TLR4-mediated NF-κB Signaling Pathways Research Articles

Ilex asprella-derived polysaccharide activates macrophage via TLR4-mediated NF-κB and MAPK signaling pathways

Ilex asprella, a valued herb in China, is recognized for its medicinal and dietary benefits, yet its active components and mechanisms of action have been largely unknown. This study focused on the polysaccharide extracted from Ilex asprella (IAP) and to investigate its structural characteristics and immunomodulatory effects. The results indicated IAP had a molecular weight of 3324 Da and was mainly composed of arabinose, galactose, glucose, and mannose. In vitro study showed 10, 50, and 100 μg/mL of IAP significantly increased the mRNA expression of inducible nitric-oxide synthase, cyclooxygenase-2, reactive oxygen, and cytokines in RAW264.7 macrophages. According to the results, IAP also facilitated the nucleus translocation of NF-κB p65 and activated the phosphorylation of p38, JNK, and ERK, revealing IAP activated RAW264.7 cells via NF-κB and MAPK signaling pathways. Furthermore, the results of membranes receptor inhibitor administration suggested toll-like receptor 4 might be the action target of IAP.

Biological role of mitochondrial TLR4-mediated NF-κB signaling pathway in central nervous system injury.

Previous studies suggested that central nervous system injury is often accompanied by the activation of Toll-like receptor 4/NF-κB pathway, which leads to the upregulation of proapoptotic gene expression, causes mitochondrial oxidative stress, and further aggravates the inflammatory response to induce cell apoptosis. Subsequent studies have shown that NF-κB and IκBα can directly act on mitochondria. Therefore, elucidation of the specific mechanisms of NF-κB and IκBα in mitochondria may help to discover new therapeutic targets for central nervous system injury. Recent studies have suggested that NF-κB (especially RelA) in mitochondria can inhibit mitochondrial respiration or DNA expression, leading to mitochondrial dysfunction. IκBα silencing will cause reactive oxygen species storm and initiate the mitochondrial apoptosis pathway. Other research results suggest that RelA can regulate mitochondrial respiration and energy metabolism balance by interacting with p53 and STAT3, thus initiating the mitochondrial protection mechanism. IκBα can also inhibit apoptosis in mitochondria by interacting with VDAC1 and other molecules. Regulating the biological role of NF-κB signaling pathway in mitochondria by targeting key proteins such as p53, STAT3, and VDAC1 may help maintain the balance of mitochondrial respiration and energy metabolism, thereby protecting nerve cells and reducing inflammatory storms and death caused by ischemia and hypoxia.

Structural characterization and immune-enhancing effects of a novel polysaccharide extracted from Sargassum fusiforme

To alleviate the adverse effects of chemotherapy and bolster immune function, a novel polysaccharide derived from Sargassum fusiforme named as SFP-αII. The structural composition of SFP-αII predominantly consisted of guluronic and mannuronic acids in a molar ratio of 33.8:66.2, with an average molecular weight of 16.5 kDa. Its structure was primarily characterized by →4)-α-GulA-(1 → and →4)-β-ManA-(1 → linkages confirmed by FT-IR, methylation, and NMR analyses. The absence of a triple-helix structure was in SFP-αII was confirmed using circular dichroism and Congo red dye assays. The dimensions varied with lengths ranging from 20 nm up to 3 μm revealed by atomic force microscopy (AFM). SFP-αII has been found to enhance immunomodulatory activity in cyclophosphamide (CTX)-induced immunosuppressed mice. This was evidenced by improvements in immune organ indices, cytokine levels, and the release of nitric oxide (NO). Specifically, SFP-αII mitigated immunosuppression by upregulating the secretion of IL-1β (167.3 %) and TNF-α (227.1 %) at a dose of 400 mg/kg, compared with the CTX group in macrophages. Ultimately, SFP-αII may serve as a mechanism for immune enhancement through modulation of TLR4-mediated NF-κB and MAPK signaling pathways. This integration of traditional Chinese and Western medicine, leveraging SFP-αII as a potential functional food could be pivotal in alleviating immunosuppressive side effects in CTX treatment.

