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The naked mole rat has unique biologic characteristics that include atypical inflammatory responses. Lipopolysaccharide induces inflammation which triggers brain centers controlling feeding, and behavior to result in “sick animal behavior”. We characterized the bodyweight, locomotor, and other behavioral responses of this rodent to lipopolysaccharide administration. Lipopolysaccharide caused weight losses, which were not prevented by TAK 242. In the open field test, lipopolysaccharide did not depress locomotion, while urination, defecation, and activity freezing were rare. The animals exhibited walling but not rearing and fast backward movements that were unaffected by lipopolysaccharide. Failure to depress locomotion suggests either a unique immunity-brain crosstalk or motor responses/centers that tolerate depressive effects of inflammation. The absence of activity freezing and rarity of urination and defecation suggests that novel environments or lipopolysaccharide do not induce anxiety, or that anxiety is expressed differently in the animal. The absence of rearing could be due to the design of the animal’s locomotor apparatus while fast backward movement could be a mechanism for quick escape from threats in the tunnels of their habitat. Our results elucidate the unique biology of this rodent, which elicits interest in the animal as a model for inflammatory research, although the findings require mechanistic corroborations.

Objectives Early acute kidney injury (AKI) after burn contributes to disastrous prognoses for severely burned patients. Burn-induced renal oxidative stress and secondary proinflammatory mediator release contribute to early AKI development, and Toll-like receptor (TLR) 4 regulates inflammation. Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that plays a vital role in protecting against ischemia-induced organ injury via its antioxidant properties and regulation of inflammation. We investigated the potential effect of HO-1 induction in preventing burn-induced early AKI and its related mechanism. Methods A classic major-burn rat model was established using a 100 °C water bath, and hemin was injected intraperitoneally immediately after the injury to induce HO-1. Histological staining and blood tests were used to assess AKI progression based on structural changes and function. Renal levels of HO-1, oxidative stress, proinflammatory mediators and TLR4-related signals were detected using ELISA, immunostaining, qRT-PCR, and western blotting. The selective TLR4 inhibitor TAK242 and TLR4 inducer LPS were introduced to determine the roles of HO-1 in burn-related renal inflammation and the TLR4 pathway. Results Hemin improved burn-induced renal histological damage and dysfunction, and this beneficial effect was related to reduced renal oxidative stress and the release of proinflammatory mediators, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6 and intracellular adhesion molecule-1 (ICAM-1). Hemin downregulated the expression of TLR4 and the subsequent phosphorylation of IKKα/β, IκBα, and NF-κB p65;. TAK242 exerted an effect similar to but weaker than hemin; and LPS reversed the antiinflammatory effect of hemin and the regulation of TLR4 signals. These results suggested that the TLR4 signaling pathway mediated the HO-1-facilitated regulation of renal inflammation after burn. Conclusion The present study demonstrated that HO-1 induction prevented burn-induced early AKI by targeting renal inflammation, which was mediated via regulation of the TLR4/NF-κB signaling pathway.

The blood-testicular barrier (BTB) is involved in spermatogenesis, protects sperm development, and plays a crucial role in the reproductive process. Tight junctions (TJs) between Sertoli cells (SCs) are the key structure of (BTB), and if its structure is damaged, BTB function is affected. The cellular inflammation caused by Gram-negative bacteria affects the structural integrity of TJs. Melatonin (MT) has anti-inflammatory effects; however, the effect of MT in newborn calf SCs is unknown. Therefore, this experiment studied the protective effect of MT. The results showed that LPS upregulated TLR4, MyD88, and NF-κB expressions, in turn, activated the TLR4/MyD88/NF-κB signaling pathway, produced a large amount of IL-6 and IL-1β, downregulated the expression of ZO-1 and Occludin, and reduced the viability of SCs, which resulted in the inflammatory response of SCs and damage of TJs. The addition of MT decreased TLR4, MyD88, and NF-κB expressions, it then inhibited the activation of TLR4/MyD88/NF-κB signaling pathway, downregulated the expression of IL-6 and IL-1β, upregulated the expression of ZO-1 and Occludin, and increased the cell viability, thereby alleviating the inflammatory response of SCs, and restored the TJs structure. Overall, our results reveal that MT can alleviate LPS-induced in newborn calf SCs Inflammation and TJs injury through TLR4/MyD88/NF-κB signaling pathway.

