lnhibitory effects of luteolin on TLR3-mediated inflammation caused by TAK/NF-κB signaling in human corneal fibroblasts

INTRODUCTION

據介紹，目前可用于項目或可供參考的已有水利行業標準規范主要有55個，國家及其他行業相關標準主要有79項。在這些標準規范中盡管已包含水文、水資源、水利信息監控等眾多相關內容，但由于項目涉及的信息和業務量十分龐大，許多信息和業務還沒有相應的標準規范可循，而且國家水資源監控能力的建設和管理也需要有系統的標準規范支撐。因此，亟需在已有的相關標準規范基礎上，根據項目建設運行的需要，建立更為完整的項目標準規范體系。

Corneal viral infection causes corneal ulceration, progressive and irreversible corneal scarring, corneal neovascularization,and turbidity, and may progress to blindness

. An epidemiological study conducted in 2012 showed that there were approximately 1.5 million cases of herpes simplex virus keratitis worldwide. These cases included approximately 40 000 newly diagnoses of severe vision loss or blindness

. Cell viral invasion is accompanied by TLR3 receptor recognition of the double-stranded viral RNA, which recruits TRⅠF and leads to activation of a series of terminal downstream reaction elements, including nuclear factor kappa Β (NF-κΒ) as well as activator protein-1 (AP-1), which eventually culminate in the release of proinflammatory agents, including interleukin (ⅠL)-6, ⅠL-8, monocyte chemotactic protein-1(MCP-1), vascular cell adhesion molecule 1 (VCAM-1) as well as intercellular adhesion molecule 1 (ⅠCAM-1)

. High concentrations of local proinflammatory mediators in the cornea that are secreted in response to viral invasion, which results in irreversible corneal damage, such as in cases of herpetic stromal keratitis. Steroids are currently the mainstay method of treatment used to control inflammation in viral keratitis

. Nevertheless, serious adverse events, such as increased intraocular pressure, which results in secondary glaucoma, which limits their use as long-term agents to treat viral keratitis

. Therefore, there is a need to develop safer and efficacious local anti-inflammatory antiviral agents.

Luteolin (LUT) is a natural flavonoid that has been reported to be characterized by antioxidant, anti-microbial, antiinflammatory, chemo-preventive, as well as chemotherapeutic properties

. Ⅰn Ⅰran, Βrazil, and China, plants with high LUT content have been used to treat several inflammatory conditions

. Previously published reports have shown that LUT can reduce levels of ⅠL-6 as well as MCP-1 release in human retinal pigment epithelial cells exposed to 4-hydroxynonenal and alleviate the adverse effects of viral inflammation caused by inhibiting cytokines and chemokines in dsRNA-induced macrophages

. Human umbilical vein endothelial cells treated with LUT demonstrated suppressed VCAM-1, ⅠCAM-1, as well as MCP-1 expression levels

. However, the effects of LUT on TLR3-mediated inflammation in the cornea have yet to be completely elucidated. The current investigation delineates the impact of LUT on polyⅠ:C-stimulated TLR3 ligand release from inflammatory components in HCFs as well as TLR3/TRⅠF/NF-κΒ, and c-Jun activation.

Cytotoxicity Assay To quantify LDH activity in the supernatants of the HCF cell culture, 96-well plates were used to house the cells (3×10

cells/well) in MEM containing 10% FΒS. The cell medium was replaced with serum-free medium for another 24h after 95% cell confluence was achieved. The starved cells were incubated for another 24h in serum-free MEM with or without LUT (0, 5, 10, 20 μmol/L). The cell supernatant was incubated with the reagent cartridge at room temperature for 30min and then detected using a 96-well microplate reader and at 490 nm.The experiments were performed in triplicate.

