Toll-like Receptor 4 Protein Levels Research Articles

Sulfate glycosaminoglycan from swim bladder exerts immunomodulatory potential on macrophages via toll-like receptor 4 mediated NF-κB signaling pathways

This study explored the immunomodulatory impact and potential mechanisms on macrophages RAW264.7 using a purified macromolecular sulfate glycosaminoglycan (SBSG) from the swim bladder, whose structure was similar to chondroitin sulfate A. The results showed that SBSG at 0.25–1 mg/mL increased the viability and phagocytosis of RAW264.7 cells. Meanwhile, SBSG promoted the secretion of tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), and nitric oxide (NO), as well as the production of reactive oxygen species (ROS). According to the RT-PCR and Western blot data, SBSG activated TLR4-nuclear factor kappa B (NF-κB) signaling pathways, which decreased the relative mRNA and protein levels of Toll-like receptor 4 (TLR4), IκB kinase β (IKKβ), NF-κB p65, and p-NF-κB p65. The molecular docking and molecular dynamic simulation findings revealed that the main binding force between TLR4 and SBSG was conventional hydrogen bond interaction, resulting in more stable ligand receptor complexes. In summary, SBSG exhibits significant immunomodulatory potential, similar to chondroitin sulfate C. The underlying molecular mechanism involved the binding of SBSG through hydrogen bonding to TLR4 receptors, triggering the NF-κB signaling pathway to downregulate the expression of related genes and proteins. This, in turn, regulated the secretion of various cytokines that were mediated by macrophages to exert the immunity of the body.

PM2.5 exposure upregulates pro-inflammatory protein expression in human microglial cells via oxidant stress and TLR4/NF-κB pathway

Exposure to ambient PM2.5 is associated with neurodegenerative disorders, in which microglia activation plays a critical role. Thus far, the underlying mechanisms for PM2.5-induced microglia activation have not been well elucidated. In this study, a human microglial cell line (HMC3) was used as the in vitro model to examine the inflammatory effect (hall marker of microglia activation) of PM2.5 and regulatory pathways. The expression of inflammatory mediators including interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) as well as the brain derived neurotrophic factor (BDNF) were determined by ELISA and/or real-time PCR, respectively. Flow cytometry was used to measure the production of intracellular reactive oxygen species (ROS). Western blot was used to measure protein levels of Toll-like receptor 4 (TLR4), NF-κB inhibitor α (IκBα) and COX-2. It was shown that PM2.5 stimulation increased IL-6 and COX-2 expression but decreased BDNF expression in a dose-dependent manner. Further studies showed that PM2.5 triggered the formation of ROS and pre-treatment with the ROS scavenger acetylcysteine (NAC) significantly suppressed PM2.5-induced IL-6 and COX-2 expression. Moreover, the nuclear factor kappa B (NF-κB) inhibitor BAY11–7085 or the TLR4 neutralizing antibody markedly blocked PM2.5-induced IL-6 and COX-2 expression. However, NAC or BAY11–7085 exhibited minimal effect on PM2.5-induced BDNF down-regulation. In addition, pre-treatment with BAY11–7085 or TLR4 neutralizing antibody reduced ROS production induced by PM2.5, and NAC pre-treatment inhibited TLR4 expression and NF-κB activation induced by PM2.5. Collectively, PM2.5 treatment induced IL-6 and COX-2 but suppressed BDNF expression. PM2.5-induced IL-6 and COX-2 expression was mediated by interactive oxidative stress and TLR4/NF-κB pathway.

ESL attenuates BLM-induced IPF in mice: Dual mediation of the TLR4/NF-κB and TGF-β1/PI3K/Akt/FOXO3a pathways

