Overexpression Of Toll-like Receptor 4 Research Articles

Involvement of circ_0029407 in Caerulein-Evoked Cytotoxicity in Human Pancreatic Cells via the miR-579-3p/TLR4/NF-κB Pathway.

Acute pancreatitis (AP) is the most prevalent gastrointestinal inflammatory disease. Circular RNAs (circRNAs) are implicated in the development of AP. Here, we identified the precise action of circ_0029407 in AP development. Human pancreatic epithelial cells (HPECs) were stimulated with caerulein. Cell viability, proliferation, and apoptosis were gauged by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays, respectively. Circ_0029407, microRNA (miR)-579-3p, and toll-like receptor 4 (TLR4) were quantified by a qRT-PCR or western blot assay. Dual-luciferase reporter and RNA pull-down assays were performed to evaluate the direct relationship between miR-579-3p and circ_0029407 or TLR4. Our results indicated that circ_0029407 was markedly overexpressed in AP serum samples and caerulein-stimulated HPECs. Reduction of circ_0029407 attenuated caerulein-imposed HPEC damage by promoting cell proliferation and repressing cell apoptosis and inflammation. Mechanistically, circ_0029407 contained a miR-579-3p binding site, and miR-579-3p downregulation reversed the effect of circ_0029407 reduction on caerulein-imposed HPEC damage. TLR4 was identified as a direct and functional target of miR-579-3p, and TLR4 overexpression reversed the impact of miR-579-3p upregulation on attenuating caerulein-imposed HPEC damage. Moreover, circ_0029407 regulated the TLR4/nuclear factor NF-kappaB (NF-κB) signaling by acting as a competing endogenous RNA (ceRNA) for miR-579-3p. Our study suggests that circ_0029407 regulates caerulein-imposed cell injury in human pancreatic cells at least in part via the TLR4/NF-κB signaling pathway by functioning as a ceRNA for miR-579-3p.

Prognostic and clinicopathological significance of TLR4 expression in patients with breast cancer: a meta-analysis.

The prognostic value of Toll-like receptor 4 (TLR4) in breast cancer remains to be determined. Therefore, this paper aims to conduct a meta-analysis to assess the correlation between TLR4 and clinicopathological indicators as well as survival outcomes in breast cancer. Related literature retrieved from Embase, PubMed, Cochrane Library, Web of Science, China National Knowledge Infrastructure (CNKI) and China Wanfang. The search deadline is April 12, 2023. The outcome measures employed in the study comprised hazard ratio (HR), odds ratio (OR), and 95% confidence interval (CI) as effective indices. The data analysis was conducted using Stata 17.0 software. High TLR4 expression was associated with lymph node metastasis (OR=2.077, 95%CI=1.160-3.717, P= 0.014), tumor size (≥2 cm) (OR=2.194, 95%CI= 1.398-3.445, P= 0.001), PR expression (OR = 0.700, 95% CI = 0.505-0.971, P= 0.033), and clinical stage (OR = 3.578, 95%CI= 3.578-5.817, P<0.05), but not with histological grade (95%CI= 0.976-1.735, P= 0.072), ER expression (OR = 1.125, 95% CI = 0.492-2.571,P= 0.781), and HER-2 status (OR = 1.241, 95% CI = 0.733-2.101, P = 0.422). In addition, TLR4 overexpression was an independent prognostic indicator of DFS (HR= 1.480, 95%CI= 1.028- 2.130, p= 0.035) in breast cancer patients, but not related to OS(HR=1.730, 95%CI= 0.979-3.057, P= 0.059). From our main analysis results, high TLR4 expression is associated with lymph node metastasis, larger tumor size (≥2 cm), later clinical stage, negative PR expression and shorter DFS, suggesting poor prognosis in breast cancer patients.

Sinigrin improves cerebral ischaemia-reperfusion injury by inhibiting the TLR4 pathway-mediated oxidative stress.

