MiR-150-5p protects rats from middle cerebral artery occlusion by inhibiting the toll-like receptor-5/nuclear factor-kappa B p65 signaling pathway / 中国组织工程研究

BACKGROUND: There is an inflammatory response in the lesion tissue of ischemic cerebral infarction, and the expression of miR-150-5p is significantly decreased. Whether miR-150-5p inhibits the release of inflammatory factors and alleviates the injury of ischemic cerebral infarction tissue through the Toll-like receptor-5/nuclear factor-KB pathway remains unclear. OBJECTIVE: To investigate the role and preliminary mechanism of miR-150-5p in ischemic cerebral infarction in rats. METHODS: (1) The rat models of middle cerebral artery occlusion were constructed and the rat models were divided into five groups: Control, miR-150-5p agomir, agomir control, miR-150-5p antagomir and antagomir control groups. (2) The rats in the control group was given the intracerebroventricular injection of normal saline, and the rats in the latter four groups were given the intracerebroventricular injection of miR-150-5p agomir (miR-150-5p agonist), agomir negative control, miR150-5p antagomir (miR150-5p inhibitor) and antagomir negative control, respectively. (3) After 7 days, the brain was graded by modified neurological severity score, the cerebral infarct volume was measured by MRI, and the histopathological changes were observed by hematoxylin-eosin staining. The expression levels of miR-150-5p, interleukin-6, tumor necrosis factor-a, Toll-like receptor-5 and nuclear factor-KB p65 in brain tissues were detected by qRT-PCR, ELISA and western blot assay, respectively. The target relationship between miR150-5p and Toll-like receptor-5 was verified by luciferase assay by retrieving the bioinformatics website Targetscan to predict the binding sites of miR-150-5p and Toll-like receptor-5. RESULTS AND CONCLUSION: (1) Compared with the control group, the modified neurological severity score, and levels of interleukin-6, tumor necrosis factor-a, Toll-like receptor-5 and nuclear factor-KB p65 proteins were significantly decreased in the miR-150-5p agomir group (P 0.05). (3) TargerScan website prediction results and luciferase reporter gene analysis results showed that miR-150-5p and Toll-like receptor-5 had a targeted binding site. (4) These results imply that miR-150-5p can inhibit the inflammatory signaling pathway of Toll-like receptor-5/nuclear factor-KB p65 in brain injury caused by ischemia and reduce the inflammatory response, thereby alleviating the damage of nerve function and playing a protective role.

A Fusion Protein of Derp2 Allergen and Flagellin Suppresses Experimental Allergic Asthma

PURPOSE: The house dust mite (HDM) is one of the most important sources of indoor allergens and a significant cause of allergic rhinitis and allergic asthma. Our previous studies demonstrated that Vibrio vulnificus flagellin B (FlaB) plus allergen as a co-treatment mixture improved lung function and inhibited eosinophilic airway inflammation through the Toll-like receptor 5 signaling pathway in an ovalbumin (OVA)- or HDM-induced mouse asthma model. In the present study, we fused the major mite allergen Derp2 to FlaB and compared the therapeutic effects of the Derp2-FlaB fusion protein with those of a mixture of Derp2 and FlaB in a Derp2-induced mouse asthma model. METHODS: BALB/c mice sensitized with Derp2 + HDM were treated with Derp2, a Derp2 plus FlaB (Derp2 + FlaB) mixture, or the Derp2-FlaB fusion protein 3 times at 1-week intervals. Seven days after the final treatment, the mice were challenged intranasally with Derp2, and airway responses and Derp2-specific immune responses were evaluated. RESULTS: The Derp2-FlaB fusion protein was significantly more efficacious in reducing airway hyperresponsiveness, lung eosinophil infiltration, and Derp2-specific IgE than the Derp2 + FlaB mixture. CONCLUSIONS: The Derp2-FlaB fusion protein showed a strong anti-asthma immunomodulatory capacity, leading to the prevention of airway inflammatory responses in a murine disease model through the inhibition of Th2 responses. These findings suggest that the Derp2-FlaB fusion protein would be a promising vaccine candidate for HDM-mediated allergic asthma therapy.

Influence of Toll-like receptor 5 on biological characteristics of mesenchymal stem cells / 军事医学

Objective To investigate the effect of Toll-like receptor 5(TLR5) on the biological characteristics of human umbilical cord-derived mesenchymal stem cells (MSCs).Methods The lentiviral vectors expressing TLR5 were constructed and MSCs were infected with related lentiviruses .The overexpression of TLR5 in MSCs was verified by FACS analysis, RT-qPCR and Western blotting before its influence on cell proliferation , surface antigens and cell differentiation was evaluated.The biological function of TLR5 was verified by detecting cytokines IL-6 and IL-8 expression after TLR5 was activated by its agonist CBLB502.Results The results showed that the expression of mRNA and protein of TLR5 was upregulated in MSCs after infection of lentivirus carrying TLR5, but their proliferation , immunophenotypes and differentiation capacity were not influenced . Moreover, IL-6 and IL-8 expression was significantly increased when MSCs-TLR5 were treated with CBLB502 ( P <0.001 ).Conclusion The biological characteristics of MSCs were not affected after infection of lentivirus/TLR5, and the expressed TLR5 was functional in MSCs , which could activate the downstream signaling pathways and then regulate immune response.