Background The etiology of food allergy is poorly understood; mouse models are powerful systems to discover immunologic pathways driving allergic disease. C3H/HeJ mice are a widely used model for the study of peanut allergy because, unlike C57BL/6 or BALB/c mice, they are highly susceptible to oral anaphylaxis. However, the immunologic mechanism of this strain’s susceptibility is not known. Objective We aimed to determine the mechanism underlying the unique susceptibility to anaphylaxis in C3H/HeJ mice. We tested the role of deleterious Toll-like receptor 4 (Tlr4) or dedicator of cytokinesis 8 (Dock8) mutations in this strain because both genes have been associated with food allergy. Methods We generated C3H/HeJ mice with corrected Dock8 or Tlr4 alleles and sensitized and challenged them with peanut. We then characterized the antibody response to sensitization, anaphylaxis response to both oral and systemic peanut challenge, gut microbiome, and biomarkers of gut permeability. Results In contrast to C3H/HeJ mice, C57BL/6 mice were resistant to anaphylaxis after oral peanut challenge; however, both strains undergo anaphylaxis with intraperitoneal challenge. Restoring Tlr4 or Dock8 function in C3H/HeJ mice did not protect from anaphylaxis. Instead, we discovered enhanced gut permeability resulting in ingested allergens in the bloodstream in C3H/HeJ mice compared to C57BL/6 mice, which correlated with an increased number of goblet cells in the small intestine. Conclusions Our work highlights the potential importance of gut permeability in driving anaphylaxis to ingested food allergens; it also indicates that genetic loci outside of Tlr4 and Dock8 are responsible for the oral anaphylactic susceptibility of C3H/HeJ mice.

Neuroinflammation has an essential role in various neurodegenerative diseases including Parkinson's disease (PD). Microglial activation as a result of neuroinflammation exacerbates the pathological consequences of the disease. The toxic effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes alpha-synuclein (α-synuclein) accumulation, which leads to dopaminergic neuron death in the MPTP-induced mouse model. Toll-like receptor 4 (TLR4) stimulates release of cytokine through NF-kB by activating glial cells, thus resulting in the death of dopaminergic neurons. Melatonin has the ability to cross the blood-brain barrier and protect neurons through anti-inflammatory properties. We hypothesized that melatonin could suppress TLR4-mediated neuroinflammation, decrease cytokine release due to the inflammatory response, and reduce dopaminergic neuron loss in the MPTP-induced mouse model. In the MPTP-induced mouse model, we aimed to assess the neuroinflammatory responses caused by TLR4 activation as well as the effect of melatonin on these responses. Three-month-old male C57BL/6 mice were randomly divided into five groups; Control (Group-C), Sham (Group-S), Melatonin-treated (Group-M), MPTP-injected (Group-P), and MPTP + melatonin-injected (Group-P + M). MPTP toxin (20 mg/kg) was dissolved in saline and intraperitoneally (i.p.) injected to mice for two days with 12 h intervals. The total dose per mouse was 80 mg/kg. Melatonin was administered (20 mg/kg) intraperitoneally to Group-M and Group-P + M twice a day for five days. Eight days after starting the experiment, the motor activities of mice were evaluated by locomotor activity tests. The effects on dopamine neurons in the SNPc was determined by tyrosine hydroxylase (TH) immunohistochemistry. TLR4, α-synuclein, and p65 expression was evaluated by immunostaining as well. The amount of TNF-alpha in the total brain was evaluated by western blot analysis. In our results seen that locomotor activity was lower in Group-P compared to Group-C. However, melatonin administration was improved this impairment. MPTPcaused decrease in TH immuno-expression in dopaminergic neurons in Group-P. TLR4 (p0.001), α-synuclein (p0.001), and p65 (p0.01) immuno-expressions were also decreased in Group-P+M compared to Group-P (using MPTP). TNF-α expression was lower in Group-C, Group-S, Group-M, and Group-P+M, when compared to Group-P (p0.0001) due to the absence of inflammatory response. In conclusion, our study revealed that melatonin administration reduced α-synuclein aggregation and TLR4-mediated inflammatory response in the MPTP-induced mouse model.

