Expression Levels Of Toll-like Receptor Research Articles

Evaluation of NFKB1 and MyD88 expression levels in a sample of non-Hodgkin lymphoma patients before and during chemotherapy

ABSTRACT Triggering of Toll-like receptors (TLRs), expressed mainly on innate immune cells, by their specific ligands (TLRLs) induces NF-κB and MyD88, resulting in stimulation of immune responses. In the tumor microenvironment, however, certain TLRs have been found to act either as pro-tumorigenic or anti-tumorigenic co-factors. This pilot prospective study aimed to explore the impact of non-Hodgkin lymphoma (NHL) on the expression levels of TLRs and if chemotherapy alters this expression. Blood mononuclear cells (PBMCs) were isolated from healthy subjects as well as from NHL patients before, during, and after chemotherapy. We used microarray to analyze the gene profiling of TLR signaling pathway and RTqPCR to confirm the gene expression of TLR9, NF-κB1, and MyD88. Regardless of chemotherapy, TLR9 gene expression was similar in NHL patients as compared to controls. The gene expression of MyD88 and NF-κB1 was higher in the patients as compared to the controls. Interestingly, there was a 11.1-fold decrease and a 3.4-fold increase, respectively, in the gene expression of NF-κB1 and Myd88 in patients after chemotherapy as compared to before chemotherapy. Our pilot study proof the concept that NHL itself induces expression of MyD88 and NF-κB1, while chemotherapy further alters this effect.

Innate immune response of bovine mammary epithelial cells in Mycoplasma bovis mastitis using an in vitro model of bovine mammary gland infection.

Mycoplasma bovis mastitisis highly contagious and disrupts lactation, posing a significant threat to the dairy industry. While the mammary gland's defence mechanism involves epithelial cells and mononuclear cells (MNC), their interaction with M. bovis remains incompletely understood. In this study, we assessed the immunological reactivity of bovine mammary epithelial cells (bMEC) to M. bovis through co-culture with MNC. Upon co-culture with MNC, the mRNA expression levels of interleukin (IL)-1β,IL-6, IL-8 and tumor necrosis factor (TNF)-α in bMEC stimulated by M. bovis showed a significant increase compared to monoculture. Additionally, when stimulated with M. bovis, the culture supernatant exhibited significantly higher concentrations of IL-6 and interferon (IFN)-γ, while IL-1β concentration tended to be higher in co-culture with MNC than in monoculture. Furthermore, the mRNA expression levels of toll-like receptor (TLR) 2 in bMEC stimulated with M. bovis tended to increase, and TLR4 significantly increased when co-cultured with MNC compared to monocultures. However, the surface expression levels in bMEC did not exhibit significant changes between co-culture and monoculture. Overall, our research indicates that the inflammatory response of bMEC is increased during co-culture with MNC, suggesting that the interaction between bMEC and MNC in the mammary gland amplifies the immune response to M. bovis in cows affected by M. bovis mastitis.

TLR2-ERK signaling pathway regulates expression of galectin-3 in a murine model of OVA-induced allergic airway inflammation

Toll-like receptor 2 (TLR2) and galectin-3 (Gal-3) are involved in the pathological process of asthma, but the underlying mechanism is not fully understood. We hypothesized that TLR2 pathway may regulate expression of Gal-3 in allergic airway inflammation. Wild-type (WT) and TLR2−/− mice were sensitized on day 0 and challenged with ovalbumin (OVA) on days 14–21 to establish a model of allergic airway inflammation, and were treated with a specific ERK inhibitor U0126. Histological changes in the lungs were analyzed by hematoxylin-eosin (HE) and Periodic Acid-Schiff (PAS) staining; cytokines and anti-OVA immunoglobulin E (IgE) were tested by ELISA; and related protein expression in lung tissues was measured by western blot. We found that the expression levels of TLR2 and Gal-3 markedly increased concomitantly with airway inflammation after OVA induction, while TLR2 deficiency significantly alleviated airway inflammation and reduced Gal-3 expression. Moreover, the expression levels of phosphorylated mitogen-activated protein kinases (p-MAPKs) were significantly elevated in OVA-challenged WT mice, while TLR2 deficiency only significantly decreased phosphorylated extracellular signal-regulated kinase (p-ERK) levels. Furthermore, we found that U0126 treatment significantly alleviated allergic airway inflammation and decreased Gal-3 levels in OVA-challenged WT mice, but had no further effect in OVA-challenged TLR2−/− mice. These above results suggested that TLR2 is an upstream signal molecule of ERK. We further demonstrated that TLR2 regulates Gal-3 expression through the ERK pathway in LTA-stimulated macrophages in vitro. Our findings showed that the TLR2-ERK signaling pathway regulates Gal-3 expression in a murine model of allergic airway inflammation.

