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 explore the effects of cytoskeleton depolymerizing agent and stabilizer on the clathrin/caveolae-mediated endocytosis, the expression of membrane vascular endothelial cadherin (VE-cad), and the vascular permeability by the transformation of cytoskeleton structure after lipopolysaccharide (LPS) treatment.
MethodsCRL-2922 cells were used in the experiments. Indexes were tested at corresponding time point according to name of group, but in blank control group indexes could be tested at any time point. CRL-2922 cells were divided into blank control group, LPS-1 h group, and LPS-4 h group to observe cytoskeleton structure; CRL-2922 cells were divided into LPS-1 h group, Cyt D+LPS-1 h group, LPS-4 h group, and Jasp+LPS-4 h group to determine the expression of membrane VE-cad, and to determine the expression of its co-immunoprecipitation with clathrin and caveolin-1 (Cav1); besides, CRL-2922 cells were divided into blank control group, LPS-1 h group, Cyt D+LPS-1 h group, LPS-4 h group, and Jasp+LPS-4 h group to detect the cumulative infiltration rate.
Results①The cytoskeleton showed a dynamic change after LPS treat-ment, the F-actin polymerized and stress fibers formed at 1 hour after LPS treatment, but depolymerized at 4 hours after LPS treatment. ②Compared with LPS-1 h group, the level of co-immunoprecipitation of VE-cad with clathrin in Cyt D+ LPS-1 h group decreased (P<0.05), the level of co-immunoprecipitation of VE-cad with Cav1 increased (P<0.05), and expression level of VE-cad in plasma membrane decreased (P<0.05); compared with LPS-4 h group, there was no significant difference in the level of co-immunoprecipitation of VE-cad with clathrin of Jasp+LPS-4 h group (P>0.05), but the level of co-immunoprecipitation of VE-cad with Cav1 decreased in Jasp+LPS-4 h group (P<0.05), and expression level of VE-cad in plasma membrane increased (P<0.05). ③Compared with blank control group, the cumulative infiltration rates of LPS-1 h group and LPS-4 h group were both higher (P<0.05); compared with LPS-1 h group, the cumulative infiltration rate of Cyt D+LPS-1 h group was higher (P<0.05); compared with LPS-4 h group, the cumulative infiltration rate of Jasp+LPS-4 h group was lower (P<0.05).
ConclusionActin cytoskeleton shifts from polymerization to depoly-merization after LPS treatment, the structural change of actin cytoskeleton is an important reason for the transformation of VE-cad endocytosis pathway from clathrin-mediated to caveolae-mediated after LPS treatment.
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 compare the different effects of ubiquitin(UB) on human umbilical vein endothelial cells (HUVECs) and macrophages under normal circumstances,and analyze whether UB could protect HUVECs from lipopolysaccharide(LPS) induced injury.
MethodsThe morphologic changes of HUVECs in vitro with up-rising concentrations of UB interventions were observed. HUVECs and human macrophages in vitro were divided into 4 groups according to UB concentration (0.01 μg/mL,0.1 μg/mL, 1 μg/mL, and 10 μg/mL). Supernatant and cells of each group were collected in 24 h after UB intervention. The levels of TNF-α and VCAM-1 in supernatant were measured by ELISA while NF-κB protein level in cells was detected by Western blot. HUVECs were divided into a LPS group(LPS 10 μg/mL) and an UB+LPS group(UB 0.1 μg/mL,LPS 10 μg/mL). The supernatant of the two groups were collected in 8,16 and 24 h after LPS and UB intervention. The levels of TNF-α and VCAM-1 in supernatant were measured by ELISA.
ResultsThe injury of HUVECs got worse with the ascending concentrations of UB.At the concentration of 50 μg/mL,UB induced HUVECs got ballooned and died massively. With the increase of UB concentration,the levels of TNF-α and VCAM-1 in HUVECs' supernatant ascended firstly and then descended,while those in human macrophages' supernatant ascended gradually. zHowever,the tendency of the NF-κB protein level in the two kinds of cells was similar when the concentration of UB increased.At the consentration of 0.1 μg/mL or 1 μg/mL,ubiquitin induced NF-κB protein level obviously increased.At the concentration of 0.01 μg/mL or 10 μg/mL,UB induced the protein level was similar with those of the control group and even decreased slightly. There was no significant difference in TNF-α or VCAM-1 levels at each time point between the LPS group and the UB+LPS group.