Influence of Toll-like receptor 5 on biological characteristics of mesenchymal stem cells / 军事医学

Objective To investigate the effect of Toll-like receptor 5(TLR5) on the biological characteristics of human umbilical cord-derived mesenchymal stem cells (MSCs).Methods The lentiviral vectors expressing TLR5 were constructed and MSCs were infected with related lentiviruses .The overexpression of TLR5 in MSCs was verified by FACS analysis, RT-qPCR and Western blotting before its influence on cell proliferation , surface antigens and cell differentiation was evaluated.The biological function of TLR5 was verified by detecting cytokines IL-6 and IL-8 expression after TLR5 was activated by its agonist CBLB502.Results The results showed that the expression of mRNA and protein of TLR5 was upregulated in MSCs after infection of lentivirus carrying TLR5, but their proliferation , immunophenotypes and differentiation capacity were not influenced . Moreover, IL-6 and IL-8 expression was significantly increased when MSCs-TLR5 were treated with CBLB502 ( P <0.001 ).Conclusion The biological characteristics of MSCs were not affected after infection of lentivirus/TLR5, and the expressed TLR5 was functional in MSCs , which could activate the downstream signaling pathways and then regulate immune response.

TLR5 Activation through NF-κB Is a Neuroprotective Mechanism of Postconditioning after Cerebral Ischemia in Mice

Postconditioning has been shown to protect the mouse brain from ischemic injury. However, the neuroprotective mechanisms of postconditioning remain elusive. We have found that toll-like receptor 5 (TLR5) plays an integral role in postconditioning-induced neuroprotection through Akt/nuclear factor kappa B (NF-κB) activation in cerebral ischemia. Compared to animals that received 30 min of transient middle cerebral artery occlusion (tMCAO) group, animals that also underwent postconditioning showed a significant reduction of up to 60.51% in infarct volume. Postconditioning increased phospho-Akt (p-Akt) levels and NF-κB translocation to the nucleus as early as 1 h after tMCAO and oxygen-glucose deprivation. Furthermore, inhibition of Akt by Akt inhibitor IV decreased NF-κB promoter activity after postconditioning. Immunoprecipitation showed that interactions between TLR5, MyD88, and p-Akt were increased from postconditioning both in vivo and in vitro. Similar to postconditioning, flagellin, an agonist of TLR5, increased NF-κB nuclear translocation and Akt phosphorylation. Our results suggest that postconditioning has neuroprotective effects by activating NF-κB and Akt survival pathways via TLR5 after cerebral ischemia. Additionally, the TLR5 agonist flagellin can simulate the neuroprotective mechanism of postconditioning in cerebral ischemia.

Relationship between polymorphism site rs5744168 of TLR5 and AAV / 实用医学杂志

Objective To investigate the relationship between putative rs5744168 of Toll-like receptors 5 (TLR5)and ANCA associated small vasculitis (AAV) in Guangxi Han nationality. Methods Polymorphism was analyzed by polymerase chain restricted fragments length polymorphism in 120 cases with AAV and 212 controls. Results (1)There were two genotypes of CC and CT in AAV group and control group. The frequencies distribution of CC and CT in 120 AAV patients were 82.50% and 17.50% respectively and the frequencies of allele C and T 91.25% and 8.75%,respectively. In controls,the genotypefrequencies of CC and CT were 88.68% and 11.3%, and frequencies of allele C and T 94.34% and 5.66%, respectively. No significant difference was found in either genotype distribution or allele frequencies between the patients and the controls ( P > 0 . 05 ) . ( 2 ) Significant reductions in the incidence of BUN, uric acid, quantitative test of 24 h urinary protein and erythrocyte sedimentation rate(ESR) were found in CC genotype (P < 0.05). (3) Binary regression model with a logit link function found total cholesterol was related with AAV. Conclusion The susceptibility of AAV in Guangxi Han population has nothing to do with the polymorphism of rs5744168.In AAV patients, polymorphism of rs5744168 may be associated with ESR, BUN, uric acid and quantitative test of 24 h urinary protein levels.

Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

PURPOSE: Invariant natural killer T (iNKT) cells play a critical role in the pathogenesis of asthma. We previously reported the association between circulating Th2-like iNKT cells and lung function in asthma patients and the suppressive effect of Toll-like receptor 5 ligand flagellin B (FlaB) on asthmatic in a mouse model. Thus, we investigated whether FlaB modulates the function of circulating iNKT cells in asthmatic patients. METHODS: Peripheral blood mononuclear cells (PBMCs) were treated with FlaB, and the secreted and intracellular cytokines of iNKT cells were evaluated by using ELISA and flow cytometry, respectively, following stimulation with alpha-galactosylceramide. Foxp3+ iNKT cells were also measured. To determine the effect of FlaB-treated dendritic cells (DCs) on iNKT cells, we co-cultured CD14+ monocyte-derived DCs and T cells from patients with house dust mite-sensitive asthma and analyzed intracellular cytokines in iNKT cells. RESULTS: A reduction of IL-4 and IL-17 production by iNKT cells in PBMCs after FlaB treatment was alleviated following blocking of IL-10 signaling. A decrease in the frequencies of IL-4+ and IL-17+ iNKT cells by FlaB-treated DCs was reversed after blocking of IL-10 signaling. Simultaneously, an increase in Foxp3+ iNKT cells induced by FlaB treatment disappeared after blocking of IL-10. CONCLUSIONS: FlaB may inhibit Th2- and Th17-like iNKT cells and induce Foxp3+ iNKT cells by DCs via an IL-10-dependent mechanism in asthmatic patients. In patients with a specific asthma phenotype associated with iNKT cells, FlaB may be an effective immunomodulator for iNKT cell-targeted immunotherapy.

Cloning, expression and functional analysis of the duck Toll-like receptor 5 (TLR5) gene

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-kappaB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation.

Decreased expression of Toll-like receptor 4 and 5 during progression of prostate transformation in transgenic adenocarcinoma of mouse prostate mice

Chronic inflammation has been considered an important risk factor for development of prostate cancer. Toll-like receptors (TLRs) recognize microbial moieties or endogenous molecules and play an important role in the triggering and promotion of inflammation. In this study, we examined whether expression of TLR4 and TLR5 was associated with progression of prostate transformation in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The expression of TLR4 and TLR5 was evaluated by immunohistochemisty in formalin-fixed paraffin-embedded prostate tissue from wild-type (WT) and TRAMP mice. Normal prostate tissue from WT mice showed strong expression of TLR4 and TLR5. However, TLR4 expression in the prostate tissue from TRAMP mice gradually decreased as pathologic grade became more aggressive. TLR5 expression in the prostate tissue from TRAMP mice also decreased in low-grade prostate intraepithelial neoplasia (PIN), high-grade PIN and poorly differentiated adenocarcinoma. Overall, our results suggest that decreased expression of TLR4 and TLR5 may contribute to prostate tumorigenesis.

Effects of all-trans retinoic acid on expression of Toll-like receptor 5 on immune cells

INTRODUCTION: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atRA, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atRA regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atRA in immune cells. MATERIALS AND METHODS: This study examined whether atRA induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atRA-treated THP-1 cells. RESULTS: atRA at a concentration of 10 nM greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atRA stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. CONCLUSION: atRA induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atRA-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atRA and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers.

Expression changes in flagellin and monocyte TLR5 mRNA in the peripheral blood of patients with sepsis / 解放军医学杂志

20/min,PaCO2 12?109 or 0.10.26 patients with sepsis infected by Gram negative bacilli were categorized as G-group,15 infected by Gram positive coccus as G+ group and 15 healthy persons as control group.All the cases were proved to have infection by body fluid examination under microscope or bacterial culture.The level of flagellin was determined by ELISA.The peripheral blood mononuclear cells(PBMC)were isolated by the density gradient centrifugation,and total RNA of PBMCs was extracted by RNA BIOzol extract.The level of TLR5 mRNA in the peripheral blood was detected by semi-quantitative RT-PCR.The statistic significance among groups was examined by Students' t test.Results Plasma flagellin levels were below the detection limit of the ELISA kit in both G+ and control groups,while the mean level of plasma flagellin amounted to 6.636?5.147ng/ml in G-group.TLR5 mRNA expression in G-group was significantly higher than that in G+ and control groups,and no significant difference was found between G+ group and control group.Conclusion Flagellin may be involved in the development of ALI through interacts with TLR5.

The expression of Toll-like receptor 5 in the lung tissues of rats with acute lung injury induced by flagellin / 解放军医学杂志

Objective To determine the expression of Toll-like receptor 5(TLR5) mRNA in the lung tissues of rats with acute lung injury induced by flagellin, and to investigate its potential role in the pathogenesis of the disease. Methods Flagellin was isolated and purified from Escherichia coli ATCC25922, subsequently identified by monoclonal antibody to flagellin. One hundred and eight SD rats were randomly divided into control group (n=36), flagellin challenged 1 group (n=36) and flagellin challenged 2 group (n=36). Rat model of ALI was reproduced by injecting flagellin. The expression of TLR5 mRNA in the lung tissues of rats was determined with in situ hybridization technique at six time points. Blood gas was monitored and pathological changes in the lung was observed at the same time. Result Flagellin was isolated and purified successfully and its molecular weight was approximately 65kD. Flagellin-induced acute lung injury model was successfully reproduced in rats. From 1h after flagellin injection, TLR5 mRNA expression was found to be increased in the lung tissues of rats with flagellin induced acute lung injury, and the expression was on the increase with the elapse of time and increase of the dose of flagellin. The PaO_2 levels in flagellin challenged 1 and 2 groups decreased. Interstitial edema, alveolar edema and inflammatory cells infiltration were also observed in flagellin challenged groups. Conclusion Flagellin can induce ALI in rats; the development of flagellin induced ALI in rats is related with TLR5 expression in the lung tissues of rats.