Overexpression Of Toll-like Receptor 4 Research Articles

Hyperbaric Oxygen Therapy Alleviates Paclitaxel-Induced Peripheral Neuropathy Involving Suppressing TLR4-MyD88-NF-κB Signaling Pathway

Paclitaxel (PAC) results in long-term chemotherapy-induced peripheral neuropathy (CIPN). The coexpression of transient receptor potential vanilloid 1 (TRPV1) and Toll-like receptor 4 (TLR4) in the nervous system plays an essential role in mediating CIPN. In this study, we used a TLR4 agonist (lipopolysaccharide, LPS) and a TLR4 antagonist (TAK-242) in the CIPN rat model to investigate the role of TLR4-MyD88 signaling in the antinociceptive effects of hyper-baric oxygen therapy (HBOT). All rats, except a control group, received PAC to induce CIPN. Aside from the PAC group, four residual groups were treated with either LPS or TAK-242, and two of them received an additional one-week HBOT (PAC/LPS/HBOT and PAC/TAK-242/HBOT group). Mechanical allodynia and thermal hyperalgesia were then assessed. The expressions of TRPV1, TLR4 and its downstream signaling molecule, MyD88, were investigated. The mechanical and thermal tests revealed that HBOT and TAK-242 alleviated behavioral signs of CIPN. Immunofluorescence in the spinal cord dorsal horn and dorsal root ganglion revealed that TLR4 overexpression in PAC- and PAC/LPS-treated rats was significantly downregulated after HBOT and TAK-242. Additionally, Western blots showed a significant reduction in TLR4, TRPV1, MyD88 and NF-κB. Therefore, we suggest that HBOT may alleviate CIPN by modulating the TLR4-MyD88-NF-κB pathway.

Silencing of Long Non-coding RNA H19 Alleviates Lipopolysaccharide (LPS)-induced Apoptosis and Inflammation Injury by Regulating miR-140-5p/TLR4 Axis in Cell Models of Pneumonia.

Mounting studies have clarified the link between long non-coding RNAs (lncRNAs) and pneumonia. This research aims to probe the function and regulatory mechanism of lncRNA H19 in lipopolysaccharide (LPS)-induced cell models of pneumonia. WI-38 cells were exposed to LPS for 12 h to mimic cell models of pneumonia. The relative expression of H19, miR-140-5p, and toll-like receptor 4 (TLR4) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability was detected by MTT assay. The protein expression of apoptosis-associated proteins (Bax and Bcl-2) and TLR4 were determined by western blot. Moreover, the content of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were measured by enzyme-linked immunosorbent assay (ELISA). The target relationship between miR- 140-5p and H19/ TLR4 was confirmed by Dual luciferase reporter (DLR) assay. LncRNA H19 and TLR4 were up-regulated, while miR-140-5p was downregulated in peripheral blood of patients with pneumonia and LPS-treated WI-38 cells compared with their controls. Silencing of H19 or miR-140-5p mimics facilitated cell viability, whereas repressed apoptosis and reduced content of TNF-α, IL-6, and IL-1β in LPS-induced WI-38 cells. H19 targeted miR-140-5p and it inversely regulated miR-140- 5p expression. MiR-140-5p targeted TLR4 and it inversely regulated TLR4 expression. H19 positively regulated TLR4 expression. Moreover, inhibition of miR-140-5p or overexpression of TLR4 reversed the effects of H19 silencing on cell viability, inflammation, and apoptosis in LPS-induced WI-38 cells. Silencing of H19 inhibited apoptosis and inflammation by miR-140- 5p/TLR4 pathway in LPS-induced WI-38 cells.

Shifts in intestinal microbiota and improvement of sheep immune response to resist Salmonella infection using Toll-like receptor 4 (TLR4) overexpression.

Toll-like receptor 4 (TLR4) identifies Gram-negative bacteria or their products and plays a crucial role in host defense against invading pathogens. In the intestine, TLR4 recognizes bacterial ligands and interacts with the immune system. Although TLR4 signaling is a vital component of the innate immune system, the influence of TLR4 overexpression on innate immune response and its impact on the composition of the intestinal microbiota is unknown. Here, we obtained macrophages from sheep peripheral blood to examine phagocytosis and clearance of Salmonella Typhimurium (S. Typhimurium) in macrophages. Meanwhile, we characterized the complex microbiota inhabiting the stools of TLR4 transgenic (TG) sheep and wild-type (WT) sheep using 16S ribosomal RNA (rRNA) deep sequencing. The results showed that TLR4 overexpression promoted the secretion of more early cytokines by activating downstream signaling pathways after stimulation by S. Typhimurium. Furthermore, diversity analysis demonstrated TLR4 overexpression increased microbial community diversity and regulated the composition of intestinal microbiota. More importantly, TLR4 overexpression adjusted the gut microbiota composition and maintained intestinal health by reducing the ratio of Firmicutes/Bacteroidetes and inflammation and oxidative stress-producing bacteria (Ruminococcaceae, Christensenellaceae) and upregulating the abundance of Bacteroidetes population and short-chain fatty acid (SCFA)-producing bacteria, including Prevotellaceae. These dominant bacterial genera changed by TLR4 overexpression revealed a close correlation with the metabolic pathways of TG sheep. Taken together, our findings suggested that TLR4 overexpression can counteract S. Typhimurium invasion as well as resist intestinal inflammation in sheep by regulating intestinal microbiota composition and enhancing anti-inflammatory metabolites.

