Expression Levels Of TLR4 Research Articles

Calcitriol modulate post-ischemic TLR signaling pathway in ischemic stroke patients

BackgroundNeuroinflammation is a significant contributor to post-ischemic neuronal death after stroke, and Toll-Like Receptors (TLRs) are one of the essential mediators in many inflammatory pathways. TLRs activate the nuclear factor kappa β (NF-kβ), which promotes the expression of various pro-inflammatory genes such as interleukin (IL-1β) and IL-6. 1,25(OH)2D3, also known as calcitriol, is an active form of vitamin D3 that acts as a neurosteroid compound with anti-inflammatory properties. This study aimed to determine the modulatory effects of calcitriol hormone on post-ischemic immunity response. MethodsNeurological tests and conventional blood factors were evaluated in patients with stroke symptoms upon arrival (n = 38) to confirm the stroke. A blood sample was taken from each stroke patient immediately upon admission and again after 24 h. The experimental group was given 10 μg calcitriol orally. The gene expression levels of TLR4, TLR2, NF-kβ, IL-1β, and IL-6 pro-inflammatory factors were measured using real-time PCR. The protein expression of TLR4 and NF-kβ markers was assessed using the flow cytometry technique. ResultsTLR4, NF-kβ, and pro-inflammatory factors IL-1β and IL-6 expression increased significantly after an ischemic stroke, and calcitriol could modulate the TLR4/NF-kβ signaling pathway 24 h after ischemia. ConclusionsCalcitriol may be considered a protective reagent after ischemia by reducing the TLR4/NF-kB activation cascade and probably plays a beneficial role in reducing and improving ischemic stroke patients' symptoms.Trial registration: Iranian Registry of Clinical Trials identifier: IRCT2017012532174N1.

Effects of puerarin on the intervertebral disc degeneration and biological characteristics of nucleus pulposus cells

Context Intervertebral disc degeneration (IDD) is the pathological basis of spinal degenerative diseases. Puerarin (PU) is an isoflavonoid with functions and medicinal properties. Objective To explore the effect of PU on IDD and its potential mechanism of action. Materials and methods Sprague-Dawley (SD) rats were divided into sham, IDD, low PU, and high PU groups. Rat nucleus pulposus cells (NPCs) were isolated and divided into control, IL-1β, 100 and 200 μmol/mL PU, TAK-242 (TLR4 inhibitor), or 200 μmol/mL PU + LPS (TLR4 activator) groups. The water content, inflammatory factors, proliferation activity, TLR4/NF-κB pathway activity, apoptosis rate, protein expression of apoptosis, and histology of the extracellular matrix (ECM) were analysed. Results In vivo: Compared with the IDD group, disorganization of intervertebral disc tissue was significantly improved, water content (2.80 ± 0.24 mg, 3.91 ± 0.31 mg vs. 2.02 ± 0.21 mg) and expression levels of collagen II and aggrecan were significantly increased, and the levels of inflammatory factors and the expression levels of TLR4, MyD88, and p-p65 were significantly decreased in IDD rats treated with PU. In vitro: Compared with the IL-1β group, the proliferation activity of IL-1β-treated NPCs and the expression of collagen II and aggrecan were significantly increased, while the apoptosis rate, levels of inflammatory factors, and the expression levels of TLR4, MyD88, and p-p65 were significantly decreased in IL-1β-treated NPCs treated with PU. LPS reversed the biological function changes of IL-1β-treated NPCs induced by PU. Conclusions PU can delay the progression of IDD by inhibiting activation of the TLR4/NF-κB pathway.

Quercetin alleviates intestinal inflammation and improves intestinal functions via modulating gut microbiota composition in LPS-challenged laying hens

