Objective To investigate the expression of granulysin ( GNLY) in lung of rats with acute lung injury ( ALI) stimulated with lipopolysaccharide ( LPS) . Methods Thirty-six healthy adult Wistar rats were randomly divided into a normal control group and a LPS group, with 18 rats in each group. LPS ( 4 mg/kg) was given intraperitoneally in the LPS group to induce ALI. The same amount of normal saline was given in the control group. The rats were randomly assigned to three subgroups ( n = 6) to be sacrificed respectively at 6, 18, and 30 hours after intraperitoneal injection. Wet/dry lung weight ratio ( W/D) and pathological changes of the lung were observed. The expression of GNLY in lung tissue was assayed by immunohistochemistry. Results In the LPS group, the W/D ratio was higher than that of the control group at each time point ( P lt;0. 05) and there were a large number of inflammatory cells infiltration and edema in interstitial spaces which suggested ALI. Compared with the control group, the expression of GNLY in the LPS group was significantly increased at all time points ( P lt;0. 05) . Conclusion GNLY may participate in ALI inflammatory process, which might play a role in preventing infection induced ALI.
Objective To investigate the influence of lipopolysaccharide(LPS) on the proliferation and collagen synthesis of normal human skin fibroblasts so as to elucidate its relation with skin wound healing. Methods Fibroblasts wereisolated and cultured in vitro, and then exposed to different doses of LPS(0.005, 0.010, 0.050, 0.100, 0.500, and 1.000 μg/ml) from E.coli055∶B5 respectively. Then the absorbance (A) value of fibroblasts was determined with the colorirneteric thiazolylblue (MTT) assay, and the cell number was counted under inverted phase contrast microscope from the 1st day to the 9th day after LPS administration, and collagen synthesis of fibroblasts in culture medium was measured with the method of pepsin digestion after incorporation of 3Hproline into stable, single-layered, confluent fibroblasts at 7 days after LPS administration. Results Compared with control group, A value increased with the increasing concentration of LPS (0.005 μg/ml 0.500 μg/ml) and LPS of 0.100 μg/mlgroup had the best effect. The difference was remarkable from the 5th day to the 9th day(P＜0.05). A value decreased when challenged with the LPS of 1.000 μg/ml and the difference was remarkable from the 3rd day to the 9th day(P＜0.05). Cell number increased with theadministration of LPS of different concentrations (0.005 μg/ml 0.500 μg/ml) and LPS of 0.100 μg/mlgroup had the best effect. The difference was remarkable from the 1st day to the 6th day(P＜0.05). Cell number decreased remarkably when challenged with LPS of 1.000 μg/ml and the difference was remarkable from the 2nd day to the 9th day(P＜0.05). Collagen synthesis increased when challenged with LPS of different concentrations (0.005 μg/ml 0.500 μg/ml) and the 0.100 μg/ml group had the best effect. However, when the dose of LPS reached 1.000 μg/ml, it inhibited collagensynthesis. Conclusion LPS could promote the proliferation andcollagen synthesis of fibroblasts within a certain range of low doses, but over-high dose ofLPS might inhibit the proliferation and collagen synthesis of fibroblasts, suggesting that LPS of certain concentrations might contribute to wound healing, while excessive LPS has negative effect on wound healing.
Objective To investigate the transduction pathway of TREM-1 during endotoxininduced acute lung injury ( ALI) in mice through the specific activating or blocking TREM-1.Methods 40 mice were randomly divided into a saline control group, an ALI group, an antibody group, and a LP17 group ( 3.5 mg/kg) . All mice except the control group were intraperitoneally injected with lipopolysaccharide ( LPS) to establish mouse model of ALI. Two hours after LPS injection, anti-TREM-1mAb ( 250 μg/kg) was intraperitoneally injected in the antibody group to activation TREM-1, and synthetic peptide LP17 was injected via tail vein in the LP17 group to blocking TREM-1. After 6,12,24, 48 hours, 3 mice in each group were sacrificed for sampling. The expression of NF-κB in lung tissue was determined by immunohistochemistry. The levels of TNF-α, IL-10, TREM-1, and soluble TREM-1 ( sTREM-1) in lung tissue and serumwere measured by ELISA. Pathology changes of lung were observed under light microscope, and Smith’s score of pathology was compared. Results Administration of anti-TREM-1mAb after ALI modeling significantly increased the NF-κB expression in lung tissue at 48h, resulting in a large number of pro-inflammatory cytokines releasing in the lung tissue and serumand lung pathology Smith score increasing. Administration of LP17 after modeling significantly down-regulated the expressions of NF-κB and pro-inflammatory cytokines, while led to a slight increase of anti-inflammatory cytokines and a decline of lung pathology Smith’s score.Conclusion TREM-1 may involve in inflammatory response by promoting the generation of inflammatory factors via NF-κB pathway, thus lead to lung pathological changes in ALI.
