ABSTRACT
Toll-like receptor 9 (TLR9) is an important component of the innate immune system and have been associated with several autoimmune diseases, such as Systemic Lupus Erythematosus (SLE). The aim of this study was to investigate polymorphisms in TLR9 gene in a Brazilian SLE patients group and their association with clinical manifestation, particularly Jaccouds arthropathy (JA). We analyzed DNA samples from 204 SLE patients, having a subgroup of them presenting JA (n=24). A control group (n=133) from the same city was also included. TLR9 single nucleotide polymorphisms (SNPs) (−1237 C>T and +2848 G>A) were identified by sequencing analysis. The TLR9 gene genotype frequency was similar both in SLE patients and the control group. In the whole SLE population, an association between the homozygosis of allele C at position −1237 with psychosis and anemia (p < 0.01) was found. Likewise, the homozygosis of allele G at position +2848 was associated with a discoid rash (p < 0.05). There was no association between JA and TLR9 polymorphisms. These data show that TLR9 polymorphisms do not seem to be a predisposing factor for SLE in the Brazilian population, and that SNPs are not associated with JA.
O receptor Toll-like 9 (TLR9) é um componente importante do sistema imunológico inato e tem sido associado a várias doenças autoimunes, como o Lúpus Eritematoso Sistêmico (LES). O objetivo deste estudo foi investigar polimorfismos no gene TLR9 em um grupo de pacientes brasileiros com LES e sua associação com a manifestação clínica, particularmente a artropatia de Jaccoud (JA). Foram analisadas amostras de DNA de 204 pacientes com LES, e um subgrupo com JA (n=24). Um grupo de controle (n=133) da mesma cidade também foi incluído. Os polimorfismos de nucleotídeos únicos TLR9 (SNPs) (−1237 C>T e +2848 G>A) foram identificados pela análise de sequenciamento. A frequência do genótipo genético TLR9 foi semelhante tanto em pacientes com LES quanto no grupo controle. Em toda a população de LES, foi encontrada associação entre a homozigose do alelo C na posição −1237 com psicose e anemia (p < 0,01). Da mesma forma, a homozigose do alelo G na posição +2848 foi associada a uma erupção cutânea discoide (p < 0,05). Não houve associação entre polimorfismos JA e TLR9. Esses dados mostram que os polimorfismos TLR9 não parecem ser um fator predisponível para o LES na população brasileira, e que os SNPs não estão associados ao JA.
Subject(s)
Humans , Joint Diseases/genetics , Lupus Erythematosus, Systemic/genetics , Toll-Like Receptor 9/analysisABSTRACT
Abstract Toll-like receptor 9 (TLR9) is an important component of the innate immune system and have been associated with several autoimmune diseases, such as Systemic Lupus Erythematosus (SLE). The aim of this study was to investigate polymorphisms in TLR9 gene in a Brazilian SLE patients group and their association with clinical manifestation, particularly Jaccouds arthropathy (JA). We analyzed DNA samples from 204 SLE patients, having a subgroup of them presenting JA (n=24). A control group (n=133) from the same city was also included. TLR9 single nucleotide polymorphisms (SNPs) (1237 C>T and +2848 G>A) were identified by sequencing analysis. The TLR9 gene genotype frequency was similar both in SLE patients and the control group. In the whole SLE population, an association between the homozygosis of allele C at position 1237 with psychosis and anemia (p 0.01) was found. Likewise, the homozygosis of allele G at position +2848 was associated with a discoid rash (p 0.05). There was no association between JA and TLR9 polymorphisms. These data show that TLR9 polymorphisms do not seem to be a predisposing factor for SLE in the Brazilian population, and that SNPs are not associated with JA.
Resumo O receptor Toll-like 9 (TLR9) é um componente importante do sistema imunológico inato e tem sido associado a várias doenças autoimunes, como o Lúpus Eritematoso Sistêmico (LES). O objetivo deste estudo foi investigar polimorfismos no gene TLR9 em um grupo de pacientes brasileiros com LES e sua associação com a manifestação clínica, particularmente a artropatia de Jaccoud (JA). Foram analisadas amostras de DNA de 204 pacientes com LES, e um subgrupo com JA (n=24). Um grupo de controle (n=133) da mesma cidade também foi incluído. Os polimorfismos de nucleotídeos únicos TLR9 (SNPs) (1237 C>T e +2848 G>A) foram identificados pela análise de sequenciamento. A frequência do genótipo genético TLR9 foi semelhante tanto em pacientes com LES quanto no grupo controle. Em toda a população de LES, foi encontrada associação entre a homozigose do alelo C na posição 1237 com psicose e anemia (p 0,01). Da mesma forma, a homozigose do alelo G na posição +2848 foi associada a uma erupção cutânea discoide (p 0,05). Não houve associação entre polimorfismos JA e TLR9. Esses dados mostram que os polimorfismos TLR9 não parecem ser um fator predisponível para o LES na população brasileira, e que os SNPs não estão associados ao JA.