Key Points TLR4 is overexpressed on MM MSCs, and its activation promotes the crosstalk with MM cells in enhancing protumoral factors.Specifically targeting TLR4 on MM MSCs strengthens conventional MM therapy and weakens MM development in vivo., Visual Abstract, Bone marrow (BM) mesenchymal stromal cells (MSCs) are abnormal in multiple myeloma (MM) and play a critical role by promoting growth, survival, and drug resistance of MM cells. We observed higher Toll-like receptor 4 (TLR4) gene expression in MM MSCs than in MSCs from healthy donors. At the clinical level, we highlighted that TLR4 expression in MM MSCs evolves in parallel with the disease stage. Thus, we reasoned that the TLR4 axis is pivotal in MM by increasing the protumor activity of MSCs. Challenging primary MSCs with TLR4 agonists increased the expression of CD54 and interleukin-6 (IL-6), 2 factors directly implicated in MM MSC-MM cell crosstalk. Then, we evaluated the therapeutic efficacy of a TLR4 antagonist combined or not with conventional treatment in vitro with MSC-MM cell coculture and in vivo with the Vk*MYC mouse model. Selective inhibition of TLR4 specifically reduced the MM MSC ability to support the growth of MM cells in an IL-6-dependent manner and delayed the development of MM in the Vk*MYC mouse model by altering the early disease phase in vivo. For the first time, we demonstrate that specific targeting of the pathological BM microenvironment via TLR4 signaling could be an innovative approach to alter MM pathology development.

Type 2 diabetes mellitus (T2DM) is known as a complex genetic disease characterized by genetic and environmental factors. The imbalanced intestinal flora and intestinal mucosal barrier are considered to be related to T2DM. Curcumin has been proved to affect the progression of T2DM. T2DM animal was established by low-dose streptozotocin intraperitoneal injection combined with high-fat diet (HFD) feeding. Hematoxylin and eosin (HE) staining and transfer electron microscopy (TEM) were used to observe morphological changes of intestinal tissues of T2DM rats. Insulin and glucose tolerance tests were performed to investigate the influence of curcumin on blood glucose. Curcumin significantly improved the intestinal integrity, hyperglycemia and insulin resistance in diabetic rats. The metabolic endotoxemia induced by HFD in diabetic rats was inhibited remarkably. Curcumin reversed gut microbiota dysbiosis in diabetic rats caused by HFD. We demonstrated that curcumin could protect intestinal mucosal barrier, improve insulin resistance and reduce blood glucose in diabetic rats. This study might provide experimental evidence for the prevention and treatment in T2DM.

Morphine tolerance poses a great challenge for clinicians, whose pathogenesis has a close connection with microglial activation and neuroinflammation. Dihydroartemisinin (DHA) that derives from artemisinin, may serve as a potential anti-inflammatory drug. In this study, the effects as well as the underlying mechanism of DHA on suppressing microglial activation and neuroinflammation were explored. The microglial cell line BV-2 cells were induced by morphine and treated with DHA or minocycline. With the application of CCK-8, the cell viability was detected. Western blot was employed to assess the expressions of Ki67, IBa-1, and TLR4 and quantitative real-time PCR (qRT-PCR) was adopted to evaluate miRNA-16 (miR-16) expression. With the adoption of ELISA kits and qRT-PCR, the release of inflammatory cytokines was evaluated. Besides, luciferase reporter assay was applied to testify the binding relationship between miR-16 and TLR4. NF-κB expression was measured by immunofluorescence. DHA reduced cell viability and decreased protein expression of Ki67 and IBa-1 in morphine-induced BV-2 cells. Additionally, DHA contributed to the declined release of pro-inflammatory cytokines. miR-16 was down-regulated by morphine but was up-regulated by DHA concentration-dependently in BV-2 cells. The inhibition of miR-16 partly abolished the inhibitory effects of DHA on morphine-induced microglial activation and neuroinflammation. Moreover, TLR4 was found to be bound to miR-16, and the inhibitory effect of DHA on TLR4/NF-κB was partly reversed by miR-16 inhibition. In conclusion, DHA remarkably suppressed microglial activation and neuroinflammation through regulating miR-16-mediated TLR4/NF-κB signaling. This study may provide a new solution to improve clinical analgesic efficacy of morphine.

Background Our previous study showed that neonatal S. pneumoniae pneumonia promoted airway smooth muscle myosin heavy chain (SMMHC) expression and AHR development. Researches demonstrated HMGB1, TLR4 and ERK are involved in smooth muscle contractile protein expression, so we hypothesis that HMGB1/TLR4/ERK pathway participated in airway SMMHC overexpression in neonatal S. pneumoniae pneumonia model. Method Neonatal (1-week-old) BALB/c mice were intranasal inoculated with D39 to establish non-lethal S. pneumoniae pneumonia model. TLR4 was inhibited 2 weeks after infection with TLR4 specific inhibitor (TAK-242). Five weeks after infection, the bronchoalveolar lavage fluid (BALF) and lungs of neonatal S. pneumoniae pneumonia and mock infection mice with or without TLR4 inhibition were collected to assess the expressions of HMGB1, TLR4 and p-ERK1/2. Airway Hyperresponsiveness (AHR) of the three groups was determined by whole-body plethysmograph. Results Our results demonstrated that neonatal S. pneumoniae pneumonia promoted HMGB1/TLR4 production, SMMHC expression and AHR development significantly, with ERK1/2 phosphorylation decreased remarkably. TLR4 inhibition after pneumonia significantly increased ERK1/2 phosphorylation, reversed airway SMMHC overexpression and alleviated AHR. Conclusion Neonatal S. pneumoniae pneumonia promotes airway SMMHC expression and AHR through HMGB1/TLR4/ERK.

