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Objective:To observe the regulatory effect of Guben Fangxiao decoction on Toll-like receptor (TLR)4, TLR7, nuclear factor-kappa B p65 (NF-κB p65) and NF-κB inhibitor protein alpha (IκBα) in mice with asthma remission, in order to explore the mechanism of Guben Fangxiao decoction in treating asthma remission. Method:Respiratory syncytial virus (RSV) combined with chicken ovalbumin (OVA) was used to build asthma remission model in 3-week-old BALB/c mice. Sixty mice were divided into blank group, model group, dexamethasone group (0.001 g·kg-1), low,medium and high-dose Guben Fangxiao decoction group (6.5,13,26 g·kg-1), respectively. Intervention was given once a day for 28 days. After administration, the mice were put to death. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung tissue and score the pulmonary inflammation. Western blot was used to detect the expressions of TLR4, TLR7, IκBα and nuclear NF-κB p65 in lung tissue of mice. Immunofluorescence was used to observe the expression of NF-κB p65 in cellnucleuses. Result:Compared with blank group, the lung tissue of model group showed obvious inflammatory cell infiltration (PκB p65 increased significantly (PκBα proteins increased significantly (PPκB p65 (PκB p65 (PκBα proteins (PConclusion:Guben Fangxiao decoction can alleviate airway inflammation, and its therapeutic effect may be achieved by regulating TLR4, TLR7, NF-κB p65 and IκBα.
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To study the effect and underlying mechanism of Mahuang Tang against influenza A virus ， the influenza virus-infected Madin-Darby canine kidney(MDCK) cells were used as the carrier in this study to detect the median tissue culture-infective dose(TCID₅₀) of influenza A virus strains(A/PR8/34) on MDCK cells with cytopathic effect(CPE) assay. Blocking influenza virus invading host cells and anti-influenza virus biosynthesis were used as two different administration methods， and then the methyl thiazolyl tetrazolium(MTT) assay was utilized to determine the antiviral effective rate(ER)， median efficacious concentration(EC₅₀) and therapeutic index(TI) of Mahuang Tang. The quantitative Real-time polymerase chain reaction(RT-PCR) was used to measure virus load and the mRNA expression levels of TLR4， TLR7， MyD88 and TRAF6 in MDCK cells at 24， 48 h after the treatment. The experiment results indicated that TCID₅₀ of A/PR8/34 for MDCK cells was 1×10-4.32/mL. The EC₅₀ values of two different treatment methods were 4.92，1.59 g·L⁻¹ respectively， the TI values were 12.53， 38.78 respectively， and when the concentration of Mahuang Tang was 5.00 g·L⁻¹， ER values were 50.21%， 98.41% respectively， showing that Mahuang Tang can block influenza virus into the host cells and significantly inhibit their biosynthesis. Meanwhile， as compared with the virus group， the virus load was significantly inhibited in Mahuang Tang groups， and Mahuang Tang high and middle doses had the significant effect on decreasing the mRNA expression of TLR4， TLR7，MyD88 and TRAF6 at 24， 48 h after the treatment. It can be demonstrated that the mechanisms of Mahuang Tang against influenza A virus are related to the inhibition of influenza virus replication and the mRNA expression of correlative genes in TLR4 and TLR7 signaling pathways.
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Animals , Dogs , Antiviral Agents , Pharmacology , Drugs, Chinese Herbal , Pharmacology , Influenza A Virus, H1N1 Subtype , Physiology , Madin Darby Canine Kidney Cells , Orthomyxoviridae Infections , Toll-Like Receptor 4 , Metabolism , Toll-Like Receptor 7 , Metabolism , Virus ReplicationABSTRACT
Objective To investigate expressions and correlations of TLR2,TLR4,TLR7 and TLR9 in eosinophil-enriched cell populations from patients with allergic rhinitis (AR),and elucidate their roles in AR. Methods Peripheral venous blood samples were collected from healthy controls (HCs)and AR patients,and then incubated with crude extracts of Artemisia pollen,dust mite,and Platanus pollen,respectively.Levels of TLR2 , TLR4,TLR7 and TLR9 in blood eosinophil-enriched cells were detected by flow cytometry.Correlations between TLR2+,TLR4+,TLR7+and TLR9+eosinophils were analyzed by SPSS.Results Levels of TLR2+eosinophils from patients with AR were reduced by 4%,mean fluorescence intensity (MFI)of TLR4+eosinophil was elevated by 20%,and TLR7+eosinophils increased up to 4.8 folds compared with HCs when cultured with medium only (P＜0.05).Artemisia pollen extracts induced approximately 7 .8 % of increase in TLR2+eosinophils from AR patients.In addition,correlations between TLR2+and TLR4+eosinophils,TLR2+and TLR7+eosinophils,and TLR7+and TLR9+eosinophils were -0.670 (P＜0.01),-0.430 (P＜0.05)and 0.446 (P＜0.05),respectively. However,allergens had few effects on TLR2,TLR4,TLR7 and TLR9 expressions in HCs.Conclusion Eosinophil-derived TLR2 ,TLR4 and TLR7 are likely to play a key role in AR.TLR2 ,TLR4 and TLR7 might become the potential targets for AR treatment.