ABSTRACT
Abstract Toll-like receptor 9 (TLR9) is an important component of the innate immune system and have been associated with several autoimmune diseases, such as Systemic Lupus Erythematosus (SLE). The aim of this study was to investigate polymorphisms in TLR9 gene in a Brazilian SLE patients group and their association with clinical manifestation, particularly Jaccoud's arthropathy (JA). We analyzed DNA samples from 204 SLE patients, having a subgroup of them presenting JA (n=24). A control group (n=133) from the same city was also included. TLR9 single nucleotide polymorphisms (SNPs) (−1237 C>T and +2848 G>A) were identified by sequencing analysis. The TLR9 gene genotype frequency was similar both in SLE patients and the control group. In the whole SLE population, an association between the homozygosis of allele C at position −1237 with psychosis and anemia (p < 0.01) was found. Likewise, the homozygosis of allele G at position +2848 was associated with a discoid rash (p < 0.05). There was no association between JA and TLR9 polymorphisms. These data show that TLR9 polymorphisms do not seem to be a predisposing factor for SLE in the Brazilian population, and that SNPs are not associated with JA.
Resumo O receptor Toll-like 9 (TLR9) é um componente importante do sistema imunológico inato e tem sido associado a várias doenças autoimunes, como o Lúpus Eritematoso Sistêmico (LES). O objetivo deste estudo foi investigar polimorfismos no gene TLR9 em um grupo de pacientes brasileiros com LES e sua associação com a manifestação clínica, particularmente a artropatia de Jaccoud (JA). Foram analisadas amostras de DNA de 204 pacientes com LES, e um subgrupo com JA (n=24). Um grupo de controle (n=133) da mesma cidade também foi incluído. Os polimorfismos de nucleotídeos únicos TLR9 (SNPs) (−1237 C>T e +2848 G>A) foram identificados pela análise de sequenciamento. A frequência do genótipo genético TLR9 foi semelhante tanto em pacientes com LES quanto no grupo controle. Em toda a população de LES, foi encontrada associação entre a homozigose do alelo C na posição −1237 com psicose e anemia (p < 0,01). Da mesma forma, a homozigose do alelo G na posição +2848 foi associada a uma erupção cutânea discoide (p < 0,05). Não houve associação entre polimorfismos JA e TLR9. Esses dados mostram que os polimorfismos TLR9 não parecem ser um fator predisponível para o LES na população brasileira, e que os SNPs não estão associados ao JA.
Subject(s)
Humans , Toll-Like Receptor 9/genetics , Lupus Erythematosus, Systemic/genetics , Brazil , Pilot Projects , Genetic Predisposition to Disease/genetics , Gene Frequency/geneticsABSTRACT
Objective:To explore the effect of Toll-like receptor 9 (TLR9) signaling pathway activation on the transcriptome in the renal tubular cells.Methods:Mouse primary renal tubular epithelial cells were extracted and cultured. When the degree of cell fusion reached 80%, they were divided into two groups, which were added with 10 μL phosphate buffered saline (PBS, PBS control group) and TLR9 activator cytosine phosphate guanidine oligodeoxynucleotide (CpG-ODN) with a final concentration of 5 μmol/L (CpG-ODN treatment group). The RNA sequencing was performed on the Illumina platform after extraction. DEGseq software was used to analyze the differential expression of genes between the two groups. Goatools and KOBAS online software were used to analyze the differential genes involved signal pathways. Homer software was used to predict transcription factors.Results:Compared with the PBS control group, there were a total of 584 differentially expressed genes in the CpG-ODN treatment group, of which 102 were up-regulated and 482 were down-regulated. The most significantly enriched gene ontology (GO) terms of differentially expressed genes included response to interferon-β, defense response to virus and other inflammatory pathway. The most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways included 2'-5'-oligoadenylate synthase activity, regulation of ribonuclease activity, negative regulation of virus life cycle, cellular response to interferon-βand defense response to protozoan. The results of transcription factor prediction showed that interferon regulatory factor 3 (IRF3) was the most significantly enriched transcription factor in the promoter sequence of differential genes; the most significant transcription factor downstream of TLR9 was IRF3, and other predicted transcription factors such as transcription factor 21 (TCF21), zinc finger protein 135 (ZNF135), and PR domain containing 4 (PRDM4) might be new candidates for TLR9 signaling pathway.Conclusion:CpG-ODN activates TLR9 signaling pathway, and primary renal tubular epithelial cells can directly respond to CpG-ODN stimulation and undergo transcriptome changes, which provides a basis for further research on the molecular mechanism of TLR9 pathway in sepsis induced acute kidney injury.