Cerebral ischaemia-reperfusion (CIR) injury occurs in stroke patients after the restoration of cerebral perfusion. Sinigrin, a phytochemical found in cruciferous vegetables, exhibits strong antioxidant activity. This study investigated the role of sinigrin in oxidative stress using a CIR injury model. The effects of sinigrin were studied in middle cerebral artery occlusion (MCAO) rats and oxygen-glucose deprivation/reoxygenation (OGD/R)-injured SH-SY5Y cells. Sinigrin treatment improved brain injury and neurological deficits induced by MCAO surgery in rats. Sinigrin inhibited apoptosis in brain tissues and SH-SY5Y cells following OGD/R induction. Additionally, sinigrin elevated the levels of superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-Px) while reducing malondialdehyde (MDA) levels. Furthermore, sinigrin inhibited the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signalling pathway. The anti-apoptotic and antioxidant activities of sinigrin in OGD/R-injured SH-SY5Y cells were reversed by TLR4 overexpression. In conclusion, sinigrin inhibits oxidative stress in CIR injury by suppressing the TLR4/MyD88 signalling pathway.

Circ_0087199 depletion attenuates lipopolysaccharides-induced human periodontal ligament cell injury through the miR-527/TLR4 axis.

Circular RNAs participate in the development of periodontitis. The present work aims to reveal the role and mechanism of circ_0087199 in human periodontal ligament cell (PDLC) injury during periodontitis. PDLCs were treated with lipopolysaccharides (LPS) to establish a periodontitis cell model. Quantitative real-time polymerase chain reaction was used to detect the expression of circ_0087199, miR-527, toll-like receptor 4 (TLR4). Western blot analysis assay was performed to assess protein expression. Cell viability, proliferation, apoptosis and inflammation were investigated by cell counting kit-8, EdU assay, flow cytometry and enzyme-linked immunosorbent assay, respectively. Oxidative stress was evaluated by malondialdehyde assay kit and superoxide dismutase activity assay kit. The interaction between miR-527 and circ_0087199 or TLR4 was confirmed by a dual-luciferase reporter assay. Circ_0087199 and TLR4 expression levels were significantly increased, while miR-527 was decreased in the periodontal ligament tissues of periodontitis patients and LPS-stimulated PDLCs when compared with controls. LPS treatment inhibited cell viability and proliferation but induced cell apoptosis, inflammation and oxidative stress, whereas these effects were attenuated after circ_0087199 knockdown. Circ_0087199 bound to miR-527 and regulated LPS-caused PDLC damage by targeting miR-527. Additionally, the overexpression of TLR4, a target gene of miR-527, rescued miR-527 mimic-mediated effects on LPS-treated PDLCs. Further, the regulation of circ_0087199 toward TLR4 involved miR-527. Circ_0087199 knockdown attenuated LPS-induced apoptosis, inflammation and oxidative stress of PDLCs by regulating the miR-527/TLR4 pathway.

Circ_0099630 knockdown alleviates lipopolysaccharide-induced injuries of human periodontal ligament cells through the inhibition of TLR4 by releasing miR-409-3p