Macleaya cordata Extract Inhibits LPS-Induced IPEC-J2 Inflammation Through TLR4-Mediated NF-κB and MAPK Signaling Pathways

Objectives: The Macleaya cordata has long been utilized as a traditional medicinal remedy for treating inflammation in certain countries, and it is also being considered as a potential alternative to antibiotic growth promoters in livestock breeding. However, the molecular mechanisms underlying its anti-inflammatory characteristics have not been thoroughly investigated. In this study, we assessed the anti-inflammatory effects of the Macleaya cordata extract (MCE) and elucidated the molecular mechanism in the porcine intestinal epithelial cell line J2 (IPEC-J2) stimulated by lipopolysaccharide (LPS). Methods: The IPEC-J2 cells were pretreated with various concentrations of MCE (50 ng/mL, 100 ng/mL, and 150 ng/mL) prior to stimulation with LPS to induce of inflammation. Subsequently, the secretion levels and mRNA expression of the inflammatory cytokines IL-6, TNF-α, IFN-γ, and IL-10 were assessed. The impact of MCE on the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways was investigated. Results: The results demonstrated that MCE significantly suppressed the production and mRNA expression levels of pro-inflammatory cytokines in LPS-induced IPEC-J2 cells, while promoting the expression of anti-inflammatory cytokines. Moreover, it down-regulated the phosphorylation of NF-κB (p65) and attenuated the expression of phosphorylated JNK, ERK, and p38 MAPKs in a concentration-dependent manner. Furthermore, MCE downregulated the expression of TLR4 and MyD88, which serve as crucial upstream signaling molecules. The findings of this study suggest that the anti-inflammatory effect of MCE is attributed to its ability to regulate inflammatory cytokines and mediators by inhibiting the TLR4-mediated NF-κB and MAPK signaling pathways. Conclusions: Therefore, MCE holds promise as a potential therapeutic and preventive agent for managing inflammatory reactions and diseases in livestock and poultry breeding, warranting further investigation.

Quercetin Alleviates Lipopolysaccharide-Induced Cell Oxidative Stress and Inflammatory Responses via Regulation of the TLR4-NF-κB Signaling Pathway in Bovine Rumen Epithelial Cells.

Subacute rumen acidosis (SARA) will cause an increase in endotoxin, which will have a negative effect on the bovine rumen epithelial cells (BREC). Flavonoids are effective in treating inflammation caused by endotoxin. Quercetin is a vital flavonoid widely occurring in fruits and vegetables and has received significant interest as a prospective anti-inflammatory antioxidant. Nonetheless, quercetin's protective machinery against such damage to BREC induced by lipopolysaccharide (LPS) remains unclear. A combined quercetin and LPS-induced BREC inflammation model was utilized to elucidate the effect of quercetin protecting BREC from LPS-induced injury. After treating BREC with different doses of LPS (1, 5, and 10 μg/mL) for 6 h or 24 h, the mRNA expression of inflammatory factors was detected. Our experimental results show the establishment of the BREC inflammation model via mRNA high expression of pro-inflammatory cytokines in BREC following 6 h treatment with 1 µg/mL LPS. The promotive effect of 80 μg/mL quercetin on BREC growth via the cell counting kit-8 (CCK8) assay was observed. The expression of pro-inflammatory cytokines and chemokines, notably tumor necrosis factor α (TNF-α), Interleukin 1β (IL-1β), IL-6, CC-motif chemokine ligand 2 (CCL2), CCL20, CCL28, and CXC motif chemokine 9 (CXCL9), etc., was significantly reduced by quercetin supplementation. We also analyzed the mRNA detection of related pathways by qRT-PCR. Our validation studies demonstrated that quercetin markedly curbed the mRNA expression of the toll-like receptor 4 (TLR4) and myeloid differentiation primary response protein (MyD88) and the nuclear factor-κB (NF-κB) in LPS-treated BREC. In addition, western blot result outcomes confirmed, as expected, that LPS significantly activated phosphorylation of p44/42 extracellular regulated protein kinases (ERK1/2) and NF-κB. Unexpectedly, this effect was reversed by adding quercetin. To complement western blot results, we assessed p-ERK1/2 and p-p65 protein expression using immunofluorescence, which gave consistent results. Therefore, quercetin's capacity to bar the TLR4-mediated NF-κB and MAPK signaling pathways may be the cause of its anti-inflammatory effects on LPS-induced inflammatory reactions in BREC. According to these results, quercetin may be utilized as an anti-inflammatory medication to alleviate inflammation brought on by high-grain feed, and it also lays out a conceptual foundation regarding the development and utilization of quercetin in the later stage.