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ObjectiveTo investigate the intervention effect of total glucosides of paeony （TGP） on the renal injury of MRL/lpr mice based on the Toll-like receptor 9 （TLR9）/myeloid differentiation factor 88 （MyD88）/nuclear transcription factor-κB （NF-κB） signaling pathway and explore the immunological mechanism of TGP in preventing and treating systemic lupus erythematosus （SLE）. MethodMRL/lpr female mice of SPF grade were randomly divided into a model group， a dexamethasone group （0.15 g·kg-1）， and high- （0.078 g·kg-1） and low-dose （0.039 g·kg-1） TGP groups， and female C57BL/6J mice were assigned to a blank group， with 7 mice in each group. Mice in each group were treated with corresponding drugs or normal saline by gavage at the same time every day. After 4 weeks， samples were collected. The kidney and spleen were weighed， and the organ index was calculated. Serum creatinine （SCr） and blood urea nitrogen （BUN） levels in each group were detected by biochemical assay. Hematoxylin-eosin （HE） staining was used to observe the histopathological changes in the kidney. The degree of renal fibrosis was evaluated by Masson staining. The serum levels of interleukin （IL）-2， interferon （IFN）-α， IL-4， and anti-nuclear antibody （ANA） were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA expression of TLR9， MyD88， and NF-κB p65 in renal tissues was detected by real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression of TLR9 and NF-κB p65 in renal tissues was detected by immunofluorescence. The protein expression of TLR9， MyD88， and NF-κB p65 in renal and spleen tissues was tested by Western blot. ResultCompared with the blank group， the model group showed increased SCr， BUN， spleen index， and kidney index （P<0.05）， deteriorated pathological injury and fibrosis in renal tissues， elevated serum levels of IFN-α， IL-4， and ANA， decreased level of IL-2 （P<0.05）， and up-regulated TLR9， MyD88， and NF-κB p65 mRNA and protein levels in the kidney and spleen （P<0.05）. Compared with the model group， the TGP groups displayed reduced SCr， BUN， spleen index， and kidney index （P<0.05）， relieved pathological damage and fibrosis in renal tissues， decreased serum levels of IFN-α， IL-4， and ANA （P<0.05）， increased level of IL-2， and declining mRNA and protein expression levels of TLR9， MyD88， and NF-κB p65 in the kidney and spleen （P<0.05）. ConclusionTGP may inhibit the expression of downstream inflammatory factors to regulate immunity and resist SLE-induced renal injury by regulating the TLR9/MyD88/NF-κB signaling pathway.
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Objective@#To investigate the effect of Toll-like receptor 9 (TLR9) on high glucose-induced retinal ganglion cells-5 (RGC-5) apoptosis and the inhibitory effects of small interfering RNA-TLR9 (si-TLR9) on apoptosis.@*Methods@#RGC-5 cells were divided into normal control group, high glucose group, high glucose+ negative control group and high glucose+ si-TLR9 group which cells were respectively dealt with normal culture medium, high glucose medium, transfection of non-specific siRNA under high glucose and transfection of siRNA-TLR9 under high glucose.The expression of TLR9 mRNA was detected by real time PCR; the survival rate of the cells was evaluated by MTT assay; the apoptotic rate of the cells was detected by flow cytometry; the caspase-3 activity in the cells was detected by related kit, and the expressions of TLR9, B cell lymphoma (bcl-2), bcl-2 associated X protein (bax), p38 mitogen-activated protein kinases (p38MAPK) and phosphorylated (p)-p38MAPK proteins were detected by Western blot.@*Results@#The expressions of TLR9 mRNA and protein in the high glucose+ si-TLR9 group were significantly decreased in comparison with the high glucose+ negative control group (both at P<0.05). The cell survival rate of the high glucose group and high glucose+ negative control group was (78.36±5.13)% and (75.12±4.25)%, respectively, which was significantly lower than (95.48±7.25)% in the normal control group, and that in the high glucose+ si-TLR9 group was (86.58±5.32)%, which was significantly reduced in comparison with the high glucose+ negative control group (all at P<0.05). The apoptotic rate of the cells in the high glucose group and high glucose+ negative control group was (13.23±1.22)% and (12.52±1.38)%, respectively, which was significantly higher than (2.26±0.15)% of the normal control group, and apoptotic rate in the high glucose+ si-TLR9 group was (7.15±0.24)%, which was significantly lower than that in high glucose+ negative control group (all at P<0.05). The expression of bax in the cells of the high glucose+ si-TLR9 group was significantly decreased, and the expression of bcl-2 in the cells of the high glucose+ si-TLR9 group was significantly increased in comparison with the high glucose+ negative control group (both at P<0.05). Caspase-3 activity in the cells was significantly decreased in the high glucose+ si-TLR9 group compared with the high glucose+ negative control group (P<0.05). The relative expression of p-p38MAPK in the cells was significantly decreased in the high glucose+ si-TLR9 group compared with the high glucose+ negative control group (P<0.05).@*Conclusions@#Down-regulation of TLR9 expression in the RGC-5 cells can inhibit the apoptosis induced by high glucose probablely by suppressing p38MAPK signaling pathway.
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Objective To investigate the effect of Toll-like receptor 9 (TLR9) on high glucose-induced retinal ganglion cells-5 (RGC-5) apoptosis and the inhibitory effects of small interfering RNA-TLR9 (si-TLR9) on apoptosis.Methods RGC-5 cells were divided into normal control group,high glucose group,high glucose+ negative control group and high glucose + si-TLR9 group which cells were respectively dealt with normal culture medium,high glucose medium,transfection of non-specific siRNA under high glucose and transfection of siRNA-TLR9 under high glucose.The expression of TLR9 mRNA was detected by real time PCR;the survival rate of the cells was evaluated by MTT assay;the apoptotic rate of the cells was detected by flow cytometry;the caspase-3 activity in the cells was detected by related kit,and the expressions of TLR9,B cell lymphoma (bcl-2),bcl-2 associated X protein (bax),p38 mitogen-activated protein kinases (p38MAPK) and phosphorylated (p)-p38MAPK proteins were detected by Western blot.Results The expressions of TLR9 mRNA and protein in the high glucose+si-TLR9 group were significantly decreased in comparison with the high glucose+negative control group (both at P＜0.05).The cell survival rate of the high glucose group and high glucose+negative control group was (78.36±5.13)％ and (75.12± 4.25) ％,respectively,which was significantly lower than (95.48± 7.25) ％ in the normal control group,and that in the high glucose+si-TLR9 group was (86.58±5.32)％,which was significantly reduced in comparison with the high glucose+negative control group (all at P＜0.05).The apoptotic rate of the cells in the high glucose group and high glucose+negative control group was (13.23 ± 1.22) ％ and (12.52± 1.38) ％,respectively,which was significantly higher than (2.26±0.15)％ of the normal control group,and apoptotic rate in the high glucose+si-TLR9 group was (7.15±0.24) ％,which was significantly lower than that in high glucose+negative control group (all at P＜0.05).The expression of bax in the cells of the high glucose+si-TLR9 group was significantly decreased,and the expression of bcl-2 in the cells of the high glucose+si-TLR9 group was significantly increased in comparison with the high glucose+ negative control group (both at P＜ 0.05).Caspase-3 activity in the cells was significantly decreased in the high glucose+si-TLR9 group compared with the high glucose+negative control group (P＜0.05).The relative expression of p-p38MAPK in the cells was significantly decreased in the high glucose+ si-TLR9 group compared with the high glucose+negative control group (P＜0.05).Conclusions Down-regulation of TLR9 expression in the RGC-5 cells can inhibit the apoptosis induced by high glucose probablely by suppressing p38MAPK signaling pathway.
