ObjectiveTo study the changes of lipopolysaccharide binding protein (LBP) in the serum of Wistar rats with obstructive jaundice and to investigate its potential mechanism.MethodsEighty male Wistar rats were randomly divided into obstructive jaundice group (OJ group, n=40） and sham operation group (SO group, n=40）. Before operation and the 5th, 10th, 15th, 20th day after common bile duct ligation, the levels of LBP, endotoxin, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in plasma were detected in all the rats. ResultsLBP levels in serum increased significantly in OJ group on the 10th day after operation compared with those of SO group. Moreover, LBP levels gradually increased in OJ group with the prolongation of obstructive time. A positive correlation existed between serum LBP and plasma endotoxin, TNF-α and IL-6.ConclusionThe study demonstrates that LBP in serum is high and plays an important role in the pathogenesis of multiple organ injury secondary to obstructive jaundice. It may be an appropriate way to treat patients with obstructive jaundice by decreasing LBP levels in serum.
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.
Objective To investigate the mRNA and protein expression of human β-defensin-2 (hBD-2) induced by lipopolysaccharide (LPS)，IL-1β and TNF-α in human airway primary epitheliums.Methods The bronchial primary epitheliums from human were stimulated with LPS，IL-1β and TNF-α respectively and then were harvested for hBD-2 expression detection.The mRNA expression of hBD-2 was detected by RT-PCR，and the protein expression by immunocytochemistry and western blot.Results There was a small expression of hBD-2 mRNA in human airway primary epitheliums before stimulation.The hBD-2 mRNA expression was significantly increased after 3 hours of LPS，IL-1β and TNF-α stimulation respectively and the expression increasement was in a dose dependent manner.The hBD-2 protein could be detected in cytoplasm after 4 hours of LPS (0.1 μg/mL)，IL-1β (1 ng/mL) and TNF-α (10 ng/mL) stimulation.Conclusions LPS and proinflammatory cytokines can induce the mRNA and protein expression of hBD-2 in a short time.The expression of hBD-2 may play an initial defense role against bacterial invasion.
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.
Objective To investigate whether P12，a kind of lipopolysaccharide（LPS）-binding protein（LBP） inhibitory peptide，could suppress the binding of LPS to alveolar macrophages（AMs） in a mouse model of endotoxemia in vivo.Methods Forty mice were randomly divided into five groups，ie.a control group，an endotoxemia group，a low dose P12-treated group，a middle dose P12-treated group and a high dose P12-treated group.Mouse model of endotoxemia was established by LPS injection intraperitoneally in the endotoxemia group and P12-treated groups.P12 was instilled via the tail vein.The effects of P12 on the binding of LPS to AMs were determined by flow cytometric analysis and quantization by mean fluorescence intensity(MFI).The productions of tumor necrosis factor α(TNF-α) in serum of mice were measured by enzyme-linked immunosorbent assay(ELISA).Results MFI in AMs from low，middle and high dose P12-treated groups was 40.08%，30.76% and 24.45%，respectively，which was higher than that of the control group(4.61%)，but less than that of the endotoxemic mice(45.31%).The concentration of TNF-α in serum of low，media and high dose P12-treated mice was (112.69±19.78)pg/mL，(86.34±9.25) pg/mL，(70.48±8.48)pg/mL respectively，which was higher than that of the control group[(24.88±5.82)pg/mL]，but less than that of the endotoxemic mice[(180.17±39.14)pg/mL].Conclusion The results suggest that P12 inhibit the binding of LPS and AMs，thus reduce the proudction of TNF-α stimulated by LPS.