Idiopathic pulmonary fibrosis (IPF) is a persistent and advanced pulmonary ailment. The roles of innate immunity and adaptive immunity are pivotal in the evolution of IPF. An ill-adjusted interaction between epithelial cells and immune cells is responsible for initiating the epithelial-mesenchymal transition (EMT) process and sustaining chronic inflammation, thereby fostering fibrosis progression. The intricacy of IPF pathogenesis has hindered the availability of efficacious agents. Elephantopus scaber Linn. (ESL) is a canonical Chinese medicine with significant immunoregulatory effects, and its aqueous extract has been proven to attenuate IPF symptoms in bleomycin (BLM)-induced mice. However, the underlying mechanism through which ESL relieves IPF remains unclear. To validate whether ESL reverses IPF by mediating the immune response and EMT. Ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) and UPLC were used to identify the components and determine the concentrations of the specific compounds in the ESL. Network pharmacology and molecular docking were applied to predict the potential mechanism underlying the anti-IPF effect of ESL. BLM-induced IPF mice were used to validate the anti-IPF effect of ESL, and lung tissue was collected to test putative pathways involved in inflammation and EMT via immunohistochemistry (ICH), real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Sixty-one compounds were identified, and thirteen main ingredients were quantified in the ESL. In silico experiments predicted that the IPF-mediated reversal of adverse effects by ESL would be related to interruption of the Toll-like receptor 4 (TLR4)/nuclear factor-k-gene binding (NF-ĸB) inflammatory pathway and the transforming growth factor-beta l (TGF-β1)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/forkhead box O3 (FOXO3a) fibrosis pathway. In vivo experiments showed that ESL alleviates BLM-induced lung inflammation and fibrosis by reducing neutrophil aggregation and fibroblast foci, similar to the effects of the positive control drug pirfenidone (PFD). ESL markedly inhibited the transcription of TNF-α, IL-1β, and IL-6, which are downstream genes of the NF-κB signaling pathway. Furthermore, the protein levels of TLR4 and p-NF-κB were correspondingly inhibited in response to ESL treatment. Additionally, ESL reverses BLM-induced changes in the expression of EMT-related biological characteristic indicators (collagen I [COLIA1], E-cadherin, and alpha smooth muscle actin [α-SMA]) at the messenger ribonucleic acid (mRNA) level and markedly inhibits the expression of EMT-related upstream proteins (TGF-β1, p-PI3K, p-Akt, and p-FOXO3a). Our research suggested that ESL attenuates BLM-induced IPF through mediating the EMT process via the TGF-β1/PI3K/Akt/FOXO3a signaling pathway and inhibiting inflammation through the TLR4/NF-κB signaling pathway, highlighting that ESL can serve as an immunoregulator for relieving the abnormal immune response and reversing the EMT in IPF.

Protective effect of modified Huangqi Chifeng decoction on immunoglobulin A nephropathy through toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B signaling pathway.

To examine the nephroprotective mechanism of modified Huangqi Chifeng decoction (, MHCD) in immunoglobulin A nephropathy (IgAN) rats. To establish the IgAN rat model, the bovine serum albumin, lipopolysaccharide, and carbon tetrachloride 4 method was employed. The rats were then randomly assigned to the control, model, telmisartan, and high-, medium-, and low-dose MHCD groups, and were administered the respective treatments via intragastric administration for 8 weeks. The levels of 24-h urinary protein, serum creatinine (CRE), and blood urea nitrogen (BUN) were measured in each group. Pathological alterations were detected. IgA deposition was visualized through the use of immunofluorescence staining. The ultrastructure of the kidney was observed using a transmission electron microscope. The expression levels of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-β1 (TGF-β1) were examined by immunohistochemistry and quantitative polymerase chain reaction. Levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappa B (NF-κB) P65, were examined by immunohistochemistry, Western blotting, and quantitative polymerase chain reaction. The 24-h urine protein level in each group increased significantly at week 6, and worsen from then on. But this process can be reversed by treatments of telmisartan, and high-, medium-, and low-dose of MHCD, and these treatments did not affect renal function. Telmisartan, and high-, and medium-dose of MHCD reduced IgA deposition. Renal histopathology demonstrated the protective effect of high-, medium-, and low-dose of MHCD against kidney injury. The expression levels of MCP-1, IL-6, and TGF-β1 in kidney tissues were downregulated by low, medium and high doses of MHCD treatment. Additionally, treatment of low, medium and high doses of MHCD decreased the protein and mRNA levels of TLR4, MyD88, and NF-κB. MHCD exerted nephroprotective effects on IgAN rats, and MHCD regulated the expressions of key targets in TLR4/MyD88/NF-κB signaling pathway, thereby alleviating renal inflammation by inhibiting MCP-1, IL-6 expressions, and ameliorating renal fibrosis by inhibiting TGF-β1 expression.

