Triggering of toll-like receptors 2 and 4 by Aspergillus fumigatus conidia in immortalized human corneal epithelial cells to induce inflammatory cytokines

Background Cornea epithelial cells play eady and crucial roles in the initiation of ocular surface responses to pathogens. Participation of toll-like receptor （TLR） 2 and TLR4, which are major forms of fungi receptors, may be involved in Aspergillus fumigatus induced immune responses. The objective of the present study was to examine whether inactive Aspergillus fumigatus conidia induce NF-KB activation and production of proinflammaory cytokines, and whether the expression of TLR2 and TLR4 were amplified by conidia in cultured immortalized human corneal epithelial cells （THCEs）. This may contribute to our knowledge of the mechanism by which the host cornea can successfully defend against invasive fungi. Methods Aspergillus fumigatus conidia were used to challenge THCE cells. THCE cells were harvested after 0.5, 1, 2 or 4 hours incubation. Real-time quantitative PCR was performed to determine the expression of TLR2, TLR4, TNF-a and IL-8. Western blotting was performed to determine the expression of NF-KB. Enzyme-linked immunosorbent assay （ELISA） was performed to determine the expression of TNF-a and IL-8. And the release of TNF-a and IL-8 in the cell supematant were also assessed by ELISA with or without pretreatment with TLR2 and TLR4 neutralizing antibodies. Results Aspergillus fumigatus conidia elicited the expression of TLR2, TLR4, TNF-a and IL-8 mRNA in THCEs. Exposure of THCE cells to Aspergillus fumigatus conidia resulted in NF-KB activation, which increased at 30 minutes （increased from 11.35±2.74 in the controls to 19.12±3.48, p〈0.05） and thereafter increased steadily up to 4 hours after challenge （P 〈0.01）. Concomitant with NF-KB activation, secretion of TNF-α and IL-8 in conidia-challenged cells was increased in a time-dependent manner. Incubation of THCE cells with TLR2 antibody or TLR4 antibody before conidia challenge resulted in inhibition of conidia-induced TNF-α and IL-8 secretion （P 〈0.05）, TLR2 antibody and TLR4 antibody together significantly increased inhibit     

Role of Toll-like receptor 4 and human defensin 5 in primary endocervical epithelial cells

Background Endocervical epithelial cells play early roles in the defense of upper female genital tract to pathogens. Toll-like receptors （TLRs） and human defensins （HD） have recently been identified as fundamental components of the innate immune responses to bacterial pathogens. We aimed to use in vitro model of human primary endocervical epithelial cells （HPECs） to investigate their roles in innate immune response of the endocervix. Methods TLR4 expression and distribution in HPECs and endocervix were investigated by immunofluorescence （IF）. Cultured HPECs were divided into lipopolysaccharide （LPS） group which were treated by LPS for 0, 24 and 48 hours, and control group without treatment. At each time point, the levels of HD5, IL-6 and TNF-a in supernants were determined by ELISA. TLR4 and HD5 expressions of cells were detected by Western blotting simultaneously. HD5 expression pattern was also compared between the HeLa cell line and HPECs. Results Endocervix tissue surface and HPECs expressed TLR4. After incubated with LPS, HPECs expressed significantly higher levels of TLR4 than control group, especially after 24 hours （P 〈0.01）, however decreased after 48 hours with a similar level of TLR4 expression compared with control group. LPS could upregulate the secretion of HD5, IL-6 and TNF-a in a time-dependent manner （24 hours： P 〈0.05; 48 hours： P 〈0.01, compared with control group）. Intracellular HD5 expression levels decreased over time. HD5 expression patterns in HPECs were different from HeLa cell line. Conclusions To respond to LPS stimulation, HPECs may function in the mucosal immune defense through TLR4 activation and HD5 secretion. HPEC is considered a significant model for immunological study.     