Quercetin, a well-known flavonoid, has been demonstrated to exert beneficial effects on intestinal functions and gut microbiota in birds. In this study, we investigated the effects of quercetin supplementation on inflammatory responses, intestinal barrier functions and gut microbial community in LPS-challenged laying hens. A total of two hundred eighty-eight 32-wk-old Jingfen No.6 laying hens were randomly assigned to 3 groups, the CON group, the LC group and the LQ group. LQ group was fed with 0.4 mg/kg quercetin and at the end of 12 wk, LC and LQ groups were challenged intraperitoneally with lipopolysaccharide (LPS). After LPS challenge, 8 birds of each group were randomly selected and sampled. LPS challenge induced an obvious intestinal mucosal injury, necrosis and shedding, while quercetin intervention maintained its structure. Quercetin significantly decreased the elevated malondialdehyde contents (P < 0.05), and increased the activity of total antioxidant capacity and glutathione peroxidase (P < 0.05) in intestinal mucosa of LPS-challenged laying hens. Quercetin rescued the LPS-induced decreases in goblet cell density and mucin2 expression levels (P < 0.05). There was a significant decline (P < 0.05) in the mRNA expression of Claudin1 and Occludin in intestinal mucosa of LPS-challenged layers, which could be alleviated (P < 0.05) by dietary quercetin. LPS challenge induced the increased expression levels (P < 0.05) of IL-1β and TLR-4 in intestinal mucosa, while these rises could be reversed (P < 0.05) following dietary quercetin supplementation. LPS challenge induced a shift in gut microenvironment, and quercetin addition could elevate the relative abundance of some short chain fatty acids (SCFA)-producing or health-promoting bacteria such as Phascolarctobacterium, Negativicutes, Selenomonadales, Megamonas, Prevotellaceae, and Bacteroides_salanitronis. In conclusion, dietary quercetin addition ameliorated the LPS challenge-induced intestinal inflammation and improved intestinal functions, possibly associated with its modulation on gut microbiota, particularly the increased population of SCFA-producing bacteria.

The evaluation of TLR1, TLR2, TLR4, TLR7, and TLR8 expression levels in the newly-diagnosed acute myeloid leukemia (AML) patients

The evaluation of TLR1, TLR2, TLR4, TLR7, and TLR8 expression levels in the newly-diagnosed acute myeloid leukemia (AML) patients

Analysis of Macrophage Chemotactic Activity and TLR9 Signaling Pathway in the Mouse Model of Viral Acute Lung Injury.

This study focused on the chemotactic activity of macrophages and the role of the TLR9 signaling pathway in the pathogenesis of viral Acute Lung Injury (ALI). For this purpose, a total of 40 male SPF mice were used, aged 5-8 weeks. They were randomly divided into an experimental group and a control group. The experimental group was further divided into S1 and S2, and the control group was further divided into D1 and D2, with 10 in each. The different groups were detected for the expression of inflammatory cytokines and chemokines and the expression of alveolar macrophages. Results showed that as for the weight, survival status, arterial blood gas analysis, lung index and wet-to-dry value of lung tissue, and lung histopathological analysis results, the S2 group showed more obvious changes versus the D2 group, and the difference was statistically significant (P<0.05). S2 had higher levels of the inflammatory factors TNF-α, IL-1β, IL-6 and the chemokine CCL3 in the BALF supernatant versus the D2 Group, and the difference is statistically significant (P<0.05). S2 had higher expression levels of chemokines CCR5, TLR9, and JMJD1A mRNA versus the D2 group, and the difference was statistically significant (P<0.05). In conclusion, the establishment of a mouse ALI model induced by poly l:C was successful; AM has a certain chemotactic activity on CCL3; polyI:C can promote the expression activity and chemotactic activity of macrophages CCR5 through signal pathways, such as TLR9.

Acupuncture ameliorates neurological function by suppressing microglia polarization and inflammatory response after cerebral ischemia in rats

To observe the effect of acupuncture on microglia polarization and inflammatory reaction in rats with cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanisms underlying improvement of CIRI. Thirty male SD rats were randomly divided into sham operation, model, and acupuncture groups, with 10 rats in each group. The CIRI model was established by occlusion of the middle cerebral artery (MCAO) for 1 h, followed by reperfusion. After modeling, rats in the acupuncture group received manual acupuncture stimulation of "Dazhui" (GV14), "Baihui"(GV20), "Shuigou" (GV26), bilateral "Zusanli" (ST36) and "Fengchi" (GB20) by twirling the needles rapidly for 10 s/acupoint every 10 min, with the needles retained for 20 min. The treatment was conducted once daily for successive 7 days. The neurological function was evaluated according to Longa's method. The state of CIRI was observed after Nissl staining, and the expression levels of Iba-1, iNOS, Arg1, BDNF, GDNF and NeuN in the ischemic cortex tissue were detected by immunofluorescence staining. The contents of TNF-α, IL-6 and IL-10 in the ischemic tissue were assayed by ELISA. The protein expression levels of BDNF, GDNF, TLR4, MyD88 and NF-κB in the ischemic tissues were detected by Western blot. The neurological deficit score on the 24 h and 7th day was considerably higher in the model group than in the sham operation group (P<0.01), and evidently lower on the 7th day in the acupuncture group than in the model group (P<0.01). The number of NeuN positive cells，the area of immunofluorescence dual labelling of Arg1, BDNF and GDNF positive staining, IL-10 content, BDNF and GDNF protein expressions were significantly decreased (P<0.01), and the immunofluorescence dual labelling area of Iba-1 and iNOS, TNF-α and IL-6 contents, the pretein expression levels of TLR4, MyD88 and NF-κB considerably increased (P<0.01) in the model group relevant to the sham operation group. In contrast to the model group, the acupuncture group had a significant increase in the number of NeuN positive cells, the immunofluorescence dual labelling area of Arg1, BDNF and GDNF positive staining, IL-10 content, and BDNF and GDNF protein expressions (P<0.05, P<0.01), and an evident decrease in Iba-1 and iNOS positive staining, contents of TNF-α and IL-6, and the protein expression levels of TLR4, MyD88 and NF-κB (P<0.01, P<0.05). Nissl staining showed a marked reduction in the number of neurons, the nucleus pyknosis and nissl bodies and loose arrangement of the neuronal cells in the model group, which was relatively milder in the acupuncture group. Acupuncture intervention can improve neurological function in CIRI rats, which may be related to its effects in regulating the polarization of microglia, reducing inflammatory reaction and increasing the secretion of neurotrophic factors in the brain, inhibiting TLR4/MyD88/NF-κB signaling pathway.