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Objective: To study the protective effect of Taoren Chengqitang on intestinal mucosal barrier in septic rats and its possible mechanism. Method: Rats were divided into sham operation group, model group (replication of septic rats with cecal ligation and perforation), low, middle and high-dose Taoren Chengqitang groups (2.85,5.70,8.55 g·kg-1), and dexamethasone group (0.01 g·kg-1),with 12 rats in each group. After last administration, rats were put to death, the morphological changes of intestinal mucosa were observed under electron microscope, the bacterial translocation rates in lymphoglandulae mesentericae, liver, kidney and spleen tissues were detected; the levels of serum tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and diamine oxidase (DAO) and intestinal fatty acid binding protein (i-FABP) levels in small intestine tissues were detected by enzyme-linked immunosorbent assay (ELISA).Expressions of Toll like receptor 9 (TLR9), myeloid differentiation factor 88 (MyD88) and nuclear factor-kappa B subunit p65 in small intestine tissues were detected by Western blot and immunohistochemistry. Result: Compared with sham operated group, the bacterial translocation rate, TNF-α and IL-1β levels in model group increased significantly, DAO, i-FABP, mucosal thickness and villus height decreased significantly, and protein expressions of TLR9, MyD88 and nucleus NF-κB p65 increased significantly (PPκB p65 protein decreased significantly. Compared with model group, the bacterial translocation rate, TNF-α and IL-1β levels in organs of low, medium and high-dose Taoren Chengqitang groups and dexamethasone group decreased significantly, DAO, i-FABP, mucosal thickness and villus height increased significantly, while the protein expressions of TLR9, MyD88 and nucleus NF-κB p65 decreased significantly (PκB p65 protein increased significantly. Conclusion: Taoren Chengqitang has a certain protective effect on intestinal mucosal barrier in septic rats, which may be related to the inhibition of TLR9 signaling pathway.
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Objective: The effect of Xiexintang on Toll like receptor 9 (TLR9) signaling pathway in macrophage derived foam cells was studied by in vitro cell experiments. Method: The fifty SPF male SD rats were randomly divided into low, medium and high dose groups (1.4,4.2,12.6 g·kg-1·d -1) and normal groups. Except 20 rats in the normal group, 10 rats in each group were given equal volume of pure water gavage in the blank group. After the last Administration for 7 days, serum was separated,and the serum containing drugs in the low, medium and high dose groups of Xiexintang was prepared. Oxidized low density lipoprotein (ox-LDL) was used to intervene the differentiation of RAW264.7 macrophages into foam cells. The cell foam was identified by oil red O staining. After observing the effect of drug containing serum on the proliferation of macrophage derived foam cells by methye thiazolye telrazlium(MTT) method,the serum containing 20%concentration of each drug was selected to act on the foam cell model. The expression of interleukin(IL) -1β and interferon (INF) -γ was determined by enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative PCR (Real-time PCR). TLR9, myeloid differentiation factor 88(MyD88)and nuclear factor(NF) -κB were detected by Western blot. Result: Oil red O staining showed that the red particles were obvious after ox-LDL intervention. The foam cell model was successfully prepared. MTT results showed that there was no significant difference in cell proliferation between the high dose group of Xiexintang in the 10%~30%concentration range and the normal group serum. Follow up selection of the serum containing 20%concentration of each dose intervened the foam cells induced by ox-LDL. Compared with the normal group,the model group after ox-LDL intervention induced the high expression of TLR9,MyD88,NF-κB p65,IL-1β,INF-γ (PPκB p65,IL-1β,INF-γ(PPConclusion: Xiexintang containing serum can inhibit ox-LDL-induced RAW264.7 macrophage foaming,and its mechanism may involve regulation of TLR9/MyD88/NF-κB p65 signaling pathway and inhibition of IL-1β and INF-γ overexpression. This may be one of its mechanisms of against atherosclerosis.