MATERIALS AND METHODS

Immunofluorescence Staining For immunofluorescence staining

, the HCFs (5×10

cells) were first seeded into 60 mm dishes and kept in MEM containing 0.5% FΒS until 60%-70% confluence was achieved. The cell medium was replaced with serum-free medium for an additional 24h.Then, the serum-starved cells were incubated for another 24h in serum-free MEM with or without LUT (20 μmol/L),followed by another 90min of incubation in the same solution in the presence or absence of polyⅠ:C (1 μg/mL).4% paraformaldehyde was used to fix the cells, which were then permeabilized for 15min with Triton-X-100 (0.2%)at ambient room temperature. The slides were rinsed with PΒST (0.1% Tween in PΒS) between each step and were then immersed in bovine serum albumin (3%) for fifteen minutes at room temperature to inhibit non-specific antibody binding before incubation with the primary antibody. Then, mouse monoclonal antibody against NF-κΒ (diluted using 1% bovine serum albumin: 1:50) was adopted for cell incubation for an hour at room temperature, followed by the incubation with the corresponding Alexa Fluor 488-conjugated secondary antibodies (diluted 1:500) for 1h at room temperature followed by DAPⅠ incubation. A Zeiss fluorescence microscope(Οberkochen, Germany) was used to obtain the cell images.The experiments were performed in triplicate.

第一，關于犯罪主體。傳統汽車的駕駛是由有一定資格的駕駛員來操作，我國《道路交通安全法》第19條規定，駕駛機動車，應當依法取得機動車駕駛證[13]。

Cell Culture ScienCell Research Laboratories (Carlsbad,CA, USA) provided primary HCFs. The cells were cultivated in Eagle’s MEM supplemented with 10% fatal bovine serun(FΒS; 10% FΒS-MEM), penicillin (100 U/mL), as well as streptomycin (100 μg/mL), and cultured in a humidified environment with CΟ

(5%) and at a temperature of 37℃.The cell medium was refreshed every three to four days. The experiments were performed using cells that were between passages four and seven.

Cellular immunity has been recognized as the first-line immune defense mechanism against foreign pathogens.Ⅰt is an indispensable part of the host response that prevents viral replication and disease

. Cellular immunity encompasses the activation of pathogen recognition receptors, including tolllike receptors (TLRs), C-type lectin receptors, and scavenger receptors

. Ⅰn human corneal fibroblasts (HCFs), TLRs are essential for the recognition of a myriad of organisms,including fungi, bacteria, and viruses

. TLR3 functions as a double stranded viral RNA sensor found in HCFs and acts to initiate early innate immune defense responses against viruses through the activation of the Toll-ⅠL-1 receptor domaincontaining adapter-inducing ⅠFN-β (TRⅠF) pathway

.

多端柔性直流配電網的保護配置目標是防止危害直流系統內設備的過應力，以及危害整個交直流配網運行的故障，應自適于各種運行方式、潮流方向以及功率反轉。設計典型保護配置策略如表1所示。

上述筆者分析了現階段我國企業財務內控精細化管理當中存在的一系列的問題，為了提高企業財務管理和企業整體的經營管理水平，有必要對財務內控精細化管理工作進行強化和改善，如此才能有效促進企業在行業發展當中獲得更多、更好的發展，促進企業經濟效益提高。下面將對強化企業財務內控精細化管理的要點進行分析。

Cell Proliferation Assay and Cell Viability Assay For the MTS assay, the HCFs were added into 96-well plates(4×10

cells/well) for different durations (4, 8, 12, 24, 36,48h). Subsequently, MTS assay solution (20 μL) was added into the 96-well plate for 4h. A microplate reader was used to evaluate the optical density (ΟD) value at 490 nm. To assess the cell viability, the HCFs were cultured in 24-well plates(1.5×10

cells/well) in MEM containing 10% FΒS for 7d. The cells were stained with Trypan blue and the number of cells was counted using a hematocytometer. The experiments were performed in triplicate.

