This study aims to explore the mechanism of "simultaneous treatment of the brain and the heart" of Naoxintong Capsules(NXT) under cerebral ischemia based on Toll-like receptor(TLR) signaling pathway.Male SD rats were randomized into sham operation group, model group, NXT group, and positive drug group.Middle cerebral artery occlusion(MCAO) model rats were used in model group, NXT group, and positive drug group, respectively.Neurological function was scored with the Bederson scale, and brain infarct rate was measured by 2,3,5-triphenyltetrazolium chloride(TTC) staining.Brain edema was detected with wet-dry weight method.Hematoxylin-eosin(HE) staining and TdT-mediated dUTP nick-end labeling(TUNEL) staining were used to observe and count apoptotic cardiocytes.In addition, serum myocardial enzymes were measured.The expression of 8 TLR signaling pathway-related proteins interferon-α(IFN-α), interferon regulatory factor-3(IRF3), interferon regulatory factor-7(IRF7), TLR2, TLR4, TLR7, TLR9, and tumor necrosis factor-α(TNF-α) in the cerebral cortex and heart of rats was detected by Western blot. Brain infarct rate, neurological function score, and brain water content in NXT group decreased significantly compared with those in the model group. At the same time, the reduction in apoptosis rate of cardiocytes and the content of serum aspartate aminotransferase(AST), alanine aminotransferase(ALT), creatine kinase(CK), and lactate dehydrogenase(LDH) were decreased in the NXT group.Systems pharmacological results and previous research showed that TLR signaling pathway played an important role in immune inflammatory response.The study of TLR signaling pathway and related proteins is helpful to elucidate the mechanism of "simultaneous treatment of the brain and the heart". Western blot results showed that NXT significantly inhibited the expression of IRF3, IRF7, TLR2, TLR7, and TNF-α in cerebral cortex and heart under cerebral ischemia.Cerebral ischemia influences cardiac functions, and TLR signaling pathway is one of the pathways for "simultaneous treatment of the brain and the heart" of NXT.
Animals ; Brain/metabolism* ; Brain Ischemia/metabolism* ; Capsules ; Drugs, Chinese Herbal ; Infarction, Middle Cerebral Artery/drug therapy* ; Male ; Myocytes, Cardiac ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 2/metabolism* ; Toll-Like Receptor 7/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

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.
Animals ; Antiviral Agents ; pharmacology ; Dogs ; Drugs, Chinese Herbal ; pharmacology ; Influenza A Virus, H1N1 Subtype ; drug effects ; physiology ; Madin Darby Canine Kidney Cells ; Orthomyxoviridae Infections ; Toll-Like Receptor 4 ; metabolism ; Toll-Like Receptor 7 ; metabolism ; Virus Replication ; drug effects

Autoimmune diseases (AID) are a group of complex disorders due to antibodies acting on self-antigens causing damage to the body. AID has long been considered as the outcome of genetic and environmental interactions. In recent years, studies have shown that increased susceptibility to AID may be associated with single nucleotide polymorphisms and copy number variations of Toll like receptor 7 (TLR7) gene, which provided a clue to further understanding of the pathogenesis of AID. This paper provides a review of the recent advances in understanding of the roles of TLR7 gene single nucleotide polymorphisms and copy number variations in AID.
Animals ; Autoimmune Diseases ; genetics ; DNA Copy Number Variations ; Genetic Predisposition to Disease ; Humans ; Polymorphism, Single Nucleotide ; Toll-Like Receptor 7 ; genetics

OBJECTIVETo investigate the expression of Toll-like receptor (TLR) mRNA in enterovirus 71(EV-A71) infected human Jurkat T cells and clarify the role of TLRs in the pathogenesis of EV-A71 infection-induced inflammation.

METHODSEV-A71 strains were isolated from feces of children patients with hand, foot and mouth disease in 2014 by Shenzhen Center for Disease Control and Prevention. Human Jurkat T cells were infected with 200 μl EV-A71 at 10(3) cell culture infective dose 50%(CCID50)/ml. The expression of TLR1-TLR10 mRNA in human Jurkat T cells was assessed at different exposure time by RT-PCR. Levels of TLR7 mRNA expression were detected by real-time PCR, and levels of myeloid differentiation factor 88 (MyD88) by western blot. The cytokine secretion of interleukin (IL)-6, IL-8 and Tumor Necrosis Factor α (TNF-α) was analyzed by ELISA assay.

RESULTSThe relative expression level of TLR7 mRNA in human Jurkat T cells were 1.26 ± 0.15, 1.75 ± 0.20, 2.26 ± 0.23 and 3.74 ± 0.62 in 6, 12, 24 and 48 h after EV-A71 infection, which the differences were significant with mock-infected group(t values were -2.96, -6.38, -9.57, -7.71; P<0.05). Western blot showed that the protein expression levels of MyD88 had increased 1.34 times and 2.17 times in 24 h and 48 h after EV-A71 infection compared with mock-infected group. After infected for 24 h and 48 h, the levels of IL-6 were (302.86 ± 38.11), (179.70 ± 14.50) pg/ml, which were significantly higher than mock-infected group (176.42 ± 9.60), (179.70 ± 14.50) pg/ml (t values were -5.57, -18.54, P<0.05). The levels of TNF-α in EV-A71 infected group (100.81 ± 9.81) pg/ml was higher than that in mock-infected group (56.19 ± 6.94) pg/ml, and the difference was significant (t=-6.43, P=0.003).