Genome-wide investigation and expression analysis of TLR gene family reveals its immune role in Vibrio tolerance challenge of Manila clam

Toll-like receptors (TLRs) play key roles in activating immune responses during infection. In this study, we identified TLR genes in Manila clam at the genome-wide level and characterized it into 9 types according to the Ruditapes philippinarum genome annotation, including TLR1 (1–10), TLR2 (1–10), TLR2–2 (1–5), TLR3 (1–3), TLR4 (1–9), TLR5, TLR6 (1–5), TLR7 (1–2), and TLR13 (1–4). The length of TLR proteins varied from 128 to 1257 amino acids. The molecular weights and theoretical isoelectric point (pI) values ranged from 14.63 to 143.32 kDa and 4.47 to 9.45, respectively. TLR genes showed universal expression levels in adductor muscle (AM), mantle (M), foot (F), gill (GI), pipe (P), digestive gland (DG), gonad (GO) and labial palp (L). Transcriptome analysis revealed that the expression level of TLR4, TLR5, TLR7 and TLR13 genes are significantly highly expressed in resistant individuals of Manila clam under Vibrio anguillarum challenge, indicating these TLR genes may play significant roles in response to invading pathogens. The results obtained in this work will provide valuable insights into the immune function of TLR gene in R. philippinarum.

Ophiopogon Polysaccharide Liposome Regulated the Immune Activity of Kupffer Cell through miR-4796.

The purpose of this article is to study the effects and mechanism of miR-4796 in the process of ophiopogon polysaccharide liposome (OPL) regulation of the immune activity of Kupffer cells (KCs). In this study, KCs were used as cell models, and were treated with OPL in different concentrations after being transfected with miR-4796 mimic or miR-4796 inhibitor. Firstly, the secretion of NO and iNOS, phagocytic activity, the expression of surface molecules CD14 and MHC II, apoptosis and ROS secretion were measured by Griess, flow cytometry, fluorescence staining and ELISA. Then, real-time PCR and Western blot were used to measure the expression of TLR4, IKKβ, MyD88 and NF-κB in the TLR4-NF-κB signaling pathway. The results showed that after transfection with miR-4796 mimic, the secretion of NO and iNOS, cell migration, cell phagocytosis and expression levels of CD14 and MHC II in the OPL group were significantly higher than those in the miR-4796 mimic control group (p < 0.05; p < 0.01). In addition, the mRNA and protein expression levels of TLR4, MyD88 and NF-κB were significantly higher than those in miR-4796 mimic control group (p < 0.05; p < 0.01). After transfection with miR-4796 inhibitor, the secretion of NO and iNOS, cell migration, cell phagocytosis, expression of CD14 and MHCII in OPL group were significantly higher than those in the miR-4796 inhibitor control group (p < 0.05; p < 0.01). These results indicated that OPL could regulate the immune activity of KCs by regulating miR-4796 and activating the TLR4-NF-κB signaling pathway.