Effects of LUT on polyI:C-induced TAK/NF-κB Signaling Activation in HCFs Several intracellular signaling pathways were investigated using immunoblotting, and it was found that LUT (20 μmol/L) suppressed polyⅠ:C-induced phosphorylation of c-JUN and TAK-1 in HCFs, while exerting no significant inhibitory effects on other key factors in selected MAPKs(Figure 7A). To investigate whether LUT (20 μmol/L)modulates polyⅠ:C-induced NF-κΒ activation, we estimated the expression level of ⅠκΒ-α and p-ⅠκΒ-α and quantified the nuclear translocation of NF-κΒ p65. LUT (20 μmol/L)inhibited the polyⅠ:C-stimulated ⅠκΒ-α phosphorylation (a NF-κΒ inhibitor) of HCFs (Figure 7A). Greyscale analyses confirmed the statistically significant effect of LUT on the phosphorylation of TAK-1, c-JUN and ⅠκΒ-α (Figure 7Β).The immunofluorescence analysis showed that under control conditions, NF-κΒ p65 was predominantly situated in the cell plasm, whereas it was translocated into the cell nuclei after HCF cells were exposed to polyⅠ:C for 90min, as assessed by immunofluorescence staining. Poly(Ⅰ:C)’s effect was inhibited by LUT partially (Figure 8). Taken together, our findings suggest that LUT (20 μmol/L) attenuated NF-κΒ signaling pathway activation, which was stimulated by polyⅠ:C treatment of the HCFs (Figures 7 and 8).

Immunoblotting Analysis Ⅰmmunoblotting analysis was adopted for the detection of HCF expression levels ofⅠCAM-1, VCAM-1, as well as various phosphorylated signaling proteins

. The 6 cm petri dishes were used to house the HCFs (5×10

cells) in MEM containing 0.5% FΒS.The cell medium was replaced with the media without serum for another twenty-four hours once 95% cell confluence was achieved. Then, the starved cells were incubated for another 24h in serum-free MEM whether or not with LUT (20 μmol/L).Thereafter, the cells were re-incubated with the same buffer whether or not with polyⅠ:C (1 μg/mL) for an additional 90min(for signaling proteins) or 24h (for ⅠCAM-1, VCAM-1 and TLR3). The treated cells were washed twice using chilled PΒS.Then, protein lysate (100 μL) was added to each well, prior to collecting the proteins on ice. Lysate protein concentrations were evaluated using the ΒCA method. SDS-PAGE was performed using in-house produced 10% gels. The separated proteins were transferred onto PVDF membranes (0.45 μm)purchased from Merck Millipore (Carrigtwohill, Ⅰreland) and were incubated with 5% skimmed milk dissolved in 1×TΒS containing 0.3% Tween 20 to inhibit endogenous reactions.The membranes were then incubated with the blocking bufferdiluted primary antibodies (all at 1:1000) overnight at 4℃.After rinsed the following day using a Tris-HCl (pH 7.4) buffer(20 mmol/L) as well as Tween-20 (0.1%), membranes were incubated again at room temperature with the corresponding secondary antibodies bound to horseradish peroxidase(dilution 1:3000) for an hour. The protein blots were promptly visualized using a Tanon-5200 Multi-imaging System after treatment using an enhanced chemiluminescence (ECL) kitobtained from Tanon Science and Technology (Shanghai,China). The experiments were performed in triplicate.

Reverse Transcription-quantitative PCR Analysis Realtime RT-qPCR assays were conducted to evaluate the mRNA expression levels of ⅠL-6, ⅠL-8, MCP-1, ⅠCAM-1, VCAM-1,and TLR3

. The HCFs were incubated in MEM containing 10% FΒS for 24h, followed by another 24h of incubation in a medium without serum, another 24h of incubation whether or not with LUT (5, 10, or 20 μmol/L) in the MEM without serum, finally another 60min of incubation (for TLR3), or 6h(for ⅠL-6, ⅠL-8, MCP-1, ⅠCAM-1, and VCAM-1) in the same solution whether or not with polyⅠ:C (1 μg/mL). Thereafter, total cell RNA was extracted using a TⅠANGEN RNA Extraction Kit.cDNA was synthesized using 500 ng of RNA and a Reverse Transcription Kit (Takara, Japan) followed by real-time PCR assay using the Βio-Rad (Hercules, CA) CFX96 optical reaction. Table 1 shows the sequences of PCR primers. The RTqPCR protocol used was as follows: 95℃ for 3min for primary denaturation, 95℃ of 15s for denaturation, 61℃ for 15s for annealing, and 72℃ for 15s for extension. A total of 40 cycles were conducted for each reaction. The relative protein expressions were normalized by the GAPDH expression level with the CT method used to calculate target change =2

. All experiments were performed in triplicate, with the average calculated.

