白藜芦醇对U937细胞基质金属蛋白酶-9转录的抑制作用

目的:观察白藜芦醇对佛波酯诱导的U937细胞中基质金属蛋白酶-9活性的影响,并从蛋白、mRNA及核转录因子激活蛋白-1(AP-1)水平对其影响进行分析。方法:酶谱法测定U937细胞培养上清中MMP-9的活性;Western blot法考察MMP-9蛋白的生成;RT-PCR法检测MMP-9 mRNA的表达;电泳迁移率变动分析法(EMSA)研究核转录因子SP-1的活性。结果:PMA 10nmol/L 可显著诱导无血清培养的U937细胞中MMP-9活性(P<0.01);白藜芦醇在1和10 μmol/L浓度下可抑制 PMA 10 nmol/L诱导的MMP-9活性(P<0.05 和P<0.01);PMA 10 nmol/L可显著诱导U937细胞中MMP-9蛋白生成(P<0.01)和MMP-9 mRNA的表达(P<0.01),白藜芦醇在1、10μmol/L浓度下可抑制PMA 10 nmol/L诱导的MMP-9蛋白生成和MMP-9 mRNA的表达(P<0.05);白藜芦醇在10、1和0.1μmol/L浓度下可抑制PMA诱导的U937细胞中AP-1的活化。结论:白藜芦醇可有效地抑制PMA诱导的U937细胞中MMP-9的活性,其作用可能是通过抑制PMA诱导的U937细胞核转录因子AP-1活化,进而降低MMP-9 mRNA表达,减少MMP-9蛋白生成而实现的。     

α-Tocopheryl succinate induces apoptosis in erbB2-expressing breast cancer cell via NF-κB pathway

Aim:

To study the molecular mechanisms underlying α-tocopheryl succinate (α-TOS)-induced apoptosis in erbB2-positive breast cancer cells and to determine whether α-TOS and the human recombinant TNF-related apoptosis-inducing ligand (hrTRAIL) act synergically to induce cell death of erbB2-expressing breast cancer cells.

Methods:

The annexin V binding method was used to measure apoptosis induced by α-TOS and/or hrTRAIL. RT-PCR and Western blotting were performed to detect gene and protein expression. A colorimetric assay was performed to detect caspase activity. The TransAM^TM NF-κB p65 kit was used to assess NF-κB activation.

Results:

α-TOS (100 μmol/L) significantly inhibited NF-κB nuclear translocation in erbB2-expressing breast cancer cells; this inhibition is expected to result in the inactivation of NF-κB. α-TOS (50 and 100 μmol/L) inhibited the expression of Flice-like inhibitory protein (FLIP) and cellular inhibitor of apoptosis protein 1 (c-IAP1) in erbB2-positive cells. α-TOS (100 μmol/L) inhibited Akt activation and augmented the activity of caspase 3 and caspase 8 in breast cancer cells expressing erbB2. α-TOS (50 μmol/L) and hrTRAIL (30 mg/mL) acted synergically to induce apoptosis in breast cancer cells. α-TOS also decreased the hrTRAIL-induced transient activation of NF-κB .

Conclusion:

Our results suggest that α-TOS mediates the apoptosis of erbB2-positive breast cancer cells and acts synergically with hrTRAIL via the NF-κB pathway.     

Okadaic acid induces matrix metalloproteinase-9 expression in fibroblasts: crosstalk between protein phosphatase inhibition and β-adrenoceptor signalling

BACKGROUND AND PURPOSE

Interactions between protein phosphatase inhibition and matrix metalloproteinase (MMP)-9 expression have implications for tissue remodelling after injury. Stimulation of β-adrenoceptors could affect such interactions as isoprenaline increases protein phosphatase 2A (PP2A) activity and MMP-9 abundance. We investigated the effect of okadaic acid (OA) on MMP-9 expression to assess interactions between phosphatase inhibition and β-adrenoceptor signalling in fibroblasts.

EXPERIMENTAL APPROACH

Fibroblasts were exposed to OA alone and in combination with isoprenaline. Effects on MMP-9 expression and intracellular signalling were studied using promoter assays, Western blot analysis and siRNA methodologies.