Role of Toll-like receptor 4 and human defensin 5 in primary endocervical epithelial cells

Background Endocervical epithelial cells play early roles in the defense of upper female genital tract to pathogens. Toll-like receptors (TLRs) and human defensins (HD) have recently been identified as fundamental components of the innate immune responses to bacterial pathogens. We aimed to use in vitro model of human primary endocervical epithelial cells (HPECs) to investigate their roles in innate immune response of the endocervix.Methods TLR4 expression and distribution in HPECs and endocervix were investigated by immunofluorescence (IF). Cultured HPECs were divided into lipopolysaccharide (LPS) group which were treated by LPS for 0, 24 and 48 hours, and control group without treatment. At each time point, the levels of HD5, IL-6 and TNF-a in supernants were determined by ELISA. TLR4 and HD5 expressions of cells were detected by Western blotting simultaneously. HD5 expression pattern was also compared between the HeLa cell line and HPECs.Results Endocervix tissue surface and HPECs expressed TLR4. After incubated with LPS, HPECs expressed significantly higher levels of TLR4 than control group, especially after 24 hours (P <0.01), however decreased after 48 hours with a similar level of TLR4 expression compared with control group. LPS could upregulate the secretion of HD5, IL-6 and TNF-α in a time-dependent manner (24 hours: P <0.05; 48 hours: P <0.01, compared with control group). Intracellular HD5 expression levels decreased over time. HD5 expression patterns in HPECs were different from HeLa cell line.Conclusions To respond to LPS stimulation, HPECs may function in the mucosal immune defense through TLR4 activation and HD5 secretion. HPEC is considered a significant model for immunological study.     

Role of Toll-like receptor 4 and human defensin 5 in primary endocervical epithelial cells

Background Endocervical epithelial cells play early roles in the defense of upper female genital tract to pathogens. Toll-like receptors (TLRs) and human defensins (HD) have recently been identified as fundamental components of the innate immune responses to bacterial pathogens. We aimed to use in vitro model of human primary endocervical epithelial cells (HPECs) to investigate their roles in innate immune response of the endocervix.Methods TLR4 expression and distribution in HPECs and endocervix were investigated by immunofluorescence (IF). Cultured HPECs were divided into lipopolysaccharide (LPS) group which were treated by LPS for 0, 24 and 48 hours, and control group without treatment. At each time point, the levels of HD5, IL-6 and TNF-a in supernants were determined by ELISA. TLR4 and HD5 expressions of cells were detected by Western blotting simultaneously. HD5 expression pattern was also compared between the HeLa cell line and HPECs.Results Endocervix tissue surface and HPECs expressed TLR4. After incubated with LPS, HPECs expressed significantly higher levels of TLR4 than control group, especially after 24 hours (P <0.01), however decreased after 48 hours with a similar level of TLR4 expression compared with control group. LPS could upregulate the secretion of HD5, IL-6 and TNF-α in a time-dependent manner (24 hours: P <0.05; 48 hours: P <0.01, compared with control group). Intracellular HD5 expression levels decreased over time. HD5 expression patterns in HPECs were different from HeLa cell line.Conclusions To respond to LPS stimulation, HPECs may function in the mucosal immune defense through TLR4 activation and HD5 secretion. HPEC is considered a significant model for immunological study.     

ROLE OF TOLL-LIKE RECEPTOR 4 IN AIRWAY INFLAMMATION OF CHRONIC OBSTRUCTIVE PULMONARY DISEASES

Objective To confirm if pulmonary epithelial cells express Toll-like receptor 4 （TLR4） and investigate the role of TLR4 in airway inflammation of chronic obstructive pulmonary diseases （COPD）. Methods The expressions of TLR4, IL-8 mRNA and NF-KB activation stimulated by differen factors E lipopolysacharides （LPS）, interleukin-lβ, cigarette smoking extract （CSE）] in pulmonary epithelial cells were investigated. Results LPS, CSE and IL-lβ induced the production of IL-8 and activation of NF-KB. The levels of 1L-8 mRNA and NF-KB protein in E1A ＋ cell were markedly higher than E1A- cell and A549 cell （ P 〈0. 05）. The TLR4 mRNA of all the cells increased along with the increase of LPS＇ stimulated time. There was significant difference among different LPS＇ doses （ 12 h： P = O. 039 ; 24 h ： P = O. 013 ）. The TLR4 mRNA of E1A ＋ cell was higher than the other two groups （ P 〈0. 05）. IL-lβ induced all the cells expressing TLR4 mRNA. CSE had no effect on the expression of TLR4 mRNA. Conclusion Pulmonary epithelial cells express TLR4. LPS and IL-lβ up-regulate IL-8 mediated via the activation of NF-KB induced by TLR4. But CSE up-regulates IL-8 mediated via the activation of NF-KB, which has no relation to TLR4 and may have another signal transduction pathway.     