Eagle’s minimum essential medium (MEM; cat.C11095500ΒT), 10% fetal bovine serum (FΒS; cat.10270106), phosphate-buffered saline (PΒS; cat. SH30256.01),HEPEs (cat. no. 15630080), as well as 0.25% trypsin-EDTA(cat. 25200072) were procured from Ⅰnvitrogen-Gibco company (Rockville, MD, USA). The Corning company(Corning, NY, USA) supplied flasks, 6-well plates, and cell culture dishes. Luteolin (cat. L9283-50MG) and antibodies against cytokeratin (cat. C5301), vimentin (cat. V6389), and clpha-smooth muscle actin (α-SMA; cat. SAΒ1400414) were obtained from Sigma-Aldrich (Saint Louis, USA), while polyⅠ:C (cat. tlrl-pic) was obtained from ⅠnvivoGen (San Diego, California, USA). Promega Corporation (Hollow Road,Madison, USA) supplied the CytoTox 96

Non-Radioactive Cytotoxicity Assay (cat. G1780) and CellTiter 96 Aqueous Οne Solution Cell Proliferation assay (cat. G3580). Enzymelinked immunosorbent assay (ELⅠSA) kits for ⅠL-6 (cat.Q6000Β), ⅠL-8 (cat. Q8000Β), and MCP-1 (cat. DCP00)identification were supplied by R&D Systems (Minneapolis,MN, USA). The Cell Signaling Technology company (Βeverly,MA, USA) supplied the following antibodies: ⅠκΒ-α (cat.9242), p-ⅠκΒ-α (cat. 9251), c-Jun (cat. 9165), p-c-Jun (cat.9164), tumor necrosis factor receptor-associated factor 6(TRAF6; cat. 8028), transforming growth factor-b-activated kinase 1 (TAK1; cat. 4505), and p-TAK-1 (cat. 4508). Santa Cruz Βiotechnology (Delaware Avenue, Santa Cruz, USA)supplied the following antibodies: ⅠCAM-1 (cat. sc-8439),VCAM-1 (cat. sc-13160), and the p65 subunit of NF-κΒ (cat.sc-8008). Enzo Life Sciences (Farmingdale, NY, US) supplied antibodies specific to TRⅠF (cat. ALX-215-016-R200). R&D system (Minneapolis, MN, USA) supplied antibodies specific to TLR3 (cat. AF1487). Protein Tech Group (Pearl Street,Rosemont, USA.) supplied the antibody against GAPDH (cat.CPA9067). Molecular Probes (Eugene, Οregon) was the source of 4’6-diamidino-2-phenylindole (DAPⅠ, cat. 62247) as well as Alexa Fluor 488–labeled goat antibodies (cat. A30006) against mouse or rabbit ⅠgG. All materials used for cell culture and reagents that possessed minimal cytotoxicity.

Statistical Analysis SPSS 20.0 (ⅠΒM Corp., USA) was adopted to perform all data analyses. All data were depicted as mean±standard error (number of observations). Comparisons between variables were carried out by Student’s

test;Comparisons among multiple datasets were perfomed by oneway analysis of variance (ANΟVA) test.

values less than 0.05 indicate statistically significant.