Hypoxia-inducible factor-1α (HIF-1α) is an important regulator of glucose metabolism and inflammatory cytokine production in innate immune responses. Viruses modulate HIF-1α to support viral replication and the survival of infected cells, but it is unclear if this transcription factor also plays an important role in regulating antiviral immune responses. In this study, we found that short and long dsRNA differentially engage TLR3, inducing distinct levels of proinflammatory cytokine production (TNF-α and IL-6) in bone marrow–derived macrophages from C57BL/6 mice. These responses are associated with differential accumulation of HIF-1α, which augments NF-κB activation. Unlike TLR4 responses, increased HIF-1α following TLR3 engagement is not associated with significant alterations in glycolytic activity and was more pronounced in low glucose conditions. We also show that the mechanisms supporting HIF-1α stabilization may differ following stimulation with short versus long dsRNA and that pyruvate kinase M2 and mitochondrial reactive oxygen species play a central role in these processes. Collectively, this work suggests that HIF-1α may fine-tune proinflammatory cytokine production during early antiviral immune responses, particularly when there is limited glucose availability or under other conditions of stress. Our findings also suggest we may be able to regulate the magnitude of proinflammatory cytokine production during antiviral responses by targeting proteins or molecules that contribute to HIF-1α stabilization.

This study is to investigate the effect of microRNA-1338 (miR-1338) on the activity of Kupffer cells (KCs) and its mechanism regulated by ophiopogon polysaccharide liposome (OPL). KCs was treated with different OPL after transfected with miR-1338 mimic and miR-1338 inhibitor. The secretion of NO and iNOS, the expression of catalase (CAT) and peroxidase (POD), the phagocytic activity, the expression of CD14 and MHC II, the apoptosis and the secretion of ROS were measured. In addition, the expressions of key signal factors TLR4, IKKβ, MyD88 and NF-κB in NF-κB signaling pathway were measured by real-time PCR and Western blot (WB). The results showed that OPL could promote the secretion of iNOS, the expression of POD, the phagocytosis, the mRNA expression of TLR4, MyD88, IKKβ and NF-κB, the protein expression of TLR4 and NF-κB, and inhibit the cell apoptosis and ROS secretion after transfected with miR-1338 mimic. After transfected with miR-1338 inhibitor, OPL could promote the secretion of NO and iNOS, the expression of POD, cell migration, phagocytosis, and inhibit cell apoptosis. Meanwhile, the mRNA expression of TLR4, MyD88, IKKβ and NF-κB and the protein expression of TLR4, MyD88 and NF-κB were promoted. These results suggested that OPL could activate TLR4-NF-κB signaling pathway and thereby improve the activity of KCs by regulating miR-1338.

Whole-cell vaccines against Gram-negative bacteria commonly display high reactogenicity caused by the endotoxic activity of lipopolysaccharide (LPS), one of the major components of the bacterial outer membrane. Underacylation of the lipid A moiety of LPS has been related with reduced endotoxicity in several Gram-negative species. Here, we evaluated whether the inactivation of two genes encoding lipid A acylases of Bordetella bronchiseptica, i.e. pagP and lpxL1, could be used for the development of less reactogenic vaccines against this pathogen for livestock and companion animals. Inactivation of pagP resulted in the loss of the secondary palmitate chain at position 3' of lipid A, but hardly affected the potency of the LPS to activate the Toll-like receptor 4 (TLR4). Inactivation of lpxL1 resulted in the loss of the secondary 2-hydroxy laurate group present at position 2 of lipid A and, unexpectedly, in the additional loss of the glucosamines that decorate the phosphate groups at positions 1 and 4' and in an increase in LPS molecules carrying O-antigen. The resulting LPS showed greatly reduced potency to activate TLR4 in HEK-Blue reporter cells expressing human or mouse TLR4 as well as in porcine macrophages. Characterization of the lpxL1 mutant revealed many pleiotropic phenotypes, including increased resistance to SDS and rifampicin, increased susceptibility to cationic antimicrobial peptides, decreased auto-aggregation and biofilm formation, and a tendency to decreased infectivity of macrophages, which are all related to the altered LPS structure. We suggest that the lpxL1 mutant will be useful for the generation of safer vaccines.