Effect of Cigarette Smoke Extract on the Proliferation of Human Airway Epithelial Cells and Expression and Activation of FAK

The effect of cigarette smoke extract (CSE) on the proliferation of human airway epithelial cells and the possible mechanism was studied. After airway epithelial cells were treated with different concentrations of CSE for 24 h, the cell proliferation was measured by MTT and the distribution of different cell cycles by flow cytometry. The FAK expression level was detected by Western blot and the degree of tyrosine phosphorylation by immunoprecipitation. The results showed that CSE could inhibit the proliferation of human airway epithelial cells, arrest the epithelial cells in G1 phase of cell cycle, dramatically decrease the number of epithelial cells in S and G2 phases； Meanwhile CSE could decrease the expression level of FAK and the degree of its tyrosine phosphorylation. The above effects of CSE were concentration-dependent. The expression of FAK and the degree of its phosphorylation was positively correlated to the increased number of epithelial cells in G1 phase, and negatively to the number of epithelial cells in S and G2 phases. It was concluded that the mechanism by which CSE could inhibit the proliferation of human epithelial cells was contributed to the increased expression and activation of FAK.     

Expression and immune effect of toll-like receptor 4 in human trophoblast cells

This study investigated the expression and immune effect of TLR4 in human trophoblast cells. The expression level of TLR4 mRNA in normal and LPS-stimulated human term trophoblast cells （1 mg/L LPS, 12 h） was detected by RT-PCR. In LPS-stimulated human term trophoblast cells of TLR4-blocked group and non-TLR4-blocked group, and normal term trophoblast cells of blank control group, apoptosis rate was measured by flow cytometry （FCM）, and the level of TNF-α determined by using enzyme linked immunosorbent assay （ELISA） respectively. RT-PCR results showed that the expression level of TLR4 mRNA in LPS-stimulated human trophoblast cells was significantly higher than that in normal cells （P〈0.01）. FCM revealed that there was significant difference in apoptosis rate of LPS-stimulated human term trophoblast cells between TLR4-blocked group and non-TLR4-blocked group （P〈0.05）, or between TLR4 antibody-blocked group and blank control group. ELISA indicated that the level of TNF-α in LPS-stimulated human trophoblast cells also had statistical differences between TLR4 antibody-blocked group and non-TLR4 antibody-blocked group （P〈0.05）. Our results suggest that TLR4 plays an important role in the immunological mechanism of apoptosis and secretion of TNF-α of human term trophoblast cells stimulated by LPS.     

Ketamine Promotes Inflammation through Increasing TLR4 Expression in RAW264.7 Cells

Summery: Ketamine(KTM), a N-methyl-D-aspartate(NMDA) receptor antagonist, was found to has an anti-inflammatory effect, but some patients suffered from exacerbated pro-inflammatory reactions after anesthesia with KTM. The present study was aimed to examine the underlying mechanism of pro-inflammatory effects of KTM. In this study, RAW264.7 cells were exposed to KTM and NMDA alone or combined for 30 min before lipopolysaccharide(LPS) stimulation. The expression levels of IL-6 and TNF-α were detected by RT-PCR and ELISA, and those of NMDA receptors by RT-PCR in RAW264.7 cells. Additionally, the TLR4 expression was determined by RT-PCR and flow cytometry, respectively. The results showed that in RAW264.7 cells, KTM alone promoted the TLR4 expression, but did not increase the expression of IL-6 or TNF-α. In the presence of LPS, KTM caused a significantly higher expression of IL-6 and TNF-α than LPS alone. NMDA could neither alter the IL-6 and TNF-α m RNA expression, nor reverse the enhanced expression of IL-6 and TNF-α m RNA by KTM in LPS-challenged cells. After TLR4-si RNA transfection, RAW264.7 cells pretreated with KTM no longer promoted the IL-6 and TNF-α expression in the presence of LPS. In conclusion, KTM accelerated LPS-induced inflammation in RAW264.7 cells by promoting TLR4 expression, independent of NMDA receptor.     