Morphine upregulates Toll-like receptor 4 expression and promotes melanomas in mice

Background Morphine and other opioids are used to manage cancer-related pain; however, the role of these drugs in cancer progression remains controversial. Emerging evidence indicates that morphine can activate Toll-like receptor 4 (TLR4) and its signaling pathways, by the way the activation and expression of TLR4 can promote melanoma. In this study, we investigated the effects of morphine on the expression of TLR4 and promotion of melanoma in mice. Methods Mice melanoma cells (B16F10) were cultured with morphine (0.1, 1 and 10 µM) for 24 h. In the other experiment, cells were treated with morphine with or without TLR4 agonist (LPS) or antagonist (TAK-242). In in-vivo model, B16F10 cells were subcutaneously injected to C57BL/6 mice, and morphine was administrated in three different treatment protocols after developing palpable tumors (acute treatment, chronic daily injections, escalating doses of morphine). In another set of experiments, B16F10 cells were pretreated with LPS (5 µg/ml) 24 h before injection into mice. Control group received normal saline. We measured cell proliferation, the expression level of Tlr4, Nuclear factor kappa-light-chain-enhancer of activated B cells 1 (Nf-κb1) genes, TLR4 protein expression, and tumor volume. Results Chronic, acute, and escalating doses of morphine increased tumor. Morphine increased the expression of Tlr4 and Nf-κb1 regardless of the treatment protocol used. Conclusion Morphine increases the progression of melanoma cancer and may be related to the increased expression of TLR4. Our results suggest that morphine should be used with caution in patients with melanoma. Highlights Morphine increases the expression of TLR4 in melanoma. Morphine increases melanoma progression. These effects are mostly observed with chronic and escalating morphine administration

Examination of the role of resveratrol in attenuating oxidative damage induced by starvation stress in the marine mussel, Mytilus coruscus, through regulation of the sirt1-mediated signaling pathway

Aquatic animals, such as the marine mussel, Mytilus coruscus, are regularly subjected to starvation due to conditions of food scarcity brought about by environmental change. Silent mating type information regulation 2 homolog 1 (sirt1) is triggered during animal starvation and participates in inflammation and stress responses. However, the function of sirt1 in Mytilus remains unknown; thus, exploring the effects of sirt1 will improve the understanding of the resistance of Mytilus to starvation. In this study, Mytilus coruscus were fasted for 9 days and treated with 20 μM resveratrol. The results indicated that sitr1 mRNA expression levels in the gonads and gills of mussels increased significantly after 3 days of starvation and significantly decreased after 9 days of starvation. 20 μM resveratrol significantly up-regulated sirt1 expression in the gonads and gills of mussels starved for 9 days. The condition factor and gonadal index considerably decreased in mussels under starvation stress. The activities of antioxidants SOD, CAT, GSH, GPX, and AKP were elevated after fasting for 3 days, but were reduced after starvation for 9 days, accompanied by histopathological damage in the gills and gonads. Resveratrol alleviates histological damage caused by starvation stress by enhancing antioxidant activity and reducing MDA production. Additionally, resveratrol decreased the mRNA expression levels of tlr4, myd88, and nfkb and increased the mRNA expression levels of ampk, foxo, and gadd45. Overall, the results suggest that resveratrol-induced sirt1 expression exerts a protective effect on mussels risking starvation by regulating TLR4/NFκB and AMPK/FOXO signaling pathways. This study might give us fresh insight into the effects of sirt1 on the response to starvation stress in mussels.

&lt;i&gt;TLR9&lt;/i&gt; gene expression in immune cells of patients with psoriasis

Background. Psoriasis (skin lesions and psoriatic arthritis PA) is a chronic inflammatory autoimmune disease that can be triggered by excessive activation of endosomal toll-like receptors (TLRs), mainly TLR9. Elevated TLR9 levels are observed in both PA and psoriasis. Therefore, studying the expression pattern of the TLR9 gene may help select therapies for patients with PA and psoriasis.&#x0D; Aim. To study the expression pattern of the TLR9 gene in blood mononuclear cells of PA and psoriasis patients without joint involvement for possible use in the transition to targeted therapy.&#x0D; Materials and methods. Mononuclear cells were isolated from the peripheral blood of 31 patients with psoriasis vulgaris without joint involvement, 45 patients with PA, and 20 healthy volunteers. TLR9 gene expression was analyzed by real-time polymerase chain reaction.&#x0D; Results. A comparison of expression levels in PA patients and healthy volunteers showed that the expression level of TLR9 in PA patients was 591 times higher than that in healthy volunteers. The expression level of TLR9 in psoriasis patients without joint involvement was also significantly (423-fold) higher than that in healthy subjects.&#x0D; Conclusion. TLR9 gene expression in mononuclear cells of psoriasis patients with severe skin lesions is similar to that in PA patients. High levels of TLR9 gene expression may be a marker of possible joint involvement in patients with psoriasis and indicate the need to reconsider the therapeutic approach to a particular patient.

