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.
ObjectiveTo study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function.MethodsBone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate resistant acid phosphatase (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area.ResultsThe flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS (P<0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant (P<0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group (P<0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant (P<0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant (P<0.05).ConclusionLPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.
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 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.
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.
Objective To compare three approaches of lipopolysaccharides ( LPS) administration for inducing acute lung injury ( ALI) in mice. Methods LPS ( 5 mg/kg) was intratracheally aerosol administered ( ITA group) , intratracheally instilled ( ITI group) , or intraperitoneally injected ( IPI group) to induce ALI in BLAB/ c mice. Evans Blue instead of LPS was intratracheally administered to observe the liquid distribution in the lungs. Two hours after LPS administration, the mice were sacrificed and the lungs were removed to determine wet-to-dry lung weight ratio ( W/D) , and the histological changes were evaluated by HE staining. Phosphorylation level of IκB-αand NF-κB p65 in lung tissue were investigated by Western blot. Transcription intensity of TNF-α and IL-1β mRNA in lung tissue were detected by real-time quantitative PCR. Results Evans Blue distributed more uniformly in the ITA group than the ITI group. The lung W/D ratio and histological changes score in three LPS administration groups were all significantly higher than the normal control group ( P lt;0. 01) , with the ITA group being the highest. The phosphorylation levels of IκB-αand NF-κB p65 were significantly higher in the ITA group than the ITI group ( P lt;0. 05) , and were significantly higher in the ITI group than the IPI group ( P lt; 0. 05) . Transcription intensity of TNF-αand IL-1βmRNA was significantly higher in the ITA group than the ITI group ( P lt;0. 05) , and were significantly higher in the ITI group than the IPI group ( P lt;0. 05) . Conclusion Being non-invasive and convenient,intratracheal LPS aerosol inhalation is an optimal method to induce ALI in mice because it induces more extensive and uniformly distributed injuries in lung.
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.
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.
目的 探討脂多糖（LPS）、白細胞介素-6（IL-6）和血小板活化因子（PAF）與重癥胸腹創傷后凝血功能紊亂發生的相關性及可能的致病機理。方法 收集2009年1月至2011年12月期間在中國人民解放軍第二五三醫院急診科就診、創傷指數≥17分且除外合并顱腦損傷及在急診科內死亡的胸腹創傷患者62例，在予以搶救、治療的同時抽血檢查血小板計數（PLT）、血漿D-二聚體（D-D）、部分活化凝血酶原時間（APTT）、凝血酶原時間（PT）、LPS、IL-6和PAF，并對其結果進行相關性分析。結果 本組患者就診時檢測的PLT為（157.73±78.11）×109/L， D-D為（1 023.88±208.72） U/L，APTT為（46.95±17.85） s，PT為（19.44±6.95） s，TT為（58.27±12.44）s，除PLT降低外，其余4項指標均升高或延長； LPS為（322.85±104.54） U/L，IL-6為（285.51±81.46） ng/mL，PAF為 （14 714.70±4 427.95） ng/L， 三者均升高； PLT與LPS、IL-6和PAF之間呈負相關關系（P＜0.001），而D-D、APTT、PT和TT與LPS、IL-6和PAF之間均呈正相關關系（P＜0.001）。結論 LPS、IL-6及PAF可能參與了重癥胸腹創傷后凝血功能障礙的發生；重癥胸腹損傷后出現的微循環障礙及內毒素血癥是凝血功能障礙發生的重要機理。針對LPS、IL-6和PAF進行早期干預，有可能改善重癥胸腹創傷患者的凝血功能障礙。