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Objective To explore the inhibitory effect of immune complex (IC) on the signal pathways of high-expressed CD40 and CD80 induced by Toll-like receptor (TLR9) agonist CpG oligodeoxynucleotide (ODN) in B lymphocytes. Methods The mice were intraperitoneally injected with CpG ODN or IC plus CpG ODN, and the spleen CD19+ B lymphocytes were sorted by magnetic-activated cell sorting (MACS). The expressions of CD40 and CD80 on the B lymphocytes were detected by flow cytometry. The spleen B lymphocytes were isolated from wild type and immunoglobulin G Fcγ receptor Ⅱb (FcγRⅡb) knockout mice by MACS. After the isolated cells were stimulated with CpG ODN or IC plus CpG ODN in vitro, the phosphorylation levels of related protein kinases were detected in the B lymphocytes by Western blotting. Following CpG ODN stimulation, the B lymphocytes were treated with JNK p38 inhibitor SP600125 (50 μmol/L) or p38 inhibitor SB203580 (20 mg/L), and then the CD40 and CD80 expression levels on the CpG ODN-activated B lymphocytes were detected by flow cytometry. Results IC inhibited CD40 and CD80 expressions on the CpG ODN-activated B lymphocytes in vivo (both P0.05). IC inhibited the phosphorylation levels of JNK and p38 induced by CpG ODN in B lymphocytes, but did not inhibit them in the B lymphocytes from FcγRⅡb－/－ mice. The CD40 and CD80 expressions on the CpG ODN-activated B lymphocytes were significantly decreased after treated with SP600125 and SB203580 (both P0.01). Conclusion IC can inhibit the CD40 and CD80 expressions induced by TLR9 agonist CpG ODN through inhibiting the JNK and p38 pathways in B lymphocytes.
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Objective The purpose of this study was to investigate the correlation between single nucleotide polymorphisms (SNP) of Toll-like receptor 9 (TLR9) and tuberculosis in the Chinese Tibetan population . Methods A total of 613 active tubercu-losis patients and 603 healthy controls were enrolled in this case -control study.Two SNPs in TLR9 (rs187084 and rs5743836) were genotyped using the multiplex ligation detection reaction technique .The association of the SNPs with the susceptibility of TB was inves -tigated using logistic regression analysis . Results There was no statistically significant difference in the allele distribution of rs 187084 G/A between the TB patients and the healthy controls (P=0.668) or correlation between the genotype of rs 187084 and the genetic suscepti-bility of TB ( P >0.05).Dominant, recessive and additive models showed no association of this SNP with TB , either (P >0.05). Conclusion The rs187084 polymorphism of TLR9 is not correlated with the genetic susceptibility of tuberculosis in the Chinese Tibetan population.
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Objective To investigate the role and mechanism of mitochondrial DNA (mtDNA) in rats with ventilator-induced lung injury (VILI) via Toll-like receptor 9 (TLR9)-myeloid differentiation factor 88 (MyD88) signaling pathway. Methods Thirty adult male Sprague-Dawley (SD) rats were randomly divided into three groups (each n = 10): spontaneous breathing group, normal tidal volume (VT) group (NVT group, VT = 8 mL/kg), and high VT group (HVT group, VT = 40 mL/kg). Rats in the NVT group and HVT group were ventilated mechanically with a positive end-expiratory pressure (PEEP) of 0 and the fraction of inspired oxygen (FiO2) at 0.50. After 4 hours of ventilation, the blood from the rats' hearts was collected and the rats were sacrificed, the levels of interleukins (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α) in serum were determined with enzyme-linked immune sorbent assay (ELISA). The bronchoalveolar lavage fluid (BALF) was collected for a determination of total protein by using the bicinchoninic acid (BCA) assay. The lung tissues were harvested to determine the wet/dry (W/D) ratio. The changes in pathobiology of lung tissue were observed with hematoxylin and eosin (HE) staining. The protein expression levels of mtDNA-encoded cytochrome C oxidase subunit Ⅳ (COX-Ⅳ), TLR9, MyD88 and nuclear factor-κB p65 (NF-κB p65) in lung tissues were determined by immunohistochemistry. Results The histopathology of lung tissues indicated that lungs from animals ventilated with HVT developed marked lung inflammation changes, whereas no major histological change was observed in animals ventilated with NVT or spontaneously breathing. The pathological score in HVT group was significantly higher than that of spontaneous breathing group and NVT group (3.50±0.41 vs. 0.25±0.09, 0.33±0.10, both P < 0.05). Compared with spontaneous breathing group and NVT group, the ratio of W/D in the HVT group was significantly increased (6.42±0.41 vs. 4.14±0.04, 4.28±0.11, both P < 0.05), the contents of total proteins in BALF were significantly increased (g/L: 0.43±0.04 vs. 0.13±0.01, 0.14±0.01, both P < 0.05), and serum IL-6, IL-1β and TNF-α levels were also increased [IL-6 (μg/L): 1.15±0.17 vs. 0.42±0.10, 0.46±0.04; IL-1β (μg/L): 6.73±0.38 vs. 2.08±0.90, 2.19±0.18; TNF-α (μg/L): 4.10±0.11 vs. 1.12±0.10, 1.14±0.04; all P < 0.05]. Immunohistochemistry results demonstrated that the proteins of COX-Ⅳ, TLR9, MyD88 and NF-κB p65 in HVT group were shown in brown, which meant strongly expressed. However, these proteins in spontaneous breathing group and NVT group were uncolored or shown in buff, which meant unexpressed or weakly expressed. The results of quantitative analysis indicated that the immunoreactive scores (IRS) of COX-Ⅳ, TLR9, MyD88 and NF-κB p65 in HVT group were significantly higher than those in spontaneous breathing group and NVT group (COX-Ⅳ IRS: 8.80±2.17 vs. 0.80±0.45, 1.40±0.55;TLR9 IRS: 8.40±2.51 vs. 1.00±0.71, 1.20±0.84; MyD88 IRS: 9.40±1.52 vs. 1.40±0.55, 1.60±0.55; NF-κB p65 IRS: 9.80±2.05 vs. 1.00±0.71, 1.20±0.84; all P < 0.05). There was no significant difference in all of the parameters between spontaneous breathing group and NVT group (all P > 0.05). Conclusion mtDNA contributes significantly to VILI by activating the TLR9-MyD88 signaling pathway, resulting in subsequent secretion of NF-κB p65 and the proinflammatory cytokines, which induce acute inflammatory injury of lung tissue.