Cerebral ischemic stroke is one of the main causes of death and long-term disability worldwide. However, the mechanism is unclear, and treatments are limited. In this study, we aimed to investigate the anti-inflammatory effect of agomelatine in a permanent middle cerebral artery occlusion (pMCAO) model. Forty-eight male Wistar rats were randomly divided into four groups: sham, pMCAO + vehicle, pMCAO + agomelatine (40 mg/kg, i.p.), and pMCAO + melatonin (10 mg/kg, i.p.) groups. On day 1 after permanent cerebral ischemia, the animals were sacrificed, and brain tissues were collected for western blot analysis, and immunohistochemistry. Agomelatine treatment ameliorated inflammatory responses by decreasing the protein levels of trigger Toll-like receptor (TLR4)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway components together with nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome components. In addition, agomelatine suppressed microglial activation and pyroptotic cell death after cerebral ischemic injury. These results suggest that agomelatine exerts an anti-inflammatory effect and attenuates brain damage by inhibiting microglial activation through the TLR4/NLRP3 signaling pathway.

TLRs reprogram macrophage metabolism, enhancing glycolysis and promoting flux through the tricarboxylic acid cycle to enable histone acetylation and inflammatory gene expression. The histone deacetylase (HDAC) family of lysine deacetylases regulates both TLR-inducible glycolysis and inflammatory responses. Here, we show that the TLR4 agonist LPS, as well as agonists of other TLRs, rapidly increase enzymatic activity of the class IIa HDAC family (HDAC4, 5, 7, 9) in both primary human and murine macrophages. This response was abrogated in murine macrophages deficient in histone deacetylase 7 (Hdac7), highlighting a selective role for this specific lysine deacetylase during immediate macrophage activation. With the exception of the TLR3 agonist polyI:C, TLR-inducible activation of Hdac7 enzymatic activity required the MyD88 adaptor protein. The rapid glycolysis response, as assessed by extracellular acidification rate, was attenuated in Hdac7-deficient mouse macrophages responding to submaximal LPS concentrations. Surprisingly however, reconstitution of these cells with either wild-type or an enzyme-dead mutant of Hdac7 enhanced LPS-inducible glycolysis, whereas only the former promoted production of the inflammatory mediators Il-1β and Ccl2. Thus, Hdac7 enzymatic activity is required for TLR-inducible production of specific inflammatory mediators, whereas it acts in an enzyme-independent fashion to reprogram metabolism in macrophages responding to submaximal LPS concentrations. Hdac7 is thus a bifurcation point for regulated metabolism and inflammatory responses in macrophages. Taken together with existing literature, our findings support a model in which submaximal and maximal activation of macrophages via TLR4 instruct glycolysis through distinct mechanisms, leading to divergent biological responses.

The role of commensal bacterial microbiota in the pathogenesis of human malignancies has been a research field of incomparable progress in recent years. Although breast tissue is commonly assumed to be sterile, recent studies suggest that human breast tissue may contain a bacterial microbiota. In this study, we used an immune-competent orthotopic breast cancer mouse model to explore the existence of a unique and independent bacterial microbiota in breast tumors. We observed some similarities in breast cancer microbiota with skin; however, breast tumor microbiota was mainly enriched with Gram-negative bacteria, serving as a primary source of lipopolysaccharide (LPS). In addition, dextran sulfate sodium (DSS) treatment in late-stage tumor lesions increased LPS levels in the breast tissue environment. We also discovered an increased expression of S100A7 and low level of TLR4 in late-stage tumors with or without DSS as compared to early-stage tumor lesions. The treatment of breast cancer cells with LPS increased the expression of S100A7 in breast cancer cells in vitro. Furthermore, S100A7 overexpression downregulated TLR4 and upregulated RAGE expression in breast cancer cells. Analysis of human breast cancer samples also highlighted the inverse correlation between S100A7 and TLR4 expression. Overall, these findings suggest that the commensal microbiota of breast tissue may enhance breast tumor burden through a novel LPS/S100A7/TLR4/RAGE signaling axis.