Immunoregulatory effects of Tetrastigma hemsleyanum polysaccharide via TLR4-mediated NF-κB and MAPK signaling pathways in Raw264.7 macrophages

Polysaccharide of Tetrastigma hemsleyanum (THP) exert antioxidant, antibacterial, lipid-lowering, and anti-inflammatory properties, especially some evidences have highlighted the efficiency of it as an anti-tumor agent. However, as a biological macromolecule with bidirectional immune regulation, the immunological enhancement effects of THP on macrophages and its underlying mechanisms are still largely unknown. In the present study, THP was prepared and characterized, and then the effect of THP on Raw264.7 cell activation was investigated. Structural characteristics of THP showed that the average molecular weight was 370.26 kDa, and the main monosaccharide composition was galactose, glucuronic acid, mannose, and glucose at a ratio of 31.56: 25.15: 19.44: 12.60, with high viscosity causing by relative high uronic acid. For immunomodulatory activity investigation, THP promoted the production of NO, IL-6 and TNF-α, as well as the expression of IL-1β, MCP-1, iNOS and COX-2, which were almost completely inhibited by TLR4 antagonist. Further study showed that THP could activate NF-κB and MAPK signaling pathways, and thus enhanced the phagocytic activity of Raw264.7 macrophages. In conclusion, the present study provided evidences that THP could be served as a new immunomodulator in both functional foods and the pharmaceutical field.

Chlamydia psittaci hypothetical inclusion membrane protein CPSIT_0842 evokes a pro-inflammatory response in monocytes via TLR2/TLR4 signaling pathways

Chlamydia psittaci (C. psittaci) is an obligate intracellular pathogen that resides within a membrane-bound compartment known as the inclusion. Upon entering the host cell, Chlamydiae secrete numerous proteins to modify the inclusion membrane. Inclusion membrane (Inc) proteins are important pathogenic factors in Chlamydia and play crucial roles in the growth and development of Chlamydia. In the present study, the C. psittaci protein, CPSIT_0842, was identified and shown to localize to the inclusion membrane. Temporal analysis revealed that CPSIT_0842 is an early expression protein of Chlamydia. Moreover, this protein was shown to induce the expression of pro-inflammatory cytokines IL-6 and IL-8 in human monocytes (THP-1 cells) via the TLR2/TLR4 signaling pathway. CPSIT_0842 increases the expression of TLR2, TLR4, and adaptor MyD88. Suppression of TLR2, TLR4, and MyD88 markedly attenuated CPSIT_0842-induced production of IL-6 and IL-8. MAP kinases and NF-κB, important downstream molecules of TLR receptors in inflammatory signaling pathways, were also confirmed to be activated by CPSIT_0842. CPSIT_0842-induced production of IL-6 was reliant on activation of the ERK, p38, and NF-κB signaling pathways while IL-8 expression was regulated by the ERK, JNK, and NF-κB signaling pathways. Specific inhibitors of these signaling pathways significantly decreased CPSIT_0842-mediated expression of IL-6 and IL-8. Together these findings demonstrate that CPSIT_0842 upregulates the expression of IL-6 and IL-8 via TLR-2/TLR4-mediated MAPK and NF-κB signaling pathways in THP-1 cells. Exploring these molecular mechanisms enhances our understanding of C. psittaci pathogenesis.