KEY RESULTS

Okadaic acid increased MMP-9 abundance in human cardiac ventricular fibroblasts, NIH3T3 fibroblasts and hepatic stellate cells. This effect was unaffected by PP2A knockdown in NIH3T3 cells. OA increased phosphorylation of NF-κB, but not NF-κB promoter activity, IκBα degradation, or nuclear translocation of p65-NF-κB. Exposure to SB202190 (p38 MAPK), U0126 (ERK1/2) and NF-κB III inhibitor revealed that OA induced MMP-9 activity through p38 MAPK. Isoprenaline inhibited OA-mediated MMP-9 expression in NIH3T3, in a β-arrestin 2- and PP2A-dependent manner. Mutation of the activator protein-1 (AP-1) and NF-κB binding sites demonstrated that OA-induced MMP-9 activity was mediated through the AP-1 but not NF-κB sites. The latter mediated the inhibitory effect of isoprenaline on OA-induced MMP-9 promoter activity.

CONCLUSION AND IMPLICATIONS

Okadaic acid induced MMP-9 activity through p38 MAPK and was inhibited by isoprenaline via a pathway involving β-arrestin 2, PP2A and an NF-κB binding motif. These findings elucidate how phosphoprotein phosphatases and adrenoceptors may modulate tissue remodelling by affecting fibroblast function.     

Z-ligustilide attenuates lipopolysaccharide-induced proinflammatory response via inhibiting NF-KB pathway in primary rat microglia

Aim:

To investigate the anti-inflammatory effect of Z-ligustilide (LIG) on lipopolysaccharide (LPS)-activated primary rat microglia.

Methods:

Microglia were pretreated with LIG 1 h prior to stimulation with LPS (1 μg/mL). After 24 h, cell viability was tested with MTT, nitric oxide (NO) production was assayed with Griess reagent, and the content of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein (MCP-1) was measured with ELISA. Protein expression of the nuclear factor-κB (NF-κB) p65 subunit, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) was detected with immunocytochemistry 1 h or 24 h after LPS treatment.

Results:

LIG showed a concentration-dependent anti-inflammatory effect in LPS-activated microglia, without causing cytotoxicity. Pretreatment with LIG at 2.5, 5, 10, and 20 μmol/L decreased LPS-induced NO production to 75.9%, 54.4%, 43.1%, and 47.6% (P<0.05 or P< 0.01), TNF-α content to 86.2%, 68.3%, 40.1%, and 39.9% (P<0.01, with the exception of 86.2% for 2.5 μmol/L LIG), IL-1β content to 31.5%, 27.7%, 0.6%, and 0% (P<0.01), and MCP-1 content to 84.4%, 50.3%, 45.1%, and 42.2% (P<0.05 or P<0.01), respectively, compared with LPS treatment alone. LIG (10 μmol/L) significantly inhibited LPS-stimulated immunoreactivity of activated NF-κB, COX-2, and iNOS (P<0.01 vs LPS group).

Conclusion:

LIG exerted a potent anti-inflammatory effect on microglia through inhibition of NF-κB pathway. The data provide direct evidence of the neuroprotective effects of LIG and the potential application of LIG for the treatment of the neuroinflammatory diseases characterized by excessive microglial activation.     

Short-term exposure to oleandrin enhances responses to IL-8 by increasing cell surface IL-8 receptors

BACKGROUND AND PURPOSE

One of the first steps in host defence is the migration of leukocytes. IL-8 and its receptors are a chemokine system essential to such migration. Up-regulation of these receptors would be a viable strategy to treat dysfunctional host defence. Here, we studied the effects of the plant glycoside oleandrin on responses to IL-8 in a human monocytic cell line.

EXPERIMENTAL APPROACH

U937 cells were incubated with oleandrin (1-200 ng mL⁻¹) for either 1 h (pulse) or for 24 h (non-pulse). Apoptosis; activation of NF-κB, AP-1 and NFAT; calcineurin activity and IL-8 receptors (CXCR1 and CXCR2) were measured using Western blotting, RT-PCR and reporter gene assays.

