Overexpression Of Toll-like Receptor 4 Research Articles

Glycolipid toxicity induces osteogenic dysfunction via the TLR4/S100B pathway

Diabetes can cause bone metabolism disorders and osteoporosis. The occurrence of both diabetes mellitus and osteoporosis increases the disability and mortality of elderly individuals due to pathological fracture. Abnormal metabolism of nutrientsis considered to be one of the important mechanisms of diabetes mellitus-induced osteoporosis. This study preliminarily explored the roles of TLR4 (Toll-like receptor 4) and S100B in osteogenic dysfunction induced by glycolipid toxicity. In this study, a diabetic rat model and TLR4-knockdown diabetic rat model were used in vivo. MC3T3-E1 cells in a high glucose and palmitic acid environment were used as glycolipid toxicity cell models in vitro. We investigated the effects of TLR4 and S100B on osteogenesis by overexpression or inhibition of TLR4 and S100B in vitro. We found that when TLR4 or S100B was inhibited, ALP and OCN were significantly up-regulated and p-ERK was significantly down regulated in the glycolipid model. These results suggest that TLR4/S100B may play a role in reducing glycolipid toxicity by regulating ERK phosphorylation.

Rat plasma protein binding of kaempferol-3-O-rutinoside from Lu'an GuaPian tea and its anti-inflammatory mechanism for cardiovascular protection.

Previous study found a high content of kaempferol-3-O-rutinoside (KR) in Lu'an GuaPian tea, however, the rat plasma protein binding and mechanism of KR for cardiovascular protection are unclear. Thus, we studied plasma protein binding using ultrafiltration followed by UPLC, and screened its inhibition against LPS-induced inflammation injury in vitro as well as the underlying mechanism by molecular docking and western blot. KR showed over 74% plasma protein binding ratio. Furthermore, KR may act on the toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). In vitro experiments showed that KR decreases the overexpression of TLR4, MyD88, and nuclear factor-κB (NF-κB), which further validates the molecular docking results, suggesting that KR could block TLR4/MyD88/NF-κB signaling. These results indicate that KR could be a potential active agent in the protection of myocardial injury. PRACTICAL APPLICATIONS: Health benefits of tea are largely dependent on the intake of flavonoids. Flavonoids are a group of compounds beneficial to cardiovascular disease and an important part of "functional foods." Lu'an GuaPian tea is mainly produced in Lu'an City, Anhui Province and is one of the top 10 famous teas in China. Kaempferol-3-O-rutinoside in Lu'an GuaPian has good hypoglycemic effect, mainly manifested in a strong inhibition of α-glucosidase and α-amylase activities. Present study showed that kaempferol-3-O-rutinoside could block TLR4/MyD88/NF-κB signaling, suggesting that it could be a potential active agent in the protection of myocardial injury.

LncRNA GAS5‑mediated miR‑23a‑3p promotes inflammation and cell apoptosis by targeting TLR4 in a cell model of sepsis.

Sepsis is a syndrome characterized by organ dysfunction and an abnormal immune response to infection. A growing body of research has shown the importance of long non-coding RNAs (lncRNAs) in tumorigenesis, virus replication, inflammatory injury and other pathological processes. The aim of the present study was to explore the role and potential mechanism of the lncRNA growth arrest-specific 5 (GAS5) in the lipopolysaccharide (LPS)-induced inflammation and apoptosis of THP-1 cells. An in vitro sepsis model was established by treating THP-1 cells with LPS. Apoptosis was detected by flow cytometry. The expression levels of IL-6, IL-1β and TNF-α were detected using reverse transcription-quantitative PCR (RT-qPCR) and ELISA, and those of GAS5, microRNA (miR)-23a-3p and Toll-like receptor 4 (TLR4) were detected by RT-qPCR. The changes in the biological activity of THP-1 cells induced by the silencing of GAS5 and overexpression of miR-23a-3p and TLR4 were investigated. The relationships among GAS5, miR-23a-3p and TLR4 were analyzed using luciferase reporter assays. The results revealed that LPS increased the expression of GAS5 in THP-1 cells, and GAS5 knockdown effectively inhibited inflammation and cell apoptosis in the LPS-induced sepsis model. In addition, the results of the luciferase reporter assays indicated that both GAS5 and TLR4 directly target miR-23a-3p. The expression of miR-23a-3p was downregulated whereas that of TLR4 was upregulated in the septic cells. Further experiments showed that the overexpression of TLR4 attenuated the suppressive effects of miR-23a-3p overexpression and GAS5 knockdown on LPS-induced inflammation and apoptosis. In conclusion, the present study indicates that GAS5 strengthens LPS-induced inflammation and apoptosis via the miR-23a-3p/TLR4 pathway.