Objective To investigate the expression of aquaporin-1( AQP1 ) in visceral and parietal pleura in SD rats and to examine the effect of AQP1 on pleural fluid turnover. Methods Five groups( n = 24 ) of SD rats were randomly assigned to received intrapleural injection of dexamethasone,lipopolysaccharide, erythromycin, hypertonic saline and normal saline, respectively. The AQP1 protein in pleural was detected with immunohistochemistry. The mRNA expression of AQP1 under stimulations at different time points was measured by real time RT-PCR. Results AQP1 was immunolocalized predominantly to the microvessels and mesothelial cells of visceral and parietal pleura. The extent of AQP1expression in parietal pleura was less than that in visceral pleura[ ( 4. 14 ±1. 12) ×104 copy /μg vs ( 7. 43 ±2. 02) ×104 copy / μg, P lt;0. 05] . AQP1 expression increased at all phases in the dexamethasone group andthe hypertonic saline group, whereas decreased in the erythromycin group and the lipopolysaccharide group.Conclusion The stimulations of dexamethasone, lipopolysaccharide, erythromycin and hypertonic saline can significantly change the AQP1 expression in pleura, which indicate that AQP1 may contribute to the accumulation and clearance of pleuritic fluids.
Objective To study the effects of two different tidal volume mechanical ventilation on lipopolysaccharide( LPS) -induced acute lung injury( ALI) , and explore the effects of glutamine on ALI.Methods Thirty male Sprague-Dawley rats were randomly divided into three groups. After anesthesia and tracheotomy were performed, the rats were challenged with intratracheal LPS ( 5mg/kg) and received ventilation for 4 hours with small animal ventilator. Group A received conventional tidal volume, while groupB received large tidal volume. Group C received large tidal volume as well, with glutamine injected intravenously 1 hour before ventilation. Arterial blood gases were measured every one hour. 4 hours later, the rats were killed by carotid artery bleeding. The total lung wetweightwas measured and lung coefficient ( total lung wet weight /body weight ×100) was counted. WBCs and neutrophils in BALF were counted. Protein concentration, TNF-α, IL-6, and cytokine-induced neutrophil chemoattractant-1 ( CINC-1) levels in BALF,myeloperoxidase ( MPO) , and superoxide dismutase ( SOD) levels in the lung were assayed respectively.Results PaO2 and SOD levels decreased more significantly in group B than those of group A. The lung coefficient, WBCs, neutrophils, protein, TNF-α, IL-6, and CINC-1 levels in BALF, MPO levels in lung increased more significantly in group B than those of group A. PaO2 and SOD levels were significantly higher in group C than those of group B. The lung coefficient, WBCs, neutrophils, protein, TNF-α, IL-6, and CINC-1 levels in BALF,MPO levels in lung were significantly lower in group C than those of group B. Conclusion Large tidal volume mechanical ventilation aggravates LPS-induced ALI, and glutamine has obviouslyprotective effects.
Objective To establish a mouse model of acute lung injury ( ALI) and pulmonary fibrosis by low dose lipopolysaccharide ( LPS) intermittent intraperitoneal injection, and to explore the pathogenesis of ALI and pulmonary fibrosis induced by endotoxin. Methods Forty C57BL/6 mice were randomly divided into a control group, a 3-days LPS group, a 2-weeks LPS group, and a 4-weeks LPS group,with 10 mice in each group. LPS was injected intraperitoneally at dose of 5 mg/ kg for three consecutive daysin the three LPS groups. Equivalent normal saline was injected by the same way in the control group. The mice lung tissues were obtained respectively 3 days ( the control group and 3-days LPS group) , 2 weeks ( the 2-weeks LPS group) , and 4 weeks ( the 4-weeks LPS group) after LPS or saline stimulation. HE staining,Van-Gieson collagen staining, and Ashcroft fibrosis score assessment were applied to evaluate the development of inflammation and fibrosis in lung tissue at various stages of ALI after LPS-stimulation. The mRNA expression of type Ⅰ procollagen and alpha smooth muscle actin ( α-SMA) were detected by realtime PCR. The deposition of collagen and fibrosis in lung tissue were detected by hydroxyproline assay. The survival condition of each group was also recorded. Results Acute inflammation occurred in mice lung tissue 3 days after intraperitoneal injection of LPS. Collagen deposited in pulmonary interstitium2 weeks afterLPS-stimulation and formed typical pulmonary interstitial fibrosis 4 weeks later accompanying with increase of Ashcroft fibrosis score. Real-time PCR and hydroxyproline assay showed that the expression of collagen and α-SMA increased 3 days after LPS-stimulation and reached the peak 4 weeks later. The animals were all survived up to the endpoint of experiment. Conclusions Accompanying with inflammation, pulmonary fibrosis initiated at early stage of ALI induced by LPS. Intraperitoneal injection of LPS at dose of 5 mg/kg for three consecutive days was able to establish the mouse model of ALI and pulmonary fibrosis with high successrate and low animal mortality, which provide an ideal experimental platform for further investigation.