KEY RESULTS

Pulse exposure to oleandrin did not induce apoptosis or cytoxicity as observed after non-pulse exposure. Pulse exposure enhanced activation of NF-κB induced by IL-8 but not that induced by TNF-α, IL-1, EGF or LPS. Exposure to other apoptosis-inducing compounds (azadirachtin, resveratrol, thiadiazolidine, or benzofuran) did not enhance activation of NF-κB. Pulse exposure to oleandrin increased expression of IL-8 receptors and chemotaxis, release of enzymes and activation of NF-κB, NFAT and AP-1 along with increased IL-8-mediated calcineurin activation, and wound healing. Pulse exposure increased numbers of cell surface IL-8 receptors.

CONCLUSIONS AND IMPLICATIONS

Short-term (1 h; pulse) exposure to a toxic glycoside oleandrin, enhanced biological responses to IL-8 in monocytic cells, without cytoxicity. Pulse exposure to oleandrin could provide a viable therapy for those conditions where leukocyte migration is defective.     

Plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the NF-κB signaling pathway

Aim:

To investigate the effects and underlying mechanisms of plumbagin, a naphthoquinone derived from medicinal plant Plumbago zeylanica, on human gastric cancer (GC) cells.

Methods:

Human gastric cancer cell lines SGC-7901, MKN-28, and AGS were used. The cell viability was examined using CCK-8 viability assay. Cell proliferation rate was determined using both clonogenic assay and EdU incorporation assay. Apoptosis was detected via Annexin V/propidium iodide double-labeled flow cytometry. Western blotting was used to assess the expression of both NF-κB-regulated gene products and TNF-α-induced activation of p65, IκBα, and IKK. The intracellular location of NF-κB p65 was detected using confocal microscopy.

Results:

Plumbagin (2.5–40 μmol/L) concentration-dependently reduced the viability of the GC cells. The IC₅₀ value of plumbagin in SGC-7901, MKN-28, and AGS cells was 19.12, 13.64, and 10.12 μmol/L, respectively. The compound (5–20 μmol/L) concentration-dependently induced apoptosis of SGC-7901 cells, and potentiated the sensitivity of SGC-7901 cells to chemotherapeutic agents TNF-αand cisplatin. The compound (10 μmol/L) downregulated the expression of NF-κB-regulated gene products, including IAP1, XIAP, Bcl-2, Bcl-xL, tumor factor (TF), and VEGF. In addition to inhibition of NF-κB p65 nuclear translocation, the compound also suppressed TNF-α-induced phosphorylation of p65 and IKK, and the degradation of IκBα.

Conclusion:

Plumbagin inhibits cell growth and potentiates apoptosis in human GC cells through the NF-κB pathway.     

Baicalin prevents LPS-induced activation of TLR4/NF-κB p65 pathway and inflammation in mice via inhibiting the expression of CD14

Previous studies have shown that baicalin,an active ingredient of the Chinese traditional medicine Huangqin,attenuates LPS-induced inflammation by inhibiting the activation of TLR4/NF-κBp65 pathway,but how it affects this pathway is unknown.It has been shown that CD14 binds directly to LPS and plays an important role in sensitizing the cells to minute quantities of LPS via chaperoning LPS molecules to the TLR4/MD-2 signaling complex.In the present study we investigated the role of CD14 in the anti-inflammatory effects of baicalin in vitro and in vivo.Exposure to LPS(1μg/mL)induced inflammatory responses in RAW264.7 cells,evidenced by marked increases in the expression of MHC II molecules and the secretion of NO and IL-6,and by activation of MyD88/NF-κB p65 signaling pathway,as well as the expression of CD14 and TLR4.These changes were dose-dependently attenuated by pretreatment baicalin(12.5–50μM),but not by baicalin post-treatment.In RAW264.7 cells without LPS stimulation,baicalin dose-dependently inhibit the protein and mRNA expression of CD14,but not TLR4.In RAW264.7 cells with CD14 knockdown,baicalin pretreatment did not prevent inflammatory responses and activation of MyD88/NF-κB p65 pathway induced by high concentrations(1000μg/mL)of LPS.Furthermore,baicalin pretreatment also inhibited the expression of CD14 and activation of MyD88/NF-κB p65 pathway in LPS-induced hepatocyte-derived HepG2 cells and intestinal epithelial-derived HT-29 cells.In mice with intraperitoneal injection of LPS and in DSS-induced UC mice,oral administration of baicalin exerted protective effects by inhibition of CD14 expression and inflammation.Taken together,we demonstrate that baicalin pretreatment prevents LPS-induced inflammation in RAW264.7 cells in CD14-dependent manner.This study supports the therapeutic use of baicalin in preventing the progression of LPS-induced inflammatory diseases.     