Overexpression of Toll-like receptor 4 contributes to the internalization and elimination of Escherichia coli in sheep by enhancing caveolae-dependent endocytosis

BackgroundGram-negative bacterial infections have a major economic impact on both the livestock industry and public health. Toll-like receptor 4 (TLR4) plays a crucial role in host defence against Gram-negative bacteria. Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections. In a previous study, we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance. The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood.ResultsIn the present study, we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model. The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection. This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis. Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4, Src signalling, or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria, weakened the ability of the monocytes to eliminate the bacteria, and increased the pH of the endosomes.ConclusionTogether, our results reveal the effects of TLR4 on the control of E. coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model, providing theoretical support for breeding disease resistance in the future. Furthermore, Src and caveolin 1 (CAV1) could be potentially valuable targets for the control of infectious diseases.

Combined protective effects of icariin and selenomethionine on novel chronic tubulointerstitial nephropathy models in vivo and in vitro.

Chronic tubulointerstitial nephropathy (CTIN) is one of the most common kidney diseases. However, treatment for CTIN has multiple limits. Adjuvant therapy through nutritional regulation has become a hot research topic at present. Icariin (ICA), an extraction of Chinese herbal medicine epimedium, has many pharmacological functions including anti-inflammation and tonifying kidney. Selenomethionine (SeMet) possesses the effects of antioxidant and lightening nephrotoxicity. However, little is known about the combined nephroprotection of them. This study was investigated to evaluate the joint effects of ICA and SeMet on CTIN and explore the mechanism. Based on a novel CTIN model developed in our previous study, mice were randomly divided into five groups (a: control; b: model; c: model + ICA; d: model + SeMet; e: model + ICA + SeMet). Renal tubule epithelial cells were treated with cyclosporine A and ochratoxin A without/with ICA or/and SeMet. The results showed that ICA or/and SeMet ameliorated CTIN by inhibiting the uptrends of blood urine nitrogen, serum creatinine, urine protein, urine gravity, histopathological damage degree and collagen I deposition. ICA or/and SeMet also increased cell proliferation and decreased apoptosis and the expression of transforming growth factor-beta 1 and α-smooth muscle actin. Emphatically, ICA and SeMet joint had better nephroprotection than alone in most indexes including fibrosis. Furthermore, ICA and SeMet joint decreased the activation of toll-like receptor 4 (TLR4)/NFκB pathway induced by CTIN. TLR4 overexpression counteracted the joint protection of ICA and SeMet. Therefore, ICA and SeMet in combination could protect against CTIN through blocking TLR4/NFκB pathway. The study will provide novel insights to explore an adjuvant therapeutic orientation.

Swimming exercise improves gene expression of PPAR-γ and downregulates the overexpression of TLR4, MyD88, IL-6, and TNF-α after high-fat diet in rat skeletal muscle cells.

Exercise training with anti-inflammatory effects can improve insulin sensitivity in muscle tissue. This study investigated the effects of eight-week swimming exercises on lipid profile, toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and peroxisome proliferator-activated receptor gamma (PPAR-γ) in gastrocnemius muscle of rats fed with high-fat diet (HFD). Thirty-two healthy male Wistar rats (8weeks, 200±20g) were randomly divided into four groups (n=8 each group): the control (C), aerobic exercise (E), HFD, and HFD+aerobic exercise (HFD & E). The exercise training protocol consisted of swimming 60min/day, 5days/week for eight weeks. Serum levels of glucose, insulin, and lipid profile were measured at end of the study. Protein expressions of TLR4, TNF-α, and IL-6 were determined by immunohistochemical method. Gene expression of TLR4/MyD88, TNF-α, IL-6, and PPAR-γ was evaluated by a real-time polymerase chain reaction in gastrocnemius muscle. HFD fed rats showed higher levels of cholesterol and LDL-c that were similar in weight gain. Meanwhile, the HFD group had a higher gene expression of TLR4, MyD88, TNF-α, IL-6, and lower gene expression of PPAR-γ compared to the control group (p<0.05). Muscle protein expression of TLR4, TNF-α, IL-6 was lower in the E and HFD&E groups (especially when compared to HFD group, P<0.05). We also showed a decrease in TLR4/MyD88 mRNA and an increase in PPAR-γ mRNA in gastrocnemius of E and HFD&E groups (compared to HFD group, p<0.05). Insulin resistance in HFD&E groups show a significant decrease compared to the HFD group (p<0.05). It seems that swimming aerobic exercise for eight weeks controlled the destructive effects of HFD on muscle inflammatory pathways along with the down-regulation of the TLR4/MyD88, inflammatory cytokine, and up-regulation PPAR-γ mRNA. It appears that the down-regulation in the expression of TLR4/MyD88 mRNA reduces the muscle pro-inflammatory cytokines, such as IL-6 and TNF-α, whose action may be caused by the adaptation of swimming aerobic exercise (an increase of PPAR-γ). Therefore, local and systemic inflammatory changes due to HFD and obesity may be affected by metabolic adaptations of aerobic exercise training, which requires further studies.