Piperine attenuates hepatic ischemia/reperfusion injury via suppressing the TLR4 signaling cascade in mice

Piperine, the major active substance in black pepper, has been shown to have anti-inflammatory and antioxidant effects in several ischemic diseases. However, the role of piperine in hepatic ischemia/reperfusion injury (HIRI) and its underlying mechanisms remain unclear. In this study, the mice were administered piperine (30 mg/kg) intragastric administration before surgery. After 24 h of hepatic ischemia-reperfusion, liver histopathological evaluation, serum transaminase measurements, and TUNEL analysis were performed. The infiltration of inflammatory cells and production of inflammatory mediators in the liver tissue were determined by immunofluorescence and immunohistochemical staining. The protein levels of toll-like receptor 4 (TLR4) and related proteins such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interleukin-1 receptor-associated kinase 1 (IRAK1), p65, and p38 were detected by western blotting. The results showed that plasma aminotransferase (ALT), aspartate aminotransferase (AST), hepatocyte apoptosis, oxidative stress, and inflammatory cell infiltration significantly increased in HIRI mice. Piperine pretreatment notably repaired liver function, improved the histopathology and apoptosis of liver cells, alleviated oxidative stress injury, and reduced inflammatory cell infiltration. Further analysis showed that piperine attenuated tumor necrosis factor-a (TNF-α) and interleukin 6 (IL-6) production and reduced TLR4 activation and phosphorylation of IRAK1, p38, and NF-κB in HIRI. Piperine has a protective effect against HIRI through the TLR4/IRAK1/NF-κB signaling pathway and may be a safer option for future clinical treatment and prevention of ischemia-related diseases.

Mechanism of aqueous extract of Strychni Semen in improving pain in rats with rheumatoid arthritis based on TLR4/TNF-α/MMP-9 signaling pathway

This study aims to explore the mechanism of aqueous extract of Strychni Semen(SA) in relieving pain in the rat model of rheumatoid arthritis(RA) via Toll-like receptor 4(TLR4)/tumor necrosis factor-α(TNF-α)/matrix metalloproteinase-9(MMP-9) signaling pathway. Firstly, the main chemical components of Strychni Semen were searched against TCMSP, TCMID, ETCM, and related literature, and the main targets of the chemical components were retrieved from TargetNet and SwissTargetPrediction. The main targets of RA and pain were searched against GeneCards, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). Venny 2.1.0 was used to obtain the common targets shared by Strychni Semen, RA, and pain, and STRING and Cytoscape 3.6.1 were used to build the protein-protein interaction network. Then, molecular docking was carried out in AutoDock Vina. Finally, the rat model of type Ⅱ collagen-induced arthritis(CIA) was established. The up-down method and acetone method were employed to examine the mechanical pain threshold and cold pain threshold of rats, and the pain-relieving effect of SA on CIA rats was evaluated comprehensively. Hematoxylin-eosin(HE) staining was employed to evaluate the histopathological changes of joints in CIA rats. The expression levels of key target proteins was determined by immunohistochemistry and Western blot, and the mRNA levels of key targets were determined by real-time fluorescence quantitative polymerase chain reaction(real-time PCR). The results of network prediction showed that Strychni Semen may act on the TLR4/TNF-α/MMP-9 signaling pathway to exert the pain-relieving effect. The results of molecular docking showed that brucine, the main active component of SA, had strong binding ability to TLR4, TNF-α, and MMP-9. The results of animal experiments showed that SA improved the mechanical and cold pain sensitivity(P<0.05, P<0.01) and reduced the joint histopathological score of CIA rats(P<0.01). In addition, medium and high doses of SA down-regulated the protein and mRNA levels of TNF-α, TLR4, and MMP-9(P<0.05,P<0.01). In conclusion, SA alleviated the mechanical pain sensitivity, cold pain sensitivity, and joint histopathological changes in CIA rats by inhibiting the over activation of TLR4/TNF-α/MMP-9 signaling pathway.