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Objective:To investigate the effect of Rab7 on cytokine induced by TLR7 (Toll like receptor-7) R848 activated in Raw264.7,and discusses the influence of Rab7 on MAPK signal transduction.Methods: TLR7 downstream cytokines such as TNF-α,IL-6,IFN-α,IFN-β and IP-10 activated by R848 were detected through Q-PCR in Rab7 silenced mouse macrophages,and then analysis of phosphorylation of MAPK determined with Western blot showed the effect of Rab7 on signal transduction of MAPK.Results: Rab7 inhibit production of cytokine activated by TLR7,and also,Rab7 had an inhibitory effect on MAPK signal pathway.Conclusion: The experimental results further illustrate that the Rab7 is the TLR7 signal transduction pathway negative regulatory factor,and to participate in MAPK signaling pathway.
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Objective To analyze the changes in the expression of Toll-like receptors 4 (TLR4) and 7 on the surface of murine bone marrow-derived dendritic cells following dengue virus type 2 (DEN2) infection.Methods DEN2 NGC strain was infected into BALB/c suckling mice through intracranial injection and injected into C6/36 cells to induce the in vivo and in vitro proliferation of DEN2, respectively.RT-PCR was performed to identify DEN2 RNA.Reed-Muench method was used to determine the 50% tissue culture infective dose (TCID50) of DEN2.Dendritic cells (DCs) were prepared by stimulating bone marrow cells isolated form C57BL/6 mice with IL-4 and GM-CSF and then identified by flow cytometry.The prepared murine bone marrow-derived DCs were infected with DEN2 and observed with direct immunofluorescence assay.Dynamic changes in the expression of CD11c, CD86 and I-A/I-E molecules on DCs after DEN2 infection were detected by flow cytometry.Levels of DEN2 RNA and the expression of TLR4 and TLR7 at mRNA level were dynamically detected by real-time quantitative PCR.Results The TCID50 of DEN2 to C6/36 cells was 10-5.8.Murine bone marrow-derived DCs were acquired with a purity of 70% and could be infected with DEN2 in vitro.The percentages of CD86 and I-A/I-E molecules on the surface of DCs infected with 1×105 TCID50 of DEN2 were statistically different from those of the negative control group.Neither of the two groups showed a significant difference in the percentages of membrane molecules over time.However, the percentages of membrane molecules on DCs increased with increasing viral load.Compared with the negative control group, the levels of DEN2 RNA in infection groups were increased with increasing virus load, while the expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level was decreased with increasing viral load.Conclusion DEN2 infection promotes the maturation of DCs.Expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level decreases with increasing viral load, which suggests that TLR4 and TLR7 are closely related to viral infection and play a certain role in the pathogenesis of DEN infection.
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@#BACKGROUND: Toll like receptors (TLRs) are a class of proteins that key role in the innate immune system. TLR7 is expressed on monocytes, macrophages and dendritic cells, T cell, B cell and eosinophiles. TLR7, originally identified as recognizing imidaquinoline, loxibrine, broprimine and ssRNA, ssRNA viruses such as vesicular stomatitis virus, influenza A virus and human immunodefiency virus. It is known that virus ssRNA affects signaling molecule of IFN-y. Objective: To determine gene and protein activation of IFN-y signal transduction by TLR7 ligand in the endothelial cells. MATERIAL: In study we used mouse aortic linear endothelial cell which is cultured (END-D) in 5% heat- inactivated fetal calf serum (FCS), medium (DMEM) containing antibiotic mix(penicillin G, streptomycin, amphotericin B) at 37°C (5% CO2). Endothelial cells treated with synthetic IFN-γ and imiquimodligands, then the NO (nitric oxide) concentration in the supernatant is determined by Griess reagent. Endothelial cells are cultured in 6 well cell culture plate and in each well 2*104cells are expected to be grown for 24 hours of culture. Then, the cells are treated with synthetic IFN-γ and имиквимод ligand for 6 hours and the NO signaling gene activation iNOS mRNA expression which is induced by IFN-γ is determined by RT-qPCR. Endothelial cells are cultured in 12 well cell culture plate and in each well 2*104 cells are expected to be grown for 18 hours of culture. Then, the cells are treated with synthetic IFN-γ and imiquimodligands for 24 hours and the NO signaling protein iNOS expression which is induced by IFN-γ is determined by western blotting. The experiment was conducted as representation mean of at least three test results. The difference between statistical probabilities is determined by the “Students” t test. The p<0.01 value is assumed to be statistically different. RESULTS: TLR7 ligand imiquimodaugmented interferon gamma induced nitric oxide production TLR7 ligand imiquimodincreased interferon gamma induced iNOS mRNA gene expression. TLR7 ilgand imiquimodup-regulated interferon gamma induced iNOS protein expression. CONCLUSIONS: TLR7 ligand imiquimod augments IFN-γ signaling in the endothelial cells. This synergistic effect has revealed in the levels of gene and protein expression.