ConclusionsUB injuries HUVECs obviously at a low concentration but injuires human macrophages at much higher concentraton. UB can not protect HUVECs from LPS-induced injury in vitro.
ObjectiveTo observe the effects of perindopril on expression level of phosphatidylinositol 3-kinase (PI3K) and lung function in rats with obstructive pulmonary disease (COPD),and investigate the therapeutic effects of perindopril on COPD.
MethodsSixty male SD rats were randomly divided into a control group,a COPD group,and a perindopril group,with 20 rats in each group. The rat model of COPD was established by intratracheal instillation of lipopolysachride and exprosure to cigarette smoke in the COPD group and the perindopril group. The rats in the perindopril group were intragastricly infused with perindopril additionally. All rats were sacrificed after 28 days. The lung function was observed and PI3K protein expression was detected using Western blot method. The pathologic changes of the lung tissue and airway were observed by HE staining.
ResultsHE staining revealed that the rat COPD model was successfully established. The COPD group appeared obvious emphysema which was allievated significantly in the perindopril group. Pulmonary function indices in the COPD group and the perindopril group were significantly decreased compared with the control group with VE value decreased by 40% and 22%,PEP value decreased by 33% and 15%,and FEV0.3 value decreased by 18% and 7%,respectively. The expression of PI3K was significantly increased in the COPD group and the perindopril group than the control group,but more significantly in the COPD group (P<0.05).
ConclusionPerindopril can significantly improve lung function in rats with COPD possibly through down-regulation of PI3K expression in the lung.
Objective To compare the in vitro inhibition activity of three mimic peptides at lipopolysaccharide binding protein (LBP)／CD14 binding sites for LPS-induced inflammatory response.Methods Enzyme-linked immunoSorbent assay(ELISA)was applied for the detection of the affinity between the mimic peptides and CD14 as well as the competitive inhibition activity of LBP.Mature U937 cells induced by PMA were co-cultured with LPS and intervened by mimic peptide.The effect of the mimic peptide on the TNF-α expression of U937 was detected by RT-PCR.Alveolar macrophages(AMs)of rats were co-cultured with FITC-LPS，and mimic peptide intervention was conducted.The effect of the mimic peptide on combination of LPS and AMs was observed by fluorescence microscope . Results Affinity between No.1 mimic peptide(FHRWPTWPLPSP，10 μg／mL)and CD14 was significantly higher than those of No.2 and No.3(20.3±4.1 11.8±2.4 and 13.7±3.3，Plt;0.01 or Plt;0.05).The competitive inhibitory activity of No.1 mimic peptide(10 μg／mL)for LBP was higher than those of No.2 and No.3[(57.2土11.2)％ vs(39.4±9.7)％ and(37.9±8.3)％ ，Plt;0.01].All of the three mimic peptides(10 μg／mL)could significantly inhibit the LPS-induced expression of TNF-α by U937 at mRNA level(Plt;0.01 or Plt;0.05)，moreover，the inhibitory activity of No.1 peptide was the highest(0.239±0.053 vs 0.406±0.112 and 0.493±0.121，Plt;0.01).In addition，No.1 mimic peptide markedly inhibited LPS combination with rat lung AMs(2157±514 vs 2763±453，plt;0.01).Conclusion No.1 mimic peptide(FHRWPTWPLPSP)have a relatively higher affinity with CD14 and high competitive inhibition activity for LBP，therefore it have the potential ability of anti-inflammatory response induced by LPS.
Objective To analyze the role of lienal polypeptide injection in acute lung injury induced by lipopolysaccharide (LPS) in rats. Methods Eighty male SD rats were randomly allocated into 4 groups: a LPS group, a control group, a lienal polypeptide group and a LPS+ lienal polypeptide group (20 rats in each group). Lienal polypeptide or normal saline was given with an intramuscular injection 30 min after an intraperitoneal injection of LPS (5 mg/kg). The severity of pulmonary injury was evaluated 4 h after LPS challenge by enzyme-linked immunosorbent assay (ELISA), wet-to-dry weight ratio, hematoxylin and eosin (HE) staining, TUNEL and Western blotting. Results Lienal polypeptide injection treatment significantly attenuated LPS-induced pulmonary histopathologic changes, alveolar hemorrhage, and neutrophil infiltration. Moreover lienal polypeptide injection significantly suppressed LPS-induced activation of metastasis-associated protein-1 (MTA1). Conclusion Lienal polypeptide injection is demonstrated to protect rats from LPS-induced acute lung injury by the expression of MTA1.
