-induced neutrophil infiltration and production of TNF-αand IL-10,and ameliorated lung pathology;mannose treatment attenuated the inflammatory reaction in wound healing and decreased leukocytes,especially neutrophils in wound fluid; mannose also inhibited the neutrophil oxidative burst,which played an important role in inflammation,but the mechanisms involved in the anti-inflammatory effect of mannose is little known.Although neutrophils plays an important role in the development of inflammation,macrophages have more important and central role in the start of the inflammation.The Inflammatory mediators such as TNF-αand IL-1 released by macrophages play a key role on the chamotaxis,adhesion,migration,infiltration,and activation of neutrophils.Mannose receptor(MR)is a kind of C-type lectin,which is predominantly expressed on the surface of macrophages.The CTLDs(C-type lectin-like domain,CTLD)of MR mediate binding of MR to sugars terminated in mannose,fucose or N-acetyl glucosamine.Dose mannose have anti-inflammatory activities on macrophage? Is MR involved in the anti-inflammatory effects of mannose?Aims:Our present aim is to evaluate the preventive effects of mannose on the lipopolysaccharide induced inflammation in murine macrophages,and to demonstrate involvement of MR in the anti-inflammatory effects of mannose.Material and methods:Peritoneal macrophages(PM)and alveolar macrophages(AM)were obtained by lavage using PBS.Two methods were used to block the function of MR,mannan competitive inhibition of the binding of MR and siRNA gene silencing of MR. Macrophages were randomly divided into these groups:control group,received DMEM media;LPS group,received LPS 1 ug/ml;mannose groups,received mannose (0.01,0.1,1,10,100mM)5 min before stimulation with LPS 1 ug/ml;mannan group, preincubated with mannan(2 mg/ml)30 min before,and received mannose 1mM 5 min before stimulation with LPS 1 ug/ml;siRNA group,macrophages were transfected with siRNA plasmid,and received mannose 1mM 5 min before stimulation with LPS 1 ug/ml.NF-κB in the nuclei were measured by Western blot after 1.5 h of incubation with drugs.The NRU(neutral red uptake)assay was performed to determine the phagocytic activity after 4 h of incubation.After 16 h of incubation with drugs,specific immunoreactivity for TNF-αand IL-1βin culture supematants was measured by ELISA;macrophages MR surface expression was analyzed by flow cytometry;TNF-α,IL-1β,and MR mRNA were detected by reverse transcriptase-PCR(RT-PCR);and the amount of MR antigen were determined by Western blotting.Results:1.SiRNA plasmid transfection in PM and AM. To examine the specific role of the mannose receptor in the effect of mannose, functional "knock-down" experiments were performed by use of sequence-specific, gene silencing with siRNA plasmid transfection target to MR.The efficient transfection ratio of PM and AM was demonstrated to be 40.5%and 43.7%, respectively,48 h post transfection under a fluorescence microscope.The MR mRNA of the transfected PM and AM was lowered to 56.1%and 59.7%of the nontransfected controls.Western-blot assay showed plasmid transfection significantly reduced PM and AM mannose receptor protein.Furthermore,flow cytometry determinated that the MR surface expression on PM and AM was significantly decreased to 56.1%and 59.7%of the control cells.The datum showed a successful transfection of SiRNA plasmid.2.LPS induced macrophages inflammation and down-regulation of MR.Phagocytic activity of PM and AM increased significantly following 1 ug/ml LPS stimulation for 4 h,and the neutral red particles in the macrophages increased markedly under the light microscope.LPS(1ug/ml)16h induced approximately 9-fold to 44-fold greater TNF-αand IL-1βproduction in macrophages culture supernatants compared with the control.Moreover,LPS 1ug/ml markedly stimulated the expression of TNF-αand IL-1βmRNA.Simultaneously,treatment with LPS caused a down-regulation of MR.A decrease of 31.2%and 17.4%of the MR surface expression on PM and AM was detected by flow cytometry with LPS stimulation for 16 hr.Similarly,western blotting analysis showed the MR protein expression in PM and AM was decreased significantly by LPS.MR mRNA of the LPS- stimulated PM and AM was also lowered to 67.3%and 66.2%of the controls,respectively.3.Mannose inhibited LPS-induced macrophages inflammation and up-regulated MR.NRU assay showed that the increased phagocytic activity of PM and AM by LPS was inhibited by mannose(0,0.01,0.1,1,10,100mM)in a dose-dependent manner. The phagocytic activity of PM and AM was inhibited by 24.2%and 11.8%, respectively,in cells pretreated with mannose 1mM compared with the LPS only groups.Mannose showed a dose-dependent inhibition on TNF-αand IL-1β production in culture supematants between 0.1 mM and 10mM both in PM and AM. A suppressive effect of mannose on TNF-αand IL-1βmRNA levels was also observed,and the dose-dependent inhibition was statistically significant.In PM pretreated with mannose 10mM,TNF-αand IL-1βmRNA was inhibited to 66.4%and 58.2%of that of LPS only group,and in AM pretreated with mannose 10mM,TNF-αand IL-1βmRNA was inhibited to 61.8%and 61.3%of that of LPS group.After a 16-hr pretreatment with mannose(0.1,1,10mM),flow cytometry,western blot and RT-PCR analysis showed the MR in mannose groups was upregulated in a dose-dependent manner in PM and AM.4.Mannan or mannose receptor gene silencing blocked the effects of mannose on LPS-induced macrophages inflammation.The inhibition of mannose(1mM)on LPS-induced phagocytic activity,TNF-αand IL-1βproduction in PM and AM,was blocked following mannan(2 mg/ml) incubation or siRNA gene silencing.Simultaneously,pretreatment of macrophages with mannan(2 mg/ml)or siRNA transfection,significantly prevented mannose-mediated MR up-regulation as determined by flow cytometry,western blot and RT-PCR analyses.And the MR expression in siRNA group was the lowest among all because of the down-regulation by LPS and siRNA gene silencing.5.Mannose inhibited the expression of NF-κB in the nuclei.The nuclear NF-κB/p65 protein level in the LPS stimulated PM or AM markedly increased to 2.4-fold or 4.5-fold greater when compared to the control.Pretreatment of macrophages with mannose(0.1,1,10 mM)significantly reduced the LPS induced expression of NF-κB/p65 in a dose-dependent manner.Mannan(2 mg/ml)or siRNA targeted against MR,significantly impaired the effect of mannose on reduction of NF-κB/p65 protein in the nuclei.Conclution:1.The results of the present study indicate that mannose is an effective inhibitor of LPS-induced macrophages inflammation,it can inhibit the increased phagocytic activity,TNF-αand IL-1βprotein and gene expression by blocking NF-κB activation in peritoneal macrophages and alveolar macrophages.Mannose appears to be a potential therapeutic agent for treating LPS-induced sepsis syndrome.2.LPS caused a down-regulation in the MR expression in peritoneal macrophages and alveolar macrophages.In contrast,mannose treatment caused an up-regulation in the MR expression as well as the anti-inflammatory effects.3.Mannan or mannose receptor gene silencing could block the effects of mannose on LPS-induced macrophages inflammation,which demonstrated that mannose inhibits LPS-induced inflammation in murine macrophages at least partially through mannose receptor.