Increased levels of circulating oxidized mitochondrial DNA contribute to chronic inflammation in metabolic syndrome, and MitoQ-based antioxidant therapy alleviates this DNA-induced inflammation

Here, the aim was to investigate the role of circulating oxidized mitochondrial DNA (ox-mtDNA) in metabolic syndrome (MetS)-associated chronic inflammation and evaluate the effect of Mito-Quinone (MitoQ)-based antioxidant therapy on inflammation. A total of 112 MetS patients and 111 healthy control individuals (HCs) were recruited. Peripheral blood was collected, and mononuclear cells (PBMCs) were separated. In a preclinical study, MitoQ, a mitochondrial-targeted antioxidant, was administered to Sprague-Dawley (SD) rats fed a high-fat diet (HFD). In vitro, H2O2- or MitoQ-treated HUVECs served as the oxidative or antioxidative cell models to detect the cell-free ox-mtDNA level. Plasma or cell-free ox-mtDNA levels were measured by qPCR. Additionally, THP-1 cells were incubated with plasma cell-free DNA (cfDNA) from MetS patients and HCs or cell-free ox-mtDNA to detect TLR9-NF-κB pathway activation. Plasma ox-mtDNA levels and TLR9 expression levels in PBMCs were increased in MetS patients. In vivo, HFD-fed rats showed elevated plasma ox-mtDNA and TLR9 expression levels in cardiac-residing immune cells, but MitoQ administration attenuated these increases. In vitro, a significant lower level of cell-free ox-mtDNA was detected in MitoQ-treated cells, compared with H2O2-treated cells. Coincubation of plasma cfDNA from MetS patients or cell-free ox-mtDNA and THP-1 cells increased TLR9-NF-κB p65 expression, and promoted IL-1β, IL-6 and IL-8 secretion in THP-1 cells. In conclusion, increased circulating ox-mtDNA contributes to chronic inflammation in MetS by activating the TLR9-NF-κB pathway. MitoQ-based antioxidant therapy effectively alleviates inflammation by reducing ox-mtDNA release.

PSUN112 Expression of Vitamin D Receptor Pathway Genes Across Tissues of Individuals with Obesity

Abstract Introduction Vitamin D deficiency is a substantial comorbidity in 50% of pediatric patients and is linked with poorer health outcomes in children. Vitamin D levels are inversely related to BMI. Therefore, the prevalence of children with low vitamin D levels is increasing as the prevalence of pediatric obesity increases. Pediatric patients with obesity and vitamin D deficiency also have a uniquely increased risk of metabolic syndrome, as compared to their lean peers. However, measured levels of vitamin D correlate with other physiological markers of vitamin D effects in lean individuals but not obese individuals. Since vitamin D levels measured as 25-hydroxyvitmain D (serum 25(OH)D) reflect a storage form of vitamin D, their levels may not be a true reflection of vitamin D action in the body in an obese population. The aim of this study was to provide foundational knowledge to understand if expression of vitamin D receptor (VDR)-target genes in different metabolic pathways correlate with each other in diverse tissues, and thus may be used as a reference standard for vitamin D action in the body. Methods We performed a secondary analysis of samples obtained from 89 obese adolescents aged 12 to 18 years old that were consented under a past IRB-approved protocol. The samples were collected at the time of bariatric surgery between 2004 and 2019. Subject data included age, gender, race/ethnicity, and BMI. Samples collected included blood, intestinal, and subcutaneous adipose tissue. The tissues were analyzed via Real Time-PCR to obtain quantitative levels of VDR-target gene expression, which included TLR4, THBD, and VDR in subcutaneous adipose tissue; and TRPV6, S100G, and VDR in intestinal tissue. Gene expression levels were normalized to the average of two housekeeping genes, GAPDH and RPLPO. Blood samples were analyzed for vitamin D levels. Results VDR-target gene expression of THBD, VDR, and TLR4 in subcutaneous adipose tissue was significantly correlated (p &amp;lt;0.05). In intestinal tissue, we also saw significant correlation between TRPV6, S100G, and VDR (p &amp;lt;0.05). THBD, TLR4, TRPV6, S100G, and VDR gene expression levels from the respective tissues did not correlate with circulating serum 25(OH)D levels (p &amp;gt;0.05). Conclusion These important preliminary findings show VDR-target genes have correlated expression patterns within different tissues – one which stores vitamin D (adipose) and one which doesn't (intestinal). Interestingly, the VDR-target gene expression levels correlated with each other despite the genes being involved in different metabolic pathways in diverse tissues. These findings also show that VDR-target gene expression does not correlate with circulating serum 25(OH)D levels. This discrepancy supports that 25(OH)D levels do not indicate levels of vitamin D action and may not be an appropriate indicator of vitamin D deficiency in the obese population. Thus, VDR-target gene expression levels may provide a better reference standard for vitamin D action in the body. This study may provide the first step in determining a new and more accurate biomarker for vitamin D deficiency and treatment in obesity. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Activity of Codonopsis canescens against rheumatoid arthritis based on TLRs/MAPKs/NF-κB signaling pathways and its mechanism