Effect and mechanism of Maxing Shigan Decoction on reducing inflammatory response in rats with cough variant asthma via TLR4/MyD88/NF-κB and p38 MAPK signaling pathways

This study aims to investigate the effect and mechanism of Maxingshigan Decoction on inflammation in the rat model of cough variant asthma(CVA). The SPF-grade SD rats of 6-8 weeks were randomized into normal, model, Montelukast sodium, and low-, medium-, and high-dose Maxing Shigan Decoction groups, with 8 rats in each group. The CVA rat model was induced by ovalbumin(OVA) and aluminum hydroxide sensitization and ovalbumin stimulation. The normal group and model group were administrated with equal volume of normal saline by gavage, and other groups with corresponding drugs by gavage. After the experiment, the number of white blood cells in blood and the levels of interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α) in the serum were measured. The lung tissue was stained with hematoxylin-eosin(HE). Western blot was employed to determine the protein levels of nuclear factor-κB(NF-κB), Toll-like receptor 4(TLR4), myeloid differentiation protein(MyD88), and mitogen-activated protein kinase(MAPK) in the lung tissue. Real-time PCR was carried out to measure the mRNA levels of TLR4 and MyD88 in the lung tissue. Compared with the normal group, the model group showed increased white blood cells, elevated IL-6 and TNF-α levels(P<0.01), lowered IL-10 level(P<0.01), up-regulated protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK(P<0.01) and mRNA levels of TLR4 and MyD88(P<0.01) in the lung tissue. HE staining showed obvious infiltration of inflammatory cells around the airway and cell disarrangement in the model group. Compared with the model group, Montelukast sodium and high-dose Maxing Shigan Decoction reduced the white blood cells, lowered the IL-6 and TNF-α levels(P<0.01), and elevated the IL-10 level(P<0.01). Moreover, they down-regulated the protein levels of TLR4, MyD88, p-p65/NF-κB p65, p-p38 MAPK/p38 MAPK in the lung tissue(P<0.01) and the mRNA levels of TLR4 and MyD88 in the lung tissue(P<0.01). HE staining showed that Montelukast sodium and high-dose Maxing Shigan Decoction reduced inflammatory cell infiltration and cell disarrangement. The number of white blood cells, the levels of IL-10 and TNF-α in the serum, the protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK, and the mRNA levels of TLR4 and MyD88 in the lung tissue showed no significant differences between the Montelukast sodium group and high-dose Maxing Shigan Decoction group. Maxing Shigan Decoction can inhibit airway inflammation in CVA rats by inhibiting the activation of TLR4/MyD88/NF-κB and p38 MAPK signaling pathways.

Ligusticum cycloprolactam inhibits IL-1β-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1β(IL-1β) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1β, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1β-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

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.

TLR4 signalling: the key to controlling EV71 replication and inflammatory response.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enterovirus 71 (EV71) that frequently affects children, leading to severe infections in some cases. In general, when infection occurs, the body upregulates inflammatory responses to eliminate pathogenic microorganisms to protect the host from infection. However, EV71 may inhibit host's innate immunity to promote virus infection. At present, it is not fully understood how EV71 hijack the host cells for its own replication. Toll-like receptor 4 (TLR4), a natural immune receptor, historically associated with bacterial endotoxin-induced inflammatory responses. However, it is still unclear whether and how TLR4 is altered during EV71 infection. In this study, we observed a reduction in both TLR4 protein and gene transcript levels in RD, GES-1, and Vero cells following EV71 infection, as detected by RT-qPCR, immunofluorescence staining and western blot. Furthermore, we observed that the TLR4 downstream molecules of MYD88, p-NF-κB p65, p-TBK1 and related inflammatory cytokines were also reduced, suggesting that antiviral innate immune and inflammatory response were suppressed. To determine the impact of TLR4 changes on EV71 infection, we interfered EV71-infected RD cells with TLR4 agonist or inhibitor and the results showed that activation of TLR4 inhibited EV71 replication, while inhibition of TLR4 promote EV71 replication. Besides, EV71 replication was also promoted in TLR4 siRNA-transfected and EV71-infected RD cells. This suggests that down-regulation the expression of TLR4 by EV71 can inhibit host immune defense to promote EV71 self-replication. This novel mechanism may be a strategy for EV71 to evade host immunity.