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Objective To investigate the effects of TLR7 on imiquimod induced apoptosis of THP-1 derived macrophages.Methods Three cell lines ( THP-1 derived macrophages, MDCK cell line and HUVEC cell line) with different capabilities of expressing TLR7 were selected.The survival rates of cells af-ter the treatment with different concentrations of imiquimod were detected by MTT assay.The levels of IL-6 in the supernatants of TLR7 inhibitor chloroquine or TLR7-siRNA treated cells were detected by enzyme-linked immunosorbent assay.The apoptosis of cells was detected by flow cytometry after inhibiting the ex-pression of TLR7.Results Imiquimod induced the apoptosis of THP-1 derived macrophages, MDCK cell lines and HUVEC cell lines.The levels of IL-6 were significantly decreased as the expression of TLR7 was inhibited by treating THP-1 derived macrophages with chloroquine or TLR7-siRNA.Treating THP-1 derived macrophages with chloroquine or TLR7-siRNA did not affect the cell apoptosis induced by imiquimod.Con-clusion Imiquimod could induce the apoptosis of THP-1 derived macrophages through TLR7 independent pathway.
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ABSTRACT:The aim of the present study was to investigate the inhibitory effects of TLR7 on Mycobacterium tuberculosis . TLR7 on infected RAW264 .7 cells was activated by chemical synthesis of TLR7 activation motif ssRNA .Activated RAW264 .7 cells were inoculated with Mycobacterium tuberculosis ,quantitative PCR method was applied to detect the phagocytosis rate of cell to bacteria at different time after infection .Cytokine production was measured by ELISA from cell supernatant .Cells were cultured on Roche medium and counted after sterile cracked with TritonX‐100 and diluted with PBS .Scanning electronic micro‐scope ( SEM ) was applied to detect the morphological changes of cells treated with TLR7 activation motif ssRNA .The highest phagocytosis rate of bacteria of RAW264 .7 cells was at 3 hours post infection (P>0 .05) .Compared with that of the control group ,treatment after 36 hours intracellular bacterial quantity in ssRNA treated group was lower (P<0 .05) ,levels of IL‐12 (P<0 .05) and IL‐4 (P<0 .05) were increased .For treatment after 48 hours ,level of IL‐4 (P<0 .05) was decreased ,and TNF‐α (P<0 .05) was increased .For treatment after 3 hours ,cell morphology of the ssRNA group was obviously better than the control group and appeared lots of phagosomes .Results suggested that TLR7 could enhance macrophages in killing Myco‐bacterium tuberculosis by forming phagosomes and regulating cytokines production ,and TLR7 activation motif ssRNA could be used in the treatment of tuberculosis .
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PURPOSE: To analyze the correlation of polymorphisms of toll-like receptor 7 (TLR7) (rs179009) and toll-like receptor 9 (TLR9) (rs187084) in hepatitis C virus (HCV) infections in the Han population. MATERIALS AND METHODS: The genotypes of TLR7IVS2-151 in HCV infection were detected by Sanger sequencing using polymerase chain reaction-restriction fragment length polymorphism to determine the TLR9 T-1486C single nucleotide polymorphisms (SNP) for all enrolled patients. RESULTS: We found no significant difference between males with spontaneous clearance of HCV versus those chronically infected [chi2=2.71, p=0.10, odd ratios (OR)=0.58, 95% confidence interval (CI) 0.31-1.11]. However, significant differences were found for the distribution of TLR7 (rs179009) in females (chi2=9.46, p=0.01). In females, a significant difference was also found between chronic hepatitis C and those with spontaneous clearance of HCV in terms of TLR7 IVS2-151G/A allele frequencies (chi2=9.50, p=0.00, OR=0.46, 95% CI 0.28-0.75). In HCV-infected patients, no significant association was found between the frequency of TLR9 genotypes and alleles. CONCLUSION: The site of TLR7 IVS2-151 (rs179009) G/A may be a factor for susceptibility of chronic HCV in the female Han population. TLR9T-1486C (rs18084) SNP may not play a major role in HCV infection. However, individual risk profiles for HCV infection did vary by sex and this relationship should be further investigated.