ObjectiveTo investigate the correlations between lipopolysaccharide(LPS), phospholipase A2 (PLA2) and platelet-activating factor (PAF) with coagulopathy after severe chest and abdominal injuries and their mechanisms. MethodsClinical data of 82 patients with severe chest and abdominal injuries whose trauma index (TI) was greater than or equal to 17 points in No. 253 Hospital of People's Liberation Army from January 2009 to June 2012 were retrospectively analyzed (severe chest and abdominal injury group). Those patients who had concomitant traumatic brain injuries or died in the Emergency Department were excluded from this study. There were 58 male and 24 female patients with their age of 16-76 (43.59±16.33)years. There were 17 patients with open injuries and 65 patients with closed injuries. There were 23 patients with fall injuries, 47 patients with traffic injuries, 8 patients with blunt force injuries, and 4 patients with penetrating injuries. Forty-two healthy volunteers who received routine medical examinations in the outpatient department of our hospital were chosen as the control group, including 27 males and 15 females with their age of 24-47 (37.32±10.45) years. Blood platelet (PLT) count, D-dimer (D-D), activated partial thromboplastin time (APTT), LPS, PLA2 and PAF were compared between the 2 groups, and linear correlation analysis was performed. ResultsPLT of the severe chest and abdominal injury group patients were significantly lower than that of the control group[(83.44±38.52)×109/L vs. (191.52±23.31)×109/L]. D-D[(1 823.89±608.02) U/L vs. (105.78±44.53) U/L], APTT [(68.24±24.12) s vs. (22.47±9.41) s], LPS[(438.66±106.02) U/L vs. (87.38±46.51) U/L], PLA2 [(41.35±14.26) ng/ml vs. (7.47±5.27)ng/ml] and PAF[(15 765.31±4 431.65) ng/L vs. (3 823.45±529.72) ng/L] of the severe chest and abdominal injury group patients were significantly higher than those of the control group(P < 0.001). PLT was significantly negatively correlated with LPS, PLA2 and PAF with all the respective correlation coefficient(r)less than-0.933 5. D-D and APTT were significantly positively correlated with LPS, PLA2 and PAF with all the respective r larger than 0.921 6. ConclusionLPS, PLA2 and PAF participate in the pathogenesis of coagulopathy in patients with severe chest and abdominal injuries. Early intervention against LPS, PLA2 and PAF may improve coagulopathy and survival rate of patients with severe chest and abdominal injuries.
Objective To investigate the relationship of pulmonary surfactant protein D( SP-D) with chronic obstructive pulmonary disease ( COPD) by measuring SP-D level in serum and lung tissue of rats with COPD.Methods The rat COPD model was established by passive smoking as well as intratracheal instillation of lipopolysaccharide ( LPS) . Thirty male SD rats were randomly divided into a control group, a LPS group, and a COPD group( n =10 in each group) . The pathologic changes of lung tissue and airway were observed under light microscope by HE staining. Emphysema changes were evaluated by mean linear intercept ( MLI) of lung and mean alveolar number ( MAN) . The level of SP-D in serum was measured by enzymelinked immunosorbent assay ( ELISA) . The expression of SP-D in lung tissue was detected by Western-blot and immunohistochemistry.Results The MLI obviously increased, and MAN obviously decreased in the COPD group compared with the control group ( Plt;0.05) . There was no significant difference in the MLI and MAN between the LPS group and the control group ( Pgt;0.05) . The serum SP-D level was ( 49.59 ±2.81) ng/mL and ( 53.21±4.17) ng/mL in the LPS group and the COPD group, which was significantly higher than that in the control group [ ( 42.14±2.52) ng/mL] ( Plt;0.05) . The expression of SP-D in lung tissue was 0.56±0.01 and 0.63±0.01 in the LPS group and the COPD group, which was also obviously ber than that in the control group ( 0.39 ±0.01) ( Plt;0.05) .Meanwhile the SP-D levels in serumand lung tissue were higher in the COPD group than those in the LPS group ( Plt;0.05) . The levels of SP-D between serum and lung tissue were positively correlated in all three groups ( r=0.93, 0.94 and 0.93, respectively, Plt;0.01) .Conclusion Both the SP-D level in serum and in lung tissue increase significantly in COPD rats and correlate well each other, which suggests that SP-D may serve as a biomarker of COPD.