Viaminate ameliorates Propionibacterium acnes-induced acne via inhibition of the TLR2/NF-κB and MAPK pathways in rats.

Viaminate, a retinoic acid derivative developed in China, has been clinically used for acne treatment to regulate and control keratinocyte cell differentiation and proliferation, inhibit keratinization, reduce sebum secretion, and regulate immune and anti-inflammatory functions; however, its potential molecular mechanism has not yet been elucidated. Therefore, we induced ear acne in rats using Propionibacterium acnes and sebum application. Symptoms of ear redness, epidermal thickening, inflammatory reaction, keratin overproduction, subcutaneous oil, and triglyceride (TG) accumulation improved significantly in acne model rats treated with viaminate for 30days. Transcriptome analysis of rat skin tissues suggested that viaminate had significant regulatory effects on fatty acid metabolism and cellular keratinization pathways. Molecular target prediction suggested that toll-like receptor 2 (TLR2) may be a key target of viaminate's therapeutic mechanism. Western blotting results confirmed that viaminate inhibited the TLR2 and its downstream pathways, nuclear factor-kappa B (NF-κB) [NF-κB inhibitor alpha (IκBα)/NF-κB-p65] and mitogen-activated protein kinases (MAPKs) [MAPK p38/c-Jun N-terminal kinase (JNK)/extracellular regulated kinase 1/2 (ERK1/2)] in acne vulgaris rats. In vitro studies revealed that viaminate treatment attenuated P. acnes proliferation and P. acnes-induced inflammatory response in human keratinocytes and has an inhibitory effect on the activation of NF-κB and MAPKs, while overexpression of TLR2 attenuated these effects. In conclusion, viaminate ameliorates P. acnes-induced acne by inhibiting the proliferation and inflammatory response of keratinocytes, ascribed to the deactivation of the TLR2-mediated NF-κB and MAPK pathways.

Atorvastatin reduces contrast media-induced pyroptosis of renal tubular epithelial cells by inhibiting the TLR4/MyD88/NF-κB signaling pathway

BackgroundContrast-induced acute kidney injury (CI-AKI) is the third most common cause of hospital-acquired renal failure. However, there is no effective treatment of CI-AKI, and its mechanism is unknown. Interestingly, atorvastatin has been reported to be effective in renal injury. Therefore, the aim of this study was to explore the effect and possible molecular mechanism of atorvastatin in CI-AKI.MethodsOn the CI-AKI in vitro model, rat tubular epithelial cells (NRK-52E) were treated with 18 mg I/ml meglumine diatrizoate (MEG) and then pretreated with atorvastatin. pcDNA3.1-TLR4 treatment was performed to overexpress toll-like receptor 4 (TLR4) in NRK-52E cells. Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) kits were used to detect NRK-52E cell viability as well as LDH release in each group, respectively; qRT-PCR to determine mRNA expression of TLR4 in cells; western blot to detect protein expression levels of pyroptosis-related proteins (NLRP3, caspase-1, ASC, and GSDMD) and TLR4/MyD88/NF-κB signaling pathway-related proteins (TLR4, MyD88, NF-κBp65, and p-NF-κB p65) in cells.ResultsMEG treatment significantly inhibited the viability of NRK-52E cells, increased pro-inflammatory factor levels and promoted pyroptosis, representing successful establishment of a rat tubular epithelial cell (NRK-52E) CI-AKI in vitro model. Notably, atorvastatin increased the activity of MEG-treated NRK-52E cells and alleviated cell injury in a concentration-dependent manner. In addition, atorvastatin significantly down-regulated the expression of TLR4 in MEG-treated NRK-52E cells. However, overexpression of TLR4 inhibited the effects of atorvastatin on increasing cell viability, alleviating cell injury, reducing pro-inflammatory factors (IL-1β, IL-6, and TNF-α) levels, and inhibiting apoptosis (by down-regulating the expression of NLRP3, caspase-1, ASC, and GSDMD). Furthermore, atorvastatin also inhibited the expression of TLR4/MyD88/NF-κB pathway-related proteins (TLR4, MyD88, and p-NF-κB p65).ConclusionAtorvastatin can attenuate CI-AKI through increasing the activity of MEG-treated renal tubular epithelial cells, relieving cell injury, as well as inhibiting pyroptosis and inflammation. More importantly, the mechanism was achieved by inhibiting the TLR4//MyD88/NF-κB signaling pathway.

