Objective To investigate influence of genders on the activity of nuclear factor-kappa B (NF-κB) in lungs of endotoxemic rats. Methods Twenty female and 20 male Wistar rats were randomly divided into four groups as follow: female control group (n=10), male control group (n=10), male endotoxemic group (n=10), and female endotoxemic group (n=10). The endotoxemic rats model was made by injecting lipopolysaccharide (5 mg/kg) into the abdominal cavity. Tissue samples were collected from the lungs in different groups and electrophoresis mobility shift assay was used to measure the activity of NF-κB. The levels of serum TNF-α and estrogen were measured at the same time. Results There was no significant difference between the activities of NF-κB in male and female control groups (1.33±0.24 vs 1.47±0.40）, and there was also no significant difference between other items in these groups as well (Pgt;0.05). Yet, the activity of NF-κB (female: 12.10±2.89; male: 19.53±2.12) and the level of TNF-α 〔female: (4.10±0.72) ng/ml; male: (6.37±1.29) ng/ml〕 were significantly increased after injection of lipopolysaccharide (Plt;0.01), and the indices in female group were significantly lower than those in male group (Plt;0.01). Correlation analysis showed that there was a positive relation between the activity of NF-κB in lungs and the level of TNF-α (female: r=0.921 1, P=0.013; male: r=0.907 2, P=0.017), and there was a negative correlation between the activity of NF-κB and the level of estrogen (female: r=-0.887 5, P=0.017; male: r=0.872 3, P=0.022) in both male endotoxemic group and female endotoxemic group (Plt;0.05). Conclusion Gender may be one of the factors that influence the activity of NF-κB in the lungs of endotoxemic rats. While on the other hand, endogenous estrogen may protect the lungs of endotoxemic rats from injury by inhibiting the activity of NF-κB.
ObjectiveTo explore the protective mechanism and effect of the resveratrol for kidney injury of obstructive jaundice. MethodsThe rats were randomly divided into three groups: sham operation group receiving laparotomy without bile duct ligation (BDL), the obstructive jaundice group with BDL, and the obstructive jaundice + resveratrol group given resveratrol following BDL. The levels of total bilirubin (TBIL), direct bilirubin (DBIL), blood urea nitrogen (BUN), and creatinine (Cr) in the serum were tested. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, glutathione (GSH) level in the renal tissues were detected. The expressions of the silent information regulator 1 (SIRT1) and nuclear factor-κB (NF-κB) proteins were tested by Western blot. The expression of SIRT1 mRNA was detected by RT-PCR and the renal cell apoptosis was examined by TUNEL staining. Results①Compared with the sham operation group, the levels of serum TBIL, DBIL, BUN and Cr were significantly higher (P < 0.05); the activity of SOD and level of GSH, and the expressions of SIRT1 mRNA and SIRT1 protein in the renal tissues were signi-ficantly lower (P < 0.05); the content of MDA, the expression of NF-κB protein, and the rate of cell apoptosis in the renal tissues were significantly higher (P < 0.05) in the obstructive jaundice group.②Compared with the obstructive jaundice group, the levels of serum TBIL, DBIL, BUN and Cr were significantly lower (P < 0.05); the activity of SOD and level of GSH, and the expressions of SIRT1 mRNA and SIRT1 protein in the renal tissues were significantly higher (P < 0.05); the content of MDA, the expression of NF-κB protein, and the rate of cell apoptosis in the renal tissues were significantly lower (P < 0.05) in the obstructive jaundice+resveratrol group. ConclusionThe resveratrol could alleviate renal damage and play a beneficial role to resist inflammation, oxidation, and apoptosis by activating the SIRT1 which probably inhibits the expression of NF-κB protein and promotes the activity of SOD in cholestatic kidney injury.
Objective To investigate the expression changes of nuclear factor kappa B (NF-κB) and matrix metalloproteinase-9 (MMP-9) in the cultured hepatocellular carcinoma cells 9204 (HCC9204) transfected with inhibitory kappa B alpha（IκB-α)vector. Methods After pcDNA3-IκB-α vector and pcDNA3 were transfected into HCC9204 by lipofectamine method, Western-blot and RT-PCR analysis were used to detect the expressions of NF-κB and MMP-9. Migration and invasion of tumor cells were assayed by fundus membrane invaded by them. Results When pcDNA3-IκB-α was transfected into HCC9204, the expression of NF-κB was decreased at the protein level, and the expression of MMP-9 mRNA and the invision and metastasis ability of transfected cells were obviously decreased. Conclusion When the activity of NF-κB is inhibited, the ability of invasion and metastasis in HCC9204 cells decrease, which could be related to the decreased the expression of MMP-9.