This paper aims to explore the activity of Codonopsis canescens extract against rheumatoid arthritis(RA) based on the Toll-like receptors(TLRs)/mitogen-activated protein kinases(MAPKs)/nuclear factor kappa B(NF-κB) signaling pathways and its mechanism. The ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) was used to identify the components of C. canescens extract. Forty-eight male SD rats were randomly divided into six groups, namely the normal group, the model group, the methotrexate(MTX) tablet group, and the low, medium, and high-dose C. canescens extract(ZDS-L, ZDS-M, and ZDS-H) groups, with 8 rats in each group. The model of collagen-induced arthritis in rats was induced by injection of bovine type Ⅱ collagen emulsion. MTX(2.5 mg·kg~(-1)), ZDS-L, ZDS-M, and ZDS-H(0.3 g·kg~(-1), 0.6 g·kg~(-1), and 1.2 g·kg~(-1)) were administrated by gavage. Rats in the normal group and the model group received distilled water. MTX was given once every three days for 28 days, and the rest medicines were given once daily for 28 days. Body weight, degree of foot swelling, arthritis index, immune organ index, synovial histopathological changes, and serum levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6) were observed. Protein expressions of TLR2, TLR4, NF-κB p65, p38 MAPK, and p-p38 MAPK in rats were determined by Western blot. Thirty-four main components were identified by UPLC-Q-TOF-MS, including 15 flavonoids, 7 phenylpropanoids, 4 terpenoids, 4 organic acids, 2 esters, and 2 polyalkynes. As compared with the normal group, the body weight of the model group was significantly decreased(P&lt;0.01), and foot swelling(P&lt;0.05, P&lt;0.01), arthritis index(P&lt;0.01), and the immune organ index(P&lt;0.01) were significantly increased. The synovial histopathological injury was obviously observed in the model group. The serum levels of inflammatory factors TNF-α, IL-1β, and IL-6 were significantly increased(P&lt;0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK in the synovial tissue were significantly increased(P&lt;0.01) in the model group. As compared with the model group, the body weights of the ZDS dose groups were increased(P&lt;0.01), and the degree of foot swelling(P&lt;0.01) and the arthritis index were decreased(P&lt;0.05, P&lt;0.01). The immune organ index was decreased(P&lt;0.01) in the ZDS dose groups, and the synovial tissue hyperplasia and inflammatory cell infiltration were alleviated. The serum levels of TNF-α, IL-1β, and IL-6 were significantly decreased(P&lt;0.05, P&lt;0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK were decreased(P&lt;0.05, P&lt;0.01) in the ZDS dose groups. C. canescens extract containing apigenin, tricin, chlorogenic acid, aesculin, ferulic acid, caffeic acid, and oleanolic acid has a good anti-RA effect, and the mechanism may be related to the inhibition of TLRs/MAPKs/NF-κB signaling pathways.

Surface layer protein A from hypervirulent Clostridioides difficile ribotypes induce significant changes in the gene expression of tight junctions and inflammatory response in human intestinal epithelial cells

BackgroundSurface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1β and TNF-α cytokines was measured by ELISA.ResultsC. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj<0.05) decreased the gene expression level of claudins family and JAM-A and increased the secretion of IL-8, TNF-α and IL1-β as compared to untreated cells. Moreover, only SlpA from RT001 could significantly induce the expression of IL-6 (padj<0.05).ConclusionThe results of the present study highlighted the importance of SlpA in the pathogenesis of CDI and C. difficile-induced inflammatory response in the gut. Further studies are required to unravel the significance of the observed results in promoting the intestinal inflammation and immune response induced by C. difficile SlpA from different RTs.