Remote ischaemic perconditioning reduces the infarct volume and improves the neurological function of acute ischaemic stroke partially through the miR-153-5p/TLR4/p65/IkBa signalling pathway.

Remote ischemic perconditioning (RIPerC) could improve neuronal damage and inhibit inflammation and apoptosis. We conducted an in-depth exploration of the protective mechanism of RIPerC in cerebral ischaemia injury. In this study, a middle cerebral artery occlusion (MCAO) mouse model was built. According to whether to undergo RIPerC treatment and the duration of cerebral infarction, mice were divided into 5 groups: Sham group, MCAO 3.0 h group, MCAO 4.5 h group, MCAO 3.0 h + RIPerC group, and MCAO 4.5 h + RIPerC group. Overexpressed or silenced miR-153-5p was transfected into the cells to analyse the effects of oxygen-glucose deprivation (OGD) treatment on Neuro-2a cell viability, apoptosis, and related gene expressions by performing quantitative real-time polymerase chain reaction (qRT-PCR), MTT assay, flow cytometry, and Western blot. Bioinformatics analysis, qRT-PCR, dual-luciferase experiment, and RNA immunoprecipitation (RIP) were used to screen and verify the miRNA and downstream mRNA-targeted Toll-like receptor 4 (TLR4). The rescue test further verified the effects of the above target genes and miR-153-5p on the apoptosis of OGD-injured cells, apoptosis-related proteins, and the p65/IkBa pathway. The plasma levels of miR-153-5p in 68 patients with ischaemic stroke were detected within 6 hours of onset, and the patients were followed up for 3 months. We found that, in in vivo studies, RIPerC treatment inhibits cerebral infarction volume and neurological damage, and promotes the expression of miR-153-5p in the MCAO animal model. The expression of miR-153-5p in OGD cells was inhibited, and its upregulation protected Neuro-2a cells. TLR4 was predicted to be the target gene of miR-153-5p and could offset the effect of miR-153-5p mimic on OGD cell protection after up-regulating TLR4. TLR4 overexpression promoted the activation of OGD on the p65/IkBa pathway. Compared with the high plasma miR-153-5p group, the 3-month overall survival rate of patients with ischaemic stroke in the low plasma miR-153-5p group was significantly lower (c2 = 5.095, p = 0.024). In conclusion, RIPerC intervention inhibits the damage caused by cerebral ischaemia partially through the miR-153-5p/TLR4/p65/IkBa signalling pathway.

Atractylenolide-I Suppresses Tumorigenesis of Breast Cancer by Inhibiting Toll-Like Receptor 4-Mediated Nuclear Factor-κB Signaling Pathway.