Toll-like receptor 4 and human defensin 5 in normal endocervical epithelial cells

BACKGROUND: Toll like receptors (TLRs) and human defensins (HD) have recently been identified as fundamental components of the innate immune responses to bacterial pathogens. We used in vitro model of human primary endocervical epithelial cells (HPEC) to investigate their role in the immune defense of the endocervix. METHODS: TLR4 expression in HPEC and endocervix were investigated by Immunofluorescence (IF). HPEC were treated with lipopolysaccharide (LPS) for 0, 24 and 48 hours, and at each time point, the supernatant was harvested and analyzed by ELISA for secretion of HD5, IL-6 and TNF-α, and the cell proteins were analyzed for TLR4 and HD5 by western blot and/or IF detection. We also compared HD5 expression pattern between the HeLa cell line and HPEC. Finally, the data was analysed with SPSS10.0. RESULTS: TLR4 expression on the surface of endocervix and HPEC were testified. LPS could upregulate the secretion of HD5, IL-6 and TNF-αin a time dependent manner (after 24hours:P<0.05;after 48hours:P<0.01 compared to unstimulated cells),while intracellular HD-5 decreased over time. TLR4 protein in HPEC increased over a 24 hour time period, and thereafter decreased (P<0.01, comparing to unstimulated). HD5 expression patterns in HPEC are different from HeLa. CONCLUSION: TLR4 in HPEC is constitutively expressed and functional. Our results show that the HPEC may take part in the mucosal immune response to LPS and play an important role. With LPS stimulation, HPEC can secret HD5 to the lumen. HPEC may be better model for immunological study.     

Expression of HLA-DR antigen in hepatocellular carcinoma and its up-regulation by interferon

Objective: To observe the expression of human leukocytic antigen DR (HLA-DR) in primary hepatocellular carcinoma (HCC) and its up-regulation by interferon (IFN). Methods: The expression of HLA-DR in 46 specimens of human HCC tissues, 4 human HCC cell lines (SMMC-7721, HCC-9204, BEL-7402 and HHCC) and a human hepatocyte cell line QZG was respectively detected by immunohistochemical ABC staining and flow cytometry. The expression of HLA-DR in the 5 cell lines was detected by ELISA before and after the cells were treated with IFN-γ or IFN-α. Results:Eighteen out of 46 HCC tissues (39.1%) expressed HLA-DR, whereas all the normal liver tissues immediately adjacent to HCC tissues were HLA-DR-negative. No obvious HLA-DR-positive staining was found in all the 5 cell lines. The expression of HLA-DR was up-regulated in all the 5 cell lines after IFN-γ or IFN-α treatment. The up-regulation of HI A-DR in QZG cells was less obvious than that in HCC cell lines. The effect of IFN-γ was more significant than that of IFN-α. Conclusion: HCC tissues can express HLA-DR to some extent, but HCC cell lines do not express detectable HLA-DR. IFN can up-regulate HLA-DR expression in HCC cells.     

Toll样受体在人眼角膜上皮和永生化人角膜上皮细胞系的表达及功能研究

目的探讨Toll样受体（TLR）1—9在人眼角膜上皮和上皮细胞系的表达及其功能性。方法以健康人外周血单个核细胞（PBMC）为阳性对照,收集20例健康青年人角膜上皮标本和培养永生化人角膜上皮细胞系（THCE）细胞,用半定量反转录聚合酶链反应检测TLR1～9mRNA表达;Western印迹检测TLR2、4的蛋白质表达;TLR3和TLR4的配体对THCE刺激后酶联免疫检测分泌IL-8的变化,结合抗体封闭实验,研究角膜上皮TLR的功能性。结果与PBMC比较,人角膜上皮强表达TLR1、2、3、5、6、9,弱表达TLR8,微弱表达TLR4;20例角膜上皮标本中发现1例TLR3、4、6、8阴性表达,1例TLR5微弱表达;人角膜上皮在蛋白质水平表达TLR2、4;THCE细胞与人角膜上皮有相似的TLR表达谱;LPS和PolyI：C刺激THCE1、4、8h后IL-8分泌增加,8h时分别达到对照组的10倍和7倍（均P〈0．05）,抗体封闭TLR4可以阻断LPS诱导的IL-8分泌。结论人角膜上皮表达TLR1—9,但不同TLRs表达水平有差异。THCE是研究人角膜上皮TLR表达和功能的良好细胞系。     