铜绿假单胞菌通过MAPK信号传导通路诱导U937细胞表达IL-8

目的探讨铜绿假单胞菌(PA)活菌对不同分化状态的U937细胞表达IL-8的诱导作用及通过MAPK信号传导通路的调控机制。方法应用人单核白血病细胞系-U937细胞,采用ELISA和RT-PCR法对PA诱导不同分化状态的U937细胞IL-8蛋白分泌和其mRNA表达进行研究,并观察MAPKs抑制剂PD98059和SB203580对IL-8表达的影响。结果PA可促进U937细胞及PMA分化的U937细胞IL-8的mRNA及蛋白分泌,而且具有明显的量效和时效关系。分别用SB203580抑制p38MAPK通路、用PD98059抑制ERK通路,均能引起抑制剂浓度依赖的IL-8的表达(P<0.01)。结论PA以浓度和时间依赖的方式感染U937细胞,促进IL-8的分泌和mRNA表达,PA可能通过MAPK信号通路启动IL-8的高效表达和分泌。     

In vitro toxicity evaluation of diesel exhaust particles on human eosinophilic cell

Diesel exhaust particles (DEPs), comprised mainly of particles less than 2.5 μm (PM 2.5) in aerodynamic diameter, have been assumed to enhance the response of asthma to allergen inhalation. Although eosinophilic infiltration is remarkable in the event of bronchial asthma induced by DEPs, the precise mechanisms leading to eosinophilia are unknown. To examine the effect of DEPs on eosinophils, we measured the cytokine products and activity of nuclear factor-kappa B (NF-κB) after addition of the proteasomal inhibitor MG132 in HL-60 clone 15 cells differentiated into eosinophils. We measured eotaxin-induced chemotaxis of cells and their activity of p38 mitogen-activated protein (MAP) kinase was analysed. Interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) were increased markedly in DEPs-treated cells. The active form of NF-κB in cells treated with DEPs was increased, and this effect was significantly decreased by the administration of MG132. Cell migration in the presence of DEPs was significantly greater, and inhibited by adding N-acetyl l-cysteine. P38 MAP kinase activity was highly influenced by DEPs-treatment. DEPs induce MCP-1 and IL-8 production by up-regulating NF-κB activity, which is inhibited in the presence of an inhibitor of proteasomal degradation. DEP also promotes eotaxin-induced chemotaxis in a p38-dependent manner.     

Manganese promotes phorbol ester-induced interleukin-2 production via AP-1 activation in Jurkat T-cells

Mn²⁺ is a minor nutrient, but is essential for numerous enzymatic activities and thus, for many cellular functions. However, its physiological roles and toxicity remain to be elucidated. In this study, we assessed the pharmacological potential and toxicity of Mn²⁺ in the immune system by examining the effects of Mn²⁺ on interleukin-2 (IL-2) production by Jurkat T-cells. Mn²⁺ at 0.1–1 mM did not significantly induce IL-2 production, whereas phorbol 12-myristate 13-acetate (PMA) at 1 μM slightly induced IL-2 production. Interestingly, Mn²⁺ at 0.3–0.7 mM promoted PMA-induced IL-2 production in a dose-dependent manner. A reporter gene assay revealed that Mn²⁺ promoted the activity of AP-1 (activator protein-1, a complex of c-Fos and c-Jun) in the presence of PMA. Western blot analysis showed that Mn²⁺ promoted the activation of JNK2 (c-Jun N-terminal kinase 2) and p38 MAPK (mitogen-activated protein kinase), which are both activators of AP-1, and upregulated the production of c-Fos and c-Jun within 4 h in the presence of PMA. These results suggest that Mn²⁺ promotes PMA-induced IL-2 production by inducing the production and activation of AP-1, at least in part.     

Geniposide inhibits high glucose-induced cell adhesion through the NF-KB signaling pathway in human umbilical vein endothelial cells

Aim:

To investigate whether geniposide, an iridoid glucoside extracted from gardenia jasminoides ellis fruits, inhibits cell adhesion to human umbilical vein endothelial cells (HUVECs) induced by high glucose and its underlying mechanisms.