BackgroundPeriodontitis triggers tooth loss and affects the health of population worldwide. Emerging evidence hints that circular RNAs (circRNAs) are involved in various diseases, including periodontitis. This study aimed to investigate the role of circ_0099630 in the progression of periodontitis.MethodsPeriodontitis cell model was constructed by treating human periodontal ligament cells (HPDLCs) with lipopolysaccharide (LPS). Quantitative real-time PCR was used to analyze the expression of circ_0099630, microRNA-409-3p (miR-409-3p) and toll-like receptor 4 (TLR4) mRNA. Western blot was used for detecting protein levels of TLR4, cleaved-caspase 3, Bcl-2, CyclinD1 and NF-κB signaling markers. For function analyses, cell proliferation was assessed by CCK-8 assay and EdU assay. The releases of pro-inflammation factors were monitored by ELISA kits. The potential relationship between miR-409-3p and circ_0099630 or TLR4 was verified by dual-luciferase reporter assay, RIP assay and pull-down assay.ResultsThe expression of circ_0099630 and TLR4 was elevated in periodontitis patients and LPS-treated HPDLCs. LPS induced HPDLC proliferation inhibition, apoptosis and inflammatory responses, while circ_0099630 knockdown or TLR4 knockdown alleviated these injuries. Besides, TLR4 overexpression reversed the inhibitory effect of circ_0099630 knockdown on LPS-induced HPDLC injuries. Mechanism analysis showed that circ_0099630 positively regulated TLR4 expression by acting as miR-409-3p sponge. MiR-409-3p restoration largely ameliorated LPS-induced HPDLC injuries by depleting TLR4. Moreover, LPS activated the NF-κB signaling pathway, while circ_0099630 knockdown inhibited the activity of NF-κB signaling via the miR-409-3p/TLR4 axis.ConclusionCirc_0099630 knockdown relieved LPS-induced HPDLC injury by miR-409-3p/TLR4 axis, suggesting that circ_0099630 might be a potential target for periodontitis treatment.

Involvement of endoplasmic reticulum and histone proteins in immunomodulation by TLR4-interacting SPA4 peptide against Escherichia coli.

Pulmonary host defense is critical for the control of lung infection and inflammation. An increased expression and activity of Toll-like receptor 4 (TLR4) induce phagocytic uptake/clearance and inflammation against Gram-negative bacteria. In this study, we addressed the mechanistic aspect of the immunomodulatory activity of the TLR4-interacting SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) against Escherichia coli. Binding of the SPA4 peptide to bacteria and direct anti-bacterial effects were investigated using flow cytometric, microscopic, and bacteriological methods. The bacterial uptake and inflammatory cytokine response were studied in dendritic cells expressing endogenous basal level of TLR4 or overexpressing TLR4. The subcellular distribution and co-localization of TLR4 and bacteria were investigated by immunocytochemistry. Furthermore, we studied the cellular expression and co-localization of endoplasmic reticulum (ER) molecules (calnexin and ER membrane protein complex subunit 1; EMC1) with lysosomal-associated membrane protein 1 (LAMP1) in cells infected with E. coli and treated with the SPA4 peptide. Simultaneously, the expression of histone H2A protein was quantitated by immunoblotting. Our results demonstrate no binding or direct killing of the bacteria by SPA4 peptide. Instead, it induces the uptake and localization of E. coli in the phagolysosomes for lysis and simultaneously suppresses the secreted levels of TNF-α. Overexpression of TLR4 further augments the pro-phagocytic and anti-inflammatory activity of SPA4 peptide. A time-dependent change in subcellular distribution of TLR4 and an increased co-localization of TLR4 with E. coli in SPA4 peptide-treated cells suggest an enhanced recognition and internalization of bacteria in conjugation with TLR4. Furthermore, an increased co-localization of calnexin and EMC1 with LAMP1 indicates the involvement of ER in pro-phagocytic activity of SPA4 peptide. Simultaneous reduction in secreted amounts of TNF-α coincides with suppressed histone H2A protein expression in the SPA4 peptide-treated cells. These results provide initial insights into the plausible role of ER and histones in the TLR4-immunomodulatory activity of SPA4 peptide against Gram-negative bacteria.

Neutrophil extracellular traps aggravate intestinal epithelial necroptosis in ischaemia-reperfusion by regulating TLR4/RIPK3/FUNDC1-required mitophagy.