The antimicrobial peptide LK2(6)A(L) exhibits anti-inflammatory activity by binding to the myeloid differentiation 2 domain and protects against LPS-induced acute lung injury in mice

Acute lung injury (ALI) is a life-threatening disease that is generally attributable to an uncontrolled inflammatory response in the lung, but there is a lack of effective treatments. At present, regulating the inflammatory response has become an important strategy for treating ALI. In the present study, LK2(6)A(L), a peptide derived from the natural antimicrobial peptide temporin-1CEa, inhibited lipopolysaccharide (LPS)-induced expression of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and NO in RAW264.7 cells. Herein, the anti-inflammatory mechanism of LK2(6)A(L) was investigated. The RNA-sequencing (RNA-seq) results showed that LK2(6)A(L) significantly inhibited the TLR4-mediated NF-κB and MAPK signaling pathways in LPS-induced RAW264.7 cells. The results of co-immunoprecipitation (Co-IP), pull-down experiment, confocal laser scanning microscopy, and surface plasmon resonance (SPR) suggested that MD2 was the direct target of LK2(6)A(L). Chemical inhibition of MD2 and its knockdown abolished the anti-inflammatory effect of LK2(6)A(L). Molecular dynamic simulation indicated that LK2(6)A(L) could bind to the active domain of the MD2 hydrophobic pocket via six hydrogen bonds. The truncated peptides were designed based on analysis of the molecular docking of LK2(6)A(L) to MD2. The truncated peptide IS-7 showed strong affinity to MD2 and a remarkable inhibitory effect on pro-inflammatory factors that was comparable to the effect of LK2(6)A(L). Finally, LK2(6)A(L) and IS-7 relieved inflammatory symptoms and lung tissue destruction in the ALI mouse model. Overall, our study suggested that LK2(6)A(L) showed promising anti-inflammatory activity by targeting MD2, and the amino acid domain 7–13 was an important area that binds with MD2 and also an anti-inflammatory active region. LK2(6)A(L) and IS-7 may be potential new treatments for ALI and other acute inflammatory diseases.

Quercetin Alleviates Lipopolysaccharide-Induced Inflammatory Response in Bovine Mammary Epithelial Cells by Suppressing TLR4/NF-κB Signaling Pathway.

Bovine mastitis is one of the most common clinical diseases in dairy cows, causing huge economic losses to the dairy industry. Quercetin is an important flavonoid existing in many food resources, which has attracted widespread attention as a potential anti-inflammatory and antioxidant. However, the molecular mechanism of quercetin on inflammatory responses and oxidative stress in bovine mammary epithelial cells (BMECs) induced by lipopolysaccharide (LPS) remains unknown. The objective of this study was to investigate the effects of quercetin on inflammation responses, oxidative stress, and barrier function of BMEC induced by LPS. Our results showed that BMEC viability was not affected by treatment with 50 and 100 μg/ml of quercetin and 1 μg/ml of LPS compared with control group. The results of oxidative stress indicators and related genes of barrier function indicated that 100 μg/ml of quercetin effectively protected the BMECs from damage of oxidative and barrier induced by 1 μg/ml of LPS. Moreover, the messenger RNA (mRNA) expressions of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and chemokines CXCL2, CXCL5, CCL5, and CXCL8 were markedly decreased in the LPS-treated bovine retinal endothelial cells (BRECs) with 100 μg/ml of quercetin relatively to LPS alone. More importantly, the mRNA expressions of toll-like receptor 4 (TLR4), CD14, myeloid differential protein-2 (MD2), and myeloid differentiation primary response protein (MyD88) genes involved in TLR4 signal pathway were significantly attenuated by the addition of quercetin in LPS-treated BMEC, suggesting that quercetin can inhibit the TLR4 signal pathway. In addition, immunocytofluorescence showed that quercetin significantly inhibited the nuclear translocation of NF-κB p65 in BMEC induced by LPS. Therefore, the protective effects of quercetin on inflammatory responses in LPS-induced BMEC may be due to its ability to suppress the TLR4-mediated NF-κB signaling pathway. These findings suggest that quercetin can be used as an anti-inflammatory reagent to treat mastitis induced by exogenous or endogenous LPS release.

Preventive effects of arctigenin from Arctium lappa L against LPS-induced neuroinflammation and cognitive impairments in mice.