Massage ameliorates lumbar disc herniation-related radicular pain in rats by suppressing TLR4/NLRP3 inflammasome signaling transduction.

Lumbar disc herniation (LDH) is a common spinal disease that can cause severe radicular pain. Massage, also known as Tuina in Chinese, has been indicated to exert an analgesic effect in patients with LDH. Nonetheless, the mechanism underlying this effect of massage on LDH remains unclarified. Forty Sprague-Dawley rats were randomly divided into four groups. A rat LDH model was established by autologous nucleus pulpous (NP) implantation, followed by treatment with or without massage. A toll-like receptor 4 (TLR4) antagonist TAK-242 was administrated to rats for blocking TLR4. Behavioral tests were conducted to examine rat mechanical and thermal sensitivities. Western blotting was employed for determining TLR4 and NLRP3 inflammasome-associated protein levels in the spinal dorsal horn (SDH). Immunofluorescence staining was implemented for estimating the microglial marker Iba-1 expression in rat SDH tissue. NP implantation induced mechanical allodynia and thermal hyperalgesia in rat ipsilateral hindpaws and activated TLR4/NLRP3 inflammasome signaling transduction in the ipsilateral SDH. Massage therapy or TAK-242 administration relieved NP implantation-triggered pain behaviors in rats. Massage or TAK-242 hindered microglia activation and blocked TLR4/NLRP3 inflammasome activation in ipsilateral SDH of LDH rats. Massage ameliorates LDH-related radicular pain in rats by suppressing microglia activation and TLR4/NLRP3 inflammasome signaling transduction.

Anti-inflammatory effects of Mentha pulegium L. extract on human peripheral blood mononuclear cells are mediated by TLR-4 and NF-κB suppression

There is great interest in evaluating the anti-inflammatory properties of new herbal products. Thus, the effects of Mentha pulegium L. extract on gene and protein expressions of pro-inflammatory mediators and transcription factors were determined.The hydro-ethanolic extract of Mentha pulegium L. was obtained and optimal non-cytotoxic concentrations of the extract were determined by MTT assay. Then, three different concentrations of Mentha pulegium L. (10, 30, and 90 μg/mL) were used to pre-treat the lipopolysaccharide (LPS)-stimulated and non-stimulated peripheral blood mononuclear cells (PBMCs) of 10 healthy individuals. Finally, the tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, Toll-like receptor-4 (TLR-4), nuclear factor-kappa B (NF-κB) p65, activator protein-1 (AP-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) gene expressions and TNF-α, IL-1β, IL-6, TLR-4, prostaglandin E2 (PGE2), and COX-2 protein levels were measured.MTT results showed that there is no significant difference in cell viability among 10, 20, 40, and 80 μg/mL concentrations of Mentha pulegium L. extract at 24, 48, and 72 h (P > 0.05). The IC50 values were 236.1, 147.0, and 118.0 μg/mL after 24, 48, and 72 h respectively. TNF-α, IL-1β, IL-6, TLR-4, iNOS, and NF-κB p65 mRNA levels in the pre-treated LPS-stimulated PBMCs were concentration-dependently reduced (P < 0.01 for TNF-α, TLR-4, and NF-κB p65; P < 0.05 for IL-1β, IL-6, and iNOS). Also, the protein levels of pro-inflammatory mediators decreased and these differences were significant for TNF-α, IL-1β, and TLR-4 (P < 0.001, P < 0.01, and P < 0.001, respectively).Mentha pulegium L. extract decreased the expression and biosynthesis of pro-inflammatory mediators. These effects are mainly mediated by TLR-4 and NF-κB suppression. Thus, Mentha pulegium L. could be useful in treating or ameliorating chronic inflammatory diseases.

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.