Gastrodin Attenuates Colitis and Prevents Tumorigenesis in Mice by Interrupting TLR4/MD2/NF-ΚB Signaling Transduction.

Chronic inflammation is one of the causative factors for tumorigenesis. Gastrodin is a main active ingredient isolated from Gastrodia elata Blume, a famous medicinal herb with a long edible history. This study aimed to explore the effects of gastrodin on colitis-associated carcinogenesis (CRC) in mice and to elucidate its potential molecular mechanisms. Balb/c mice were induced with azoxymethane (AOM) and dextran sulfate sodium (DSS) for 12 weeks. Gastrodin (50 mg/kg) was administered via oral gavage three times per week until the end of the experiment. Disease indexes, including body weight, bloody diarrhea, colon length, histopathological score, and tumor size, were measured. Tumor cell proliferation was evaluated by BrdU incorporation assay and tumor cell cytotoxicity was assessed by cell counting kit (CCK-8). The expression levels of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-ΚB) signaling molecules, NF-ΚB luciferase, and pro-inflammatory cytokines were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), immunoblotting, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), or reporter gene assays. The binding affinity between gastrodin and myeloid differentiation protein-2 (MD2) was analyzed by molecular docking and cellular thermal shift assay (CETSA). Gastrodin administration was demonstrated to mitigate various CRC-related symptoms in mice, including weight loss, diarrhea, and tissue abnormalities. Notably, gastrodin suppressed tumor cell growth during colitis- associated tumorigenesis, resulting in fewer and smaller adenomas in the colon. Unlike irinotecan, a broadspectrum antitumor drug, gastrodin did not exhibit apparent cytotoxicity in various colorectal adenocarcinoma cell lines. Additionally, gastrodin downregulated TLR4/NF-ΚB signaling molecules and pro-inflammatory mediators in mice and macrophages. Molecular docking and CETSA experiments suggested that gastrodin binds to the MD2 protein, potentially interfering with the recognition of lipopolysaccharide (LPS) by TLR4, leading to NF-ΚB pathway inhibition. This study provides evidence for the first time that gastrodin attenuated colitis and prevented colitisrelated carcinogenesis in mice, at least partially, by diminishing tumor-promoting cytokines through the interruption of TLR4/MD2/NF-ΚB signaling transduction.

Pudilan Anti-inflammatory Oral Liquid and Organic Acid Component from Taraxaci Herba Attenuate Allergic Asthma in Young Mice through Toll-like Receptor 2/Toll-like Receptor 4 Signaling Pathway

Abstract Objective: Allergic asthma (AA) is a chronic airway inflammatory disease characterized by airway hyper-responsiveness (AHR). Pudilan anti-inflammatory oral liquid (PDL) along with its main medicinal material, Taraxaci Herba (Taraxacum mongolicum Hand.-Mazz, TH) has been widely used to treat upper respiratory tract infections. Research has shown that the major ingredient of TH, the organic acid component (OAC), possesses favorable AA activity. However, the attenuated effects of PDL and OAC from TH (TH-OAC) on AA and their possible mechanisms remain poorly understood. This study analyzed the attenuating effects of PDL and TH-OAC on AA and the underlying mechanisms. Methods: Young BALB/c mice were sensitized and stimulated to develop asthma using ovalbumin. Histological examinations were performed by hematoxylin and eosin staining. Western blotting, immunohistochemistry, and protein expression detection of toll-like receptor 2 (TLR2), TLR4, and orosomucoid 1-like protein 3 (ORMDL3) were performed to detect the presence of inflammatory components in the lung tissue. The messenger RNA (mRNA) expression levels were determined using quantitative real-time polymerase chain reaction. Results: Results showed that PDL and TH-OAC alleviated augmented AHR and typical asthmatic pathological changes, including inflammatory infiltration and thickening of the alveolar wall. They also significantly reduced the levels of the immunoglobulin E, IL-4, IL-5, IL-6, tumor necrosis factor-α, and Nitric oxide (NO) in lung tissues of mice. Protein and mRNA expression levels of TLR2, TLR4, and ORMDL3 were downregulated following treatment with PDL and TH-OAC. Conclusions: PDL and TH-OAC can reduce asthma-induced inflammatory damage to the bronchi. These results provide a theoretical basis for the treatment of asthma in clinical settings.

Yemazhui () ameliorates lipopolysaccharide-induced acute lung injury modulation of the toll-like receptor 4/nuclear factor kappa-B/nod-like receptor family pyrin domain-containing 3 protein signaling pathway and intestinal flora in rats.