CONCLUSIONTLR7 is the main pattern recognition receptor responsible for EV-A71 recognition in immune cells, which then leads to the activation of TLR7 downstream signaling and the production of proinflammatory cytokines.

Blotting, Western ; Cell Line ; Enterovirus A, Human ; pathogenicity ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Myeloid Differentiation Factor 88 ; metabolism ; Real-Time Polymerase Chain Reaction ; T-Lymphocytes ; immunology ; virology ; Toll-Like Receptor 7 ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study the significance of toll-like receptors (TLR) -7 and -8 in the pathogenesis of infection caused by Enterovirus type 71 (EV71) through measuring the expression of TLR7 and TLR8 in brain and lung tissues from the death cases caused by EV71 infection.

METHODSNine children who died of EV71 infection (EV71 group) were selected as study subjects, and 7 children who died of accidents or non-infectious diseases were used as the control group. Brain and lung tissues from the death cases in both groups at autopsy were collected, and immunohistochemistry was applied to detect the expression of TLR7 and TLR8 in lung and brain tissues in both groups. Integrated optical density (IOD) was applied for semi-quantitative analysis of the expression of TLR7 and TLR8.

RESULTSImmunohistochemical results showed that the expression of TLR7 and TLR8 in lung and brain tissues was strongly positive in the EV71 group, and the IOD values in the EV71 group were also significantly higher than those in the control group (P<0.05). There was no significant difference in the expression of TLR7 and TLR8 between lung and brain tissues in the EV71 group (P>0.05).

CONCLUSIONSTLR7 and TLR8 are highly expressed in lung and brain tissues from the patients who die of severe EV71 infection, suggesting that TLR7 and TLR8 may be involved in the pathogenesis of brain and lung damages caused by severe EV71 infection.

Brain ; immunology ; Child ; Cytokines ; physiology ; Enterovirus A, Human ; Enterovirus Infections ; etiology ; immunology ; Humans ; Lung ; immunology ; Toll-Like Receptor 7 ; analysis ; physiology ; Toll-Like Receptor 8 ; analysis ; physiology

Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56(+) DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56(+) DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56(+) DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56(+) DCs represent a novel mDC subset mixed with some pDC features. A CD4(+)CD56(+) hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56(+) DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4(+)CD56(+) neoplasm may be a tumor counterpart of CD56(+) mDCs but not pDCs.
Biomarkers ; metabolism ; CD56 Antigen ; genetics ; immunology ; Cell Lineage ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Gene Expression ; Hematologic Neoplasms ; genetics ; immunology ; pathology ; Humans ; Immunophenotyping ; Interferon Type I ; biosynthesis ; metabolism ; Interleukin-12 ; biosynthesis ; metabolism ; Interleukin-3 Receptor alpha Subunit ; genetics ; immunology ; Lectins, C-Type ; genetics ; immunology ; Membrane Glycoproteins ; genetics ; immunology ; Myeloid Cells ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; genetics ; immunology ; Terminology as Topic ; Toll-Like Receptor 4 ; genetics ; immunology ; Toll-Like Receptor 7 ; genetics ; immunology ; Toll-Like Receptor 9 ; genetics ; immunology

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.
Alleles ; China* ; Confidence Intervals ; Female ; Gene Frequency ; Genotype ; Hepacivirus* ; Hepatitis C* ; Hepatitis C, Chronic ; Hepatitis* ; Humans ; Male ; Methods ; Polymorphism, Single Nucleotide* ; Toll-Like Receptor 7* ; Toll-Like Receptor 9* ; Toll-Like Receptors*

OBJECTIVETo investigate the expression of Toll-like receptor 7 (TLR7) in gastric cancer cell lines and the effect of imiquimod, a TLR7 agonist, on the proliferation and apoptosis of SGC-7901 cells.

METHODSThe protein expression levels of TLR7 were detected with Western blotting in 3 human gastric cancer cell lines (SGC-7901, HGC-27 and MKN-28). The cell line expressing the highest TLR7 level was exposed to different doses of imiquimod for 12-72 h and the cell viability was assessed with MTT assay. The cell apoptosis rate after 100 µg/ml imiquimod treatment for 12 or 24 h was quantified by flow cytometry, and the ultrastructual changes of the cells were observed under electron microscope. The expression of apoptosis-related genes Bcl-2 and Bax were analyzed with real-time PCR.

