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 whether p38 mitogen activated protein kinase (p38MAPK) inhibitor can reduce acute lung injury (ALI) caused by lipopolysaccharide (LPS) by regulating Th17/Treg balance. Methods Balb/c mice were randomly divided into a control group, an ALI group and an intervention group. The mice in the control group were injected with phosphate-buffered saline, the mice in the ALI group were intraperitoneally injected with 40 mg/kg LPS, and the mice in the intervention group were injected with SB203580 (0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg) intraperitoneally 1 h prior to the intraperitoneal injection of LPS. All mice were killed on 12 h later respectively. Hematoxylin-eosinstin staining was used to observe the pathological changes of lung tissue, and cell classification, counting, and total protein levels in bronchoalveolar lavage fluid (BALF) were detected. Transcript expression of forkhead box p3 (Foxp3) and retinoic acid receptor-related orphan receptor-γt (RORγt) was detected by real-time polymerase chain reaction. Interleukin (IL)-6, IL-10, IL-17, IL-23 and transforming growth factor-β (TGF-β) in lung tissue and IL-6, tumor necrosis factor-α (TNF-α) in serum were measured by enzyme-linked immunosorbent assay. The Th17 and Treg subset distribution in spleen was determined by flow cytometry. Results Histopathological examination showed that LPS induced inflammatory cell infiltration in lung tissue, increased cell count and protein levels in BALF (P<0.05), and increased proportion of neutrophils and monocytes in the ALI mice. SB203580 significantly attenuated tissue injury of the lungs in LPS-induced ALI mice. Serum levels of IL-6 and TNF-α in the ALI group were significantly higher than those in the control group, and inflammatory cytokines were decreased after SB203580 intervention. Compared with the ALI group, the production of inflammatory cytokines associate with Th17, including IL-17, IL-23, RORγt was inhibited, and the production of cytokines associate with Treg, such as IL-10 and Foxp3 in lung tissue was increased in the intervention group in a concentration-dependent manner with SB203580. After SB203580 intervention, Th17/Treg ratio was significantly decreased compared with the LPS group (P<0.05). Conclusion p38MAPK inhibitor can reduce LPS-induced ALI by regulating the imbalance of Treg cells and Th17 cells.
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ObjectiveTo investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide. Methods Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR). ResultsELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05). ConclusionMT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.
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 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 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.
目的 探讨脂多糖（LPS）、白细胞介素-6（IL-6）和血小板活化因子（PAF）与重症胸腹创伤后凝血功能紊乱发生的相关性及可能的致病机理。方法 收集2009年1月至2011年12月期间在中国人民解放军第二五三医院急诊科就诊、创伤指数≥17分且除外合并颅脑损伤及在急诊科内死亡的胸腹创伤患者62例，在予以抢救、治疗的同时抽血检查血小板计数（PLT）、血浆D-二聚体（D-D）、部分活化凝血酶原时间（APTT）、凝血酶原时间（PT）、LPS、IL-6和PAF，并对其结果进行相关性分析。结果 本组患者就诊时检测的PLT为（157.73±78.11）×109/L， D-D为（1 023.88±208.72） U/L，APTT为（46.95±17.85） s，PT为（19.44±6.95） s，TT为（58.27±12.44）s，除PLT降低外，其余4项指标均升高或延长； LPS为（322.85±104.54） U/L，IL-6为（285.51±81.46） ng/mL，PAF为 （14 714.70±4 427.95） ng/L， 三者均升高； PLT与LPS、IL-6和PAF之间呈负相关关系（P＜0.001），而D-D、APTT、PT和TT与LPS、IL-6和PAF之间均呈正相关关系（P＜0.001）。结论 LPS、IL-6及PAF可能参与了重症胸腹创伤后凝血功能障碍的发生；重症胸腹损伤后出现的微循环障碍及内毒素血症是凝血功能障碍发生的重要机理。针对LPS、IL-6和PAF进行早期干预，有可能改善重症胸腹创伤患者的凝血功能障碍。
Objective To investigate the effect of microRNA-27a (miR-27a) on the apoptosis of human lung adenocarcinoma cells A549 induced by lipopolysaccharide (LPS) by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway, and its mechanism is discussed preliminarily. Methods The complementary binding sites of miR-27a and phosphatidylinositol-3 kinase catalytic subunit delta (PIK3CD) were analyzed by Starbase and verified by double luciferase. The A549 cells were divided into normal group, LPS group, LPS+miR-27a mimic negative control group, LPS+miR-27a mimic group, LPS+miR-27a mimic+PI3K activator group. In the LPS+miR-27a mimic negative control group, LPS+miR-27a mimic group and LPS+miR-27a mimic+PI3K activator group, the cells were transfected with miR-27a mimic negative control, miR-27a mimic and miR-27a mimic, respectively, and were cultured for 6 h. After that, the cells were cultured