To investigate the impact of Yemazhui (Herba Eupatorii Lindleyani, HEL) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) and explore its underlying mechanism in vivo. The chemical constituents of HEL were analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method. Then, HEL was found to suppress LPS-induced ALI in vivo. Six-week-old male Sprague-Dawley rats were randomly divided into 6 groups: control, LPS, Dexamethasone (Dex), HEL low dose 6 g/kg (HEL-L), HEL medium dose 18 g/kg (HEL-M) and HEL high dose 54 g/kg (HEL-H) groups. The model rats were intratracheally injected with 3 mg/kg LPS to establish an ALI model. Leukocyte counts, lung wet/dry weight ratio, as well as myeloperoxidase (MPO) activity were determined followed by the detection with hematoxylin and eosin staining, enzyme linked immunosorbent assay, quantitative real time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. Besides, to explore the effect of HEL on ALI-mediated intestinal flora, we performed 16s rRNA sequencing analysis of intestinal contents. HEL attenuated LPS-induced inflammation in lung tissue and intestinal flora disturbance. Mechanism study indicated that HEL suppressed the lung coefficient and wet/dry weight ratio of LPS-induced ALI in rats, inhibited leukocytes exudation and MPO activity, and improved the pathological injury of lung tissue. In addition, HEL reduced the expression of tumor necrosis factor-alpha, interleukin-1beta (IL-1β) and interleukin-6 (IL-6) in bronchoalveolar lavage fluid and serum, and inhibited nuclear displacement of nuclear factor kappa-B p65 (NF-κBp65). And 18 g/kg HEL also reduced the expression levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88, NF-κBp65, phosphorylated inhibitor kappa B alpha (phospho-IκBα), nod-like receptor family pyrin domain-containing 3 protein (NLRP3), IL-1β, and interleukin-18 (IL-18) in lung tissue, and regulated intestinal flora disturbance. In summary, our findings revealed that HEL has a protective effect on LPS-induced ALI in rats, and its mechanism may be related to inhibiting TLR4/ NF-κB/NLRP3 signaling pathway and improving intestinal flora disturbance.

MicroRNA-630 alleviates inflammatory reactions in rats with diabetic kidney disease by targeting toll-like receptor 4.

Diabetic kidney disease (DKD) is a major complication of diabetes mellitus. Renal tubular epithelial cell (TEC) damage, which is strongly associated with the inflammatory response and mesenchymal trans-differentiation, plays a significant role in DKD; However, the precise molecular mechanism is unknown. The recently identified microRNA-630 (miR-630) has been hypothesized to be closely associated with cell migration, apoptosis, and autophagy. However, the association between miR-630 and DKD and the underlying mechanism remain unknown. To investigate how miR-630 affects TEC injury and the inflammatory response in DKD rats. Streptozotocin was administered to six-week-old male rats to create a hyperglycemic diabetic model. In the second week of modeling, the rats were divided into control, DKD, negative control of lentivirus, and miR-630 overexpression groups. After 8 wk, urine and blood samples were collected for the kidney injury assays, and renal tissues were removed for further molecular assays. The target gene for miR-630 was predicted using bioinformatics, and the association between miR-630 and toll-like receptor 4 (TLR4) was confirmed using in vitro investigations and double luciferase reporter gene assays. Overexpression of miR-630 in DKD rats led to changes in body weight, renal weight index, basic blood parameters and histopathological changes. The expression level of miR-630 was reduced in the kidney tissue of rats with DKD (P < 0.05). The miR-630 and TLR4 expressions in rat renal TECs (NRK-52E) were measured using quantitative reverse transcription polymerase chain reaction. The mRNA expression level of miR-630 was significantly lower in the high-glucose (HG) and HG + mimic negative control (NC) groups than in the normal glucose (NG) group (P < 0.05). In contrast, the mRNA expression level of TLR4 was significantly higher in these groups (P < 0.05). However, miR-630 mRNA expression increased and TLR4 mRNA expression significantly decreased in the HG + miR-630 mimic group than in the HG + mimic NC group (P < 0.05). Furthermore, the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 were significantly higher in the HG and HG + mimic NC groups than in NG group (P < 0.05). However, the levels of these cytokines were significantly lower in the HG + miR-630 mimic group than in the HG + mimic NC group (P < 0.05). Notably, changes in protein expression were observed. The HG and HG + mimic NC groups showed a significant decrease in E-cadherin protein expression, whereas TLR4, α-smooth muscle actin (SMA), and collagen IV protein expression increased (P < 0.05). Conversely, the HG + miR-630 mimic group exhibited a significant increase in E-cadherin protein expression and a notable decrease in TLR4, α-SMA, and collagen IV protein expression than in the HG + mimic NC group (P < 0.05). The miR-630 targets TLR4 gene expression. In vivo experiments demonstrated that DKD rats treated with miR-630 agomir exhibited significantly higher miR-630 mRNA expression than DKD rats injected with agomir NC. Additionally, rats treated with miR-630 agomir showed significant reductions in urinary albumin, blood glucose, TLR4, and proinflammatory markers (TNF-α, IL-1β, and IL-6) expression levels (P < 0.05). Moreover, these rats exhibited fewer kidney lesions and reduced infiltration of inflammatory cells. MiR-630 may inhibit the inflammatory reaction of DKD by targeting TLR4, and has a protective effect on DKD.