Methods:

HUVECs were isolated from human umbilical cords and cultured. The adhesion of monocytes to HUVECs was determined using fluorescence-labeled monocytes. The mRNA and protein levels of vascular cell adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) were measured using real-time RT-PCR and ELISA. Reactive oxygen species (ROS) production was measured using a fluorescent probe. The amounts of nuclear factor-kappa B (NF-κB) and inhibitory factor of NF-κB (IκB) were determined using Western blot analysis. The translocation of NF-κB from the cytoplasm to the nucleus was determined using immunofluorescence.

Results:

Geniposide (10–20 μmol/L) inhibited high glucose (33 mmol/L)-induced adhesion of monocytes to HUVECs in a dose-dependent manner. This compound (5–40 μmol/L) also inhibited high glucose-induced expression of VCAM-1 and E-selectin at the gene and protein levels. Furthermore, geniposide (5–20 μmol/L) decreased ROS production and prevented IκB degradation in the cytoplasm and NF-κB translocation from the cytoplasm to the nucleus in HUVECs.

Conclusion:

Geniposide inhibits the adhesion of monocytes to HUVECs and the expression of CAMs induced by high glucose, suggesting that the compound may represent a new treatment for diabetic vascular injury. The mechanism underlying this inhibitory effect may be related to the inhibition of ROS overproduction and NF-κB signaling pathway activation by geniposide.     

白藜芦醇抑制巨噬细胞细胞外基质金属蛋白酶诱导物的表达

目的探讨白藜芦醇对细胞外基质金属蛋白酶诱导物(EMMPRIN)表达的影响。方法将人类单核细胞系THP-1和MCF-7细胞共培养,测定上清液中MMP-9活性。用PMA诱导THP-1为巨噬细胞，加入白藜芦醇，观察EMMPRIN表达和MMP-9活性变化。细胞共转染实验测定白藜芦醇对PPARγ的激动作用。用PPARγ的拮抗剂GW9662预处理细胞后，测定白藜芦醇对EMMPRIN表达的影响。结果PMA诱导使单核细胞EMMPRIN表达明显增强，与EMMPRIN高表达的MCF-7细胞共培养显著增加单核细胞表达MMP-9。白藜芦醇显著抑制EMMPRIN和MMP-9生成。白藜芦醇明显激动PPARγ，GW9662大幅减弱白藜芦醇对EMMPRIN和MMP-9的作用。结论单核细胞向巨噬细胞分化过程中表达明显增强的EMMPRIN可能是促进MMPs表达的主要因子。白藜芦醇通过激动PPARγ抑制EMMPRIN的表达，可能是其抑制巨噬细胞MMPs产生的机制。     

Anti-inflammatory mechanisms of resveratrol in activated HMC-1 cells: Pivotal roles of NF-κB and MAPK

Resveratrol is a phytoalexin polyphenolic compound found in various plants, including grapes, berries, and peanuts. Recently, studies have documented various health benefits of resveratrol including cardiovascular and cancer-chemopreventive properties. The aim of the present study was to demonstrate the effects of resveratrol on the expression of pro-inflammatory cytokines, as well as to elucidate its mechanism of action in the human mast cell line (HMC-1). Cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus A23187 in the presence or absence of resveratrol. To study the possible effects of resveratrol, ELISA, RT-PCR, real-time RT-PCR, Western blot analysis, fluorescence, and luciferase activity assays were used in this study. Resveratrol significantly inhibited the PMA plus A23187-induction of inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8. Moreover, resveratrol attenuated cyclooxygenase (COX)-2 expression and intracellular Ca²⁺ levels. In activated HMC-1 cells, phosphorylation of extra-signal response kinase (ERK) 1/2 decreased after treatment with resveratrol. Resveratrol inhibited PMA plus A23187-induced nuclear factor (NF)-κB activation, IκB degradation, and luciferase activity. Resveratrol suppressed the expression of TNF-α, IL-6, IL-8 and COX-2 through a decrease in the intracellular levels of Ca²⁺and ERK 1/2, as well as activation of NF-κB. These results indicated that resveratrol exerted a regulatory effect on inflammatory reactions mediated by mast cells.