Neutrophil extracellular trap (NET) has been confirmed to be related to gut barrier injury during intestinal ischaemia-reperfusion (II/R). However, the specific molecular regulatory mechanism of NETs in II/R-induced intestinal barrier damage has yet to be fully elucidated. Here, we reported increased NETs infiltration accompanied by elevated inflammatory cytokines, cellular necroptosis and tight junction disruption in the intestine of human II/R patients. Meanwhile, NETs aggravated Caco-2 intestinal epithelial cell necroptosis, impairing the monolayer barrier in vitro. Moreover, Pad4-deficient mice were used further to validate the role of NETs in II/R-induced intestinal injury. In contrast, NET inhibition via Pad4 deficiency alleviated intestinal inflammation, attenuated cellular necroptosis, improved intestinal permeability, and enhanced tight junction protein expression. Notably, NETs prevented FUN14 domain-containing 1 (FUNDC1)-required mitophagy activation in intestinal epithelial cells, and stimulating mitophagy attenuated NET-associated mitochondrial dysfunction, cellular necroptosis, and intestinal damage. Mechanistically, silencing Toll-like receptor 4 (TLR4) or receptor-interacting protein kinase 3 (RIPK3) via shRNA relieved mitophagy limitation, restored mitochondrial function and reduced NET-induced necroptosis in Caco-2 cells, whereas this protective effect was reversed by TLR4 or RIPK3 overexpression. The regulation of TLR4/RIPK3/FUNDC1-required mitophagy by NETs can potentially induce intestinal epithelium necroptosis.

Downregulation of circ_0035292 Alleviates LPS-Induced WI-38 Cell Injury via Targeting miR-494-3p/TLR4 Pathway in Infantile Pneumonia.

Circular RNAs (circRNAs) have been confirmed to mediate infantile pneumonia development. In this, we investigated the role and new mechanism of circ_0035292 regulating infantile pneumonia progression. Lipopolysaccharide (LPS)-treated WI-38 cells were used to mimic infantile pneumonia cell injury models. Quantitative real-time PCR was used to measure circ_0035292, microRNA (miR)-494-3p and toll-like receptor 4 (TLR4). Cell proliferation and apoptosis were assessed by MTT assay, EdU assay, and flow cytometry. Protein expression was tested using western blot analysis. Inflammation and oxidative stress were evaluated by measuring IL-6, IL-1β, MDA and SOD levels using ELISA assay and corresponding kits. RNA interaction was confirmed by dual-luciferase reporter assay and RIP assay. Circ_0035292 had elevated expression in infantile pneumonia patients and LPS-induced WI-38 cells. Silenced circ_0035292 could enhance WI-38 cell proliferation, while suppress apoptosis, inflammation and oxidative stress under LPS treatment. Mechanically, circ_0035292 targeted miR-494-3p to positively regulate TLR4. The rescue experiments indicated that miR-494-3p inhibitor abolished the function of circ_0035292 knockdown, and TLR4 overexpression reversed the inhibitory effect of miR-494-3p on LPS-induced WI-38 cell injury. Circ_0035292 might be a potential target for infantile pneumonia treatment, which knockdown could relieve LPS-induced cell injury via the regulation of miR-494-3p/TLR4 axis.

TLR4 Overexpression Aggravates Bacterial Lipopolysaccharide-Induced Apoptosis via Excessive Autophagy and NF-κB/MAPK Signaling in Transgenic Mammal Models.

Gram-negative bacterial infections pose a significant threat to public health. Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and induces innate immune responses, autophagy, and cell death, which have major impacts on the body's physiological homeostasis. However, the role of TLR4 in bacterial LPS-induced autophagy and apoptosis in large mammals, which are closer to humans than rodents in many physiological characteristics, remains unknown. So far, few reports focus on the relationship between TLR, autophagy, and apoptosis in large mammal levels, and we urgently need more tools to further explore their crosstalk. Here, we generated a TLR4-enriched mammal model (sheep) and found that a high-dose LPS treatment blocked autophagic degradation and caused strong innate immune responses and severe apoptosis in monocytes/macrophages of transgenic offspring. Excessive accumulation of autophagosomes/autolysosomes might contribute to LPS-induced apoptosis in monocytes/macrophages of transgenic animals. Further study demonstrated that inhibiting TLR4 downstream NF-κB or p38 MAPK signaling pathways reversed the LPS-induced autophagy activity and apoptosis. These results indicate that the elevated TLR4 aggravates LPS-induced monocytes/macrophages apoptosis by leading to lysosomal dysfunction and impaired autophagic flux, which is associated with TLR4 downstream NF-κB and MAPK signaling pathways. This study provides a novel TLR4-enriched mammal model to study its potential effects on autophagy activity, inflammation, oxidative stress, and cell death. These findings also enrich the biological functions of TLR4 and provide powerful evidence for bacterial infection.