Inhibition of Ferulic Acid on U937 Cell Growth and Its Related Mechanism

To investigate the effects of ferulic acid (FA) on proliferation, apoptosis and Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway of human acute myeloid leukemia (AML) U937 cells. Different concentrations of FA (0、10、25、50、100、200 μmol/L) was used to treat human AML cell lines Kasumi-1, HL-60, and U937 cells respectively for 24 h. The cell survival rate was detected by cell counting kit-8 method, and the 50% inhibitory concentration (IC50) of each cell line was calculated. The U937 cells were divided into control group, FA group (50 μmol/L FA), FA+pcDNA group (50 μmol/L FA+transfected with empty plasmid), FA+pcDNA-TLR4 group (50 μmol/L FA+transfected with TLR4 overexpression plasmid). Flow cytometry was used to detect U937 cell cycle and cell apoptosis; plate clone formation test was used to detect U937 cell clone formation ability; fluorescence quantitative PCR was used to detect the TLR4, NF-κB p65 mRNA levels in U937 cells; Western blot was used to detect the expression levels of CyclinD1, CyclinE, Bcl-2 related X protein (Bax), B cell lymphoma-2 (Bcl-2), Caspase-3, TLR4, and NF-κB p65 protein in U937 cells. With the increase of FA concentration, the survival rates of Kasumi-1, HL-60 and U937 cells gradually decreased(r=-0.919, r=-0.909, r=-0.900), the IC50 of U937 cells was 50.25±2.23 μmol/L. Compared with the control group, after drug treatment of U937 cells, the ratio of G0/G1 phase cells, apoptosis rate, expression levels of Bax and Caspase-3 proteins in FA group were significantly increased (P<0.05), the number of cell clones, the ratios of S phase and G2/M phase cells, expression levels of CyclinD1, CyclinE and Bcl-2 proteins, and TLR4, NF-κB p65 mRNA and protein were significantly decreased (P<0.05); compared with FA group and FA+pcDNA group, the ratio of G0/G1 phase cells, apoptosis rate, expression levels of Bax and Caspase-3 proteins in FA+pcDNA-TLR4 group were significantly decreased (P<0.05), the number of cell clones, the ratios of S phase and G2/M phase cells, expression levels of expression levels of CyclinD1, CyclinE and Bcl-2 proteins, and TLR4, NF-κB p65 mRNA and proteins were significantly increased (P<0.05). FA inhibits U937 cell proliferation and promotes cell apoptosis by inhibiting the TLR4/NF-κB signaling pathway.

LncRNA SNHG16 regulates RAS and NF-κB pathway-mediated NLRP3 inflammasome activation to aggravate diabetes nephropathy through stabilizing TLR4.

LncRNA SNHG16 and Toll-like receptor-4 (TLR4) participate in diabetes nephropathy. This study investigated whether SNHG16 regulates diabetic renal injury (DRI) via TLR4 and its related mechanism. Diabetic mice and high glucose (HG)-induced HRMCs were used to examine the expressions of SNHG16 and TLR4. The SNHG16 expression, cytokines, reactive oxygen species, MDA, SOD, GSH, and fibrosis-related proteins were evaluated in HG-induced HRMCs transfected with sh-NC or sh-SHNG16. RNA immunoprecipitation and RNA pull-down determined the interaction between SNHG16 and EIF4A3 or TLR4 and EIF4A3. We used HG-treated HRMCs or diabetic mice to investigate the roles of TLR4 or SNHG16 in renal injuries. Both SNHG16 and TLR4 were upregulated in diabetic conditions. HG increased serum Scr and BUN, led to significant fibrosis, increased inflammation- and renal fibrosis-related proteins in mice, and increased ROS, MDA, and decreased SOD and GSH in HRMCs. SNHG16 silencing diminished HG-upregulated SNHG16, decreased HG-increased cytokines secretion, ROS, MDA, and fibrosis but increased SOD and GSH. RIP and RNA pull-down confirmed that SNHG16 recruits EIF4A3 to stabilize TLR4 mRNA. TLR4 knockdown alleviated HG-induced renal injuries by suppressing RAS and NF-κB-mediated activation of NLRP3 inflammasomes. SNHG16 knockdown alleviated HG-induced renal injuries in HG-induced HRMCs or diabetic mice. Interestingly, TLR4 overexpression reversed the effects of SNHG16 knockdown. Mechanistically, SNHG16 knockdown alleviated HG-induced renal injuries by suppressing TLR4. SNHG16 accelerated HG-induced renal injuries via recruiting EIF4A3 to enhance the stabilization of TLR4 mRNA. The SNGHG16/ELF4A3/TLR4 axis might be a novel target for treating DRI.

Circ-PTTG1IP/miR-671-5p/TLR4 axis regulates proliferation, migration, invasion and inflammatory response of fibroblast-like synoviocytes in rheumatoid arthritis.

Circular RNAs (circRNAs) are related to rheumatoid arthritis (RA) development. However, the function and mechanism of circRNA pituitary tumor-transforming 1 interacting protein (circ- PTTG1IP) in RA are unknown. The expression of circ-PTTG1IP in synovial tissues of RA patients and fibroblast-like synoviocytes from RA patients (RA-FLSs) were detected by RT-qPCR. The results uncovered that circ-PTTG1IP was overexpressed in RA patients and RA-FLSs, and circ-PTTG1IP knockdown suppressed cell proliferation, migration, invasion and inflammatory response in RA-FLSs. Besides, we found that circ-PTTG1IP could directly bind to miR-671-5p, and toll-like receptor 4 (TLR4) was a target of miR-671-5p, which was confirmed by dual-luciferase reporter assay. miR-671-5p inhibitor attenuated the effects of circ-PTTG1IP knockdown on RA-FLSs, while the effects of miR-671-5p mimic on RA-FLSs were partly reversed by TLR4 overexpression. Furthermore, circ-PTTG1IP could upregulate TLR4 expression by miR-671-5p. Thus, circ-PTTG1IP knockdown repressed cell proliferation, migration, invasion and inflammatory response in RA-FLSs by regulating the miR-671-5p/TLR4 axis.