Background: Toll-like receptor 4 (TLR4) is an essential sensor related to tumorigenesis, and overexpression of TLR4 in human tumors often correlates with poor prognosis. Atractylenolide‐I (AT-I), a novel TLR4-antagonizing agent, is a major bioactive component from Rhizoma Atractylodes Macrocephalae. Emerging evidence suggests that AT-I exerts anti-tumor effects on various cancers such as colorectal cancer, bladder cancer and melanoma. Nevertheless, the effects of AT-I on mammary tumorigenesis remain unclear. Methods: In order to ascertain the correlation of TLR4/NF-κB pathway with breast cancer, the expression of TLR4 and NF-κB in normal breast tissues and cancer tissues with different TNM-stages was detected by human tissue microarray and immunohistochemistry technology. The effects of AT-I on tumorigenesis were investigated by cell viability, colony formation, apoptosis, migration and invasion assays in two breast cancer cells (MCF-7 and MDA-MB-231), and N-Nitroso-N-methylurea induced rat breast cancer models were developed to evaluate the anti-tumor effects of AT-I in vivo. The possible underlying mechanisms were further explored by western blot and ELISA assays after a series of LPS treatment and TLR4 knockdown experiments. Results: We found that TLR4 and NF-κB were significantly up-regulated in breast cancer tissues, and was correlated with advanced TNM-stages. AT-I could inhibit TLR4 mediated NF-κB signaling pathway and decrease NF-κB-regulated cytokines in breast cancer cells, thus inhibiting cell proliferation, migration and invasion, and inducing apoptosis of breast cancer cells. Furthermore, AT-I could inhibit N-Nitroso-N-methylurea-induced rat mammary tumor progression through TLR4/NF-κB pathway. Conclusion: Our findings demonstrated that TLR4 and NF-κB were over expressed in breast cancer, and AT-I could suppress tumorigenesis of breast cancer via inhibiting TLR4-mediated NF-κB signaling pathway.

Silencing TLR4/MyD88/NF-κB Signaling Pathway Alleviated Inflammation of Corneal Epithelial Cells Infected by ISE.

The regulatory role of toll-like receptor 4 (TLR4) in the inactivate staphylococcus epidermidis (ISE)-induced cornea inflammation is not well investigated. Here, TLR4 silence could decrease inflammatory cytokines in corneal epithelial cells treated with ISE. The mouse corneal epithelial cells were exposed to ISE for 24 h, either alone or with the NF-κB inhibitor, TLR4 lentivirus to bilaterally (knock-down or and overexpression). The expression of TLR4 in mouse corneal epithelial cells was investigated using western blot and qRT-PCR assay. The inflammatory cytokine levels were evaluated by qRT-PCR and ELISA, respectively. The relative impact factors of TLR4-mediated NF-κB signaling detected using western blot assay. Results show the expression levels of TLR4 and some inflammatory cytokines were significantly increased in corneal epithelial cells treated with ISE. TLR4 Silence markedly decreased ISE-induced production of IL12, TNF-α, CCL5, and CCL9 in corneal epithelial cells. Furthermore, the nuclear translocation of NF-κB p65 and myeloid differentiation protein 88 (MyD88) in the cells treated with ISE were further reduced by silencing TLR4. Inhibition of TLR4-mediated NF-κB signaling by using BAY11-7082 also alleviated ISE-induced inflammation. In the rescue experiment, transfected the stable TLR4 silenced corneal epithelial cells with TLR4 overexpression lentivirus, we found that TLR4 overexpression can restore the down-regulation of TLR4 and inflammatory cytokines (IL12, TNF-α, CCL9) caused by TLR4 knocked down. Therefore, ISE-induced cornea inflammation was due to the activation of the TLR4/MyD88/NF-κB signaling pathway, and dramatically stimulated IL12, TNF-α, CCL9 secretion. TLR4 silence presented mitigates damage in corneal epithelial cells treated with ISE.

Inflammation of Conduction Tissue in Patients with Arrhythmic Phenotype of Myocarditis.

Background: Myocarditis can manifest with lone ventricular tachyarrhythmias (LVT). Elective inflammation of conduction tissue (CT) is supposed but unproved. Methods: Forty-two of 420 patients with biopsy proven myocarditis presented with LVT. Twelve of them included CT sections in endomyocardial biopsies. Real-time polymerase chain reaction (PCR) for viral genomes, immunohistochemistry for viral antigens and Toll like receptor 4 (TLR4) were performed. Twelve myocarditis patients with infarct-like or cardiomyopathic phenotype and CT included in tissue section were used as controls. Results: Four of the 12 patients presented non-sustained ventricular tachycardia (nsVT), six with sustained ventricular tachycardia (sVT), two with ventricular fibrillation. CT was inflamed in all LVT patients and not in controls (p < 0.001). PCR was positive for influenza-A virus in two, herpes simplex virus type 2 (HSV2) in one and adenovirus in one with positive CT immunostaining for viral antigens. In eight patients, negative PCR and TLR4 overexpression suggested an immune-mediated pathway. Patients with influenza-A myocarditis and CT infection responded to oseltamivir, those with HSV2 (Herpes Virus 2) and adenovirus infection died. The eight patients with immune-mediated myocarditis were treated with steroids and azathioprine. Seven of them had no more VT(ventricular tachyarrhythmias)during six-month follow-up. Conclusions: Arrhythmic phenotype of myocarditis is associated with CT inflammation/infection. Molecular characterization of CT damage may lead to pharmacologic control of arrhythmias in 75% of cases.

