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.
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 investigate the effects of cytokines on the expression of syndecan-1 in cultured human retinal pigment epithelial (RPE) cells and the signal transduction pathway.
Methods
Reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of syndecan-1 mRNA and protein in normal RPE cells. The expression of syndecan-1 in RPE cells stimulated by different cytokines was detected and quantitatively analyzed by image process of immunofluorescence. The stimulation included 7 and 35 ng/ml tumor necrosis factor (TNF)-alpha; for 24 hours, 1 and 6 mu;g/ml lipopolysaccharide (LPS) for 11 hours, 7 ng/ml TNF-alpha; for 0 to 24 hours (once per 2 hours, and 13 times in total), and 30% supernatant of monocyte/macrophage strain (THP-1 cells) for 3, 14 and 43 hours. The effect of 30% supernatant of THP-1 cells was assayed after pretreated by PD098059［the specific inhibitor of extracellular signal regulated kinase(ERK) 1/2］for 2 hours. After exposed to 30% supernatant of THP-1 cells for 3 hours and treated by 0.25% trypsin for 5 minutes, RPE cells attaching was evaluated by methyl thiazolyl tetrazolium assay.
Results
In normal human RPE cells, expressions of syndecan-1 mRNA and protein were detected, and b syndecan-1 positive yellowish green fluorescence was found in the cell membrane and cytoplasm while light green fluorescence was in the nucleus. As the concentration and stimulated time of TNF-alpha; or LPS increased, the fluorescence intensity decreased（Plt;0.01）, and after exposed to 30% supernatant of THP-1 cells, weaker fluorescence intensity was detected (Plt;0.001). Pretreatment with 50 mu;mol/L PD098059 for 2 hours partly inhibited the effect of THP-1 cells supernatant. After exposed to 30% supernatant of THP-1 cells for 3 hours, the number of attached cells decreased compared with the controls（Plt;0.05）.
Conclusions
TNF-alpha; and LPS down-regulate the expression of syndecan-1 in cultured human RPE cells. The supernatant of THP-1 cells down-regulates the expression of syndecan-1 and lessens the cells attaching, which is at least mediated by ERK 1/2 pathway.
(Chin J Ocul Fundus Dis, 2006, 22: 113-116)
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.
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 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.
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 the effects of interleukin (IL)-26 on the late phase of lipopolysaccharides (LPS)-induced lung inflammation in mouse model.MethodsThirty-two mice were equally and randomly divided into four groups: blank control group, IL-26 control group, LPS model group, and IL-26 intervention group. The blank control group was given intranasal administration of phosphate buffered solution (PBS, 40 μl) and PBS (40 μl) 10 minutes apart. The IL-26 control group was given recombinant human interleukin-26 (rhIL-26; 50 μg/kg, dissolved in 40 μg PBS) and PBS successively. The LPS model group was given intranasal administration of PBS (40 μl) and LPS (10 mg/kg, dissolved in 40 μl PBS) at 10 minutes interval. The IL-26 intervention group was given intranasal administration of rhIL-26 and LPS at 10 minutes interval. Seventy-two hours later after treatment, bronchoalveolar lavage fluid (BALF) cell count, cytokine assay and pathological staining of lung tissue were performed in each group. The gene expression of inflammatory pathway in lung tissue was detected by RT-PCR. One-way ANOVA was used for comparison between groups. ResultsCompared with the blank control group, the expression of tumor necrosis factor-α and activating transcription factor 3 in IL-26 control group increased significantly (all P < 0.05). The number of peripheral blood mononuclear cells, total BALF cells, lymphocytes and neutrophils, and the content of macrophage inflammatory protein-1a in BALF were significantly increased in IL-26 intervention group comparing with LPS model group (all P < 0.05). IL-26 intervention group had more inflammatory subsidence in interstitial, perivascular, peribronchial and mean values than LPS model group (all P < 0.05). The expressions of Toll-like receptor 4, Toll-like receptor 2 and interferon γ induced protein 10 in IL-26 intervention group were significantly higher than those in LPS model group (all P < 0.05).ConclusionIL-26 can significantly alleviate the late inflammatory reaction of lung tissue in LPS-induced mouse inflammation model.
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.