Three Artemisia pollens trigger the onset of allergic rhinitis via TLR4/MyD88 signaling pathway.

The prevalence of allergic rhinitis is high, making it a relatively common chronic condition. Countless patients suffer from seasonal Allergic rhinitis (AR). The objective of this investigation is to examine the potential involvement of common pollen allergens in seasonal allergic rhinitis, and study the proposed mechanism of Toll-like receptor 4 (TLR4)/Myeloid differentiation primary response gene 88 (MyD88) signaling pathway in the induction of AR. A mouse AR model (sensitized group) was constructed with pollen extracts and ovalbumin(OVA) of Artemisia annua (An), Artemisia argyi (Ar) and Artemisia Sieversiana (Si), and thereafter, AR symptom score was performed. After successful modeling, mouse serum and nasal mucosa tissues were extracted for subsequent experiments. The expression levels of immunoglobulin E (IgE), Interleukin (IL)-4, IL-5, IL-13 and Tumor Necrosis Factor-α (TNF-α) in serum were detected using Enzyme-linked immunosorbent assay (ELISA); Hematoxylin-eosin (H&E) staining methods were used to observe the pathological changes of the nasal mucosal tissue; Utilizing immunohistochemistry (IHC) staining, the expression levels of TLR4, MyD88 and Nuclear factor kappa B (NF-κB) p65 in mouse nasal mucosa were quantified; The mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of sensitized mice were detected with Quantitative reverse transcription PCR (qRT-PCR) and Western Blot. Finally, the in vitro culture of Human nasal mucosal epithelial cells (HNEpC) cells was conducted, and cells were treated with 200µg/ml Artemisia annua pollen extractand OVA for 24h. Western Blot assay was used to detect the expression level of TLR4, MyD88 and NF-κB p65 proteins before and after HNEpC cells were treated with MyD88 inhibitor ST-2825. On the second day after AR stimulation, the mice showed obvious AR symptoms. H&E results showed that compared to the control group, the nasal mucosal tissue in the sensitized group was significantly more inflamed. Furthermore, ELISA assay showed increased expression levels of IgE, IL-4, IL-5, IL-13 and TNF-α in serum of mice induced by OVA and Artemisia annua pollen, Artemisia argyi pollen and Artemisia Sieversiana pollen than those of the control group. However, the expression level of IL-2 was lower than that of the control group (P < 0.05). Using Immunohistochemistry staining visually observed the expression levels of TLR4, MyD88 and NF-κB p65 in mouse nasal mucosa tissues and quantitatively analyzed. The expression levels of TLR4, MyD88 and NF-κB p65 in the sensitized group were higher than those in the control group, and the differences were statistically significant (P < 0.05). The results from qRT-PCR and Western Blot showed that the mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of the sensitized group were significantly higher than those in the control group (P < 0.05). Finally, HNEpC cells were cultured in vitro and analyzed using Western Blot. The expression levels of TLR4, MyD88 and NF-κB p65 in OVA and An groups were significantly increased (P < 0.05). After ST-2825 treatment, TLR4 protein expression was significantly increased (P < 0.05) and MyD88 and NF-κB p65 protein expression were significantly decreased (P < 0.05). To sum up, the occurrence and development of AR induced by OVA and pollen of Artemisia annua, Artemisia argyi and Artemisia Sieversiana were related to TLR4/MyD88 signal pathway.

Mechanism of Chinese sturgeon IFN-γ inhibition on Mycobacterium marinum (Acipenser sinensis)

IFN-γ plays a crucial role in both innate and adaptive immune responses and is a typical Th1 cytokine that promotes Th1 response and activates macrophages. When macrophages were incubated with IFN-γ, their phagocytosis ratio against Mycobacterium marinum increased significantly, as observed under fluorescence microscopy. The macrophages engulfed a large number of M. marinum. The proliferative ability of macrophages treated with IFN-γ was significantly weaker on the 4th and 7th day after phagocytosis and subsequent re-infection with marine chlamydia (P < 0.001). This suggests that IFN-γ enhances the phagocytosis and killing ability of macrophages against M. marinum. IFN-γ protein also significantly increased the production of reactive oxygen species (H2O2) and nitric oxide (NO) by macrophages. Additionally, the expression levels of toll-like receptor 2 (tlr2) and caspase 8 (casp8) were significantly higher in macrophages after IFN-γ incubation compared to direct infection after 12 h of M. marinum stimulation. Apoptosis was also observed to a higher degree in IFN-γ incubated macrophage. Moreover, mRNA expression of major histocompatibility complex (MHC) molecules produced by macrophages after IFN-γ incubation was significantly higher than direct infection. This indicates that IFN-γ enhances antigen presentation by upregulating MHC expression. It also upregulates tlr2 and casp8 expression through the TLR2 signaling pathway to induce apoptosis in macrophages. The pro-inflammatory cytokine showed an initial increase followed by a decline, suggesting that IFN-γ enhances the immune response of macrophages against M. marinum infection. On the other hand, the anti-inflammatory cytokine showed a delayed increase, significantly reducing the expression of pro-inflammatory cytokines. The expression of both cytokines balanced each other and together regulated the inflammatory reaction against M. marinum infection.