Toll样受体4在人肺泡上皮细胞株中的表达及其在细胞炎症反应中的作用

目的 明确人Ⅱ型肺泡上皮细胞是否表达Toll样受体4(TLR4),探讨TLR4在慢性阻塞性肺疾病(COPD)气道炎症中的作用.方法 将人Ⅱ型肺泡上皮细胞系A549细胞分为正常对照组、转染腺病毒E1A质粒(E1A+组)和转染不含有腺病毒E1A的空白质粒组(E1A-组),分别以不同浓度的脂多糖(LPS,0、0.1、1、10μg/ml)、白细胞介素1β(IL-1β,0、0.1 ng/ml)、香烟提取物(CSE,0、10%、20%、40%)刺激.刺激后12、24 h,用反转录聚合酶链反应技术检测各组细胞IL-8 mRNA和TLR4 mRNA的表达,用蛋白质印迹技术测定核因子κB(NF-κB)亚单位P65蛋白的表达.结果 10μg/ml LPS、0.1 ng/ml IL-1β或20%CSE刺激24 h后,E1A+组IL-8 mRNA表达量(2.82、1.87、4.70)明显高于正常对照组(0.95、0.78、1.02,均P<0.05)和E1A-组(0.97、0.81、1.12,均P<0.05).10μg/ml的LPS刺激后12、24 h,E1A+组细胞TLR4 mRNA表达水平分别为4.52、7.99,明显高于正常对照组(1.91、3.81,均P<0.05)和E1A-组(2.00、3.88,均P<0.05);IL-1β也可增加TLR4 mRNA表达.CSE刺激对各组细胞TLR4 mRNA表达水平均无明显影响.3种刺激因子均可引起各组细胞NF-κB亚单位P65蛋白表达水平增高.结论 人Ⅱ型肺泡上皮细胞表达TLR4,LPS、IL-1β引起IL-8释放可能与TLR4介导的NF-κB激活有关.     

TLR4表达水平与COPD大鼠肺动脉平滑肌细胞合成分泌功能的关系

目的 探讨Toll样受体4(TLR4)表达水平与慢性阻塞性肺疾病(COPD)大鼠肺动脉平滑肌细胞(PASMCs)合成分泌功能的关系.方法 建立大鼠COPD模型,HE染色判定模型建立,免疫组织化学染色观察对照组与COPD模型组肺组织动脉平滑肌层TLR4的表达.分离培养鉴定原代PASMCs,脂多糖(LPS)、TLR4的特异性抑制剂TAK-242干预细胞,实验分组:空白组、LPS组、TAK-242组、LPS+ TAK-242组,Western blot检测各组PASMCs中TLR4的表达,ELISA法检测各组细胞培养上清液中Th1细胞因子干扰素(IFN)-γ、Th2细胞因子白介素(IL)-6及血小板衍化生长因子(PDGF)的浓度;并将TLR4的表达水平与上清液中炎性因子的分泌水平进行相关性分析.结果 COPD模型组大鼠肺组织动脉平滑肌层TLR4的表达明显高于正常大鼠;与空白组比较,LPS组PASMCs中TLR4的表达明显升高(P＜0.05);LPS组PASMCs中合成分泌IFN-γ、IL-6、PDGF的水平较对照组明显升高,TAK-242阻断TLR4,PASMCs中TLR4的表达明显降低(P＜0.05);细胞培养上清液中IFN-γ、IL-6、PDGF的表达水平较空白组明显降低(P＜0.05);TLR4表达水平与细胞培养上清液IFN-γ、IL-6及PDGF的浓度呈显著正相关性(r =0.95、0.87、0.83,P ＜0.05).结论 TLR4可能参与调控PASMCs的合成分泌功能.     