Objective To observe the effects of exogenous pulmonary surfactant (PS) on ventilation-induced lung injury (VILI) in rats, and to investigate its possible mechanisms. Methods A total of 40 Wistar rats were divided into 4 groups with randomized blocks method: control group, high tidal volume (HV) group, VILI group, and PS group, with 10 rats in each group. The control group was subjected to identical surgical procedure but was never ventilated. After 30 min of mechanical ventilation (MV) with Vt 45 ml/kg, the rats in HV group were killed immediately; rats in the VILI group were continually ventilated for up to 150 min with Vt 16 ml/kg; in the PS group, 100 mg/kg of PS administered intratracheally and with the same settings as VILI group. Mean artery pressure (MAP), blood gas analysis, lung wet to dry weight ratios (W/D), thorax-lung compliance, and cell counts in bronchoalveolar lavage fluid (BALF) were determined. Nuclear factor-κB(NF-κB) activity in lungs was measured by enzyme-linked immunosorbent assay (ELISA), interleukin-8(IL-8) in serum and BALF was determined by radioimmunoassay (RIA). Pathological examination of the lung was performed. Results Injurious ventilation significantly decreased MAP and PaO2/FiO2, but increased NF-κB activity and W/D. MAP and PaO2/FiO2 improved, but NF-κB activity, IL-8 in serum and BALF, and cell counts in BALF reduced significantly in PS group compared with those in VILI group. Histological studies showed reduced pulmonary edema and atelectasis in the PS group. Conclusion PS administered intratracheally can suppress the increased activity of NF-κB induced by VILI, exogenous PS can be used to treat VILI.
【 Abstract 】 Objective Overexpressions of epidermal growth factor (EGF) and EGF receptor have been associated with progression and invasive phenotype of pancreatic cancer. However, the underlying molecular mechanism by which EGF worked in pancreatic cancer cells has not been completely understood. In this study, effect of EGF on the invasion and metastasis of pancreatic cancer cells and its regulatory mechanism were investigated. Methods The effects of EGF on the proliferation, adhesion and invasion of pancreatic cancer cells were detected by WST-1 proliferation assay, adhesion assay and invasive assay, respectively. The activity and expression of MMP-2 and MMP-9 were examined by zymography, Western blot and RT-PCR, respectively. The activity of NF- κ B was examined by EMSA. Results EGF could significantly promote the invasiveness of pancreatic cancer cells but did not affect cell proliferation or adhesion. The expressions of NF- κ B and MMP-9 were significantly increased by EGF, but EGF did not affect the activity and expression of MMP-2. Furthermore, EGF stimulated the NF- κ B binding activity. Pretreatment with NF- κ B inhibitors, pyrrolidine dithiocarbamate (PDTC), could significantly inhibit the activity of NF- κ B induced by EGF. Meanwhile, the EGF-induced expression and activity of MMP-9, as well as cell invasiveness were also inhibited by NF- κ B inhibitor. Conclusion EGF could increase the expression and promote the invasiveness of MMP-9 via the activation of NF- κ B in pancreatic cancer cells, which implies that NF- κ B inhibitant, such as PDTC, may diminish the invasiveness of pancreatic cancer cells.