M2 macrophage-derived exosomal miR-145-5p protects against the hypoxia/reoxygenation-induced pyroptosis of cardiomyocytes by inhibiting TLR4 expression.

Exosomes carrying micro ribonucleic acids (miRNAs) protect against myocardial ischemic injury. In the study, we sought to investigate the protective effect mechanism of M2 macrophage-derived exosome miR-145-5p in hypoxia-reoxygenation (H/R)-induced cardiomyocytes. M2 macrophages were isolated and induced from blood donated by healthy donors. M2 macrophages were transfected with or without miR-145-5p. Exosomes derived from M2 macrophages were isolated and identified by flow cytometry, nanoparticle tracking analysis, and transmission electron microscopy (TEM). AC16 cells were used to establish an H/R model, and cell activity was detected using a Cell Counting Kit 8 (CCK-8). Western blot was used to detect the expression of gasdermin D (GSDMD), nucleotide-binding domain-like receptor protein 3 (NLRP3), and caspase-1 in the H/R-induced AC16 cells to evaluate pyroptosis. Immunofluorescence staining was used to detect the positive rates of PKH26 and caspase-1. Combined with database prediction, dual luciferase reporter assays were used to validate toll-like receptor 4 (TLR4) as a downstream target molecule of miR-145-5p. A real-time quantitative polymerase chain reaction (RT-qPCR) analysis and western blot were used to detect the expression of TLR4 in the AC16 cells. Flow cytometry, western blot, nanoparticle tracking and TEM results confirmed the successful isolation of M2 macrophage-derived exosomes. CCK-8 results showed M2 macrophage-derived exosomes decreased the viability of the H/R-induced cells. Western blot results showed the expressions of GSDMD, caspase-1, and NLRP3 were significantly downregulated in the H/R group. Moreover, CCK-8 results showed the M2 macrophage-derived exosome miR-145-5p significantly ameliorated H/R-induced AC16 cellular activity. Western blot results confirmed the expressions of GSDMD, NLRP3, and caspase-1 were significantly downregulated in the macrophage-derived exosome miR-145-5p group compared to the M2 macrophage-derived exosome NC (normal control) group. Immunofluorescence staining results displayed the same trend in terms of the caspase-1 positivity rate. Further, we demonstrated overexpression of TLR4 partially reversed the protective effect of M2 macrophage-derived exosome miR-145-5p in the H/R-induced AC16 cells. Additionally, overexpression of TLR4 reversed the protein expression associated with pyroptosis in M2 macrophage-derived exosome miR-145-5p in the H/R-induced AC16 cells. Our study indicated M2 macrophage-derived exosomes carrying miR-145-5p inhibited H/R-induced cardiomyocyte pyroptosis by downregulating the expression of TLR4.

Radix Glycyrrhizae extract and licochalcone a exert an anti-inflammatory action by direct suppression of toll like receptor 4

Ethnopharmacological relevanceRadix Glycyrrhizae (GL), a herbal medicine that is widely available, has shown advantages for a variety of inflammatory diseases. Toll like receptor 4 (TLR4) pathway has been shown to play a key role in the progression of inflammation. Aim of the studyThe purpose of this study was to investigate the involvement of TLR4 in the anti-inflammatory mechanism of GL extract and its active constituent on acute lung injury (ALI). Materials and methodsA model of inflammation produced by lipopolysaccharide (LPS) was established in C57BL/6 mice and macrophages derived from THP-1. To screen the active components of GL, molecular docking was used. Molecular dynamics and surface plasmon resonance imaging (SPRi) were used to study the interaction of a specific drug with the TLR4-MD2 complex. TLR4 was overexpressed by adenovirus to confirm TLR4 involvement in the anti-inflammatory activities of GL and the chosen chemical. ResultsWe observed that GL extract significantly reduced both LPS-induced ALI and the production of pro-inflammatory factors including TNF-α, IL-6 and IL-1β. Additionally, GL inhibited the binding of Alexa 488-labeled LPS (LPS-488) to the membrane of THP-1 derived macrophages. GL drastically reduce on the expression of TLR4 and the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-κB). Furthermore, molecular docking revealed that Licochalcone A (LicoA) docked into the LPS binding site of TLR4-MD2 complex. MD2-LicoA binding conformation was found to be stable using molecular dynamic simulations. SPRi indicated that LicoA bound to TLR4-MD2 recombinant protein with a KD of 3.87 × 10−7 M. LicoA dose-dependently reduced LPS-488 binding to the cell membrane. LicoA was found to significantly inhibit LPS-induced lung damage and inflammation. Furthermore, LicoA inhibited TLR4 expression, MAPK and NF-κB activation in a dose-dependent manner. The inhibitory effects of GL and LicoA on LPS-induced inflammation and TLR4 signaling activation were partly eliminated by TLR4 overexpression. ConclusionOur findings imply that GL and LicoA exert inhibitory effects on inflammation by targeting the TLR4 directly.