Porcine Mandibular Bone Marrow-Derived Mesenchymal Stem Cell (BMSC)-Derived Extracellular Vesicles Can Promote the Osteogenic Differentiation Capacity of Porcine Tibial-Derived BMSCs.

Existing research suggests that bone marrow-derived mesenchymal stem cells (BMSCs) may promote endogenous bone repair. This may be through the secretion of factors that stimulate repair processes or directly through differentiation into osteoblast-progenitor cells. However, the osteogenic potential of BMSCs varies among different tissue sources (e.g., mandibular versus long BMSCs). The main aim of this study was to investigate the difference in osteogenic differentiation capacity between mandibular BMSCs (mBMSCs) and tibial BMSCs (tBMSCs). Bioinformatics analysis of the GSE81430 dataset taken from the Gene Expression Omnibus (GEO) database was performed using GEO2R. BMSCs were isolated from mandibular and tibial bone marrow tissue samples. Healthy pigs (n = 3) (registered at the State Office for Nature, Environment, and Consumer Protection, North Rhine-Westphalia (LANUV) 81-02.04.2020.A215) were used for this purpose. Cell morphology and osteogenic differentiation were evaluated in mBMSCs and tBMSCs. The expression levels of toll-like receptor 4 (TLR4) and nuclear transcription factor κB (NF-κB) were analyzed using quantitative polymerase chain reaction (qPCR) and Western blot (WB), respectively. In addition, mBMSC-derived extracellular vesicles (mBMSC-EVs) were gained and used as osteogenic stimuli for tBMSCs. Cell morphology and osteogenic differentiation capacity were assessed after mBMSC-EV stimulation. Bioinformatic analysis indicated that the difference in the activation of the TLR4/NF-κB pathway was more pronounced compared to all other examined genes. Specifically, this demonstrated significant downregulation, whereas only 5-7 upregulated genes displayed significant variances. The mBMSC group showed stronger osteogenic differentiation capacity compared to the tBMSC group, confirmed via ALP, ARS, and von Kossa staining. Furthermore, qPCR and WB analysis revealed a significant decrease in the expression of the TLR4/NF-κB pathway in the mBMSC group compared to the tBMSC group (TLR4 fold changes: mBMSCs vs. tBMSCs p < 0.05; NF-κB fold changes: mBMSCs vs. tBMSCs p < 0.05). The osteogenic differentiation capacity was enhanced, and qPCR and WB analysis revealed a significant decrease in the expression of TLR4 and NF-κB in the tBMSC group with mBMSC-EVs added compared to tBMSCs alone (TLR4 fold changes: p < 0.05; NF-κB fold changes: p < 0.05). Our results indicate that mBMSC-EVs can promote the osteogenic differentiation of tBMSCs in vitro. The results also provide insights into the osteogenic mechanism of mBMSCs via TLR4/NF-κB signaling pathway activation. This discovery promises a fresh perspective on the treatment of bone fractures or malunions, potentially offering a novel therapeutic method.

Acupoint application therapy alleviates pain by regulating immune function through inhibiting TLR4/MyD88/NF-κB p65 signaling in a primary dysmenorrhea rat model.