RESULTSAll the 3 cell lines expressed TLR7, among which SGC-7901 cells showed the highest expression level. TLR7 agonist imiquimod dose- and time-dependently reduced the viability of SGC-7901 cells. Exposure to 100 µg/ml imiquimod for 24 h resulted in SGC-7901 cell apoptosis as shown by an increased ratio of early apoptotic cells and significant ultrastructural changes of the cells. Real-time PCR demonstrated that imiquimod treatment for 24 h caused a dose-dependent reduction of Bcl-2 mRNA expression and increment of Bax mRNA expression.

CONCLUSIONSTLR7 protein is expressed in all the 3 gastric cancer lines and its agonist imiquimod can inhibit cell proliferation and induce apoptosis in SGC-7901 cells.

Aminoquinolines ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Real-Time Polymerase Chain Reaction ; Stomach Neoplasms ; metabolism ; pathology ; Toll-Like Receptor 7 ; agonists ; metabolism

Psoriasis is a chronic inflammatory disease related to genome-wide and surroundings, it is important to develop a suitable animal model to research psoriasis pathogenesis and evolve pharmacotherapeutics. With the development of transgenetic technology in the past few years, psoriasis virulence gene animal model become a hotspot. Research of animal model of human psoriasis genes is reviewed in the paper.
Aminoquinolines ; toxicity ; Amphiregulin ; Animals ; Disease Models, Animal ; EGF Family of Proteins ; genetics ; metabolism ; Humans ; Keratin-14 ; genetics ; metabolism ; Keratin-5 ; genetics ; metabolism ; Keratinocytes ; metabolism ; Membrane Glycoproteins ; agonists ; Mice, Transgenic ; Psoriasis ; etiology ; genetics ; metabolism ; Receptor, TIE-2 ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Toll-Like Receptor 7 ; agonists ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo observe effect of Shufeng Xuanfei Recipe (SXR) and Jiebiao Qingli Recipe (JQR) on mRNA and protein expressions of Toll-like receptor 7 (TLR7), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappaB (NF-kappaB) in mice infected with influenza virus FM1.

METHODSOne hundred and eight mice were randomly divided into nine groups, i.e., the normal control group, the model group, the Oseltamivir group (at the daily dose of 2.5 g/mL), the high dose SXR group (at the daily dose of 3.762 g/kg), the middle dose SXR group (at the daily dose of 1.881 g/kg), the low dose SXR group (at the daily dose of 0.941 g/kg), the high dose JQR group (at the daily dose of 4.368 g/kg), the middle dose JQR group (at the daily dose of 2.184 g/kg), and the low dose JQR group (at the daily dose of 1.092 g/kg), 12 in each group. All mice were mildly anesthetized by ether. Mice in the normal control group were treated by nasal drop of 0.05 mL normal saline, while mice in the rest groups were infected by nasal drop of 0.05 mL influenza virus strain FM1 (LD50). The successful modeling rate was 100%. All medication was performed by gastrogavage 2 h after infection. Distilled water was given by gastrogavage to mice in the normal control group and the model group at the daily dose of 0.2 mL, each time per day for 4 successive days. mRNA expressions of TLR7, MyD88, and NF-kappaB in the lung tissue were determined by Western blot.

RESULTSCompared with the normal control group, mRNA expressions of TLR7, MyD88, and NF-kappaB increased in the model group (P < 0.01). Compared with the model group, mRNA and protein expressions of TLR7, MyD88, and NF-kappaB decreased in the Oseltamivir group, the high, middle, and low dose SXR groups (P < 0.05, P < 0.01); mRNA and protein expressions of TLR7 and NF-kappaB decreased in the high and middle dose JQR groups (P < 0.05, P < 0.01); mRNA expressions of MyD88 decreased in the high and middle dose JQR groups (P < 0.05); protein expressions of MyD88 decreased in the middle dose JQR group (P < 0.05); protein expressions of TLR7 and NF-kappaB decreased in the low dose JQR group (P < 0.05). Compared with the Oseltamivir group, protein expressions of MyD88 decreased in the low dose SXR group (P < 0.05); protein expressions of NF-kappaB decreased in the middle and low dose SXR groups (P < 0.01); mRNA and protein expressions of TLR7 (P < 0.05, P < 0.01), and protein expressions of MyD88 (P < 0.01) decreased in the high, middle, and low dose JQR groups; mRNA and protein expressions of NF-kappaB decreased in the low dose JQR group (P < 0.05, P < 0.01).

CONCLUSIONSEach dose SXR and middle dose JQR could down-regulating the activity of NF-kappaB through adjusting MyD88 dependent TLR signal pathway, thus fighting against influenza virus. SXR was more effective than JQR.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Lung ; metabolism ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Mice ; Mice, Inbred ICR ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; NF-kappa B ; genetics ; metabolism ; Orthomyxoviridae ; Orthomyxoviridae Infections ; drug therapy ; metabolism ; Pneumonia, Viral ; drug therapy ; metabolism ; RNA, Messenger ; genetics ; Signal Transduction ; drug effects ; Toll-Like Receptor 7 ; genetics ; metabolism