The role and mechanism of autophagy in lipopolysaccharide-induced inflammatory response of A549 cells

To study the role and mechanism of autophagy in lipopolysaccharide (LPS)-induced inflammatory response of human alveolar epithelial A549 cells. A549 cells were stimulated with LPS to establish a cell model of inflammatory response, and were then grouped (n=3 each) by concentration (0, 1, 5, and 10 μg/mL) and time (0, 4, 8, 12, and 24 hours). The A549 cells were treated with autophagy inhibitor 3-methyladenine (3-MA) to be divided into four groups (n=3 each): control, LPS, 3-MA, and 3-MA+LPS. The A549 cells were treated with autophagy agonist rapamycin (RAPA) to be divided into four groups (n=3 each): control, LPS, RAPA, and RAPA+LPS. The A549 cells were transfected with the Toll-like receptor 4 (TLR4) overexpression plasmid to be divided into four groups (n=3 each): TLR4 overexpression control, TLR4 overexpression, TLR4 overexpression control+LPS, and TLR4 overexpression+LPS. The A549 cells were transfected with TLR4 siRNA to be divided into four groups (n=3 each): TLR4 silencing control,TLR4 silencing, TLR4 silencing control+LPS, and TLR4 silencing+LPS. CCK-8 assay was used to measure cell viability. Western blot was used to measure the protein expression levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4. After stimulation with 1 μg/mL LPS for 12 hours, the levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4 increased and reached the peak (P<0.05). Compared with the LPS group, the 3-MA+LPS group had reduced expression of autophagy-related proteins and increased expression of inflammation-related proteins and TLR4, while the RAPA+LPS group had increased expression of autophagy-related proteins and reduced inflammation-related proteins and TLR4 (P<0.05). The TLR4 overexpression+LPS group had reduced autophagy-related proteins and increased inflammation-related proteins compared with the TLR4 overexpression control+LPS group, and the TLR4 silencing+LPS group had increased autophagy-related proteins and reduced inflammation-related proteins compared with the TLR4 silencing control+LPS group (P<0.05). In the LPS-induced inflammatory response of human alveolar epithelial A549 cells, autophagic flux has a certain protective effect on A549 cells. TLR4-mediated autophagic flux negatively regulates the LPS-induced inflammatory response of A549 cells.

Loureirin C ameliorates ischemia and reperfusion injury in rats by inhibiting the activation of the TLR4/NF-κB pathway and promoting TLR4 degradation.

Ischemic stroke is a leading cause of death and disability worldwide. Post-ischemia, microglia respond immediately to the alternations in neuronal activity and mediate inflammation. Toll-like receptor 4 (TLR4) plays a key role in this phenomenon. To explore the effect of loureirin C, an effective compound from Chinese Dragon's blood, on ischemic stroke, Sprague-Dawley rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) with/without intragastric administration of loureirin C (7, 14, and 28 mg/kg). Loureirin C alleviated MCAO/R-induced brain impairment evaluated by neurological scores (p < 0.001), brain water content (p < 0.001), and cerebral infarct volume (p=0.001). The neuroprotective (p < 0.001) and inhibitory effects on microglial activation (p < 0.001) of loureirin C were revealed by immunofluorescence. Rescue studies with TLR4 overexpression in BV-2 microglia showed that the antiinflammatory effect of loureirin C was attributable to the inhibition of TLR4 protein expression. Moreover, co-immunoprecipitation assays showed that the binding of Triad3A, an E3 ubiquitin ligase of TLR4, was increased by loureirin C (p=0.003). Our study demonstrates that loureirin C could be a promising therapeutic agent for the management of ischemic stroke by inhibiting microglial activation, potentially by Triad3A-mediated promotion of TLR4 ubiquitination and degradation.

Circular BANP knockdown inhibits the malignant progression of residual hepatocellular carcinoma after insufficient radiofrequency ablation.