To investigate the effects of graphene-based warm uterus acupoint paste on uterine Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear transcription factor-kappa B p65 (NF-κB p65) signaling pathway and Th1/Th2 immune balance in primary dysmenorrhea ( PD ) model rats, so as to reveal its immunological mechanisms of relieving dysmenorrhea. Thirty SD female rats were randomly divided into 3 groups：normal group, model group and acupoint paste group, with 10 rats in each group. PD rat model was established by subcutaneous injection of estradiol benzoate for 10 consecutive days. At the same time of modeling, graphene-based warm uterus acupoint paste was applied to the acupoints of "Guanyuan" (CV4), bilateral "Zigong" (EX-CA1) and "Sanyinjiao" (SP6) of rats in the acupoint paste group. The application was continuously applied once daily for 10 d, 5 h each time. On the 11th day, oxytocin was injected intraperitoneally to observe the writhing latency, writhing times within 30 min and writhing score of rats in each group. The spleen and thymus indexes were calculated. The pathological changes of spleen and thymus tissue were observed after HE staining. The contents of serum immunoglobulin (Ig) A, IgG, tumor necrosis factor-α (TNF-α), interleukin (IL)-2, interferon-γ (IFN-γ), IL-4 and IL-10 were detected by ELISA . The protein and mRNA expression levels of TLR4, MyD88 and NF-κB p65 in rat uterine tissue were detected by Western blot and real-time quantitative PCR, respectively. Compared with the normal group, the writhing times and writhing scores within 30 min of rats in the model group were significantly increased(P<0.001), and the rats showed writhing reaction (P<0.01). The spleen index and thymus index were significantly decreased(P<0.01, P<0.05). The spleen and thymus had obvious pathological changes. The contents of IgA, IgG, TNF-α, IL-2 and IFN-γ in serum were significantly increased, while the contents of serum IL-4 and IL-10 were significantly decreased(P<0.001, P<0.01). The expression levels of TLR4, MyD88, NF-κB p65 protein and corresponding mRNA in uterine tissue were significantly increased(P<0.001). Following intervention, compared with the model group, the writhing latency time of rats in the acupoint paste group was prolonged, and the writhing times and writhing scores within 30 min were significantly decreased (P<0.001). The spleen index and thymus index were significantly increased(P<0.01, P<0.05). The pathological changes of spleen and thymus were improved. The contents of serum IgA, IgG, TNF-α, IL-2 and IFN-γ were significantly decreased, while the contents of IL-4 and IL-10 were significantly increased(P<0.001, P<0.05, P<0.01). The expression of TLR4, MyD88, NF-κB p65 protein and the corresponding mRNA levels in uterine tissue were decreased(P<0.001, P<0.01). Graphene-based warm uterus acupoint paste can regulate the immune balance of Th1/ Th2 by regulating TLR4/ MyD88/ NF-κB p65 signaling pathway, repair the pathological damage of immune tissue, improve immune function, and effectively relieve the pain symptoms of PD rats.

Effect of running exercise training on inflammatory mediators and cytokines expression in testicular tissue; effect of exercise intensity

The aim of this study is to investigate the impact of running exercise training protocols (ETPs) with varying intensities on inflammatory responses, with a specific focus on the interactions between inflammatory mediators, cytokines, and Leydig cell steroidogenic activity, as well as testosterone secretion. To this end, 24 Wistar rats were subdivided into sedentary control, low (LICT), moderate (MICT), and high (HICT) intensity continuous running ETP groups. After 8 weeks, the expression levels of Toll-like receptor-4 (TLR-4), nuclear factor-kappa-B (NF-KB), interleukin-6 (IL-6), interleukin-10 (IL-10), Tumor necrosis factor alpha (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and the testicular nitric oxide (NO) content were assessed and compared between groups. Moreover, the mean distributions of Leydig cells/mm2 of interstitial connective tissue, their steroidogenic activity, and serum level of testosterone were assessed. The LICT did not show any significant (p > 0.05) change in the expression levels of all aforementioned biomarkers. In contrast, both the MICT and HICT groups demonstrated a significant (p < 0.05) increase in the expression levels of TLR-4, NFK-B, IL-6, TNF-α, iNOS, and COX-2 at both the mRNA and protein levels. The testicular NO has increased in HICT and MICT groups. Despite a decrease in the distribution of Leydig cells in both the MICT and HICT groups, the HICT group exhibited a significant (p < 0.05) reduction in Leydig cell steroidogenic activity and serum testosterone levels. In conclusion, our findings revealed that ETPs can influence Leydig cell steroidogenic activity and testosterone secretion, contingent on their intensity. These effects are attributed to alterations in the expression levels of pro-inflammatory mediators and cytokines.

[Yeast extract improves the inflammatory response of HepG2 cells induced by ethyl alcohol and lipopolysaccharide].

To explore the ameliorative effect of yeast extract(YE) on the inflammatory response of human hepatoma cells(HepG2) induced by ethyl alcohol(EtOH) and lipopolysaccharide(LPS), and further explore the potential molecular mechanism based on Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB) signaling pathway. HepG2 cells were induced by 50 mmol/L EtOH and 1 μg/mL LPS combined with YE intervention. The expression level of inflammatory cytokines was detected by ELISA. The expression level of TLR4 and the nuclear translocation of NF-κB were detected by immunofluorescence staining. The expression levels of TLR4, NF-κB, phospho-NF-κB-P65(P-NF-κB-p65), nucleus-phospho-NF-κB-p65(N-P-NF-κB-p65), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β) were detected by Western blot. Compared with the control group, the cells in EtOH+LPS group produced a large number of inflammatory factors and had a significant inflammatory response. YE intervention significantly alleviated EtOH+LPS induced hepatocyte inflammatory response. Further molecular mechanism studies showed that YE significantly reduced TLR4 expression level and inhibited NF-κB nuclear translocation in hepatocytes. YE can effectively inhibit the inflammatory response of HepG2 cells induced by EtOH and LPS, and its molecular mechanism may be related to the down-regulation of TLR4/NF-κB pathway.