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Abstract Local administration of toll-like receptor 9 (TLR9), agonist cytidine-phosphate-guanosine oligodeoxynucleotide (CpG ODNs), and CD40 ligand (CD40L) can decrease ligature-induced periodontal inflammation and bone loss in wild type (WT) mouse. Objective: This study aimed to explore whether such effect is dependent on TLR9 signaling. Material and Methods: Purified spleen B cells isolated from WT C57BL/6J mice and TLR9 knockout (KO) mice were cultured for 48 hours under the following conditions: CD40L, CpG+CD40L, CpG at low, medium and high doses. We determined B cell numbers using a hemocytometer at 24 h and 48 h. Percentages of CD1dhiCD5+ B cells were detected by flow cytometry. Interleukin-10 (IL-10) mRNA expression and protein secretion were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and by ELISA, respectively. The silk ligature was tied around the maxillary second molars for 14 days, during which the CpG+CD40L mixture or PBS was injected into palatal gingiva on days 3, 6, and 9. Results: For both WT and TLR9 KO mice, CpG significantly induced B cell proliferation, increased IL-10 mRNA expression and protein secretion of IL-10 but reduced CD1dhiCD5+ B cells population; local injection of CpG+CD40L mixture significantly decreased alveolar bone loss and the number of TRAP-positive cells adjacent to the alveolar bone surface, and significantly increased the gingival mRNA expression of IL-10 and decreased RANKL and IFN-γ mRNA expression. Conclusions: These results indicated that CpG plus CD40L decreased periodontal inflammation and alveolar bone loss in a TLR9-independent manner in ligature-induced experimental periodontitis.
Subject(s)
Animals , Oligodeoxyribonucleotides/pharmacology , Periodontitis/drug therapy , Alveolar Bone Loss/drug therapy , CD40 Ligand/pharmacology , Cytidine/pharmacology , Toll-Like Receptor 9/drug effects , Guanine Nucleotides/pharmacology , Reference Values , Time Factors , Enzyme-Linked Immunosorbent Assay , B-Lymphocytes/drug effects , Cells, Cultured , Adjuvants, Immunologic/pharmacology , Reproducibility of Results , Interleukin-10/analysis , Disease Models, Animal , Toll-Like Receptor 9/analysis , Real-Time Polymerase Chain Reaction , Flow Cytometry , Gingiva/drug effects , Gingiva/pathology , Mice, Inbred C57BLABSTRACT
OBJECTIVE:To study the relationship between cisplatin-resistant of human ovarian cancer cells (SKOV3) and Toll-like receptor 9 (TLR9) under hypoxia. METHODS:Immunofluorescence method was used to detect the TLR9 expression in rupture and intact membranes SKOV3. SKOV3 was taken,adding cisplatin,then CCK-8 was used to detect cell inhibition rate af-ter 6,12,24 h of added into TLR9 specific agonists CpG-ODN 2006 (group A) and its isotype control CpG-ODN 2006 control (group B). SKOV3 or SKOV3 pretreated by 0(not added),1,10,102,103 μmol/L TLR9 specific antagonist chloroquine for 3 h were taken,adding cisplatin,cell inhibition rate was detected after 24 h of added into SKOV3 normoxia(21% O2),hypoxia(1%O2)incubating 6,12,24 h,HICR under hypoxia in pretreatment was calculated. Western blot method was used to detect the multi-drug resistance associated protein(MRP)expression in SKOV3 under the conditions of normoxia 24 h cell supernatant(group C), hypoxia 24 h cell supernatant(group D),hypoxia 24 h cell supernatant+10 μmol/L chloroquine(group E). RESULTS:TLR9 was expressed in both cell membrane and cytoplasm of SKOV3. Compared with group A,cell inhibition rate in group B was decreased (P<0.01). Compared with normoxia cell supernatant,cell inhibition rate was decreased after hypoxia cell supernatant (P<0.05). Compared with not added chloroquine,adding 10,102 μmol/L chloroquine can obviously decrease HICR(P<0.01),and the most significant decrease was showed when using hypoxia 24 h cell supernatant+10 μmol/L chloroquine. Compared with group C,MRP expression in group D was strengthened (P<0.01);compared with group D,MRP expression in group E was weakened (P<0.01). CONCLUSIONS:TLR9 receptors can be activated under hypoxia,and cause SKOV3 to cisplatin-resistance,the effect may be related with up-regulating MRP expression.