Objective To explore the expression of myeloid differentiation protein2 ( MD-2) in rat lung and its role in acute lung injury ( ALI) induced by lipopolysaccharide ( LPS) . Methods Twenty male SD rats were randomly divided into a LPS group and a control group. The wet/dry ratios of lung tissues were measured and the histological changes of lung tissues were observed under microscope. Alveolar macrophages were collected from bronchial alveolar lavage fluid ( BALF) . The MD-2 mRNA and protein expressions were detected by RT-PCR, Western blot, and immunohistochemistry respectively. The MD2-siRNA oligo were transfected into NR8383 cells and 1 μg/mL LPS was used to stimulate the cells. The expressions of MD-2 mRNA and protein were detected by RT-PCR and Western blot. The levels of TNF-αin rat serum and cell culture supernatant were detected by ELISA. Results Compared with the control group, the expressions of MD-2 mRNA and protein in alveolar macrophages and lung tissue were elevated ( P lt;0. 01) , as well as the level of TNF-αin rat serum. The expressions of MD-2 mRNA and protein in NR8383 cell and the level ofTNF-αin supernatant increased obviously after LPS stimulation ( P lt;0. 01) . There were no changes of MD-2 mRNA and protein expressions and TNF-α of NR8383 cells treated by MD-2 siRNA with or without LPS stimulation ( P gt;0. 05) . Conclusions The expression of MD-2 in lung increases obviously after challengedby LPS. KnockdownMD-2 gene of NR8383 cell byMD-2 siRNA can inhibit TNF-αsecretion induced by LPS stimulation.MD-2 may play an important role in rat ALI induced by LPS.
Objective To explore the protective effects of liver X receptor-αactivator ( LXRα)T0901317 on rats with acute lung injury ( ALI) . Methods Seventy-two male Wistar rats were randomly divided into three goups, ie. a control group, a LPS group, and a T0901317 group. Artery blood gas analysis,lung tissue wet/dry weight ratio,myeloperoxidase activity, and lung histopathological changes were measured.The expressions of LXRαand TNF-αmRNA in lung tissue were detected by RT-PCR. The protein levels ofTNF-αand LXRαwere examined with ELISA and immunohistochemistry, respectively. Results In the ALI rats, PaO2 decreased, lung W/D weight ratio and myeloperoxidase activity increased significantly compared with the control group ( P lt; 0. 05) . Histopathological examination also revealed obvious lung injury. In theLPS group, the expression of TNF-αmRNA in lung tissue and the level of TNF-αprotein in lung homogenate and serum increased markedly( all P lt; 0. 05) while the expression of LXR-αmRNA declined significantly ( P lt; 0. 05) . Immunohistochemical staining showed that lung tissues of the normal rats expressed LXRαsignificantly but in the LPS group the expression of TNF-αand LXR-αin lung tissue decreased markedly ( P lt;0. 05) . After the treatment with T0901317, the expressions of LXR-αin lung tissues were significantly higher than those in the LPS group both at the mRNA and the protein level ( P lt; 0. 05) . Conclusion T0901317 plays an anti-inflammatory effect through up-regulating the expression of LXR-αand suppressing the expression of TNF-α, thus reduces the infiltration and aggregation of inflammatory cells in lung tissue.