Circular RNAs (circRNAs) are endogenous non-coding RNAs, some of which have pathological roles. The current study aimed to explore the role of circRNA BTG3-associated nuclear protein (circ-BANP) binding with let-7f-5p and its regulation of the toll-like receptor 4 (TLR4)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in residual hepatocellular carcinoma (HCC) after insufficient radiofrequency ablation (RFA). Circ-BANP, let-7f-5p, and TLR4 expressions in HCC samples were assessed using reverse transcription- quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Bioinformatics prediction, RNA pull-down assay, and dual luciferase reporter gene assay were used to analyze the relationships among circ-BANP, let-7f-5p, and TLR4. Huh7 cells were used to generate an in vitro model of residual HCC, defined as Huh7-H cells, which were transfected with either a plasmid or the sequence of circ-BANP, let-7f-5p, or TLR4. Expression of circ-BANP, let-7f-5p, and TLR4 mRNA was determined by RT-qPCR. TLR4, STAT3, p-STAT3, vascular endothelial growth factor A, vascular endothelial growth factor receptor-2, and epithelial-mesenchymal transformation (EMT)-related factors proteins were determined by Western blotting. Cell proliferation was determined by cell counting kit-8 and 5-Ethynyl-2'-deoxyuridine (EdU) assay and cell migration and invasion by Transwell assay. Animal studies were performed by inducing xenograft tumors in nude mice. Circ-BANP and TLR4 mRNAs were upregulated in HCC tissues (the fold change for circ-BANP was 1.958 and that for TLR4 was 1.736 relative to para-tumors) and expression further increased following insufficient RFA (fold change for circ- BANP was 2.407 and that of TLR4 was 2.224 relative to para-tumors). Expression of let-7f-5p showed an opposite tendency (fold change for let-7f-5p in HCC tissues was 0.491 and that in tumors after insufficient RFA was 0.300 relative to para-tumors). Competitive binding of circ-BANP to let-7f-5p was demonstrated and TLR4 was identified as a target of let-7f-5p (P < 0.01). Knockdown of circ-BANP or elevation of let-7f-5p expression inhibited the TLR4/STAT3 signaling pathway, proliferation, invasion, migration, angiogenesis, and EMT in Huh7 and Huh7-H cells (P < 0.01). The effects induced by circ-BANP knockdown were reversed by let-7f-5p inhibition. Overexpression of TLR4 reversed the impact of let-7f-5p upregulation on the cells (P < 0.01). Silencing of circ-BANP inhibited the in vivo growth of residual HCC cells after insufficient RFA (P < 0.01). Knockdown of circ-BANP upregulated let-7f-5p to inhibit proliferation, migration, and EMT formation in residual HCC remaining after insufficient RFA. Effects occur via regulation of the TLR4/STAT3 signaling pathway.

CircSCAPER knockdown attenuates IL-1β-induced chondrocyte injury by miR-127-5p/TLR4 axis in osteoarthritis

Purpose Osteoarthritis (OA) is a chronic inflammatory degenerative disease characterized by articular cartilage degradation. Circular RNAs have been shown to play significant roles in OA process. Herein, this work aimed to investigate the potential role and mechanism of circSCAPER in OA progression. Methods Levels of circSCAPER, miR-127-5p and toll-like receptor 4 (TLR4) were detected by qRT-PCR or western blotting. Cell apoptosis was determined by flow cytometry. The expression of Aggrecan and Matrix metallopeptidase was examined using western blot to assess extracellular matrix (ECM) degradation. Inflammatory response and oxidative stress were determined by measuring the release of inflammatory factors, along with the generation of intracellular reactive oxygen species and malondialdehyde. The interaction between miR-127-5p and circSCAPER or TLR4 was determined by dual-luciferase reporter, RNA immunoprecipitation and pull-down assays. Results Chondrocytes were treated with interleukin-1β (IL-1β) to mimic OA condition in vitro. CircSCAPER was increased in OA cartilages and IL-1β-induced chondrocytes. Functionally, knockdown of circSCAPER attenuated IL-1β-evoked apoptosis, ECM degradation, inflammation and oxidative stress in vitro. CircSCAPER up-regulation in OA cartilages was discovered to be accompanied by decreased miR-127-5p and increased TLR4. Mechanistically, circSCAPER acted as a sponge for miR-127-5p to positively regulate TLR4 expression in chondrocytes. IL-1β treatment reduced miR-127-5p expression but up-regulated TLR4 expression, re-expression of miR-127-5p suppressed IL-1β-caused chondrocyte injury, which was abolished by TLR4 overexpression. Moreover, miR-127-5p inhibition reversed the protective action of circSCAPER knockdown on chondrocytes under IL-1β treatment. Conclusion CircSCAPER silencing protected against IL-1β-induced apoptosis, ECM degradation, inflammation and oxidative stress in chondrocytes via miR-127-5p/TLR4 axis.

Role of PIM2 in acute lung injury induced by sepsis.

Pediatric sepsis can cause lung damage leading to death in children. In addition, its complicated pathogenesis currently presents a difficult problem in the medical field. Proviral integrations of Moloney virus 2 (PIM2) is a prognostic marker of pediatric sepsis; therefore, the aim of the present study was to investigate the role of PIM2 in lung injury caused by pediatric sepsis. To meet this aim, the expression of PIM2 in lipopolysaccharide (LPS)-induced BEAS-2B pulmonary epithelial cells was detected using reverse transcription-quantitative (RT-q)PCR and western blotting. Subsequently, the expression of PIM2 was inhibited using the cell transfection technique. Cell Counting Kit-8, TUNEL and western blotting, use of a fluorescence kit, ELISA detection kits were used to detect the expression of inflammatory- and cell injury-associated indicators following PIM2 inhibition. In addition, the expression of proteins known to be associated with the Toll-like receptor 2 (TLR2)/myeloid differentiation primary response 88 (MyD88) pathway were also assessed using western blotting. Finally, the simultaneous inhibition of PIM2 expression and overexpression of TLR2 were investigated in an attempt to elucidate the underlying mechanism. The expression level of PIM2 was revealed to be increased in LPS-induced BEAS-2B cells. Interference with PIM2 expression led to an increase in BEAS-2B cell viability, the inhibition of apoptosis and a reduction in oxidative stress and the inflammatory response. These processes were also revealed to be accomplished via downregulation of the TLR2/MyD88 signaling pathway. Overall, the present study demonstrated that knockdown of PIM2 alleviated LPS-induced bronchial epithelial cell injury by inhibiting the TLR2/MyD88 pathway.