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Objective To compare expression of Toll?like receptors 7 and 9(TLR7, TLR9)as well as their regulatory molecules myeloid differentiation factor 88(MyD88)and nuclear factor?κB(NF?κB)in peripheral blood mononuclear cells(PBMCs)between patients with vitiligo and healthy individuals, and to explore their significance. Methods Flow cytometry was performed to measure expression of TLR7 and TLR9 in PBMCs among 36 patients with vitiligo and 22 healthy controls, and real?time fluorescence?based quantitative PCR(RT?PCR)was conducted to determine mRNA expression of MyD88 and NF?κB in the above blood samples. Results Compared with healthy controls, patients with vitiligo showed higher expression of TLR7 and mRNA expression of MyD88 and NF?κB, but lower expression of TLR9. However, significant differences were only observed in the mRNA expression of NF?κB(t=2.814, P=0.008), but not in the expression of TLR7 and TLR9 or the mRNA expression of MyD88 between patients and controls (t = 1.477, 1.761, 0.058, all P > 0.05). Conclusion NF?κB, as a key signaling molecule of TLR7 and TLR9 regulation pathways, increases obviously in patients with vitiligo, suggesting that NF?κB may be involved in the pathogenesis of vitiligo.
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Objective To evaluate the therapeutic effect of Maxing-Shigan decoction combined with salmeterol/fluticasone inhalation for acute exacerbation of chronic obstructive pulmonary disease (COPD). Methods A total of 86 patients with acute exacerbation of COPD were enrolled and randomly divided into a salmeterol/fluticasone group (41 patients) and a combined treatment group (45 patients). The salmeterol/fluticasone group was treated by salmeterol/fluticasone inhalation, and the combined treatment group by Maxing-Shigan decoction combined with salmeterol/fluticasone inhalation. Serum C-reactive protein (CRP) was detected by a immunonephelometric assay, and Toll-like receptor 9 (TLR9) in hemocytes was detected by flow cytometry. The score of the syndromes in traditional Chinese medicine (TCM), such as cough, sputum, gasping and shortness of breath, as well as pulmonary function and therapeutic effect were evaluateds. Results After the treatment, the serum C-reactive protein in the combined treatment group was significantly lower than that in the salmeterol/fluticasone group (4.3 ± 1.2 mg/L vs. 8.4 ± 2.5 mg/L;t=5.417, P<0.01), and the TLR9 expression was significantly higher (1.9 ± 0.7 vs. 1.6 ± 0.4;t=3.418, P<0.05). The scores of the syndromes in TCM, such as cough (1.7 ± 0.6 vs. 3.8 ± 1.1;t=2.859, P<0.05), sputum (1.6 ± 0.4 vs. 3.9 ± 1.2;t=3.027, P<0.05), gasping (1.2 ± 0.5 vs. 3.4 ± 1.3;t=3.416, P<0.05) and shortness of breath (1.5 ± 0.7 vs. 3.7 ± 1.6;t=3.468, P<0.05) in the combined treatment group were significantly lower than those in the salmeterol/fluticasone group. The forced expiratory volume in first second (75.4 ± 5.8 L vs. 62.8 ± 6.9 L;t=3.526, P<0.05) and the percentage of forced expiratory volume in first second to forced vital capacity (85.7%± 10.3%vs. 71.9%± 15.4%;t=5.648, P<0.01) in the combined treatment group were significantly higher than those in the salmeterol/fluticasone group. The time to symptoms alleviated (3.4 ± 0.7 d vs. 5.6 ± 1.2 d; t=3.256, P<0.05) and the use dose was (1.8 ± 0.2) ×103μg vs. (5.3 ± 0.4)×103μg, and use times of salmeterol/fluticasone (7.4 ± 1.3 vs. 16.5 ± 3.4;t=4.574, P<0.05) in the combined treatment group were significantly decreased than those in the salmeterol/fluticasone group. The total effective rate in in the combined treatment group were significantly decreased than those in the salmeterol/fluticasone group (84.4% vs. 73.2%; χ2=4.519, P<0.05). Conclusion Maxing-Shigan decoction combined with salmeterol/fluticasone inhalation can improve the pulmonary function in patients with acute exacerbation of COPD, its effiency is suppior to salmeterol/fluticasone inhalation alone.
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AIM:To probe whether CpG oligodeoxyribonucleotides 7909 (CpG ODN7909) combined with Toll like receptor (TLR)9 affected the chemotherapeutic sensitivity of doctaxel (DOC) in human lung cancer A549 and H520 cell lines.METHODS:Sequences of TLR9 siRNAs were designed.A549 and H520 cells were transfected with TLR9 siR-NA by lipofectamine.The expression of TLR9 was detected by Western blot .The cell activity was measured by CCK-8 as-say.The experiments were divided into blank control group , control siRNA group and TLR9 siRNA interference group.The cell cycle distribution and cell apoptosis were analyzed by flow cytometry .The expression of P38 and Bax was determined by Western blot .The cells in each group were exposed to CpG ODN 7909 and/or DOC.RESULTS: In A549 cells and H520 cells, CpG ODN7909 alone had no obvious effect on the cell activity , G2/M phase arrest and apoptosis , but in-creased the protein expression of P 38 and Bax ( P<0.01) .In addition, there was no significant changes of the above inde-xes in CpG ODN7909 treated-TLR9 siRNA group was observed .DOC alone significantly inhibited the cell activity , higher the G2/M phase fractions, apoptotic rates and Bax expression (P<0.01), but didn’t affect the expression of P38 in all 3 groups.Compared with the cells treated with DOC alone , the cells treated with CpG ODN7909 combined with DOC exhibi-ted lower cell activity, higher G2/M phase fractions, apoptosis rates and more Bax expression (P<0.01), showed no sig-nificant change of P38 expression.In addition, there was no significant change of the above indexes in CpG ODN 7909 com-bined with DOC treated-TLR9 siRNA group was observed .CONCLUSION:CpG ODN7909 may enhance the chemothera-peutic sensitivity of DOC in human lung cancer cells by combining with TLR 9.The mechanism might be related to enhan-cing the inhibitory effect and apoptosis of DOC on the cell activity in vitro, arresting the cells at G 2/M phase of the cells .