Arctigenin (Arc) is a phenylpropanoid dibenzylbutyrolactone lignan in Arctium lappa L, which has been widely applied as a traditional Chinese herbal medicine for treating inflammation. In the present study, we explored the neuroprotective effect and the potential mechanisms of arctigenin against LPS-evoked neuroinflammation, neurodegeneration, and memory impairments in the mice hippocampus. Daily administration of arctigenin (50mg/kg per day, i.g.) for 28days revealed noticeable improvements in spatial learning and memory deficits after exposure to LPS treatment. Arctigenin prevented LPS-induced neuronal/synaptic injury and inhibited the increases in Abeta (Aβ) generation and the levels of amyloid precursor protein (APP) and β-site amyloid precursor protein cleavage enzyme 1 (BACE1). Moreover, arctigenin treatment also suppressed glial activation and reduced the production of proinflammatory cytokines. In LPS-treated BV-2 microglial cells and mice, activation of the TLR4 mediated NF-κB signaling pathway was significantly suppressed by arctigenin administration. Mechanistically, arctigenin reduced the LPS-induced interaction of adiponectin receptor 1 (AdipoR1) with TLR4 and its coreceptor CD14 and inhibited the TLR4-mediated downstream inflammatory response. The outcomes of the current study indicate that arctigenin mitigates LPS-induced apoptotic neurodegeneration, amyloidogenesis and neuroinflammation as well as cognitive impairments, and suggest that arctigenin may be a potential therapeutic candidate for neuroinflammation/neurodegeneration-related diseases.

Echinacea purpurea extract inhibits LPS-induced inflammatory response by interfering with TLR4-mediated NF-κB and MAPKs signaling pathways

Echinacea purpurea (Asteraceae family) is widely used in the European countries and the United States due to its proven immune enhancement and anti-inflammatory effects. Echinacea purpurea has been reported prevent and treat upper respiratory tract infections and common cold, but the underlying molecular mechanisms are not well understood. In the present study, we examined the anti-inflammatory effects and molecular mechanisms of Echinacea purpurea (EP) extract using lipopolysaccharide (LPS)-stimulated signal pathways in RAW264.7 cells. Our results suggest that EP extract exerts anti-inflammatory effects by down-regulating the expression of LPS-induced toll-like receptor 4 (TLR4), subsequently inhibiting the activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling pathways and suppression of the release of pro-inflammatory cytokines. These results suggest that EP extract is a potential therapeutic agent for inflammatory diseases.

Mechanisms of RAW264.7 macrophages immunomodulation mediated by polysaccharide from mung bean skin based on RNA-seq analysis

In this study, the immunomodulation properties and the possible molecular mechanisms of mung bean skin polysaccharide (MBP) on RAW264.7 were investigated. The results showed that MBP could enhance the phagocytic activity, promote intracellular reactive oxygen species (ROS) production as well as the release of nitric oxide (NO) and cytokines in macrophages. The results of global screening of all transcripts by RNA-seq revealed 927 differentially expressed genes between the control and MBP-treated groups, including 196 up-regulated genes and 731 down-regulated genes. From GO analysis, there were 5 cellular components terms (CC), 5 biological processes terms (BP) and 10 molecular functions terms (MF). Toll-like receptor 4 (TLR4 receptor), mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways were selected by KEGG analysis to elucidate the mechanism of MBP action on macrophages. Western blot analysis showed that MBP could increase the expression of TLR4, MAPKs and NF-κB signaling pathway-related proteins, which confirmed that TLR4, MAPKs and NF-κB signaling pathways were essential to MBP-mediated mocrophage activation. Moreover, the production of both NO and cytokine TNF-α was found to be significantly reduced after the addition of TLR4, MAPKs and NF-κB inhibitors. These results suggest that MBP can control the immune response of macrophages through TLR4-mediated MAPKs and NF-κB signaling pathways. This work concluded MBP could serve as a potential natural immunomodulator, providing a molecular basis for its application in functional foods.

Ginsenoside Rb1 Mitigates Escherichia coli Lipopolysaccharide-Induced Endometritis through TLR4-Mediated NF-κB Pathway.