Effect of cigarette smoke extract on lipopolysaccha- ride-activated mitogen-activated protein kinase signal transduction pathway in cultured cells

Background  Lipopolysaccharide (LPS) forms outer membrane of the wall of Gram-negative cells. LPS can directly cause damage to epithelia of respiratory tract and is the major factor responsible for the chronic inflammation of respiratory passage. The mitogen-activated protein kinase (MAPK) signal transduction pathway of the airway epithelia is intimately associated with the action of LPS. The chronic inflammation of respiratory tract and smoking are interrelated and entwined in the development and progression of chronic lung diseases. This study was designed to examine the effects of cigarette smoke extract (CSE) and LPS on MAPK signal transduction pathway in order to further understand the roles CSE and LPS play in chronic lung inflammation.
Methods  Cultured primary human epithelial cells of airway were divided into four groups according to the stimulants used: blank control group, LPS-stimulation group, CSE-stimulation group and CSE plus LPS group. Western blotting was employed for the detection of phosphorylation level of extracellular-signal-regulated-kinase (ERK_1/2), p38 MAPK and c-Jun N-terminal kinase (JNK). The expression of cytokines of MAPK transduction pathway (granulocyte-macrophage colony stimulating factor (GM-CSF) and mRNA of IL-8) in the primary epithelial cells of respiratory tract was also determined.
Results  Western blotting revealed that the phosphorylation levels of ERK_1/2, p38 MAPK and JNK were low and 2 hours after the LPS stimulation, the phosphorylation of ERK_1/2, p38 MAPK and JNK were all increased. There was a significant difference in the phosphorylation between the LPS-stimulation group and blank control group (P<0.05); no significant difference was found between CSE-stimulation group and blank control group (P>0.05); there was a significant difference between CSE + LPS group and blank control group and between CSE + LPS group and LPS group (P<0.05). The phosphorylation of CSE-LPS group was higher than that of blank control group but lower than that of LPS group. In blank control group, the expression of IL-8 and GM-CSF mRNA was low in the epithelial cells of airway and the release of IL-8 and GM-CSF was also at a low level. One hour after LPS stimulation, the level of IL-8 mRNA increased (P<0.05) and reached a peak after 2 hours. On the other hand, GM-CSF mRNA level increased 2 hours after the stimulation (P<0.05) and reached the highest level 4 hours after the stimulation. Two hours after LPS stimulation, IL-8 and GM-CSF protein level began to rise (P<0.05), and the level was the highest 8 hours after the stimulation (P<0.01). Stimulation with CSE alone had no effect on the release of IL-8 and GM-CSF and expression of IL-8 mRNA (P>0.05), but pre-treatment with CSE could delay the LPS-induced release of IL-8 and GM-CSF and the expression of IL-8 mRNA and its peak was lower.
Conclusions  LPS stimulation can significantly increase the phosphorylation of ERK_1/2, p38 MAPK and JNK in the epithelial cells of airway and activate the MAPK transduction pathway, thereby can activate the downstream signal transduction pathway, and can ultimately result in the release of cytokines by the epithelial cells of airway. CSE can partially abolish the LPS-induced activation of MAPK signal transduction pathway and the expression of cytokines of the pathway, which might contribute to the development and progression of the inflammatory reactions in COPD patients.

     

Lipopolysaccharide upregulates the expression of Toll-like receptor 4 in human vascular endothelial cells

目的：探讨内皮细胞TLR2和TLR4的表达以及内毒素对其表达的影响。方法：按RNA提取试剂盒说明书提取内毒素作用后的ECV-304(人脐静脉内皮细胞株)和人脐静脉内皮细胞总RNA,运用逆转录PCR(RT-PCR)检测细胞TLR2和TLR4mRNA的表达。结果：人脐静脉内皮细胞和ECV304细胞株均能表达TLR2和TLR4mRNA,但是,TLR4的表达水平明显强于TLR2的表达,LPS能明显上调TLR4的表达,但对TLR2的表达无影响。结论：本研究提示TLR4能明显上调LPS作用的信号转导分子,在LPS对内皮细胞的激活效应中具有重要的作用;ECV-304是内皮细胞研究的良好的实验模型,可代替人脐静脉内皮细胞而较为广泛应用。     

TLR4 mAb对人正常肝内胆管上皮细胞LPS-TLR4-NF-κB通路的影响

目的对脂多糖( LPS)及Toll样受体4单克隆抗体( TLR4 mAb)作用于体外培养的人肝内胆管细胞( HiBEC)后其TLR4 mRNA和核转录因子-κB( NF-κB) mRNA的表达情况进行了研究。方法体外培养HiBEC,采用 RT-PCR 法检测经LPS和不同浓度的TLR4 mAb作用后,HiBEC细胞株中TLR4 mRNA和NF-κB mRNA的表达情况。结果 LPS可以上调HiBEC中TLR4 mRNA和NF-κB mRNA的表达( P<0．05);而 LPS 作用于 TLR4 mAb 处理后的 HiBEC 中其TLR4 mRNA和NF-κB mRNA的表达下调;高质量浓度TLR4 mAb使NF-κB mRNA的表达下调更明显( P<0．05)。结论LPS 能上调HiBEC中TLR4 mRNA和NF-κB mRNA的表达;而 TLR4 mAb 则能拮抗 LPS 对 HiBEC 的刺激作用,下调TLR4 mRNA和NF-κB mRNA 的表达。     