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PURPOSE: The -1237T/C polymorphism of the Toll-like receptor 9 (TLR9) gene has been implicated in the susceptibility of inflammatory bowel diseases (IBDs), but the results remain conflicting. We further investigated this association via meta-analysis. MATERIALS AND METHODS: Multiple electronic databases were extensively searched until February, 2015. The strength of association was evaluated by calculating the pooled odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: A total of 2987 cases and 2388 controls from eight studies were analyzed. Overall, association was found between TLR9 -1237T/C polymorphism and the risk of IBDs when all the studies were pooled (recessive model, OR: 1.59, 95% CI: 1.02-2.47, p=0.04; homozygote comparison, OR: 1.62, 95% CI: 1.04-2.52, p=0.03; allele model, OR: 1.13, 95% CI: 1.00-1.27, p=0.05). Stratification by ethnicity indicated an association between TLR9 -1237T/C polymorphism and IBDs risk in Caucasians (recessive model, OR: 1.59, 95% CI: 1.02-2.47, p=0.04; homozygote comparison, OR: 1.62, 95% CI: 1.04-2.52, p=0.03; allele model, OR: 1.12, 95% CI: 1.00-1.27, p=0.05). When stratified by disease type, significant correlation were only found in the Crohn's disease subgroup (recessive model, OR: 1.69, 95% CI: 1.05-2.73, p=0.03; homozygote model, OR: 1.74, 95% CI: 1.07-2.82, p=0.02; allele model, OR: 1.15, 95% CI: 1.01-1.32, p=0.04). CONCLUSION: The present study suggested that the TLR9 -1237T/C polymorphism might act as a risk factor in the development of IBDs, particularly in Caucasians.
Subject(s)
Humans , Alleles , White People/genetics , Genetic Predisposition to Disease/genetics , Homozygote , Inflammatory Bowel Diseases/ethnology , Odds Ratio , Polymorphism, Genetic/genetics , Risk Factors , Toll-Like Receptor 9/geneticsABSTRACT
AIM:To observe the Toll-like receptor 9 (TLR9) activation in microglia BV-2 cells after oxygen-glucose deprivation and reoxygenation ( OGDR) , and its effects on neuronal apoptosis.METHODS:The BV-2 cell super-natants were collected after the corresponding treatment and added to mouse primary cortical neurons after OGDR for 4 h, followed by normal culture for 24 h.The cells were divided into normal BV-2 group, NC-siRNA group, TLR9-siRNA group, OGDR group, OGDR+NC-siRNA group, OGDR+TLR9-siRNA group and control group (without adding BV-2 cell supernatant) .The changes of the neuronal morphology were observed under an inverted phase-contrast microscope, and the neuronal apoptosis was detected by TUNEL.The protein expression of cleaved caspase-3 was detected by Western blot-ting.RESULTS:After OGDR, the axon turned thin, twisted and broken, and neuronal swelling, decrease in refraction and vacuolar degeneration were observed.The green-stained apoptotic bodies in the neurons in all groups were positive. Compared with control group, the caspase-3 protein levels in other groups were increased.Compared with the normal BV-2 group, the caspase-3 protein in OGDR group and TLR9-siRNA group was increased.Compared with OGDR+TLR9-siRNA group, the caspase-3 protein in TLR9-siRNA group and OGDR group was decreased.CONCLUSION: After OGDR, TLR9 activation in BV-2 cells induces neuronal apoptosis with the increase in caspase-3 protein level.Inhibition of TLR9 expression reduces neuronal damage.
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Objective To investigate the expression of TLR9 mRNA of peripheral blood mononuclear cells (PBMCs) in patients with acute pancreatitis .Methods Fifty two AP patients with the disease duration in 24 h were collected ,peripheral EDTAK2 coag‐ulation vein blood were collected on the first ,third and fifth day ,then plasma were cryop reserved to detect pancreatic elastase , proinflammatory cytokines and anti‐inflammatory cytokines .Then the peripheral EDTAK2 coagulation vein blood two to three months after treatment were collected in the same method to undertake these tests ,and act as the reference level value .Peripheral blood was collected from 36 acute pancreatitis patients .Three months later ,peripheral blood was collected again from these 36 peo‐ple as controls .PBMCs were isolated by Ficoll‐Hypaque gradient centrifugation .RT‐PCR was adopted to determine the relative con‐tent of the expression of TLR9 mRNA of the PBMCs .Results The relative content of expression of TLR9 mRNA were significant‐ly up‐regulated in the patients with acute pancreatitis ,compared with that of controls (P<0 .05) .The up‐regulated expression of TLR9 mRNA was related to expression of TNF‐a and IL‐1 .Conclusion The up‐regulated expression of TLR9 mRNA in acute pan‐creatitis patients indicates that infective factors might be mediated by TLR 9 .