Arctium lappa L. roots inhibit the intestinal inflammation of dietary obese rats through TLR4/NF-κB pathway

Long-term consumption of Arctium lappa L. roots can lead to weight loss. To explore the relationship between anti-obesity and anti-inflammation, the effects and mechanism of A. lappa L. root powder (ARP) on intestinal inflammation in obese rats were investigated. Dietary obese rats were successfully established by feeding a high-fat and high-sugar diet. The control group (n = 6) consumed a normal diet. The intestines were compared among the groups (each n = 6) with and without the administration of ARP (intragastric 7.5 g/kg·bw/d). Real-time quantitative reverse transcription-polymerase chain reaction and western blotting analysis revealed that ARP effectively inhibited the expression of pro-inflammatory and inflammatory cytokines in the colons of obese rats. These cytokines included interleukin (IL)-1β, IL-8, IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. The inhibition rates for all these cytokines exceeded 88 %. Moreover, ARP demonstrated the ability to down-regulate key genes involved in Toll-like receptor 4 (TLR4) complexes, namely Tlr4, myeloid differentiation protein-2 (Md2), and myeloid differentiation factor 88 (Myd88), along with downstream signaling molecules such as tumor necrosis factor receptor associated factor 6 (TRAF6) and nuclear factor-κB (NF-κB), with inhibition rates over 81 %. Additionally, ARP was observed to inhibit protein levels of TLR4, NF-κB, IL-1β, and TNF-α in the colons of obese rats, with inhibition rates of 65.6 ± 10.9 %, 84.4 ± 19.9 %, 80.8 ± 14.4 %, and 68.4 ± 17.5 %, respectively. This study confirmed the effectiveness of ARP in inhibiting intestinal inflammation through the blockade of the TLR4/NF-κB signaling pathway. It also suggested that ARP holds potential in improving intestinal health in the context of obesity, implying its possible application in the prevention and treatment of obesity and related metabolic diseases.

Analysis of Influence of Cigarette Smoke on Signaling Pathways of Innate Immune System in Monocytes of Peripheral Blood

INTRODUCTION: Tobacco smoking is an important medical problem since it has a significant impact on the development and progression of chronic obstructive pulmonary disease (COPD). The components of tobacco smoke can initiate and support local and systemic inflammation with participation of monocytes and macrophages.&#x0D; AIM: To study molecular mechanisms associated with the impact of cigarette smoke on signaling pathways of the innate immune system in monocytes of peripheral blood.&#x0D; MATERIALS AND METHODS: The methods of in silico analysis was used to identify genes associated with the impact of tobacco smoke. On the basis of the data obtained, a cellular model of inflammation was created in vitro using tobacco smoke extract and monocytes of peripheral blood isolated by immunomagnetic separation. An enzyme-linked immunoassay (ELISA) kit was used to measure the concentration of tumor necrosis factor- (TNF-), interleukin-1 (IL-1) in cell supernatants, and of Toll-like receptor 4 (TLR4), ATP-binding cassette A1 (ABCA1) in homogenates of cell membranes of native monocytes and monocytes exposed to 4% tobacco smoke extract. These data were compared with the levels of TNF-, IL-1, TLR4 and ABCA1 in monocytes of peripheral blood of patients with COPD with frequent exacerbation phenotype and with obliterating atherosclerosis of lower limb arteries (OALLA). For statistical processing and visualization of the data, MedCalc 20.1.4 and R (version 4.2.2) software was used.&#x0D; RESULTS: Tobacco smoke influences TLR4, TNF- signaling pathways and lipid metabolism. Cigarette smoke extract enhanced the expression of proinflammatory cytokines TNF- and IL-1 in cell supernatants, increased the level of TLR4 and decreased that of ABCA1 in plasmolemma of monocytes of peripheral blood. In patients with COPD with frequent exacerbation phenotype and with OALLA, there were shown increase in the levels of proinflammatory TNF- and IL-1 cytokines in cell supernatants, increase in the level of TLR4 and reduction of the level of ABCA1 in plasmolemma of monocytes of peripheral blood compared to native monocytes of healthy individuals.&#x0D; CONCLUSION: Cigarette smoke enhances the production of proinflammatory TNF- and IL-1 cytokines, increases the levels of TLR4 protein and reduces the amount of ABCA1 transporter in membranes of monocytes of peripheral blood. This may partially explain the cause of the influence of cigarette smoke on development of the pulmonary and cardiovascular diseases. COPD with frequent exacerbation phenotype and OALLA are characterized by enhancement of inflammation with participation of monocytes.