Endometritis is the inflammatory response of the endometrial lining of the uterus and is associated with low conception rates, early embryonic mortality, and prolonged inter-calving intervals, and thus poses huge economic losses to the dairy industry worldwide. Ginsenoside Rb1 (GnRb1) is a natural compound obtained from the roots of Panax ginseng, having several pharmacological and biological properties. However, the anti-inflammatory properties of GnRb1 in lipopolysaccharide (LPS)-challenged endometritis through the TLR4-mediated NF-κB signaling pathway has not yet been researched. This study was planned to evaluate the mechanisms of how GnRb1 rescues LPS-induced endometritis. In the present research, histopathological findings revealed that GnRb1 ameliorated LPS-triggered uterine injury. The ELISA and RT-qPCR assay findings indicated that GnRb1 suppressed the expression level of pro-inflammatory molecules (TNF-α, IL-1β and IL-6) and boosted the level of anti-inflammatory (IL-10) cytokine. Furthermore, the molecular study suggested that GnRb1 attenuated TLR4-mediated NF-κB signaling. The results demonstrated the therapeutic efficacy of GnRb1 in the mouse model of LPS-triggered endometritis via the inhibition of the TLR4-associated NF-κB pathway. Taken together, this study provides a baseline for the protective effect of GnRb1 to treat endometritis in both humans and animals.

Identification, semisynthesis, and anti-inflammatory evaluation of 2,3-seco-clavine-type ergot alkaloids from human intestinal fungus Aspergillus fumigatus CY018.

Intestinal commensal fungi are vital to human health, and their secondary metabolites play a key role in the reciprocal relationship. In the present study, the first example of 2,3-seco ergot alkaloids belonging to clavine-type were isolated from the fermentation of human intestinal fungus Aspergillus fumigatus CY018, including two pairs of diastereoisomers, secofumigaclavines A (3) and B (4) and secofumigaclavines C (5) and D (6), one analogue features a highly unsaturated skeleton, secofumigaclavine E (7), along with two known ones, fumigaclavines C (1) and D (2). Their structures were identified based on extensive spectroscopic data in a combination of quantum chemical calculations. Moreover, a single-step operation of semi-synthetic reaction based on riboflavin (RF)-dependent photocatalysis was performed to obtain the novel 2,3-seco ergot alkaloids 3 and 5 from their biosynthetic precursors 1 and 2. All the isolated compounds were evaluated for their anti-inflammatory activity. Among them, secofumigaclavine B (4) could bind to MD2 with a low micromole level of the equilibrium dissociation constant measured by surface plasmon resonance (SPR), and suppress TLR4-mediated NF-κB signaling pathway in RAW264.7cells, resulting in its anti-inflammatory effect. Molecular dynamics revealed that amino acid residue Tyr131 played a key role in the interaction of secofumigaclavine B (4) with MD2. These findings suggested that secofumigaclavine B (4) could be considered as a potential candidate for the development of MD2 inhibitors.

Exploring the Mechanism of Resveratrol in Reducing the Soft Tissue Damage of Osteoarthritis Based on Network Pharmacology and Experimental Pharmacology.