Objective To explore whether microRNA-203 (miR-203) targets and regulates the Toll-like receptor 4 (TLR4)/nuclear transcription factor kappa B (NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) to protect alveolar epithelial cells from lipopolysaccharide (LPS)-induced apoptosis and inflammation injury. Methods The alveolar epithelial A549 cells were used as the research objects and divided into: Control group (normal culture), LPS group (LPS treatment), LPS+miR-NC mimics group (LPS treatment after transfection of miR-NC mimics), LPS+ miR-203 mimics group (LPS treatment after transfection of miR-203 mimics), LPS+miR-203 mimics+pcDNA group (LPS treatment after transfection of miR-203 mimics and pcDNA), LPS+miR-203 mimics+pcDNA-TLR4 group (LPS treatment after transfection of miR-203 mimics and pcDNA-TLR4). Dual luciferase reporter gene was used to detect the targeting relationship between miR-203 and TLR4; Real-time quantitative reverse transcription-polymerase chain reaction was used to detect the relative expression levels of miR-203 and TLR4 mRNA; enzyme-linked immunosorbent assay was used to measure the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6; flow cytometry was used to detect the apoptosis rate of A549 cells; Western blot was used to detect the expression of B-cell lymphoma/leukemia-2 gene (Bcl-2) and Bcl-2 associated X protein (Bax), TLR4, NF-κB and NLRP3 proteins in A549 cells. Results There was a targeted regulation relationship between miR-203 and TLR4. Compared with the Control group, the expression of miR-203, TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS group increased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant increased, the apoptosis rate increased, the level of Bcl-2 protein in cells decreased (P<0.05). Compared with the LPS+miR-NC mimics group, the expression of TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS+miR-203 mimics group decreased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant decreased, the apoptosis rate decreased, the expression level of miR-203 and the level of Bcl-2 protein in cells increased (P<0.05). Compared with the LPS+miR-203 mimics+pcDNA group, the expression of miR-203, TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS+miR-203 mimics+pcDNA-TLR4 group increased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant increased, the apoptosis rate increased, the expression level of miR-203 and the level of Bcl-2 protein in cells decreased (P<0.05). Conclusion MiR-203 can target TLR4/NF-κB/NLRP3 to protect alveolar epithelial cells from apoptosis and inflammation induced by LPS.
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 detect the effects of cytokines on the expression of early growth response gene-1 (Egr-1) in cultured human retinal pigment epithelial (RPE) cells. Methods Immunofluorescence staining, Western blotting and reverse transcription polymerase chain reaction (RT-PCR) were used to detect and quantitatively analyze the expression of Egr-1 protein and mRNA in cultured human RPE cells which were exposed to stimulants, including 20 mu;g/ml lipopolysaccharide (LPS), 40 ng/ml tumor necrosis factor (TNF)-alpha;, 10 U/ml interferon (IFN)gamma;, 30% supernatant of monocyte/macrophage strain (THP1 cells) and the vitreous humor from healthy human eyeballs, for 0, 10, 20, 30, 40 and 60 minutes, respectively. Results The RPE cells stimulated for 0 minute revealed faint green fluorescence of Egr-1 in the cytoplasm. With exposure to the stimulants, the expressionof Egr-1 increased obviously and b green fluorescence was found in cytoplasm in some nuclei of RPE cells. Compared with the untreated RPE cells, after stimulated by 20 mu;g/ml LPS, 40 ng/ml TNFalpha;, 10 U/ml IFNgamma;, 30% supernatant of THP-1 cells and the vitreous humor, the approximate ultimate amplitudes of Egr-1 mRNA enhanced 1.9, 1.3, 14, 1.2, and 1.4 times, respectively; the greatest amplitudes of Egr-1 protein increased 3.4, 1.2, 1.7, 32, and 1.3 times, respectively. Conclusion LPS, TNF-alpha;, IFN-gamma;, supernatant of THP-1 cells and the vitreous humor can upregulate the expression of Egr-1 mRNA and protein in cultured human RPE cells, and induce its nuclear transposition, which suggests the activation of Egr-1.