Taxifolin ameliorates abdominal aortic aneurysm by preventing inflammation and apoptosis and extracellular matrix degradation via inactivating TLR4/NF-κB axis.

Abdominal aortic aneurysm (AAA) is a serious aortic disease with high mortality. Vascular smooth muscle cells (VSMCs) loss is a prominent feature of AAA. Taxifolin (TXL) is a natural antioxidant polyphenol and possesses therapeutic functions in numerous human diseases. This study aimed to investigate TXL's impact on VSMC phenotype in AAA. In vitro and in vivo of VSMC injury model was induced by angiotensin II (Ang II). The potential function of TXL on AAA was determined using Cell Counting Kit-8, flow cytometry, Western blot, quantitative reverse transcription-PCR, and enzyme-linked immunosorbent assay. Meanwhile, TXL mechanism on AAA was checked by a series of molecular experiments. Also, TXL function on AAA in vivo was further evaluated using hematoxylin-eosin staining, TUNEL assay, Picric acid-Sirius red staining and immunofluorescence assay in C57BL/6 mice. TXL alleviated Ang II-induced VSMC injury mainly by enhancing VSMC proliferation and weakening cell apoptosis, alleviating VSMC inflammation, and reducing extracellular matrix (ECM) degradation of VSMCs. Furthermore, mechanistic studies corroborated that TXL reversed the high levels of Toll-like receptor 4 (TLR4) and p-p65/p65 induced by Ang II. Also, TXL facilitated VSMC proliferation and reduced cell apoptosis, repressed inflammation, and ECM degradation of VSMCs, while these effects were reversed by TLR4 overexpression. In vivo studies further confirmed that TXL owned the function of alleviating AAA, such as alleviating collagen fiber hyperplasia and inflammatory cell infiltration in AAA mice, and repressing inflammation and ECM degradation. TXL protected VSMCs against Ang II-induced injury through activating TLR4/noncanonical nuclear factor-kappaB(NF-κB).

Effects of toll like receptor 4 overexpression on blood parameters, body composition, and gastrointestinal microbial diversity in sheep

Genetic modifications can improve the health status and disease resistance of farm animals. In the previous study, genetically modified (GM) sheep overexpressing toll like receptor 4 (TLR4) were produced. The GM sheep had stronger ability to eliminate invasive microbes compared to the wild-type (WT) sheep. Physiological status and immune homeostasis, blood parameters, body composition, and gastrointestinal microbial diversity were evaluated in this study to elucidate the effects of TLR4 overexpression. In a set of 10 GM rams, the TLR4 mRNA and protein expression levels in the peripheral blood mononuclear cells, muscle, kidney, and spleen were higher than those of the WT (n = 10; P < 0.05). No significant differences in the parameters of red blood cells, white blood cells, platelets and in the composition of circulating T lymphocyte subsets were observed between the GM and WT sheep (P > 0.05). Of the biochemical parameters, only total protein levels differed (were higher) in the GM sheep than in the WT sheep (P < 0.05) while no significant differences were observed for the other indices (P > 0.05). GM and WT sheep showed similar offal weights and muscle fatty acid and amino acid compositions (P > 0.05). Furthermore, TLR4 overexpression did not affect the community structure of the gastrointestinal tract microbiota. Eleven KEGG pathways associated with the TLR4 gene, physiology and biochemistry, growth and metabolism, fatty acids, and amino acids did not demonstrate significant differences between the GM and WT sheep (P > 0.05). In conclusion, TLR4 overexpression had no principal or adverse effects on the physiological development and health of sheep, except for the general mRNA and protein expression levels.