Perfluorocarbon attenuates lipopolysaccharide-mediated inflammatory responses of alveolar epithelial cells in vitro

Background  Toll-like receptor-4 (TLR-4) is integrally involved in lipopolysaccharide (LPS) signaling and has a requisite role in the activation of nuclear factor-κB (NF-κB). The exact mechanisms that lend perfluorocarbon (PFC) liquids a cytoprotective effect have yet to be elucidated. Therefore we examined in an in vitro model the cytoprotective effect of PFC on LPS-stimulated alveolar epithelial cellls (AECs). 

Methods  AECs (A549 cells, human lung adenocarcinoma cell line) were divided into four groups: control, PFC, LPS and LPS + PFC (coculture group) groups. Intercellular adhesion molecule-1 (ICAM-1) was detected by ELISA, tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) were detected by radioimmunological methods. The expression of TLR-4 mRNA and protein was detected by real time PCR and Western blotting, respectively. The activation of NF-κB was detected by Western blotting (proteins of I-κBα and NF-κB p65). 

Results  ICAM-1, TNF-α and IL-8 were significantly increased in LPS-stimulated AECs groups. The expression of TLR-4 mRNA and protein in LPS-stimulated groups was markedly increased. Meanwhile, NF-κB was activated as indicated by the significant degradation of IκB-α and the significant release of NF-κB P65 and its subsequent translocation into the nucleus. There were no significant effects of PFC alone on any of the factors studied while the coculture group showed significant downregulation of the secretion of ICAM-1, TNF-α and IL-8, the expression of TLR-4 mRNA and the activity of NF-κB. 

Conclusions  Taken together, our results demonstrate that LPS can induce AEC-related inflammatory injury via the activation of TLR-4 and subsequent activation of NF-κB. PFC is able to protect AECs from LPS-induced inflammatory injury by blocking the initiation of the LPS signaling pathway, which is indicated by the significant decrease of TLR-4 expression and NF-κB activation.

     

TLR9 expression and its role in chemosensitivity to DDP in human cervical cancer cells in vitro

Inflammation and infection play an important role in the pathogenesis of many cancers.Toll-like receptors(TLRs) are a class of pattern recognition receptors that recognize conserved components of microbes and trigger the immune response against invading microorganisms.Toll-like receptor 9(TLR9) recognizes non-methylated cytosine-phosphateguanosine(CpG) DNA sequences which are the surrogate for viral DNA.TLR9 may react to tumor development and progression during chronic inflammation that involves the tumor microenvironment.In order to study the role of TLR9 in cervical cancer,we analyzed the TLR9 expression in different types of HPV infection cervical cancer cells.Then we detected if CpG sequences influenced the TLR9 expression and the sensitivity to cisplatin(DDP) of these cervical cancer cells in vitro.The expression of TLR9 mRNA and protein in SiHa,Hela and C33A cells was detected by RT-PCR and Western blotting.Real-time PCR was used to examine the TLR9 expression changes induced by CpG.Chemosensitivity of the cervical cancer cells to cisplatin(DDP) was measured by MTT.It was observed that the expression of TLR9 mRNA and protein was increased gradually in SiHa(HPV16+),Hela(HPV18+) and C33A(HPV-) cells.Low doses of CpG increased the TLR9 expression only in C33A(HPV-) cells,but not in SiHa(HPV16+) and Hela(HPV18+) cells.Furthermore,low dose of CpG significantly increased the sensitivity of C33A(HPV-) cells,but not that of SiHa(HPV16+) and Hela(HPV18+) cells.These results indicated that TLR9 may serve as a protective agent in HPV negative cervical cancer cells.It was concluded that TLR9 could improve the sensitivity to DDP in HPV negative cervical cancer cells and might represent a potential therapeutic option in clinical practice.