ObjectiveTo investigate the effect of curcumin on lipopolysaccharide (LPS)-induced inflammation and apoptosis in alveolar macrophage via microRNA-132 (miR-132)/high mobility group protein B1 (HMGB1).MethodsThe cultured mouse alveolar macrophage line (RAW264.7 cells) were divided into the control group, the LPS group, the LPS+50 μmol/L curcumin group, and the LPS+100 μmol/L curcumin group. Forty-eight hours after drug treatment, the levels of miR-132/HMGB1, inflammatory mediator and apoptotic were detected. Secondly, the empty vector, synthetic miR-132 mimics and inhibitors were transfected into another cultured mouse alveolar macrophage line (RAW264.7 cells) to detect the inflammation and apoptosis of alveolar macrophage after transfection.ResultsCompared with the control group, in the LPS group, the apoptosis of alveolar macrophage, the levels of interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α, and the expression of miR-132 increased, while the expression of HMGB1 decreased (P<0.05); compared with the LPS group, in the two curcumin groups, the apoptosis of alveolar macrophage, the levels of IL-6, IL-8 and TNF-α, and the expression of miR-132 decreased, while the expression of HMGB1 increased (P<0.05); and the greater the drug concentration, the more obvious the effect (P<0.05). In addition, up-regulation of miR-132 reduced the expression of HMGB1 in alveolar macrophage, increased inflammatory factor, and induced apoptosis in alveolar macrophage; however, down-regulation of miR-132 increased the expression of HMGB1 in alveolar macrophage, reduced inflammatory factor, and inhibited apoptosis in alveolar macrophage (P<0.05).ConclusionCurcumin could decrease LPS-induced inflammation and apoptosis in alveolar macrophage via decreasing miR-132 and increasing HMGB1.
Objective To investigate the effects of inhaled prostaglandin E1 （PGE1）on Th1/Th2 polarity in rat model of lipopolysaccharide（LPS） induced acute lung injury（ALI）.Methods Healthy adult male Wistar rats [weight （200±20）g] were randomly divided into normal control（NS） group，LPS group and PGE1 group.The model of ALI were established by injecting LPS of 5 mg/kg into caudal vein.The rats in PGE1 group inhaled aerosolized PGE1（2 μg/mL）for 30 minutes after LPS injection,then repeat the procedure 12 hours later. 1 h，6 h，12 h and 24 h after last PGE1 inhalation,enzyme linked immunosorbant assay （ELISA） was empolyed to measure the level of interferon-γ（IFN-γ）and interleukin-4（IL-4）in the serum and bronchoalveolar lavage fluid（BALF）and the ratio of IFN-γ/IL-4（Th1/Th2）was calculated.Pathological examination was made under light microscope.Results Pathological examination of lung tissue demonstrated success ALI model.Compared to NS group,the ratio of IFN-γ/IL-4（Th1/Th2）both in serum and BALF in LPS group elevated significantly（Plt;0.01）. PGE1 administration significantly decreased the ratio IFN-γ/IL-4 in serum after 6h（Plt;0.01）and in BALF at all time points（Plt;0.01）.Conclusion The imbalance of was found in the LPS induced ALI，inhaled PGE1 aerosol inhalation could restore Th1/Th2 cytokine balance in the rats model induced by LPS.