Aim To explore the mechanism of resveratrol in reducing the soft tissue damage of osteoarthritis (OA) based on network pharmacology. Methods Pharmmapper was used to predict the target of resveratrol, OMIM and Genecards were used to collect OA-related disease genes, and David ver 6.8 was used for enrichment analysis. Then, animal experiments were carried out for verification. The rat OA model was established and the rats were randomly divided into 4 groups: model group, resveratrol low-dose group, resveratrol high-dose group, and blank control group for follow-up experiments. Hematoxylin-eosin (HE) staining was used to detect the degree of pathological damage of rat bones and joints. Enzyme-linked immunosorbent assay (ELISA) was used for the content of inflammatory factors. Western blot was used to detect the expression of Toll-like receptor 4 (TLR4), Myeloid differentiation factor 88 (MyD88), nuclear factor kappa B protein (NF-κB), cysteine protease-9 (CASP-9), Bcl-2 protein, and Bax protein. Results Through network pharmacological analysis, this study found that resveratrol may regulate the TLR4 signaling pathway, PI3K-Akt signaling pathway, FoxO signaling pathway, Osteoclast differentiation, Rheumatoid arthritis, etc. Animal experiments showed that compared with the model group, the pathological damage of bone and joint in the resveratrol low-dose and high-dose groups was significantly improved. Compared with the model group, the serum levels of IL-1beta, IL-6, IL-17, TNF-α, and MCP-1 in the resveratrol low-dose and high-dose groups were significantly reduced (P < 0.05); protein levels of TLR-4, MyD88, and NF-κB p65 were significantly reduced (P < 0.05); caspase-9 and Bax protein levels were significantly reduced (P < 0.05), and Bcl-2 was significantly increased (P < 0.05). Conclusion Resveratrol may inhibit the activation of the TLR4-mediated NF-κB signaling pathway and has a repairing effect on soft tissue damage in OA.

Nanocurcumin alleviates inflammation and oxidative stress in LPS-induced mastitis via activation of Nrf2 and suppressing TLR4-mediated NF-κB and HMGB1 signaling pathways in rats.

Coliform mastitis is a worldwide serious disease of the mammary gland. Curcumin is a pleiotropic polyphenol obtained from turmeric, but it is hydrophobic and rapidly eliminated from the body. However, nanoformulation of curcumin significantly improves its pharmacological activity by enhancing its hydrophobicity and oral bioavailability. Our study aimed to investigate the possible antioxidant and anti-inflammatory effects of nanocurcumin as a prophylactic against LPS-induced coliform mastitis in rat model, where LPS was extracted from a field strain of Escherichia coli (bovine mastitis isolate). The study was conducted on twenty lactating Wistar female rats divided into four equal groups, and the mastitis model was initiated by injection of LPS through the duct of the mammary gland. The results showed that nanocurcumin significantly attenuated the lipid peroxidation (MDA), oxidized glutathione, the release of pro-inflammatory cytokines (TNF-α and IL-1β), and the gene expression of TLR4, NF-κB p65, and HMGB1. Meanwhile, it improved the reduced glutathione level and Nrf2 activity and preserved the normal alveolar architecture. These findings suggested that nanocurcumin supplementation can be a promising potential protective approach for coliform mastitis.

Ibudilast Attenuates Folic Acid-Induced Acute Kidney Injury by Blocking Pyroptosis Through TLR4-Mediated NF-κB and MAPK Signaling Pathways.

Folic acid (FA)-induced renal tubule damage, which is characterized by extensive inflammation, is a common model of acute kidney injury (AKI). Pyroptosis, a pro-inflammatory form of cell death due to the activation of inflammatory caspases, is involved in AKI progression. Ibudilast, a TLR4 antagonist, has been used in the clinic to exert an anti-inflammatory effect on asthma. However, researchers have not explored whether ibudilast exerts a protective effect on AKI by inhibiting inflammation. In the present study, ibudilast reversed FA-induced AKI in mice, as indicated by the reduced serum creatinine and urea nitrogen levels, and improved renal pathology, as well as the downregulation of kidney injury marker-1. In addition, ibudilast significantly increased the production of the anti-inflammatory factor IL-10 while suppressing the secretion of the pro-inflammatory cytokine TNF-α and macrophage infiltration. Moreover, in the injured kidney, ibudilast reduced the levels of both inflammasome markers (NLRP3) and pyroptosis-related proteins (caspase-1, IL1-β, IL-18, and GSDMD cleavage), and decreased the number of TUNEL-positive cells. Further mechanistic studies showed that ibudilast administration inhibited the FA-induced upregulation of TLR4, blocked NF-κB nuclear translocation, and reduced the phosphorylation of NF-κB and IκBα, p38, ERK, and JNK. Thus, this study substantiates the protective effect of ibudilast on FA-induced AKI in mice and suggests that protection might be achieved by reducing pyroptosis and inflammation, likely through the inhibition of TLR4-mediated NF-κB and MAPK signaling pathways.