Expression of Toll-like receptors in the cerebellum during pathogenesis of prion disease.

Prion diseases, such as scrapie, entail the accumulation of disease-specific prion protein (PrPSc) within the brain. Toll-like receptors (TLRs) are crucial components of the pattern recognition system. They recognize pathogen-associated molecular patterns (PAMPs) and play a central role in orchestrating host innate immune responses. The expression levels of Toll-like receptors (TLRs) in the central nervous system (CNS) were not well-defined. To establish a model of prion diseases in BALB/C mice, the 22L strain was employed. The features of the 22L strain were analyzed, and the cerebellum exhibited severe pathological changes. TLR1-13 levels in the cerebellum were measured using quantitative polymerase chain reaction (qPCR) at time points of 60, 90, 120, and the final end point (145 days post-infection). During the pathogenesis, the expression levels of Toll-like receptors (TLRs) 1, 2, 7, 8, and 9 increased in a time-dependent manner. This trend mirrored the expression patterns of PrPSc (the pathological isoform of the prion protein) and glial fibrillary acidic protein. Notably, at the end point, TLR1-13 levels were significantly elevated. Protein level of TLR7 and TLR9 showed increasing at the end point of the 22L-infected mice. A deeper understanding of the increased Toll-like receptors (TLRs) in prion diseases could shed light on their role in initiating immune responses at various stages during pathogenesis. This insight is particularly relevant when considering TLRs as potential therapeutic targets for prion diseases.

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.

Sodium dichloroacetate improves migration ability by suppressing LPS-induced inflammation in HTR-8/SVneo cells via the TLR4/NF-κB pathway.

Inadequate cytotrophoblast migration and invasion are speculated to result in preeclampsia, which is a pro-inflammatory condition. Sodium dichloroacetate (DCA) exerts anti-inflammatory actions. Thus,we sought to investigate the effect of DCA on the migration function of the lipopolysaccharide (LPS)-stimulated human-trophoblast-derived cell line (HTR-8/SVneo). HTR-8/SVneo cells were treated with LPS to suppress cell migration. Cell migration was examined by both scratch wound healing assay and transwell migration assay. Western blotting was used to analyze the expression levels of toll-like receptor-4 (TLR4), nuclear factor-κB (NF-κB), TNF-α, IL-1β, and IL-6 in the cells. DCA reversed LPS-induced inhibition of migration in HTR-8/SVneo cells. Furthermore, DCA significantly suppressed LPS-induced activation of TLR4, phosphorylation of NF-κB (p65), translocation of p65 into the nucleus, and the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). Treatment with inhibitors of TLR4 signal transduction (CLI095 or MD2-TLR-4-IN-1) reduced LPS-induced overexpression of pro-inflammatory cytokines, and a synergistic effect was found between TLR4 inhibitors and DCA in HTR-8/SVneo cells. DCA improved trophoblast cell migration function by suppressing LPS-induced inflammation, at least in part, via the TLR4/NF-κB signaling pathway. This result indicates that DCA might be a potential therapeutic candidate for human pregnancy-related complications associated with trophoblast disorder.

Study on the effect of ginaton on reducing cerebral vasospasm and early brain injury after hemorrhagic stroke by inhibiting inflammatory response

Objective: The objective of this study was to investigate the effects and possible mechanisms of action of ginseng on cerebral vasospasm and early brain injury (EBI) following hemorrhagic stroke. Materials and Methods: Sprague-Dawley (SD) rats (n = 48) were randomly divided into sham operation (sham group), subarachnoid hemorrhage (SAH) model (SAH group), normal saline (NS group), and Ginaton (Extract of Ginkgo Biloba Leaves Drops) intervention (gin group) groups. MCP-1 mRNA and tumor necrosis factor levels were detected using reverse transcription–polymerase chain reaction. The relative expression of mRNA was detected by Western blotting. Results: (1) Compared with the sham group, the SAH, NS, and gin groups had different degrees of neurological dysfunction. Compared with the SAH and NS groups, the neurological deficits in the gin group were significantly improved (P &lt; 0.05). (2) Compared with the sham group, the relative expression levels of MCP-1 mRNA in the SAH, NS, and gin groups were 5.1 ± 0.9, 3.4 ± 0.6, and 2.5 ± 0.4, respectively; the relative expression levels of mRNA were 13.3 ± 2.4, 11.2 ± 1.8, and 3.8 ± 0.6, respectively. (3) The apoptosis rates of brain tissue in the sham, SAH, NS, and gin groups were 4.8 ± 0.7, 54.2 ± 10.3, 50.1 ± 7.4, and 28.4 ± 4.5, respectively. (4) Western blot showed that the relative expression levels of toll-like receptor-4 (TLR-4) protein in the sham, SAH, NS, and gin groups were 0.29 ± 0.03, 0.87 ± 0.15, 0.65 ± 0.13, and 0.41 ± 0.17, respectively; the relative expression levels of B protein were 0.21 ± 0.04, 0.96 ± 0.14, 0.73 ± 0.18, and 0.30 ± 0.05, respectively. Gin treatment could inhibit TLR-4 and nuclear factor-κB (NF-κB) protein expression. Conclusions: Dona tablets may inhibit activation of the NF-κB signaling pathway, and SAH-induced inflammatory response, so as to reduce cerebral vasospasm and EBI.