MiR-145 Regulates the Chemoresistance of Hepatic Carcinoma Cells Against 5-Fluorouracil by Targeting Toll-Like Receptor 4.

Background5-fluorouracil (5-FU) is a common drug for hepatic carcinoma (HCC), but the drug resistance of clinical chemotherapy restricts its use. Studies have demonstrated that miRNA molecules can act as a chemoresistance regulator in drug resistance of tumors, whereas the role of miR-145 in the 5-FU-resistant HCC remains unclear.ObjectiveTo explore the prognostic value of miR-145 in HCC and its molecular mechanism in 5-FU-resistant HCC cells.MethodsA qRT-PCR assay was conducted to quantify miR-145 in HCC tissues and 5-FU-resistant HCC cells. The Cell Counting Kit-8 (CCK-8) and flow cytometry were adopted to analyze the proliferation and apoptosis of 5-FU-resistant HCC cells. The Western blot was adopted to quantify toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and apoptosis-related proteins. Moreover, an in vivo tumor xenotransplantation of nude mice was conducted to determine the effect of miR-145 on 5-FU-resistant HCC cells.ResultsMiR-145 was expressed lowly in HCC tissues and cells, and linked to high TNM staging and lymph node metastasis of HCC patients. Down-regulation of miR-145 indicated a poorer prognosis and it promoted drug resistance of HCC cells and inhibited cell apoptosis. In contrast, miR-145 overexpression improved the sensitivity of HCC cells to 5-FU and enhanced the inhibition of 5-FU on tumor growth. The luciferase reporter gene assay showed that TLR4 was the direct target of miR-145, and the Western blot assay revealed that overexpression of TLR4 reversed the inhibitory effect of miR-145 overexpression on TLR4 and MyD88 protein and the effects of it on apoptosis-related proteins.ConclusionMiR-145 is an inhibiting factor in HCC and can target TLR4 to mediate the chemoresistance of HCC, which may provide novel ideas for treating HCC.

Autophagy inhibits TLR4-mediated invasiveness of oral cancer cells via the NF-κB pathway.

Toll-like receptor 4 (TLR4) is abnormally expressed in oral cancer tissues and promotes cancer cell invasion. The purpose of this study was to clarify the mechanism by which autophagy regulates oral cancer invasion through the TLR4-NF-κB pathway. We examined TLR4 expression in oral cancer tissues and analysed the relationship between its expression and clinicopathological features. The invasion and migration of LPS-stimulated oral cancer cells with up- or downregulation of TLR4 expression was detected in addition to NF-κB signalling and autophagy levels. Furthermore, the role of autophagy in regulating TLR4-mediated cell invasiveness was explored by silencing the expression of key autophagy genes ATG7 and p62. We found that TLR4 overexpression was closely related to cervical lymphatic metastasis and poor survival. TLR4 activated the NF-κB pathway to promote the invasiveness of OSCC cells, and autophagy partly inhibited invasiveness by suppressing the NF-κB pathway. We observed that p62 translocated from the cytoplasm to the nucleus when autophagy was activated by LPS. Finally, silencing p62 further promoted LPS-mediated cell invasiveness. Toll-like receptor 4 significantly enhanced the invasiveness of OSCC cells. Autophagy may regulate cell invasiveness through the NF-κB pathway by modulating both the cytoplasmic and nuclear levels of p62.

Macrophage Membrane-Coated Nanoparticles Alleviate Hepatic Ischemia-Reperfusion Injury Caused by Orthotopic Liver Transplantation by Neutralizing Endotoxin.