O-GlcNAcylation of TLR4 inhibits osteogenic differentiation of periodontal ligament stem cells.

Toll-like receptor 4 (TLR4)-mediated inflammatory responses are associated with diabetes and periodontitis, which are dysregulated by O-GlcNAcylation. This study aimed to investigate the effects of O-GlcNAc transferase (OGT)-mediated TLR4 O-GlcNAcylation on the osteogenesis of periodontal ligament stem cells (PDLCs). PDLCs were treated with high glucose (HG) to establish a cell model. Osteogenic differentiation was evaluated using western blotting, an alkaline phosphatase activity assay, and an alizarin red S staining assay. The regulation of OGT on the O-GlcNAcylation of TLR4 was analyzed using co-immunoprecipitation, immunoprecipitation, western blotting, and immunofluorescence staining. The results showed that HG inhibited osteogenic differentiation and promoted inflammatory response. Knockdown of OGT promoted osteogenic differentiation of HG-treated PDLCs. OGT interacted with TLR4 and increased the O-GlcNAcylation and protein levels of TLR4 in the cytomembrane of PDLCs. Moreover, silenced TLR4 reversed the effects on osteogenic differentiation induced by OGT in HG-treated PDLCs. O-GlcNAcylation of TLR4 induced by OGT suppresses osteogenic differentiation of PDLCs after HG stimulation. The findings suggest a promising strategy for treating DP.

Elevated expression of Toll-like receptor 4 and cytokines in both serum and myometrium at term may serve as promising biomarkers for uterine activation preceding labor.

Increased inflammation and cytokine levels are considered risk factors and promoters of preterm birth (PTB). However, the regulatory mechanism of pregnancy-related inflammation remains unclear. Toll-like receptor 4 (TLR4) plays a critical role in inflammatory responses in various diseases. Therefore, our study aimed to investigate whether TLR4 is involved in the inflammatory responses during uterine activation for labor, with the goal of identifying potential biomarkers for uterine activation at term. We used flow cytometry to detect TLR4 expression on CD14+ maternal blood monocytes in the first, second, and third trimesters. ELISA was employed to measure TLR4 and cytokines levels in the maternal serum of term non-labor (TNL), term labor (TL) women and LPS induced preterm labor and PBS injected controls. TLR4siRNA was transfected into the human myometrial smooth muscle cells (HMSMCs), which were subsequently treated with IL-1β. The mRNA and protein levels of TLR4, uterine contraction-related protein connexin 43 (CX43), oxytocin receptor (OTR), MAPK/NF-κB signaling pathway, and cytokines were analyzed using qRT-PCR, western blotting, and immunohistochemistry. The study revealed TLR4 expression on CD14+ maternal blood monocytes was higher in the third trimester group compared to the first and second trimester groups (p<0.001). Maternal serum concentrations of TLR4 and cytokines were significantly higher in the TL group than the TNL group (p<0.001). TLR4, OTR, CX43, activated MAPK/NF-κB expression, and cytokines levels were upregulated in TL group, and similarly significantly higher in the LPS-induced preterm group than in the control group. Using the HMSMCs we demonstrated that TLR4siRNA transfection suppressed contractility. Interfering with TLR4 expression reduced the expression of OTR, CX43, cytokines, and MAPK/NF-κB activation. There was a significant positive relationship between TLR4 expression and the inflammatory status in the myometrium. ROC analysis indicated that TLR4 and cytokines may serve as potential biomarkers for predicting uterine activation for labor. Our data suggest that TLR4 and cytokines can act as stimulators of uterine activation for labor at term. Furthermore, the MAPK/NF-κB pathway appears to be one of the potential signaling pathways mediating TLR4's regulation of parturition initiation.

Aucubin combined with ADSCs-exos protects TBHP-induced nucleus pulposus cells via TLR4/NF-κB pathway

This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-β-galactosidase(SA-β-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1β(IL-1β), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1β and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1β and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.