Therapeutic Effects of (+)-Afzelechin on Particulate Matter-Induced Pulmonary Injury.

Particulate matter (PM) constitutes a hazardous blend of organic and inorganic particles that poses health risks. Inhalation of fine airborne PM with a diameter of ≤ 2.5 μm (PM2.5) can lead to significant lung impairments. (+)-afzelechin (AZC), a natural compound sourced from Bergenia ligulata, boasts a range of attributes, including antioxidant, antimicrobial, anticancer, and cardiovascular effects. However, knowledge about the therapeutic potential of AZC for patients with PM2.5-induced lung injuries remains limited. Thus, in this study, we investigated the protective attributes of AZC against lung damage caused by PM2.5 exposure. AZC was administered to the mice 30 min after intratracheal instillation of PM2.5. Various parameters, such as changes in lung tissue wet/dry (W/D) weight ratio, total protein/total cell ratio, lymphocyte counts, levels of inflammatory cytokines in bronchoalveolar lavage fluid (BALF), vascular permeability, and histology, were evaluated in mice exposed to PM2.5. Data demonstrated that AZC mitigated lung damage, reduced W/D weight ratio, and curbed hyperpermeability induced by PM2.5 exposure. Furthermore, AZC effectively lowered plasma levels of inflammatory cytokines produced by PM2.5 exposure. It reduced the total protein concentration in BALF and successfully alleviated PM2.5-induced lymphocytosis. Additionally, AZC substantially diminished the expression levels of Toll-like receptors 4 (TLR4), MyD88, and autophagy-related proteins LC3 II and Beclin 1. In contrast, it elevated the protein phosphorylation of the mammalian target of rapamycin (mTOR). Consequently, the anti-inflammatory attribute of AZC positions it as a promising therapeutic agent for mitigating PM2.5-induced lung injuries by modulating the TLR4-MyD88 and mTOR-autophagy pathways.

Exploration of the molecular mechanism guiding Xinfeng capsule regulatory mechanism for rheumatoid arthritis inflammation.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joint synovium. The traditional Chinese medicine Xinfeng capsule (XFC) has a remarkable alleviating effect on inflammatory symptoms, such as joint pain and swelling, in patients with RA. However, the underlying mechanism of action remains to be elucidated. This study intended to conduct network pharmacology, animal experiments, data mining, and molecular docking to explore the molecular mechanism through which XFC can improve the inflammatory symptoms of RA. The Apriori association rules and a random walk model were employed to evaluate the effect of XFC on the clinical inflammatory indexes of RA. The active ingredients and the potential target genes of XFC were obtained from public databases. Based on the search tool for recurring instances of neighboring genes (STRING) database, the Database for Annotation, Visualization and Integrated Discovery (DAVID) database, Cytoscape software, and molecular docking method, the molecular mechanism by which XFC acts on RA was also analyzed. Finally, an adjuvant arthritis rat model was established to verify the effects of XFC on inflammation-related signaling pathways and inflammatory factors. XFC significantly reduced the level of C-reactive protein (CRP), vascular endothelial growth factor (VEGF), and the erythrocyte sedimentation rate (ESR). The docking space structures of the active ingredients in XFC, namely triptolide and quercetin, and the key targets were stable. Inflammation-related biological processes were identified as the key factors involved in the development of RA, and the regulation of the toll-like receptor (TLR) signaling pathway may be the key link for XFC toward improving the inflammatory state of RA. The expression levels of toll-like receptor 4 (TLR4), myeloid differentiation primary response protein MyD88 (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK1), TNF receptor-associated factor 6 (TRAF6), TGF-beta-activated kinase 1 (TAK1), phospho-Inhibitor of NF-κB kinaseβ (p-IKKβ), phospho-Nuclear factor-k-gene binding (p-NF-κB), and interleukin-1β (IL-1β) can all be decreased by XFC. XFC improves joint inflammation symptoms by lowering pro-inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-γ (INF-γ) levels. XFC could effectively improve the clinical inflammatory indexes of RA. The active ingredients of XFC improved the inflammatory state of RA by regulating the TLR-signaling pathway.

Toll-like receptor expression and functional behavior in platelets from patients with systemic lupus erythematosus

BackgroundMultiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. Materials and MethodsThirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. ResultsIn resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. ConclusionOur study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.