PRIMARY OPEN ANGLE GLAUCOMA: MECHANISMS OF PATHOGENESIS AND GENETIC PREDISPOSITION. Review

Relevance. Primary open-angle glaucoma (POAG) is a progressive optic neuropathy with loss of retinal ganglion cells (RGCs) and narrowing of the visual fields in the eyes with a gonioscopic open angle. The main mechanisms of this are increased intraocular pressure (IOP), circulatory disorders, trabecular meshwork (TM), ischemic metabolic disorders and chronic inflammation. However, questions about the role of POAG genetic predisposition remain open.&#x0D; Objective: analysis of current data on the mechanisms of pathogenesis of progressive neuropathy in POAG and the role of genetic predisposition.&#x0D; Methods. The analysis of scientific publications in open international electronic scientometric databases: Scopus, PubMed, Web of Science, Google Scholar, SID, MagIran, IranMedex, IranDoc, ScienceDirect, Embase by keywords (a total of 67 sources). Search depth – 10 years (2012-2022).&#x0D; Results. There are more than 60 million glaucoma patients in the world, 20% of whom have an incurable stage. By 2040, the number of patients is projected to increase to 112 million, with POAG accounting for 75% of cases. Among the main mechanisms of glaucoma, an important role belongs to chronic inflammation and immune damage, which occur in response to ischemic injury. Prolonged inflammatory process leads to hypersecretion of inflammatory mediators and infiltration of inflammatory cells into ischemic tissue, which aggravates the effects of increased IOP and ischemia. It is known that mutations in the gene of Toll-like receptor 4 (TLR4) are associated with both infectious and non-infectious diseases, including POAG: activation of TLR4 initiates TM fibrosis, causes increased IOP, activates RGCs apoptosis in the model of acute glaucoma. TLR4 ligands, such as heat shock proteins and lipopolysaccharides are candidate antigens for glaucoma. TLR4 overexpression at retinal microglia and astrocytes induce an innate immune response through NF-κB activation, which enhances the expression of proinflammatory cytokines.&#x0D; Conclusions. A promising direction is to study the contribution of TLR4 mutations to the POAG mechanisms, which will identify the mechanisms of immune disorders and establish the genetic risk of individual mutations in different ethnic groups.

Molecular docking and in vivo studies of liquiritin against acute myocardial infarction via TLR4/MyD88/NF-κB signaling

Licorice (Glycyrrhiza glabra L.) is an essential herb in Chinese medicine, as well as a common ingredient in health foods and natural sweeteners. Liquiritin, the primary constituent of licorice, possesses a wide range of pharmacological and biological properties. This research aims to study the protective mechanism of liquiritin in the myocardium. The potential therapeutic efficacy of liquiritin against acute myocardial infarction (AMI) was tested using molecular docking and verified using an AMI rat model caused by the ligation of the LAD coronary artery. Molecular docking between liquiritin and toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) was predicted using SystemsDock. Then, for experimental validation, in vivo studies were employed. Rats with the AMI model established by ligation of left anterior descending coronary artery were divided into four groups—sham group, model group, captopril group, and liquiritin group. LVSP, LVEDP, +dp/dtmax, and -dp/dtmax were detected and analyzed. HE and Masson staining were used to observe the pathological changes. The protein expressions of TLR4, MyD88, and nuclear factorκB p65 (NF-κB p65) were detected by Western blotting. Molecular docking showed that liquiritin may act on the TLR4 and MyD88, and, therefore, liquiritin was predicted to exert anti-inflammatory effects by regulating the TLR4/MyD88 signaling pathway. Liquiritin improved LVSP, +dp/dtmax, -dp/dtmax, and LVEDP levels, and alleviated pathological changes and cardiac fibrosis. Further study found that liquiritin could decrease the overexpression of TLR4, MyD88, and NF-κB, which validated the molecular docking study. Hence, liquiritin ameliorates AMI by reducing inflammation, and blocking TLR4/MyD88/NF-κB signaling. These results indicate that liquiritin as a potential compound could alleviate AMI and broaden its application.