Objective To investigate the changes in osteoprotegerin (OPG) / receptor activator of nuclear factor-κB ligand (RANKL) ratio in sepsis-associated acute lung injury (SA-ALI) and the role of regulation of this ratio on the inflammatory response in SA-ALI. Methods Eighteen C57BL/6 male mice were randomly divided into sham operation group, cecal ligation and perforation (CLP) group and RANKL group, with 6 mice in each group. Before the experiment, the RANKL group was intraperitoneally injected with 5 μg (0.2 mL) of recombinant RANKL antibody, whereas both the sham operation group and the CLP group were intraperitoneally injected with a volume-matched normal saline. One hour later, the sham operation group underwent only abdominal exploration and repositioning, while the other groups underwent the CLP surgery to induce the SA-ALI model. After 24 h of modelling, all mice were sacrificed and samples were collected. Pathological evaluation of lung tissues was performed by haematoxylin-eosin staining; enzyme-linked immunosorbent assay was used to detect serum concentrations of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β; while the mRNA and protein expression of OPG and RANKL, along with their ratio values, were detected by real-time polymerase chain reaction for quantitative analysis and protein immunoblotting. Results The SA-ALI mouse model was successfully established. Compared with the sham operation group, mice in the CLP group showed disturbed alveolar structure, obvious alveolar and interstitial haemorrhage and inflammatory cell infiltration, elevated serum levels of IL-6, TNF-α and IL-1β (P<0.05), significantly increased mRNA and protein expression of OPG and elevated OPG/RANKL ratio in lung tissue (P<0.05), whereas RANKL mRNA and protein expression was significantly decreased (P<0.05). Compared with the CLP group, the pathological damage of lung tissue in the RANKL group was reduced, the infiltration of alveolar and interstitial inflammatory cells was significantly improved, and the alveolar structure and morphology were more regular, with lower serum levels of IL-6, TNF-α and IL-1β (P<0.05), significantly lower mRNA and protein expression of OPG and OPG/RANKL ratio in lung tissue (P<0.05), and significantly higher mRNA and protein expression of RANKL in lung tissue (P<0.05). Conclusion The alteration of OPG/RANKL ratio may be related to the pathophysiological process of SA-ALI, and the decrease in its level may reflect the attenuation of the inflammatory response in SA-ALI.
ObjectiveTo investigate the role and mechanism of P-selectin glycoprotein ligand-1 (PSGL-1) in hydrochloric acid-induced acute lung injury (ALI) in mice.MethodsWild-type mice (WT) and PSGL-1 knockout mice (PSGL-1 -/-) were randomly subjected to normal saline (NS) or hydrochloric acid (HCl) challenged group. The mice were intratracheally instilled with NS or HCl (1 μl/g weight) into the left lung with a catheter. After 2 hours, respiratory function index enhanced pause (Penh), PaO2 and PaO2 were analyzed. The wet to dry weight ratio (W/D) of the left lung and total protein concentration in bronchoalveolar lavage fluid (BALF) were measured. The number of leukocytes in BALF was counted too. Targeted lung tissue was processed for further HE or immunohistochemistry staining. Meanwhile, the expressions of interleukin-6 (IL-6), IL-1β, nuclear factor-κB (NF-κB), IκBa and p-IκBa in lung tissue were measured.ResultsThe Penh (4.77±1.22 vs. 5.80±0.84) and PaCO2 [(63.7±3.9) mm Hg vs. (74.4±7.4) mm Hg] in the PSGL-1 knockout mice were significantly lower than those in the WT mice after HCl stimulation (P<0.05), while the PaO2 was higher than that in the WT mice [(81.0±7.1) mm Hg vs. (62.0±8.9) mm Hg, P<0.05)]. The lung W/D ratio (4.86±0.15 vs. 5.22±0.20), protein concentration [(3.71±0.64) μg/μl vs. (4.74±0.98) μg/μl] and total leukocyte count [(13.00±2.18) ×107/L vs. (49.42±3.35) ×107/L] in BALF were significantly lower in the PSGL-1 knockout mice challenged with HCl than those in the WT mice (P<0.05). Besides, the protein expressions of IL-6, IL-1β, p65 and p-IκBa in the PSGL-1 knockout mice were lower than those in the WT mice after HCl instillation, while the IκBa expression was higher than that in the WT mice (P<0.05). More numbers of neutrophils and macrophages were found in the lung of the WT mice than the PSGL-1 knockout mice challenged with HCl. However, the differences of above values between the WT mice and the PSGL-1 knockout mice instilled with NS were not found, all of which were significantly lower than the correspongding HCl group except for IκBa (P<0.05).ConclusionPSGL-1 may play important roles in the development of HCl-induced ALI via the NF-κB signaling pathway and inflammation.