Toll-Like Receptor 4 Induction During Renal Ischemia Correlates with Serum Creatinine in a Pig Model.

Objectives: To determine whether toll-like receptor 4 (TLR4), a mediator of organ ischemia-reperfusion injury, is overexpressed during warm ischemia in a porcine solitary kidney model, and whether its expression correlates with creatinine, a surrogate for kidney function. Materials and Methods: Eight adult Yorkshire pigs underwent initial laparoscopic nephrectomy. After 1 week, animals were randomized into two groups: group 1 underwent laparoscopic renal hilar dissection, renal ischemia by cross-clamping, and reperfusion (ischemia group); group 2 underwent laparoscopic renal hilar dissection alone (sham group). Animals were survived to day 7 postrandomization. Peripheral blood was sampled for serum creatinine (sCr) and TLR4 expression at the following time points or corresponding intervals: prenephrectomy, 1-week postnephrectomy (preischemia), after 90 minutes of ischemia, 30 minutes postreperfusion, and at sacrifice. Intragroup TLR4 expression changes were analyzed using repeated measures ANOVA. Intergroup TLR4 expression was compared using Mann-Whitney's test. Correlation between sCr and TLR4 was assessed using Spearman's test. Results: Seven animals completed the experiment (four ischemia and three sham). Relative TLR4 expression significantly increased from baseline levels during ischemia, reperfusion, and sacrifice time points only in the ischemia group, and was significantly higher for the ischemia group after 90 minutes of ischemia (p = 0.034). sCr was significantly higher for the ischemia group during the reperfusion phase (p = 0.048). Relative TLR4 expression level significantly correlated with sCr in the overall cohort (Spearman's rho = 0.69) and in the ischemia group (Spearman's rho = 0.82; p < 0.0001 for each). Conclusions: Warm ischemia in a porcine solitary kidney induces acute overexpression of TLR4 in peripheral blood leukocytes, which is detectable. Relative TLR4 expression level strongly correlated with sCr but had an observable change sooner than change in sCr. Pending further investigation, TLR4 overexpression during renal ischemia may represent a sensitive quantitative marker of unilateral renal injury sustained during nephron-sparing surgery.

HMGB1-mediated-TLR4/MyD88 signaling regulates cell proliferation, migration and invasion in esophageal squamous cell carcinoma

Purpose: To study the effect of high mobility group box protein 1 (HMGB1)-mediated toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) on esophageal squamous cell carcinoma (ESCC). &#x0D; Methods: Cell viability was determined using CCK-8 test and colony formation assay, while cell migration and invasion were assessed by Transwell assay. Western blotting was used to determine protein and mRNA expression levels. The effect of TLR4/MyD88 signaling pathway on proliferation of ESCC cells was investigated via in vitro knockdown of HMGB1. &#x0D; Results: Data from in vitro experiments indicated that HMGB1 knockdown significantly decreased the proliferation, migration, and invasion of EC9706 cells in siHMGB1 group, when compared with siNC group (number of invasive cells in siHMGB1 vs. corresponding number in siNC: 26.7 ± 4.5 vs. 68.7 ± 2.5; p &lt; 0.01), and also decreased proliferation, migration and invasion of TE-9 cells in siHMB1, relative to siNC group (number of invasive cells in siHMGB1 vs. corresponding number in siNC: 29.3 ± 3.5 vs. 55.7 ± 3.1; p &lt; 0.01). Moreover, MGB1 knockdown via siTLR4 transfection significantly down-regulated the expressions of TLR4/MyD88 and epithelial-mesenchymal transition (EMT), but over-expression of TLR4 reversed the inhibition by HMGB1 knockdown on ESCC cells (p &lt; 0.01). &#x0D; Conclusion: These findings suggest that HMGB1-mediated TLR4/MyD88 signal pathway is a potential treatment route for ESCC.

