To investigate the expression of Toll-like receptor (TLR) 2 and 4 mRNA in local tissues of model of oropharyngeal candidiasis in mice and to explore the potential role of TLR2 and TLR4 in earlier period of immune response, a murine model of oropharyngeal candidiasis inoculated by cotton wool balls saturated with Candida albicans was established. Mice were sacrificed at the indicated time points and the oropharyngeal tissues were excised. The expression of TLR2 and TLR4 mRNA was detected by RT-PCR. The results showed that low level of TLR2/4 mRNA could be detected in oropharyngeal tissues, but they were markedly up-regulated 6 h after inoculation, peaking after 12-24 h. Tissue TLR4 mRNA was gradually down-regulated 24-48 h, while TLR2 mRNA levels remained high up to the 72nd h. These data suggested that oropharyngeal infection of Candida albicans could result in up-regulation of TLR2/4 mRNA expression in local tissues, which might play important roles in earlier period of immune response.
Candidiasis/metabolism ; Candidiasis, Oral/*metabolism ; Mouth Mucosa/*metabolism ; Pharyngitis/metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Random Allocation ; Toll-Like Receptor 2/*biosynthesis ; Toll-Like Receptor 2/genetics ; Toll-Like Receptor 4/*biosynthesis ; Toll-Like Receptor 4/genetics

OBJECTIVETo investigate the effect of RNA interfering TLR4 signal pathway on phagocytosis of Kupffer cells.

METHODSRAW2647 mice mononuclear macrophage leukemia cells were observed. The tested group was interfered by Tlr4-mus-1567 RNA which had the best result confirmed by QPCR, cells interfered by Negative Control RNA as NC group, and normal cell as control. We perform the phagocytosis test on each group.

RESULTSThe tested group has lower phagocytes percentage than control (17.67% +/- 3.51% vs 32.00% +/- 3.00%, P < 0.01), and lower phagocytic index (46.33% +/- 7.51% vs 82.00% +/- 6.08%, P < 0.01).

CONCLUSIONSDecreased phagocytic activity was observed on Kupffer cells by RNA interference.

Animals ; Kupffer Cells ; immunology ; Mice ; Phagocytosis ; RNA Interference ; Signal Transduction ; Toll-Like Receptor 4 ; genetics ; immunology

Acetylcysteine ; pharmacology ; Animals ; Cell Line ; Gene Expression ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; Mice ; Toll-Like Receptor 2 ; genetics ; Toll-Like Receptor 4 ; genetics

OBJECTIVETo investigate the toll-like receptors (TLR) profile of human epidermal keratinocytes.

METHODSWe cultured the immortalized human epidermal keratinocyte cell line HaCaT cells and normal human epidermal keratinocytes (NHEK) and separated epidermis with dispase from foreskins. TLR 1-10 mRNA expression was detected with reverse transcription polymerase chain reaction (RT-PCR). TLR 2 and 4 protein expressions on surface of HaCaT cells and NHEK were detected using flow cytometry.

RESULTSHaCaT cells, NHEK, and epidermis all expressed TLR 1-10 mRNA with different intensity. TLR 4 protein was detected on the surfaces of HaCaT cells and NHEK, while the expression of TLR 2 protein was few.

CONCLUSIONHuman epidermal keratinocytes constitutively express all TLR 1-10 mRNA, which may enable human keratinocytes to respond to a wide range of pathogenic micro-organisms.

Cell Line ; Cell Line, Tumor ; Epidermis ; cytology ; Flow Cytometry ; Humans ; Keratinocytes ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Toll-Like Receptors ; genetics ; metabolism

The liver and kidney fibrosis model was established by thioacetamide(TAA) and unilateral ureteral obstruction(UUO) in SD rats. The rats were randomly divided into three groups: model group, low and high-dose groups of C21 steroidal glycosides of Cynanchum auriculatum. Another blank control group was set. Four weeks later, serum was taken to detect the biochemical indexes of liver and kidney function. Urine protein and urine creatinine were detected by kits. Liver and kidney tissue samples were stained with HE and Masson staining, and hydroxyproline content was detected. Western blot was used to detect expressions of fibrotic proteins, inflammatory factors and TLR4 signaling pathways, so as to observe the preventive and therapeutic effects of C21 steroidal glycosides from C. auriculatum on hepatic and renal fibrosis and explore its molecular mechanism. Four weeks later, serum biochemical results showed that liver and kidney functions were seriously damaged, and pathological sections showed that inflammatory cell infiltration, decrease of parenchymal cells, and increase of interstitial fibrosis in liver and kidney tissues. The results showed that low and high doses(150, 300 mg·kg~(-1)) of C21 steroidal glycosides could significantly reduce the collagen deposition and the pathological changes of liver and kidney fibrosis compared with the model group. At the same time, we found that the expression levels of TLR4 and MyD88 signaling pathway proteins were significantly increased in the liver and kidney tissues of the model group, and a large number of NF-κB signaling pathway proteins migrated into the nucleus. On the contrary, the expression levels of TLR4, MyD88 signaling pathway proteins and the nuclear migration of NF-κB were significantly inhibited in the low and high dose groups of C21 steroidal glycosides from C. auriculatum. Therefore, it was speculated that the mechanism of C21 steroidal glycoside for preventive and therapeutic effect on hepatic and renal fibrosis was related to inhibit TLR4/MYD88/NF-κB inflammatory pathway, thus preventing hepatic and renal fibrosis.
Animals ; Cynanchum ; Fibrosis ; Glycosides ; Kidney/pathology* ; Liver ; NF-kappa B/genetics* ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 4/genetics*