PurposeTo investigate the effect and mechanism of macrophage membrane-coated nanoparticles (M-NPs) on hepatic ischemia-reperfusion injury (I/RI) caused by orthotopic liver transplantation. In addition, the advantages of TLR4+/M-NPs compared to M-NPs are discussed.Materials and MethodsWe prepared biomimetic M-NPs and identified their characteristics. M-NPs were injected into an SD rat model of orthotopic liver transplantation, and the anti-inflammatory and anti-I/RI activities of M-NPs were studied in vivo and in vitro. In addition, we overexpressed macrophage membrane Toll-like receptor 4 (TLR4) in vitro and prepared TLR4+/M-NPs. Then, we assessed the characteristics and advantages of TLR4+/M-NPs.ResultsThe M-NPs neutralized endotoxin, inhibited the overactivation of Kupffer cells (KCs) and suppressed the secretion of inflammatory factors by inhibiting the endotoxin-mediated TLR4/MyD88/IRAK1/NF-κB signaling pathway. In an orthotopic liver transplantation model in SD rats, M-NPs showed significant therapeutic efficacy by neutralizing endotoxin and suppressing the secretion of inflammatory factors. Moreover, overexpression of TLR4 on the macrophage membrane by using a TLR4+-plasmid in vitro effectively reduced the amount of M-NPs needed to neutralize an equivalent dose of endotoxin, reducing the potential risks of NP overuse.ConclusionThis study indicates that M-NPs can effectively alleviate I/RI induced by liver transplantation.

MiR‑199a‑3p suppresses cervical epithelial cell inflammation by inhibiting the HMGB1/TLR4/NF‑κB pathway in preterm birth.

Preterm birth (PTB) is the primary cause of neonatal mortality worldwide. Infection and inflammation are considered to be the primary causes of PTB. Cervical remodeling is an important step in the process of preterm delivery, and the destruction of the cervical epithelial barrier and inflammation are important triggers of cervical remodeling. The aim of the present study was to determine the effect and underlying mechanism of microRNA (miR)-199a-3p/high-mobility group box 1 protein (HMGB1) signaling in cervical epithelial inflammation in PTB. The results of this study revealed that miR-199a-3p was significantly decreased in cervical epithelial tissue samples from patients in both the preterm labor and preterm premature rupture of membrane groups. This decrease was also observed in tissue samples from a lipopolysaccharide (LPS)-induced PTB mouse model and in LPS-induced ectocervical and endocervical cells. Whereas, the expression of HMGB1 and toll-like receptor 4 (TLR4) was significantly increased, which was associated with the upregulation of interleukin (IL)-1β and tumor necrosis factor (TNF)-α expression. Furthermore, overexpression of miR-199a-3p significantly suppressed the expression and activation of HMGB1 and TLR4/NF-κB signaling, and decreased the levels of IL-1β and TNF-α in vitro and in vivo. Additionally, overexpression of HMGB1 and/or TLR4 reversed the anti-inflammatory effects of miR-199a-3p mimics in vitro and in vivo. These results indicate that miR-199a-3p acts as a negative inflammatory regulator in PTB by targeting HMGB1 to regulate the TLR4/NF-κB pathway.

Overexpression of Toll-Like Receptor 4 Affects Autophagy, Oxidative Stress, and Inflammatory Responses in Monocytes of Transgenic Sheep.

Toll-like receptor 4 (TLR4) is a critical pattern recognition receptor that plays a critical role in the host innate immune system’s recognition of Gram-negative bacteria. Since it is the lipopolysaccharide (LPS) receptor, it links the activated inflammatory response with autophagy and oxidative stress. Autophagy, or type II programmed cell death, was reported to have defensive functions in response to the production of inflammatory cytokines and oxidative stress. To explore the relationship between autophagy, inflammation, and oxidative stress, a TLR4-enriched transgenic (Tg) animal model (sheep) was generated. Autophagy activity in the Tg blood monocytes was significantly higher than in the wild-type animal under LPS stress, and it returned to normal after transfection of TLR4 siRNA. Pretreatment with 3-methyladenine (3-MA) inhibited autophagy and enhanced oxidative stress and the production of TNF-α. The LPS-induced reactive oxygen species (ROS) level was markedly increased in the Tg group at an early stage before quickly returning to normal values. In addition, suppressing ROS production by N-acetyl-L-cysteine down-regulated the number of intracellular autophagosomes and the expression of Beclin-1, ATG5, and cytokines IL-1β, IL-6, and TNF-α. Further mechanistic investigation suggested that the TLR4-associated p38 mitogen-activated protein kinase (MAPK) signaling pathway was involved in autophagy and oxidative stress. P38 MAPK promotes intracellular autophagy, ROS production, and inflammatory response. Moreover, TLR4 over-expression suppressed oxidative stress and the production of inflammatory cytokines and increased autophagy activity in vivo. Taken together, our results showed that LPS induced autophagy, which was related to TLR4-mediated ROS production through the p38 MAPK signaling pathway. In addition, our study also provided a novel transgenic animal model to analyze the effects of TLR4 on autophagy, oxidative stress, and inflammatory responses.