Propolis ameliorates restenosis in hypercholesterolemia rabbits with carotid balloon injury by inhibiting lipid accumulation, oxidative stress, and TLR4/NF-κB pathway.

Neointima formation and atherosclerosis are the main complications after the endovascular intervention and vascular surgery, and there are no effective drugs. Propolis is a kind of resin substance produced by honeybees and has numerous health-beneficial effects. In this study, we evaluated the effects of propolis (125 and 250mg·kg-1 ·day-1 , 6weeks) on carotid restenosis in hypercholesterolemia rabbits. Propolis significantly ameliorated the degree of carotid restenosis, inhibited neointima hyperplasia, reduced serum lipids profile, and enhanced the anti-oxidative activities in hypercholesterolemia rabbits. Furthermore, propolis reduced the plasma levels of C-reactive protein, interleukin-6, and tumor necrosis factor-α (TNF-α), and inhibited the expression of CD68, TLR4, NF-κB p65, MMP-9, and TNF-α in the carotid arteries. The results indicate that propolis has a protective effect on carotid restenosis in rabbits, which is associated with regulating blood lipids, inhibiting oxidative stress and inflammation, and its anti-inflammatory activity may be related to inhibit TLR4-mediated NF-κB signaling pathway. PRACTICAL APPLICATIONS: Restenosis is a primary challenge in angioplasty and atherosclerotic treatment. Hyperlipidemia can induce inflammation and accelerate the formation of restenosis. Recently, natural products have been widely used to prevent intimal hyperplasia of common cardiovascular diseases. Propolis is currently a popular functional food, but the role of propolis on carotid restenosis after angioplasty and its underlying mechanism remains unclear. This study showed that propolis inhibits the effect of carotid restenosis in hypercholesterolemia rabbits. The results of this study may provide a basis for propolis to prevent and treat vascular restenosis.

IL-1 Receptor Antagonist Protects the Osteogenesis Capability of Gingival-Derived Stem/Progenitor Cells under Inflammatory Microenvironment Induced by Porphyromonas gingivalis Lipopolysaccharides.

Mesenchymal stem cells (MSCs) have been considered to be a future treatment option for periodontitis due to their excellent regenerative capability. However, it is still a challenge to protect MSCs' biological properties from multiple bacterial toxins in local inflammatory environment. The present study is aimed at investigating the treatment effect of interleukin-1 receptor antagonist (IL-1ra) on cell proliferation, migration, and osteogenic differentiation of gingival-derived mesenchymal stem cells (GMSCs) under an inflammatory microenvironment induced by Porphyromonas gingivalis lipopolysaccharides (P. gingivalis-LPS). GMSCs derived from Sprague-Dawley (SD) rats' free gingival tissues were treated with P. gingivalis-LPS (10 μg/mL) to create in vitro inflammatory environment. Different concentrations of IL-1ra (0.01-1 μg/mL) were used to antagonize the negative effect of LPS. Cell behaviors including proliferation, cloning formation unit (CFU), cell migration, osteogenic differentiation, mineral deposition, and cytokine production were assessed to investigate the protection effect of IL-1ra on GMSCs under inflammation. The toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) pathway activated by LPS was evaluated by real-time quantitative polymerase chain reaction (RT-PCR) and western blot. In response to P. gingivalis-LPS treatment, cell numbers, cloning formation rate, cell migration rate, proinflammatory cytokine production, and osteogenic differentiation-associated protein/mRNA expressions as well as mineralized nodules were suppressed in a time-dependent manner. These negative effects were effectively attenuated by IL-1ra administration in a time- and dose-dependent manner. In addition, mRNA expressions of TLR4 and IkBα decreased dramatically when IL-1ra was added into LPS-induced medium. IL-1ra also reversed the LPS-induced TLR4/NF-κB activation as indicated by western blot. The present study revealed that IL-1ra decreased inflammatory cytokine production in a supernatant, so as to protect GMSCs' osteogenesis capacity and other biological properties under P. gingivalis-LPS-induced inflammatory environment. This might be explained by IL-1ra downregulating TLR4-mediated NF-κB signaling pathway activation.

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.