Anti-inflammatory effects of theaflavin-3′-gallate during influenza virus infection through regulating the TLR4/MAPK/p38 pathway

Severe pathological damage caused by the influenza virus is one of the leading causes of death. However, the prevention and control strategies for influenza virus infection have certain limitations, and the exploration for new influenza antiviral drugs has become the major research direction. This study evaluated the antiviral activities of four theaflavin derivatives (TFs). Cytopathic effect (CPE) reduction assay revealed that theaflavin-3′-gallate (TF2b) and theaflavin (TF1) could effectively inhibit the replication of influenza viruses H1N1-UI182, H1N1-PR8, H3N2, and H5N1, and TF2b exhibited the most significant antiviral activity in vivo. Intraperitoneal injection of TF2b at 40 mg/kg/d effectively alleviated viral pneumonia, maintained body weight, and improved the survival rate of mice infected with a lethal dose of H1N1-UI182 to 55.56%. Hematological analysis of peripheral blood further showed that TF2b increased the number of lymphocytes and decreased the number of neutrophils, monocytes, and platelets in the blood of infected mice. RT-qPCR results showed that TF2b reduced the mRNA expression levels of inflammatory cytokines (IL-6, TNF-α, and IL-1β), chemokines (CXCL-2 and CCL-3), and interferons (IFN-α and IFN-γ) after influenza virus infection. In addition, TF2b significantly down-regulated the expression levels of TLR4, p-p38, p-ERK, and cytokines IL-6, TNF-α, IL-1β, and IL-10. These results suggest that TF2b not only significantly inhibits viral replication and proliferation in vitro, but also alleviates pneumonia injury in vivo. Its antiviral effect might be attributed to the down-regulation of influenza virus-induced inflammatory cytokines by regulating the TLR4/MAPK/p38 signaling pathway.

The salmonella effector Hcp modulates infection response, and affects salmonella adhesion and egg contamination incidences in ducks.

Salmonella Entertidis (SE) often causes persistent infections and egg contamination in laying ducks. Hcp, the core structural and effector proteins of the Type VI Secretion System (T6SS) in SE, contributes to bacterial invasion, adhesion and virulence. However, little is known about the effect of Hcp on the host’s infection responses and egg contamination incidences in duck. Herein, we generated an hcp deletion mutant SE MY1△hcp and detected its ability to invade duck granulosa cells (dGCs) and contaminate eggs. In comparison with MY1-infected group, the SE adhesion decreased by 15.96% in MY1△hcp-infected dGCs, and the apoptosis in MY1△hcp-infected dGCs decreased by 26.58% and 30.99% at 3 and 6 hours postinfection, respectively. However, the expression levels of immunogenic genes TLR4, NOD1, TNFα, IL-1β and proinflammatory cytokines IL-6, IL-1β, TNF-α release were markedly lower in the dGCs inoculated with MY1△hcp than that of the wild type. Besides, the laying ducks were challenged with MY1 or MY1△hcp in vivo, respectively. The lower egg production and higher egg contamination were observed in MY1-infected ducks in comparison with MY1△hcp-infected birds. Furthermore, the host’s infection response of differentially abundant proteins (DAPs) to Salmonella effector Hcp was identified using quantitative proteomics. A total of 164 DAPs were identified between the MY1- and MY1△hcp-infected cells, which were mainly engaged in the immune, hormone synthesis, cell proliferation and cell apoptotic process. Among them, STAT3, AKT1, MAPK9, MAPK14, and CREBBP were the center of the regulatory network, which might serve as key host response regulators to bacterial Hcp. In conclusion, we demonstrated that effector Hcp contributed to not only SE invasion, induction of dGCs apoptosis, and trigger of immune responses, but also enhanced contamination incidences. Also, the STAT3, AKT1, MAPK9, MAPK14, and CREBBP were identified as host’s infection response regulators of bacterial Hcp in duck. Overall, these results not only offered a novel evidence of SE ovarian transmission but also identified some promising candidate regulators during SE infection.

MiR-146 Relieves Acute Asthma via Decreasing Epidermal Growth Factor Receptor/Toll-Like Receptor 4 (EGFR/TLR4) and Enhancing Autophagy

There is a close relationship between acute asthma and autophagy. In addition, some studies claim that miR-146 can regulate autophagy and participate in acute asthma. This study further explores the role of miR-146 in acute asthma and underlying mechanism. Twenty BALB/c mice were selected and randomly divided into two groups, the model group and the control group, each with 10 mice. Lung tissues, peripheral blood, alveolar lavage fluid, and primary lymphocytes were separated into miR-146 over expression group (miR-146 mimic), miR-146 low expression group (miR-146 inhibitor), negative control group (NC), blank group, or SBI-0206965 group. Acute asthma was established and the expression levels of miR-146, EGFR, TLR4, LC3, beclin1, and ATG5 in each group was measured. The targeting relationship and correlation between miR-146 and EGFR were also investigated. The expression of IL-4 in model group was increased compared to control arm while the expression of IFN-γ was opposite (P &lt; 0.05). The expressions of miR-146, LC3, beclin1, and the expression of ATG5 were decreased (P &lt; 0.05). The expressions of miR-146 gene and LC3, beclin1, ATG5 mRNA and protein in the miR-146 mimic group were the highest, while the expressions of EGFR and TLR4 were the lowest. The SBI-0206965 group and the miR-146 inhibitor group are opposite to the miR-146 mimic group, the SBI-0206965 group and the miR-146 inhibitor group have significant differences (P &lt; 0.05). miR-146 has a directly targeted EGFR and TLR4, and both showed a negative correlation (rEGFR=−0.397, P = 0.013; rTLR4=−0.402, P = 0.021). During the onset of asthma, miR-146 was abnormally decreased. miR-146 directly targets and negatively regulates EGFR. In addition, miR-146 down-regulates TLR4 gene to increase CD4+ lymphocytes’ aphagocytosis-related markers (LC3, beclin1, ATG5) which further promotes the autophagy process and ultimately alleviates the degree of acute asthma. Its main mechanism is related to the down-regulation of the EGFR/TLR4 through regulated the expression of autophagy. Our study provided a scientific reference for further understanding of acute pathogenesis of asthma.