MicroRNA-421 protects against chronic intermittent hypoxia-induced vascular endothelial cell injury by targeting TLR4

Purpose: To investigate the role of miR-421 in endothelial cell injury caused by chronic intermittent hypoxia (CIH), and to unravel the mechanism of action.&#x0D; Methods: A rat aortic endothelial cell model of CIH was established by 18-h exposure to hypoxic treatment. Cell viability was evaluated by MTT while cell apoptosis was determined by flow cytometry. Cellular reactive oxygen species (ROS) levels were assessed by cellular reactive oxygen species (ROS) assay kit. The mRNA and protein levels were also determined. Sprague Dawley rats were used to establish a rat CIH model over a 6-week hypoxic exposure. The degree of lung and renal injuries in the rats were observed by HE staining.&#x0D; Results: MiR-421 was downregulated and toll-like receptor 4 (TLR4) upregulated in CIH cells compared to control cells. Treatment of CIH cells decreased their viability, increased cellular ROS levels, promoted cell apoptosis, and caused changes in protein levels of apoptosis- and inflammation-related genes. However, miR-421 mimics reversed these results caused by CIH treatment. Dual luciferase reporter assay verified that TLR4 was targeted by miR-421. Moreover, TLR4 overexpression suppressed the protective effect of miR-421 on CIH cells. Finally, miR-421 agomiR inhibited CIH-induced TLR4 upregulation in rats. Histopathological examinations confirmed that miR-421 agomiR inhibited CIHinduced injury and collagen deposition in rat lungs and kidneys.&#x0D; Conclusion: MiR-421 protects vascular endothelial cells against CIH-induced injury in rats by targeting TLR4, which may provide a biomarker for the diagnosis and treatment of CIH-induced injury.&#x0D; Keywords: Obstructive sleep apnea syndrome, MiR-421, TLR4, Chronic intermittent hypoxia, Vascular endothelial cells

Antiaging Factor Klotho Retards the Progress of Intervertebral Disc Degeneration through the Toll-Like Receptor 4-NF-κB Pathway.

Antiaging protein Klotho exhibits impressive properties of anti-inflammation, however is declined early after intervertebral disc injury, making Klotho restoration an attractive strategy of treating intervertebral disc inflammatory disorders. Here, we have found that Klotho is enriched in nucleus pulposus (NP) cells and Klotho overexpression attenuates H2O2-induced acute inflammation essentially via suppressing Toll-like receptor 4 (TLR4). The proinflammatory NF-κB signaling and cytokine expressions paralleled with Klotho repression and TLR4 elevation in both NP cells (H2O2 treatment) and rat intervertebral disc (needle puncture treatment). Overexpression of TLR4 downregulated expression of Klotho, whereas interfering TLR4 expression diminished the inhibitory effects of H2O2 on Klotho in NP cells. Consistently, Klotho knockdown by RNA interferences largely diminished the anti-inflammatory and intervertebral disc protective effects in an Intervertebral Disc Degeneration (IDD) model. Thus, our study indicates that TLR4-NF-κB signaling and Klotho form a negative-feedback loop in NP cells. Also, we demonstrate that the expression of Klotho is regulated by the balance between upregulation and downregulation of TLR4-NF-κB signaling.

Exendin‑4 inhibits lipotoxicity‑induced oxidative stress in β‑cells by inhibiting the activation of TLR4/NF‑κB signaling pathway.