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Female , Humans , Male , Alleles , China , Confidence Intervals , Gene Frequency , Genotype , Hepacivirus , Hepatitis C , Hepatitis C, Chronic , Hepatitis , Methods , Polymorphism, Single Nucleotide , Toll-Like Receptor 7 , Toll-Like Receptor 9 , Toll-Like ReceptorsABSTRACT
Objective: To study the anti-influenza virus A/PR/8/34 (H1N1) activity of the volatile oil in Cinnamomi Ramulus (VOCR) and cinnamaldehyde in vitro, and to reveal the effect of TLR7 signaling pathway in anti-influenza. Methods: The IC50 and therapeutic index (TI) of VOCR and cinnamaldehyde in vitro were determined using influenza virus-infected Madin-Darby canine kidney (MDCK) cell line. ELISA was conducted to determine the level of interferon-β (IFN-β) in MDCK cells. The real-time RT-PCR was used to determine the mRNA expresstion of TLR3, TLR7, TRAF-3, interleukin-1 related acceptor kinase-4 (IRAK-4), and IFN-β. Results: Both VOCR and cinnamaldehyde had the direct virucidal activities on influenza virus; The IC50 values were 1.85 × 10-7 and 5.77 × 10-7 g/mL, respectively. The TI values were 27.04 and 9.51, respectively. Compared with the virus group, VOCR and cinnamaldehyde (0.25 × 10-5 g/mL) had the significant effects on increasing the serum level of IFN-β in infected MDCK cells (P < 0.05) and the mRNA expression of TLR7, IRAK-4, and IFN-β (P < 0.05). Conclusion: VOCR and cinnamaldehyde have good anti-influenza virus activities in the cellular level. The mechanisms are related to the activiation of TLR7 signaling pathway and IRAK-4, and leading to the high expression of IFN-β.
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The present study aims to evaluate the anti-inflammatory effect of methanol extract from leaves of Carpinus tschonoskii (CE) on R848-stimulated primary bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs). Primary BMDMs and BMDCs were used for pro-inflammatory cytokine production. Human embryonic kidney cell line 293T (HEK293T) was used to access NF-kappaB activity. In all cases, R848 was used to stimulate the cells. The CE (0~150 microg/ml) was treated to BMDMs, BMDCs, and HEK293T cells. CE pre-treatment in R848-stimulated BMDMs and BMDCs showed a dose-dependent inhibitory effect on pro-inflammatory cytokine (e.g., IL-12 p40, IL-6, and TNF-alpha) production as compared to non-treated controls. In NF-kappaB reporter gene assay, the CE pre-treatment inhibited NF-kappaB-dependent luciferase activity in a dose-dependent manner. Overall, our findings suggest that CE has significant inhibitory effect on pro-inflammatory cytokine production and deserve further studies concerning potentials of CE for medicinal uses.
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Humans , Betulaceae , Cell Line , Corynebacterium , Dendritic Cells , Genes, Reporter , Interleukin-12 , Interleukin-6 , Kidney , Luciferases , Macrophages , Methanol , NF-kappa BABSTRACT
Objective To examine the expression of TLR7/8 in monocytes purified from HIV-1 infected individuals and to study its association with disease progression. Methods Sixty-three HIV-1 infected individuals and 18 normal controls were enrolled. Monocytes were purified by MACS system and RNA of them was extracted by RNA mini kit of QIAGEN company. TLR7/8 expression was tested by real-time RT-PCR with ABI7500. Results It was found that the expression of TLR7 was strongly correlated with absolute CD4 count (r =0.614, P＜0.01) , so was TLR8 (r =0.419, P＜0.01). The expression of TLR7 in slow progressor (SP) group was higher than that in HIV-1 infected patients group, AIDS patients group and normal group (P ＜ 0. 05 ) . HIV group and normal group were strongly higher than AIDS group (P ＜ 0. 05). It was no significant differentiation of expression of TLR7 between HIV infection group and normal control group. The expression of TLR8 in SP group and normal group were significantly higher than that in AIDS group (P ＜ 0. 05). The expression of TLR8 was no singnificantly difference between SP group and HIV group or normal control group, so was it between HIV group or normal control group and AIDS group. Conclusion The expression of TLR7/8 in monocytes from HIV-1 infected patients significantly correlated with disease progression.
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0.05). Compared with control cells, the relative luciferase activity of NF-?B in virus-infected cells was apparently up-regulated (P