Objective To investigate the effects of glutathione S-transferase M5 (GSTM5) on the inflammation in human bronchial epithelial 16HBE cells and its possible molecular mechanisms. Methods Acute lung injury cell model was constructed with 16HBE cells induced by tumour necrosis factorα (TNF-α, 10 ng/mL). The cells were devided into a control group, a TNF-α group (TNF-α), a GSTM5 group (GSTM5+TNF-α), a negative control group (negative control plasmid+TNF-α). GSTM5-GFP plasmid and negative control plasmid were respectively transfected to the cells of the GSTM5 group and the negative control group using Lipofectamine2000. The contents of interleukin-6(IL-6), IL-8, IL-10 in the cell supernatant were measured by ELISA.The expression of nuclear factor-κB (NF-κB) mRNA was detected by RT-PCR, and the expression of NF-κB, phospho-NF-κB, p38, phospho-p38 protein were detected by Western blot. Results The GSTM5-GFP eukaryotic expression vector was successfully constructed and transfected successfully confirmed by fluorescence microscope. The contents of IL-6, IL-8, IL-10 in the TNF-α-induced cell supernatant were significantly higher than those in the control group(P < 0.05), and the contents of IL-6, IL-8, IL-10 in the GSTM5 group were lower than those in the TNF-α group (P < 0.05)with statistically significant difference. At the same time, the total NF-κB mRNA, phospho -NF-κB and phospho-p38 protein were increased in TNF-α stimulated cells compared with the control group (P < 0.05), while the GSTM5 group was lower than that in the TNF-α group and the negative control group (P < 0.05). Conclusion Overexpression of GSTM5 inhibits the phosphorylation of p38MAPK and NF-κB and down-regulates the inflammation of TNF-α-induced human bronchial epithelial 16HBE cells.
Objective To investigate the effects of nuclear factor kappa B decoy oligodeoxynucleotides ( NF-κB decoy ODN) transfection on biological characteristics of mature dendritic cells ( mDCs) in mice. Methods Immature DCs were harvested from Balb / c mice bone marrow, followed by the incubation with antigen OVA and LPS, and mature DCs were evaluated by the expressions of CD11c and MHC-Ⅱ detected by FACS. Mature DCs were transfected with NF-κB decoy ODN and the changes of NF-κB activity after the transfection were detected by EMSA. The expressions of the costimulatory molecules( CD40,CD80 and CD86) on DCs were detected by FACS and the proliferation of T cells was tested by mixed lymphocyte reaction( MLR) . Results The mature DCs were cultured successfully. The NF-κB activity of NF-κB decoy ODN transfected DCs was decreased significantly( P lt; 0. 05) . There was no difference in the expressions of CD40 and CD80, but the expression of CD86 was decreased significantly in NF-κB decoy ODN transfection group( P lt; 0. 05) . MLR test showed that the proliferation of T lymphocyte cells was inhibited by NF-κB decoy ODN transfected DCs, but was stimulated bly by the DCs of other groups. Conclusions Mature DCs transfected with NF-κB decoy ODN could inhibit the proliferation and activation of antigenspecical T cells, which was probably related to the down-regulation of CD86 on DCs. This modified DCs might be a promising vaccine for the treatment of asthma in the future.
目的探讨髓样细胞分化蛋白88（MyD88）在重症急性胰腺炎（SAP）发病机理的作用。 方法将48只小鼠按随机数字表法随机分为SAP组（32只）与正常对照组（16只）；再将2组小鼠随机（随机数字表法）分为6、12、24及48 h组，SAP组各亚组每组8只，正常对照组每亚组4只。SAP组小鼠腹腔注射20% L-精氨酸以诱导SAP模型，正常对照组小鼠仅腹腔注射生理盐水。分别于建模术后6、12、24及48 h处死小鼠，取其动脉血，采用ELISA方法检测血清中白细胞介素-1β（IL-1β）、白细胞介素-10（IL-10）及肿瘤坏死因子-α（TNF-α）浓度；同时取其胰腺组织（正常对照组仅术后6 h取材），用逆转录-聚合酶链反应（RT-PCR）方法检测胰腺组织中MyD88 mRNA和核因子-κB（NF-κB）mRNA的表达水平，并进行HE染色。 结果镜下见SAP组小鼠的胰腺组织随时间进展其炎症逐渐加重。各时点SAP组小鼠的IL-1β、IL-10及TNF-α浓度均高于正常对照组（P＜0.05）；各时点SAP组与正常对照组（6 h组）相比较，其胰腺组织中MyD88 mRNA及NF-κB mRNA的表达水平均较高（P＜0.05）。各时点SAP组小鼠MyD88 mRNA的表达水平与血清IL-1β、IL-10及TNF-α的浓度和NF-κB mRNA的表达水平均呈正相关（P＜0.01）。 结论MyD88的表达对SAP的发生和发展可能均具有重要的作用。