Circ_ZNF652 regulates the proliferation and apoptosis of LPS-induced WI-38 cells via miR-302e/TLR4 axis

BackgroundCircular RNAs (circRNAs) play an important regulatory role in multiple human diseases, including organ allograft rejection. Infantile pneumonia (IP) is a common disease that seriously threatens the health of infants and young children. CircRNAs have been shown to be involved in the advance of IP. However, the function of circ_ZNF652 in IP has not been fully studied. MethodsLipopolysaccharide (LPS)-treated WI-38 cells were used as cell injury models of IP. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circ_ZNF652, miR-302e and Toll-like receptor 4 (TLR4). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium Bromide (MTT) assay, thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) assay, and flow cytometry assay were utilized to explore cell functions. Western blot was employed to examine the protein levels of PCNA, Bcl-2, Bax, and TLR4. ELISA was used to detect the release of inflammatory cytokines. The relationship between miR-302e and circ_ZNF652 or TLR4 was verified by dual-luciferase reporter assay and RNA pull down assay. ResultsCirc_ZNF652 was significantly up-regulated in serum of IP patients and LPS-induced WI-38 cells. Silencing circ_ZNF652 enhanced cell proliferation and inhibited cell apoptosis in LPS-induced WI-38 cells. MiR-302e was identified as a target of circ_ZNF652, and knockdown of circ_ZNF652 alleviated LPS-induced WI-38 cell injuries by up-regulating miR-302e. In addition, TLR4 was a downstream target of miR-302e. Overexpression of TLR4 recovered cell apoptosis and inflammation that were repressed by miR-302e enrichment in LPS-induced WI-38 cells. ConclusionCirc_ZNF652 regulates the expression of TLR4 by regulating miR-302e, thereby mediating cell proliferation, apoptosis and inflammation. The results provide a novel targeted therapy for IP.

Vitamin D Improves the Effect of Glucocorticoids on Attenuating Lipopolysaccharide-Induced IL-6 Production via TLR4/NF-κB Pathway in Human Respiratory Epithelial Cells

Background: Glucocorticoid (GC) resistance results in unsatisfactory outcomes in chronic rhinosinusitis with nasal polyps (CRSwNP) patients. Previous studies have shown that calcitriol, the active form of vitamin D, alone or in combination with corticosteroids, exerts crucial immunomodulatory effects on inflammatory responses. However, whether vitamin D can improve the effect of GCs to attenuate inflammation in the epithelium of CRSwNP remains unclear. Methods: A human bronchial epithelial cell line (Beas-2B) and primary human nasal epithelial cells (HNECs) obtained from 10 patients with CRSwNP were exposed to lipopolysaccharide (LPS) for 24 h to establish an inflammation model. LPS-stimulated HNECs and Beas-2B cells were treated with/without dexamethasone in the presence or absence of calcitriol pretreatment for 24 h. The expression levels of interleukin-6 (IL-6) mRNA and protein were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Toll-like receptor 4 (TLR4)/NF-κB pathway related markers were examined by western blotting. One-way ANOVA or the Kruskal-Wallis test with post hoc analysis were used for multiple comparisons among groups. Results: The production of IL-6 in Beas-2B cells and primary HNECs after LPS stimulation was significantly increased, which could be inhibited by dexamethasone or calcitriol alone. However, significant inhibition of IL-6 production was observed in the calcitriol plus dexamethasone group. Further analysis showed that calcitriol could enhance the effect of dexamethasone in inhibiting LPS-induced overexpression of TLR4, Myd88, and phosphorylation of p65. Conclusion: Our findings demonstrated that vitamin D could improve the effect of GCs to alleviate the level of IL-6 induced by LPS via the TLR4/NF-κB pathway in human respiratory epithelial cells.

Matrine inhibits advanced glycation end products-induced macrophage M1 polarization by reducing DNMT3a/b-mediated DNA methylation of GPX1 promoter

Advanced glycation end products (AGEs) are characterized diabetic metabolites inducing macrophage M1 polarization which is crucial in diabetes-exacerbated atherosclerosis. Matrine was proved anti-atherosclerotic. The current study was aimed to investigate the inhibitory effects of matrine on AGEs- induced macrophage M1 polarization and underlying molecular mechanisms. Primary mouse macrophages were exposed to AGEs. Receptor for AGEs (RAGE) and toll-like receptor 4 (TLR4) were over-expressed by vectors. Matrine was used to treat these cells. Inducible nitric oxide synthase (iNOS) expression and pro-inflammatory cytokine production were used to evaluate macrophage M1 polarization. Oxidative stress was assessed by intracellular reactive oxygen species (ROS) generation, total antioxidant capacity (TAC) and malondialdehyde (MDA) contents. Relative mRNA expression level was determined by real-time PCR. Western blotting was used to evaluate protein and protein phosphorylation levels. Bisulfite sequencing PCR (BSP) was used to evaluate DNA methylation. Matrine reduced AGEs exposure-elevated expressions of DNA methyltransferase (DNA MTase, DNMT)3a and DNMT3b in macrophages which were not affected by RAGE or TLR4 over expressions. DNA methylation rate of GPX1 promoter was reduced from 97.22% to 66.67% in AGEs- exposed macrophages treated by matrine. GPX1 expression was up-regulated by matrine, which further suppressed AGEs/RAGE-mediated oxidative stress. Thus, the activation of down-stream TLR4/STAT1 signaling pathway was inhibited by matrine treatment which eventually suppressed AGEs- induced macrophage M1 polarization. However, these effects of matrine were impaired by RAGE and TLR4 overexpression. Results from this study suggested that matrine inhibited AGEs- induced macrophage M1 polarization by suppressing RAGE-induced oxidative stress-mediated TLR4/STAT1 signaling pathway. Matrine exerted anti-oxidant effects via increasing GPX1 expression by inhibiting DNMT3a/b-induced GPX1 promoter DNA methylation.