The present study aimed to investigate the relationship between the protective effects of exendin‑4 (EX‑4) on lipotoxicity‑induced oxidative stress and meta‑inflammation in β‑cells and the toll‑like receptor 4 (TLR4)/NF‑κB signaling pathway. Lipotoxicity, hydrogen peroxide (H2O2)‑induced oxidative stress in β cells, obese Sprague Dawley rats and TLR4 truncation rats were utilized in the present study. The expression levels were detected by western blotting; cell apoptosis was detected by TUNEL assay; and the intracellular reactive oxygen species (ROS) levels were analyzed using a ROS assay kit. The findings of the present study showed that EX‑4 inhibited the expression of TLR4, NF‑κB p65 subunit and p47phox in a concentration‑dependent manner, and decreased the intracellular level of ROS. Additionally, silencing of TLR4 expression enhanced the protective effects of EX‑4, while overexpression of TLR4 attenuated these protective influences. Simultaneously, it was demonstrated that TLR4 was involved in the process of EX‑4 intervention to inhibit H2O2‑induced oxidative stress in islet β‑cells. Moreover, it was found that EX‑4 also inhibited TLR4‑ or NF‑κB agonist‑induced oxidative stress. These results were also confirmed in an animal model of obese rats, in which EX‑4 was able to improve the function of β‑cells, attenuate oxidative stress, and inhibit the expression levels of TLR4 and NF‑κB p65 subunit in the pancreas of the diet‑induced obese rats. Furthermore, truncation of the TLR4 gene in SD rats delayed the aforementioned damage. In summary, EX‑4 may inhibit lipotoxicity‑induced oxidative stress in β‑cells by inhibiting the activation of the TLR4/NF‑κB signaling pathway.

MicroRNA-542-3p Regulates P-glycoprotein Expression in Rat Epilepsy via the Toll-like Receptor 4/Nuclear Factor-kappaB Signaling Pathway.

The function of microRNA-542-3p (miR-542-3p) in rat epilepsy is still unclear. The levels of miR-542-3p and toll-like receptor 4 (TLR4) were determined through quantitative real-time PCR. The protein levels were examined via the western blot analysis. The relationship between miR-542-3p and TLR4 was confirmed through luciferase assay. Pathological changes were analyzed via Hematoxylin-eosin (HE) and Nissl staining. The rats and hippocampal cells were treated with kainic acid (KA) in vivo and in vitro. miR-542-3p was low in KA-treated rats, hippocampal cells and cerebrospinal fluid of patients with epilepsy. Further functional analysis showed that miR-542-3p overexpression inhibited KAinduced average seizure frequency, damage of hippocampal neuron and cell apoptosis, leading to the alleviation of the brain injury in epilepsy rats. miR-542-3p was determined to downregulate TLR4 expression. The relationship between miR-542-3p and TLR4 was confirmed. TLR4 knockdown reduced KA-induced nuclear factor-kappa B p65 (NF-κB p65), multidrug resistance 1 (MDR1), P-glycoprotein (P-gp) and apoptosis-associated protein levels. Further, for NF-κB p65, MDR1, P-gp and apoptosis-associated protein levels detection, miR-542-3p mimic showed a suppressive effect on these KA-induced protein levels, whereas TLR4 overexpression ameliorated the miR-542-3p-induced these protein levels in KA-treated epilepsy rats. We identified that miR-542-3p attenuated seizure-induced brain injury and the expression of P-gp in epilepsy rats through inhibiting TLR4/NF-κB signaling pathway, which might contribute to improved epilepsy therapy.

The upregulation of microRNA-223 promoted the apoptosis of liver cancer cells via TLR4

ABSTRACT Liver cancer (LC) is the third largest contributor to mortality related to malignancies worldwide. The study was directed toward exploring the effects of miR-223 on LC proliferation and apoptosis. Dual luciferase reporter assay (DLRA) was carried out to verify the direct relationship of TLR4 mRNA 3′-UTR and miR-223-3p. HepG2 cells subsequently were transfected with miR-223 mimics. MTT assay and flow cytometric assay were carried out to evaluate proliferation and cell death, respectively. Toll-like receptor 4 (TLR4) transcription and translation were examined via qRT-PCR and western blotting, respectively. We observed that the miR-223 expression in LC tissues was inhibited, accompanied by reinforced TLR4 expression. Excessive miR-223 expression noticeably inhibited cell growth and promoted apoptosis in HepG2 and Bel-7402 HCC cell lines. Additionally, knockdown of TLR4 expression could suppress cell growth and promote apoptosis in HepG2 and Bel-7402 HCC cell lines. Moreover, TLR4 expression was suppressed via miR-223 transfection. T DLRA verified that miR-223 directly targeted TLR4. TLR4 over-expression canceled the effects of miR-223 on LC proliferation and apoptosis. Collectively, our findings suggest that miR-223 is an inhibitor of malignancy and participates in the inhibition of malignancy generation and reinforcement of LC cell death by targeting TLR4.