Impacts of an 8-week regimen of aged garlic extract and aerobic exercise on the levels of Fetuin-A and inflammatory markers in the liver and visceral fat tissue of obese male rats

Obesity-induced chronic low-grade systemic inflammation is linked to the development of numerous diseases. Fetuin-A is known to affect inflammation and insulin resistance in obesity conditions. Free fatty acid (FFA)-induced proinflammatory cytokine expression in adipocytes occurs only in the presence of both Fetuin-A and toll-like receptor 4 (TLR4) and removing either of them prevented FFA-induced insulin resistance. Aged garlic extract (AGE) and exercise training have anti-inflammatory effects; however, the impact of AGE on Fetuin-A is unknown. We examined the effects of AGE with or without aerobic training (AT) on Fetuin-A and inflammatory markers. Forty healthy male Sprague Dawley rats were randomly assigned to normal diet (ND) (n=8) or high-fat diet (HFD) groups (n=32) and fed for 9 weeks. After 9 weeks ND group continued normal diet, and the HFD group was randomly assigned to the HFD, HFD+AGE (600mg/kg, once daily), HFD+AT (5 days/week), and HFD+AGE+AT groups that were continued for 8 weeks (n=8). The significance of differences among groups was assessed using one-way analysis of variance followed by the post-hoc Tukey test. Statistically significant differences were considered for p<0.05. AGE, AT, and AGE+AT significantly decreased body weight, plasma Fetuin-A, HOMA-IR, mRNA and protein levels of Fetuin-A and NFƙB in the liver and mRNA and Protein levels of Fetuin-A, TLR4 and NFƙB in visceral adipose tissue (VAT) compared to HFD. However, only AGE+AT significantly decreased TLR4 protein levels in the liver. Although AT and AGE reduce Fetuin-A and inflammatory markers, a combination of the two may be more effective at lowering inflammation.

Arctiin Inhibits Inflammation, Fibrosis, and Tumor Cell Migration in Rats With Ehrlich Solid Carcinoma.

ESC or Ehrlich solid carcinoma is a type of tumor originating from a spontaneous mammary adenocarcinoma in mice. It is a highly aggressive and fast-growing carcinoma that can create a solid mass when inserted under the skin. Its solid, undifferentiated form makes it an ideal model for researching cancer biology, tumor immunology, and testing various anti-cancer treatments. Additionally, arctiin has multiple beneficial properties, such as anti-proliferative, anti-oxidative, anti-adipogenic, and anti-bacterial. This study aimed to explore the potential anti-cancer benefits of arctiin in rats with ESC while also analyzing its effects on cell fibrosis markers, tumor cell migration, and inflammasome pathways. Rats were given a tumor in their left hind limb via an intramuscular injection consisting of 2×106 cells. After eight days, some of the rats received a daily oral dose of 30 mg/kg of arctiin for three weeks. Muscle samples were observed under an electron microscope or stained with hematoxylin/eosin. Additionally, gene expression and protein levels of toll-like receptor 4 (TLR4), NLR family pyrin domain containing 3 (NLRP3), signal transducer and activator of transcription 3 (STAT3), transforming growth factor (TGF)-β, endothelial growth factor (VEGF), and cyclin D1 were assessed in another part of the muscle samples. When ESC rats were given arctiin as a treatment, their mean survival time increased and their tumor volume and weight decreased. Additionally, when tumor tissue was examined under an electron microscope, it showed signs of pleomorphic cells, necrosis, nuclear fragmentation, membrane damage with cytoplasmic content spilling, and loss of cellular junction. The stained sections with hematoxylin/eosin showed a dense cellular mass and compressed, degenerated, and atrophied muscle. However, treatment with arctiin improved all these effects. Finally, the expression of TLR4, NLRP3, STAT3, TGF-β, VEGF, and cyclin D1 was significantly reduced with arctiin treatment. Through the use of arctiin, tumor sizeand weight were effectively reduced, leading to an increase in the average survival time of rats and an improvement in muscle structure. Additional research has shown that arctiin is able to suppress inflammation, fibrosis, and the migration of tumor cells by inhibiting STAT3, TGF-β1, TLR4, NLRP3, VEGF, and cyclin D1.