OBJECTIVETo investigate the expression of Toll-like receptors (TLRs) in thymus of myasthenia gravis (MG) patients and the relationship with clinical features.

METHODSThymic specimens of 36 patients received extended thymectomy for MG were divided into three groups by pathological type: 13 thymoma tissues (thymoma group) and 13 thymic tissues adjacent to thymomas (parathymoma group) from 13 cases of MG patients with thymomas, and 23 thymic tissues from MG patients without thymomas (MG nonthymoma group). Twenty-one normal thymic specimens from cardiac surgery were used as controls. The levels of TLR2-4 mRNA were examined by RT-PCR, then the levels of TLR4 mRNA were assayed by real time RT-PCR and their relationship with clinical features were analyzed.

RESULTSThe levels of TLR4 mRNA among the different groups had significant differences, while there was no difference in TLR2 and TLR3 levels. The real time RT-PCR showed that the level of TLR4 mRNA in nonthymoma group was significantly higher than that in control group(0.8544+/- 0.1200 vs 0.6851+/- 0.1524, P=0.018). And so is parathymoma group compared with the thymoma group (0.8214+/- 0.1019 vs 0.7101+/- 0.0916, P=0.005). No significant difference of TLR4 mRNA level was found between the parathymoma and nonthymoma groups. Nevertheless, the expression of TLR4 in both groups was increased compared with control group. The levels of TLR4 mRNA had positive correlation with Osserman type(R=0.609; P=0.004) .

CONCLUSIONTLR4 may play a key role in the pathogenesis of MG. It was the thymic tissues adjacent to thymomas but not thymomas themselves participated in the onset of MG.

Adolescent ; Adult ; Female ; Gene Expression Regulation ; Humans ; Male ; Middle Aged ; Myasthenia Gravis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Thymus Gland ; metabolism ; Toll-Like Receptor 2 ; genetics ; Toll-Like Receptor 3 ; genetics ; Toll-Like Receptor 4 ; genetics ; Toll-Like Receptors ; genetics ; Young Adult

The innate immune response in patients who develop inflammatory bowel disease (IBD) may be abnormal. However, the exact role of Toll-like receptors (TLRs) / CD14 gene in the pathogenesis of IBD has not been fully elucidated. We aimed to investigate the association between polymorphisms of TLR1, 2, 4, 6, and CD14 gene and susceptibility to IBD in Korean population. A total 144 patients of IBD (99 patients with ulcerative colitis, 45 patients with Crohn's disease) and 178 healthy controls were enrolled. Using a PCR-RFLP, we evaluated mutations of TLR1 (Arg80Thr), TLR2 (Arg753Gln and Arg677Trp), TLR4 (Asp299Gly and Thr399Ile), TLR6 (Ser249Pro) genes and the -159 C/T promoter polymorphism of CD14 gene. No TLR polymorphisms were detected in Korean subjects. T allele and TT genotype frequencies of CD14 gene were significantly higher in IBD patients than in healthy controls. In subgroup analysis, T allelic frequency was higher in pancolitis phenotype of ulcerative colitis. In Korean population, the promoter polymorphism at -159 C/T of the CD14 gene is positively associated with IBD, both ulcerative colitis and Crohn's disease.
Adult ; Aged ; Alleles ; Antigens, CD14/*genetics ; Asian Continental Ancestry Group/*genetics ; Colitis, Ulcerative/genetics ; Crohn Disease/genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Inflammatory Bowel Diseases/*genetics ; Male ; Middle Aged ; Phenotype ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Republic of Korea ; Toll-Like Receptor 1/genetics ; Toll-Like Receptor 2/genetics ; Toll-Like Receptor 4/genetics ; Toll-Like Receptor 6/genetics ; Toll-Like Receptors/*genetics