ObjectiveTo investigate changes of lipopolysaccharidebinding protein (LBP) and its clinical significance in activation of Kupffer cells (KCs) during endotoxemia.MethodsWistar rat endotoxemia model was established by injection of a dose of LPS (5 mg/kg, Escherichia coli O111∶B4) via the tail vein of rats, then sacrificed 1, 3, 6 and 12 hour respectively. Hepatic tissue was collected to measure LBP mRNA expression by reverse transcritasepolymerase chain reaction (RTPCR). The levels of plasma endotoxins, LBP, TNFα and IL6 were determined. The pathological changes of hepatic tissue were observed under electron microscope.ResultsWhen the levels of plasma LPS elevated, expression of LBP mRNA in hepatic tissue were ber than that in control rats. The levels of plasma LBP, TNFα and IL6 were increased markedly also in rat with endotoxemia when compared with that in control groups (P＜0.01). KCs were seen to be enlarged in size, their surface projections were increased in number, and their cytoplasm was full of phagocytic vacuoles or electron dense phagosomes which indicated active phagocytosis.ConclusionLPS can markedly upregulate LBP mRNA expression in hepatic tissue, the levels of plasma LBP also increased. LBP may be a critical factor of LPS which stimulates KCs to produce and release different proinflammary mediators.
ObjectiveTo evaluate the combination of lipopolysaccharide-amine nanopolymersomes (LNPs), as a gene vector, with target gene and the transfection in bone marrow mesenchymal stem cells (BMSCs) so as to provide a preliminary experiment basis for combination treatment of bone defect with gene therapy mediated by LNPs and stem cells. MethodsPlasmid of bone morphogenetic protein 2 (pBMP-2)-loaded LNPs (pLNPs) were prepared. The binding ability of pLNPs to pBMP-2 was evaluated by a gel retardation experiment with different ratios of nitrogen to phosphorus elements (N/P). The morphology of pLNPs (N/P=60) was observed under transmission electron microscope (TEM) and atomic force microscope (AFM). The size and Zeta potential were measured by dynamic light scattering (DLS). The resistance of pLNPs against DNase I degradation over time was explored. The viability of BMSCs, transfection efficiency, and expression of target protein were investigated after transfection by pLNPs in vitro. ResultsAt N/P≥1.5, pLNPs could completely retard pBMP-2; at N/P of 60, pLNPs was uniform vesicular shape under AFM; TEM observation demonstrated that pLNPs were spherical nano-vesicles with the diameter of (72.07±11.03) nm, DLS observation showed that the size of pLNPs was (123±6) nm and Zeta potential was 20 mV; pLNPs could completely resist DNase I degradation within 4 hours, and such protection capacity to pBMP-2 decreased slightly at 6 hours. The cell survival rate first increased and then decreased with the increase of N/P, and reached the maximum value at N/P of 45; the cytotoxicity was in grade I at N/P≤90, which meant no toxicity for in vivo experiment. While the transfection efficiency of pLNPs increased with the increase of N/P, and reached the maximum value at N/P of 60. So it is comprehensively determined that the best N/P was 60. At 4 days, transfected BMSCs expressed BMP-2 continuously at a relatively high level at N/P of 60. ConclusionLNPs can compress pBMP-2 effectively to form the nanovesicles complex, which protects the target gene against enzymolysis. LNPs has higher transfection efficiency and produces more amount of protein than polyethylenimine 25k and Lipofectamine 2000.
ObjectiveTo investigate the mechanism of lung tissue apoptosis in LPS-induced mice ARDS via TNF-α neutralization. MethodsThirty-six mice were randomly divided into a control group,a LPS group,and TNF-α neutralization group.LPS(5 mg/kg) was intratracheally nebulized to induce ARDS in the LPS group and the TNF-α neutralization group.Twenty-four hours before LPS treatment,etanercept (0.4 mg/kg) was abdominal injected to the mice in the TNF-α neutralization group.Mice were sacrificed 2 hours after LPS treatment.PCR were used to detected the expression of NF-κB p65,Bax and Bcl-2 in lung tissue.Western blot were used to detected protein level of NF-κB p65,Erk1/2 and their phosphorylation and Bax,Bcl-2.The lung dry-to-wet ratio was measured.The lung histological changes were evaluated by HE staining. ResultsActivation level of NF-κB p65 and Erk1/2 was elevated,the ratio of Bcl-2 and Bax was decreased in the LPS group(P<0.05).After TNF-α neutralization,the activation level of NF-κB p65 and Erk1/2 were reduced,the ratio of Bcl-2 and Bax was increased (P<0.05).Compared with the LPS group,the lung dry-to-wet ratio and lung injury semi-quantitative score were significantly decreased in the TNF-α neutralization group (P<0.05). ConclusionTNF-α neutralization can suppress lung injury in LPS-induced ARDS mice by inhibiting activation of NF-κB p65 and Erk1/2,increasing the ratio of Bcl-2 and Bax ratio,and eventually reducing apoptosis.