Deletions of MGF110-9L and MGF360-9L from African swine fever virus are highly attenuated in swine and confer protection against homologous challenge

African swine fever, caused by a large icosahedral DNA virus (African swine fever virus, ASFV), is a highly contagious disease in domestic and feral swine, thus posing a significant economic threat to the global swine industry. Currently, there are no effective vaccines or the available methods to control ASFV infection. Attenuated live viruses with deleted virulence factors are considered to be the most promising vaccine candidates; however, the mechanism by which these attenuated viruses confer protection is unclear. Here, we used the Chinese ASFV CN/GS/2018 as a backbone and used homologous recombination to generate a virus in which MGF110-9L and MGF360-9L, two genes antagonize host innate antiviral immune response, were deleted (ASFV-ΔMGF110/360-9L). This genetically modified virus was highly attenuated in pigs and provided effective protection of pigs against parental ASFV challenge. Importantly, we found ASFV-ΔMGF110/360-9L infection induced higher expression of Toll-like receptor 2 (TLR2) mRNA compared with parental ASFV as determined by RNA-Seq and RT-PCR analysis. Further immunoblotting results showed that parental ASFV and ASFV-ΔMGF110/360-9L infection inhibited Pam3CSK4-triggered activating phosphorylation of proinflammatory transcription factor NF-κB subunit p65 and phosphorylation of NF-κB inhibitor IκBα levels, although NF-κB activation was higher in ASFV-ΔMGF110/360-9L-infected cells compared with parental ASFV-infected cells. Additionally, we show overexpression of TLR2 inhibited ASFV replication and the expression of ASFV p72 protein, whereas knockdown of TLR2 had the opposite effect. Our findings suggest that the attenuated virulence of ASFV-ΔMGF110/360-9L might be mediated by increased NF-κB and TLR2 signaling.

MiR-21 alleviates renal tubular epithelial cells injury induced by ischemia by targeting TLR4

Renal ischemia is the initial stage of kidney damage, leading to mitochondrial metabolism disorders and cell necrosis. In this study, we aimed to investigate the biological functions and potential mechanisms of miR-21 in protecting renal tubular epithelial cells from oxidative stress and apoptosis following oxygen glucose deprivation (OGD). Following an OGD injury, miR-21 levels increased in HK-2 renal tubular epithelial cells. Overexpression of miR-21 decreased the protein expressions of cleaved caspase-3, BAX, P53, cell apoptosis and increased Bcl-2 expression in HK-2 cells with OGD injury. In vivo studies found that miR-21 agomir reduced renal tissue apoptosis, while miR-21 antagomir increased it. In addition, overexpression of miR-21 reduced levels of reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) in HK-2 cells with OGD injury. However, miR-21 inhibition exhibited the opposite effect. A dual-luciferase reporter assay demonstrated that miR-21 directly regulates Toll-like receptor 4 (TLR4) by targeting the 3′-UTR of TLR4 mRNA. Overexpression of miR-21 led to decreased TLR4 protein expression, and TLR4 knockdown was shown to greatly increase AKT activity in HK-2 cells by in vitro kinase assay. Additionally, TLR4 knockdown promoted AKT phosphorylation and hypoxia-inducible factor-1α (HIF-1α) expression, while TLR4 overexpression inhibited these processes. Furthermore, AKT activation abolished the effect of TLR4 on HIF-1α, while AKT inhibition decreased the expression of TLR4 on HIF-1α in TLR4 knockdown HK-2 cells. Further study revealed that HIF-1α inhibition abolished the protective effect of miR-21 overexpression on ROS, LDH levels and cell apoptosis in HK-2 cells after OGD injury, which is indicated by increased levels of ROS and LDH, as well as increased cell apoptosis after HIF-1α inhibition in miR-21-treated HK-2 cells. In conclusion, miR-21 defends OGD-induced HK-2 cell injury via the TLR4/AKT/HIF-1α axis.

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.