This study investigated the mechanism of baicalin on lipopolysaccharide(LPS)/interferon γ(IFN-γ)-induced inflammatory microglia based on the triggering receptor expressed on myeloid cells 2(TREM2)/Toll-like receptor 4(TLR4)/nuclear factor kappaB(NF-κB) pathway. Specifically, LPS and IFN-γ were used to induce inflammation in mouse microglia BV2 cells. Then the normal group, model group, low-dose(5 μmol·L~(-1)) baicalin group, medium-dose(10 μmol·L~(-1)) baicalin group, high-dose(20 μmol·L~(-1)) baicalin group, and minocycline(10 μmol·L~(-1)) group were designed. Cell viability was detected by CCK-8 assay and cell morphology was observed under bright field. The expression of interleukin-1β(IL-1β), interleukin-4(IL-4), inducible nitric oxide synthase(iNOS), interleukin-6(IL-6), interleukin-10(IL-10), and arginase-1(Arg-1) mRNA was detected by real-time quantitative PCR, the protein expression of tumor necrosis factor-α(TNF-α), IL-1β, TREM2, TLR4, inhibitor kappaB-alpha(IκBα), p-IκBα, NF-κB p65 and p-NF-κB p65 by Western blot, and transfer of NF-κB p65 from cytoplasm to nucleus by cellular immunofluorescence. Compared with the normal group, most of the BV2 cells in the model group tended to demonstrate the pro-inflammatory M1 amoeba morphology, and the model group showed significant increase in the mRNA levels of IL-1β, IL-6, and iNOS, decrease in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), rise of the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.01), reduction in TREM2 protein expression, and increase in the expression of NF-κB p65 in nucleus. Compared with the model group, baicalin groups and minocycline group showed the recovery of BV2 cell morphology, significant decrease in the mRNA levels of IL-1β, IL-6 and iNOS, increase in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), reduction in the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.05), rise of TREM2 protein expression, and decrease in the expression of NF-κB p65 in nucleus. In summary, these results suggest that baicalin can regulate the imbalance between TREM2 and TLR4 of microglia and inhibit the activation of downstream NF-κB, thus promoting the polarization of microglia from pro-inflammatory phenotype to anti-inflammatory phenotype.
Animals ; Flavonoids ; Inflammation/genetics* ; Interferon-gamma ; Lipopolysaccharides/adverse effects* ; Mice ; NF-kappa B/metabolism* ; Toll-Like Receptor 4/metabolism*

Animals ; Glomerulonephritis, IGA/drug therapy* ; Hydroxychloroquine/therapeutic use* ; Ki-67 Antigen ; Kidney ; Rats ; Toll-Like Receptor 4/genetics* ; Vitamin D

OBJECTIVE: To investigate the inflammatory effects of Cinobufotalin on monocytes in resting state and macrophages in activated state and its molecular mechanism. METHODS: THP-1 cells were stimulated with Phorbol 12-myristate 13-acetate to induce differentiation into macrophages. Lipopolysaccharides was added to activate macrophages in order to establish macrophage activation model. Cinobufotalin was added to the inflammatory cell model for 24 h as a treatment. CCK-8 was used to detect cell proliferation, Annexin V /PI double staining flow cytometry was used to detect cell apoptosis, flow cytometry was used to detect macrophage activation, and cytometric bead array was used to detect cytokines. Transcriptome sequencing was used to explore the gene expression profile regulated by Cinobufotalin. Changes in the significantly regulated molecules were verified by real-time quantitative polymerase chain reaction and Western blot. RESULTS: 1∶25 concentration of Cinobufotalin significantly inhibited the proliferation of resting monocytes(P<0.01), and induced apoptosis(P<0.01), especially the activated macrophages(P<0.001, P<0.001). Cinobufotalin significantly inhibited the activation of macrophages, and significantly down-regulated the inflammatory cytokines(IL-6, TNF-α, IL-1β, IL-8) released by activated macrophages(P＜0.001). Its mechanism was achieved by inhibiting TLR4/MYD88/P-IκBa signaling pathway. CONCLUSION Cinobufotalin can inhibit the inflammatory factors produced by the over-activation of macrophages through TLR4/MYD88/P-IκBa pathway, which is expected to be applied to the treatment and research of diseases related to the over-release of inflammatory factors.
Humans ; Toll-Like Receptor 4/metabolism* ; Myeloid Differentiation Factor 88/genetics* ; Macrophages/metabolism* ; Cytokines/metabolism* ; Lipopolysaccharides/pharmacology* ; NF-kappa B