Meta-analysis of TLR4 pathway-related protein alterations induced by arsenic exposure.

Arsenic is a toxic metal, which ultimately leads to cell apoptosis. TLR4 signaling pathway played a key role in immunomodulatory. Therefore, alterations in related proteins on the TLR4 signaling pathway induced by arsenic exposure was systematically reviewed and analyzed by meta-analysis. Some databases were searched including PubMed, Web of Science, China National Knowledge Infrastructure (CNKI), and WANFANG MED ONLINE. The results of NF-κB, IKK, NF-κBp65, phospho-NF-κBp65, and TLR4 expressions were analyzed by Review Manage 5.3. In the arsenic intervention group, NF-κB, phospho-NF-κBp65, and TLR4 expression levels were higher than the control group, respectively. SMD and 95%CI were 11.29 (6.34, 16.24), 4.71(1.73, 7.68), and 5.79 (-4.22, 15.80). Compared to controls, in the exposed group, IKK levels were found to be 38.11-fold higher (Z = 0.97; P = 0.33); NF-κBp65 levels were found to be 0.92-fold higher (Z = 3.33; P = 0.0009) for normal cells and tissue, while IKK levels were found to be 5.18-fold lower (Z = 5.34; P < 0.0001); NF-κBp65 levels were found to be 2.01-fold lower (Z = 3.87; P = 0.0001) for abnormal cells. With comparing of low dose, high dose of arsenic exposure was found to reduce the expression of NF-κB, but increase the expression of NF-κBp65. This review supports the alterations in related proteins on the TLR4 signaling pathway induced by arsenic exposure, which is helpful to provide theoretical basis for the mechanism of toxicity of arsenic-induced immune system damage.

The Transcription Factor Otc4A Stimulates the Proliferation, Invasion, and Stemness of Colorectal Cancer Cells by Inhibiting the Regulation of miR-7-5p on TLR4.

Background To investigate the effects and mechanism of octamer-binding transcription factor 4 (Otc4A) on proliferation, invasion, and stemness of colorectal cancer (CRC) cells. Methods Firstly, normal fetal human cells (FHC, colon epithelial cells) and HT29 cells (CRC cells) were cultured. The expression levels of Otc4A, miR-7-5p, and TLR4 in cells were then detected by qRT-PCR. CCK-8 was adopted to measure cell proliferation rate after Otc4A, miR-7-5p, and TLR4, respectively, were either knocked out or overexpressed in HT29 cells. Later, the cell viability was detected by cell cloning assay; cell invasion by transwell; cell sphere-forming ability by sphere-formation assay; protein expression level of Otc4A, p65, p-p65, and TLR4 by western blot; and the targeting relationships between miR-7-5p and Otc4A as well as miR-7-5p and TLR4 by dual-luciferase reporter assay. Finally, chromatin immunoprecipitation was applied to verify the interaction between Otc4A and miR-7-5p. Results In HT29 cells, Otc4A expression was significantly increased. Additionally, the knockdown of Otc4A prevented HT29 cells from proliferating, migrating, forming spheres, and activating NF–B signaling. Otc4A could negatively regulate miR-7-5p, and miR-7-5p could target TLR4 expression. Besides, a negative correlation was found between Otc4A and miR-7-5p. Finally, the knockdown of miR-7-5p or overexpression of TLR4 could significantly reverse the effect of the knockdown of Otc4A on HT29 cells. Conclusion The transcription factor Otc4A can regulate the level of TLR4 by inhibiting the expression of miR-7-5p and then promote the proliferation and invasion of CRC cell HT29 as well as enhance cell stemness.