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.
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.
ObjectiveTo investigate the effect of curcumin on the expression regulation of endogenousβ-glucoronidase (β-GD) induced by lipopolysaccharide (LPS).Methods① Human normal intrahepatic biliary epithelial cell line (HiBEpiC) cells in the logarithmic growth phase were divided into blank control group (0 h group) and 7 different stimulation time groups. The cell density was adjusted to 1×104/mL, and the cells were stimulated with 100 mg/mL LPS for 1, 3, 6, 18, and 24 hours respectively, including another two groups where the cells were cultured with LPS-free medium for 18 and 24 hours after LPS stimulation for 24 h. ② HiBEpiC cells in the logarithmic growth phase were divided into blank control group, LPS+low, medium, and high concentration curcumin group. The cell density was adjusted to 1×104/mL. In the blank control group, cells were not stimulated with any reagent; in the LPS group, cells were stimulated with 100 mg/mL LPS, in the other three groups, the cells were stimulated with 100 mg/mL LPS and simultaneously 20, 40, and 80 μmol/L curcumin, respectively, for 24 hours. The expressions of c-myc and endogenous β-GD were detected by Western blot method.Results① The expressions of endogenous β-GD and c-myc in HiBEpiC cells gradually increased with the prolongation of treatment time by LPS, and the expression levels of β-GD and c-myc at each time point group were significantly different from those in the 0 h group (P<0.05). ② There were significant difference between any two groups of the blank control group, LPS group, LPS+low concentration of curcumin group, LPS+medium concentration of curcumin group, and LPS+high concentration of curcumin group (P<0.05).ConclusionCurcumin is able to inhibit the increased expression of endogenous β-GD induced by LPS, possibly via inhibiting expression of c-myc.
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 role of the p38 MAPK signaling pathway in sTREM-1 expression of RAW264.7 cells induced by lipopolysaccharide (LPS). MethodsMacrophage cell line RAW264.7 cells were cultured in vitro and induced with the same concentration of LPS at different time. The protein expressions of p38 MAPK and phosphorylation of p38 MAPK(p-p38 MAPK) were detected by Western blot. The mRNA expression of p38 MAPK was detected by RT-PCR. The level of sTREM-1 was detected by enzyme linked immunosorbent assay method.The RAW264.7 cells were treated by SB203580 at different concentration,the changes of above indexes were observed. ResultsThe p-p38 MAPK and p38 MAPK mRNA could be inducted by LPS in a time-dependent manner. The p-p38 MAPK and p38 MAPK mRNA could be inhibited by SB203580. After SB203580 blocking p38 MAPK signal transduction pathway,the sTREM-1 expression was significantly inhibited in a dose dependent manner. ConclusionLPS can induce sTREM-1 expression in RAW264.7 cells by activating the p38 MAPK signaling pathway.
目的：建立存活時間較長的急性重癥肝炎小鼠模型。方法：將50只BALB/c小鼠平均分成5組，其中A、B、C、D 4組為實驗組，E組為對照組；實驗組分別給予D氨基半乳糖（D-GalN）和脂多糖（LPS），劑量分別為800 mg/kg和10 μg/kg、500 mg/kg和10 μg/kg、500 mg/kg和5 μg/kg、300 mg/kg和5 μg/kg，用生理鹽水稀釋至1 mL，行腹腔注射；對照組E腹腔注射生理鹽水2 mL。以12 h死亡率、24 h死亡率及肝組織學改變為觀察指標。結果：A、B、C、D及E組小鼠12 h死亡率分別為80%%、30%、10%、0和0；24 h死亡率分別為90%、60%、30%、0和0；A和B組小鼠肝組織學均呈急性重癥肝炎表現，C組中有5只小鼠肝組織學符合重癥肝炎的表現，而該組其余小鼠及D組所有肝組織學雖有炎癥改變，但達不到重癥肝炎的程度，E組肝組織學表現正常。結論：LPS 10 μg/kg聯合D- GalN 500 mg/kg可成功建立12 h存活率較高的急性重癥肝炎小鼠模型。
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 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.
Objective To investigate the ability of gene-loaded lipopolysaccharide-amine nanopolymersomes (LNPs) in inducing osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by in vitro gene transfection, where LNPs were used as a non-viral cationic carrier, and their properties were optimized during synthesis. Methods LNPs were synthesized by a graft-copolymerization method, and the effects of different pH environments during synthesis on physicochemical properties of LNPs and LNPs/plasmid of bone morphogenetic protein 2-green fluorescent protein (pBMP-2-GFP) complexes were explored. Then, optimized LNPs with maximum transfection efficiency and safe cytotoxicity in rat BMSCs were identified by cytotoxicity and transfection experiments in vitro. Thereafter, the optimized LNPs were used to mediate pBMP-2-GFP to transfect rat BMSCs, and the influences of LNPs/pBMP-2-GFP on osteogenic differentiation of BMSCs were evaluated by monitoring the cell morphology, concentration of BMP-2 protein, activity of alkaline phosphatase (ALP), and the formation of calcium nodules. Results The nitrogen content, particle size, and zeta potential of LNPs synthesized at pH 8.5 were lower than those of the other pH groups, with the lowest cytotoxicity (96.5%±1.4%) and the highest transfection efficiency (98.8%±0.1%). After transfection treatment, within the first 4 days, BMSCs treated by LNPs/pBMP-2-GFP expressed BMP-2 protein significantly higher than that treated by Lipofectamine2000 (Lipo)/pBMP-2-GFP, polyethylenimine 25K/pBMP-2-GFP, and the blank (non-treated). At 14 days after transfection, ALP activity in BMSCs treated by LNPs/pBMP-2-GFP was higher than that treated by Lipo/pBMP-2-GFP and the blank, comparable to that induced by osteogenic medium; with alizarin red staining, visible calcium nodules were found in BMSCs treated by LNPs/pBMP-2-GFP or osteogenic medium, but absent in BMSCs treated by Lipo/pBMP-2-GFP or the blank with apoptosis. At 21 days after transfection, transparent massive nodules were discovered in BMSCs treated by LNPs/pBMP-2-GFP, and BMSCs exhibited the morphologic features of osteoblasts. Conclusion LNPs synthesized at pH 8.5 has optimal transfection efficiency and cytotoxicity, they can efficiently mediate pBMP-2-GFP to transfect BMSCs, and successfully induce their directional osteogenic differentiation, whose inducing effect is comparable to the osteogenic medium. The results suggest that gene transfection mediated by LNPs may be a convenient and effective strategy in inducing directional differentiation of stem cells.
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.