in complete medium for 24 h, and then, except for the normal group, the cells in the other groups were stimulated with 10 mg/L LPS for 24 h, and the PI3K activator 740 Y-P was added to the LPS+miR-27a mimic+PI3K activator group, and cells in normal group were cultured in complete medium for the same time. Real-time quantitative polymerase chain reaction was used to detect the expression level of miR-27a in cells; cell counting kit 8 was used to detect cell proliferation; Hoechst33342 staining and flow cytometry was used to detect apoptosis; autophagy of A549 cells was observed by transmission electron microscope; Western blot was used to detect the expression of PIK3CD, phosphorylated-AKT (p-AKT), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3 and microtubule-associated protein 1 light chain 3 II (LC3II) protein. Results There was a binding site between miR-27a and PIK3CD, which was verified by double luciferase. Compared with those in normal group, the expression level of miR-27a, proliferation rate and protein expression level of Bcl-2 in LPS group and LPS+miR-27a mimic negative control group were lower (P<0.05), the apoptosis rate, protein expression levels of PIK3CD, p-AKT, Bax, cleaved caspase-3, LC3Ⅱ were higher (P<0.05); compared with those in LPS group and LPS+miR-27a mimic negative control group, the expression level of miR-27a, proliferation rate and protein expression level of Bcl-2 in LPS+miR-27a mimic group were higher (P<0.05), the apoptosis rate, protein expression levels of PIK3CD, p-AKT, Bax, cleaved caspase-3, LC3Ⅱ were lower (P<0.05); compared with those in LPS+miR-27a mimic group, the expression level of miR-27a and proliferation rate in LPS+miR-27a mimic+PI3K activator group were lower (P<0.05), the apoptosis rate, protein expression levels of PIK3CD, p-AKT, cleaved caspase-3, LC3Ⅱ were higher (P<0.05). The number of cells in the normal group was more, the cells were closely arranged, the nucleus size was uniform, and the organelle structure was normal; in LPS group and LPS+miR-27a mimic negative control group, cells became round, nuclei pyknosis, formed clumps, and showed multiple round autophagic vesicles of different sizes; the number of nuclear pyknotic cells in LPS+miR-27a mimic group decreased, and the number of nuclear pyknotic cells in LPS+miR-27a mimic+PI3K activator group increased compared with LPS+miR-27a mimic group, a small number of circular autophagic vesicles were observed, but the number was different. Conclusion Overexpression of miR-27a can inhibit PI3K/Akt pathway and reduce LPS induced apoptosis of human lung adenocarcinoma cells A549, which may be related to the reduction of autophagy.
ObjectiveTo study the protective effect and mechanism of ophiopogonin D (OP-D) on lipopolysaccharide induced acute lung injury (ALI) in mice.MethodsFifty SPF C57BL/6 mice were randomly divided into five groups, ie. a control group, a sham operation group, a model group, an OP-D group (10 mg·kg–1·d–1), and a dexamethasone group (2 mg·kg–1·d–1), with 10 mice in each group. One day before the establishment of the model, the OP-D group and the dexamethasone group received the corresponding drugs by gavage. The model group, the OP-D group and the dexamethasone group received lipopolysaccharide (2 mg/kg, 30 μL) through the trachea to establish the ALI model. The sham operation group received the same volume of normal saline. The blank control group was not treated. Six hours after the operation, the mice were weighed and then killed for peripheral blood and lung tissue. The weight of lung tissue was measured to evaluate the degree of pulmonary edema; the pathological changes of lung tissue were observed by hematoxylin-eosin staining; the mRNA expressions of interleukin (IL)-6, IL-10, and IL-17 in lung tissue were detected by qPCR; the percentage of Th17 and Treg cells in peripheral blood was detected by flow cytometry.ResultsCompared with the model group, the degree of pulmonary edema in the OP-D group decreased significantly (P<0.05), the lung tissue injury decreased, the mRNA expressions of IL-6 and IL-17 in the lung tissue and the proportion of Th17 cells in the peripheral blood decreased significantly (P<0.05), the proportion of Treg cells in the peripheral blood and the mRNA expression of IL-10 in the lung tissue increased significantly (P<0.05).ConclusionOP-D may have therapeutic effect on LPS induced ALI in mice by regulating the balance of Th17/Treg cells.