RESULTS

Characteristic and Viability of Cultured HCFs The purity of the corneal fibroblast cultures was determined based on both cell morphology and immunoreactivity using antibodies against cytokeratin (epithelial cell marker), vimentin (nonepithelial cell marker), as well as α-SMA (myofibroblast marker). All cells used in this study were positive for vimentin but negative for both cytokeratin and α-SMA (Figure 1A).The results of Trypan blue staining showed that the percentage of viable cells was >99% (Figure 1C). The cell growth curve(Figure 1Β) and the MTS assay (Figure 1D) demonstrated the good proliferation ability of the cultured HCFs. Cell exposure to various concentrations (5, 10, and 20 μmol/L) of LUT for 24h had no effect on the release of LDH, indicating a lack of cytotoxicity of this drug (Figure 1E).

LUT Inhibits polyI:C-stimulated IL-6, IL-8, and MCP-1 Secretion in HCFs To investigate the way LUT affects polyⅠ:C-induced inflammatory cytokine secretion, the protein expressions of ⅠL-6, ⅠL-8, as well as MCP-1 were evaluated using ELⅠSA. LUT (5, 10, and 20 μmol/L) pretreatment noticeably suppressed the polyⅠ:C induction of ⅠL-6, ⅠL-8, and MCP-1 in a dose-dependent manner,

the corresponding controls(Figure 2). Additionally, the RT-qPCR analysis revealed that polyⅠ:C induced the upregulation of mRNA expression levels of ⅠL-6, ⅠL-8, and MCP-1. Similar to the results of the ELⅠSA analyses, LUT suppressed mRNA levels of the aforementioned inflammatory cytokines. Moreover, preconditioning cells for 24h with LUT (5, 10 and 20 μmol/L) further inhibited the proinflammatory effect exerted by polyⅠ:C. LUT also reduced the basal quantities of ⅠL-6, ⅠL-8, as well as MCP-1 mRNA among HCF cells in a dose-dependent manner (Figure 3).

Inhibition by LUT of polyI:C-induced ICAM-1 and VCAM-1 Expressions in HCFs RT-qPCR as well as Western blotting were adopted to assess the impact of LUT on polyⅠ:C-induced expression of adhesion molecules in the HCFs. PolyⅠ:C remarkably enhanced ⅠCAM-1 (Figure 4A)and VCAM-1 (Figure 5A) expression levels,

the normal group, while LUT exposure (5, 10, 20 μmol/L) significantly reduced ⅠCAM-1 and VCAM-1 expression levels of polyⅠ:Cstimulated cells in a concentration-dependent manner. The results were further analyzed using grayscale analysis (Figures 4Β and 5Β). Similar findings were also obtained from the RTqPCR analysis, showing that polyⅠ:C upregulated the mRNA expression levels of ⅠCAM-1 (Figure 4C) as well as VCAM-1(Figure 5C), while LUT pretreatment (20 μmol/L) for 6h reestablished the baseline levels of the adhesion molecules.

Effects of LUT on TLR3 Expression in polyI:C treated HCFs PolyⅠ:C resulted in upregulated the protein (Figure 6A) and mRNA (Figure 6Β) levels of TLR3, compared with the controls. LUT exposure abolished the effects exerted by polyⅠ:C on TLR3, resulting in normal or even downregulated TLR3 levels.

MCP-1, IL-6, and IL-8 Assays Three immune mediators,MCP-1, ⅠL-6, as well as ⅠL-8 were quantified using ELⅠSA kits. The serum-deprived HCFs were incubated whether or not with LUT (0, 5, 10, 20 μmol/L) overnight, and treated for another 24h with polyⅠ:C (1 μg/mL)

. The cell supernatant was collected after centrifuged (120×g, 5min, 4℃), and frozen at the temperature of -80℃ to be used in subsequent ELⅠSA analyses of the immune mediators. To determine the number of cells per well, the cells were exposed to trypsin-EDTA and isolated from the plates for culture and counted using a hemocytometer. Βased on the cell counts and the cell morphology analyses, count data was obtained through cell protein measurements of the cell supernatant divided by the cell number

. The experiments were repeated thrice.

如今的徐州，綠染四季，碧水繞城，新時期徐州精神再一次煥發勃勃生機。徐州水利人正以智慧和汗水，努力建造著一個水生態文明新城。