Acute lung injury (ALI) is a sudden onset systemic inflammatory response. ALI causes severe morbidity and death and currently no effective pharmacological therapies exist. Natural products represent an excellent resource for discovering new drugs. Screening anti-inflammatory compounds from the natural product bank may offer viable candidates for molecular-based therapies for ALI. In this study, 165 natural compounds were screened for anti-inflammatory activity in lipopolysaccharide (LPS)-challenged macrophages. Among the screened compounds, flavokawain B (FKB) significantly reduced LPS-induced pro-inflammatory IL-6 secretion in macrophages. FKB also reduced the formation of LPS/TLR4/MD2 complex by competitively binding to MD2, suppressing downstream MAPK and NF-κB signaling activation. Finally, FKB treatment of mice reduced LPS-induced lung injury, systemic and local inflammatory cytokine production, and macrophage infiltration in lungs. These protective activities manifested as increased survival in the ALI model, and reduced mortality upon bacterial infection. In summary, we demonstrate that the natural product FKB protects against LPS-induced lung injury and sepsis by interacting with MD2 and inhibiting inflammatory responses. FKB may potentially serve as a therapeutic option for the treatment of ALI.

The preventive effect and molecular mechanism of lycopene (LP) in dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice were evaluated. Compared to the DSS group, the LP prevention groups not only significantly inhibited the DSS-induced weight loss, decreased the disease activity index (DAI) score, increased the colon length, and improved inflammation in the colon but also significantly increased the levels of superoxide dismutase (SOD),catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione (GSH) in the colon and reduced inflammatory cytokine, myeloperoxidase (MPO), and malondialdehyde (MDA) levels. Notably, when compared to the DSS group, the protein expression levels of TLR4, TRIF, and p-NF-κB p65 in the mice colon tissue were downregulated and those of tight junction-related proteins were upregulated in the LP + DSS group, with the most significant effect observed in the 10 mg/kg LP + DSS group. These results confirmed that the upregulation of tight junction-related protein expression after blocking the TLR4/TRIF/NF-κB signaling pathway may be one of the mechanisms through which LP prevents UC.

A balance between the positive and negative regulation of toll-like receptor (TLR) signaling pathways is required to avoid detrimental and inappropriate inflammatory responses. Although some protein post-translational modifications (PTMs) such as phosphorylation and ubiquitination have been demonstrated to potently modulate innate immune responses, the role of methylation, an important PTM, control of TLR4 signaling pathway remains unclear. In this study, we found that protein arginine methyltransferase 1, 2 and 3 (PRMT1, 2 and 3) were recruited to methylate TLR4-CD (cytoplasmic domain) after lipopolysaccharide (LPS) stimulation respectively, but the effect of PRMT2 on arginine methylation of TLR4-CD is the most significant among above three PRMTs, which prompted us to focus on PRMT2. Reduction of PRMT2 expression down-regulated arginine (R) methylation level of TLR4 with or without LPS treatment. Methionine 115 (M115) mediated PRMT2 catalyzed-arginine methylation of TLR4 on R731 and R812. Furthermore, PRMT1, 2 and 3 was recruited to methylate interferon regulatory factor 3 (IRF3) after LPS stimulation respectively, but the effect of PRMT2 on arginine methylation of IRF3 is the most significant among the above three PRMTs. Arginine methylation of TLR4 on R812 or arginine methylation of IRF3 on R285 mediated the interaction between TLR4 and IRF3 respectively. Arginine methylation of IRF3 on R285 induced by LPS led to its dimerization and promoted its translocation from the cytoplasm to the nucleus. In addition, the enhancement of arginine methylation of TLR4 induced by PRMT1 or 2 increased IRF3 transcription activity with or without LPS treatment, while PRMT2 with histidine 112 glutamine (H112Q) or methionine 115 isoleucine (M115I) mutation and TLR4 with arginine 812 lysine (R812K) mutation decreased it. Arginine methylation of TLR4 on R812 or PRMT2 enhanced interferon-β (IFN-β) production. Our study reveals a critical role for PRMT2 and protein arginine methylation in the enhancement of IFN-β production via TLR4/IRF3 signaling pathway and may provide a therapeutic strategy to control endotoxemia.

This study investigated the effect and mechanism of chrysosplenol D (CD) on LPS-induced acute lung injury in mice. Histological changes in the lungs were measured by hematoxylin-eosin staining. The levels of IL-6, IL-1β, and TNF-α in the bronchoalveolar lavage fluid were detected by ELISA. The levels of oxidative stress were detected by the cuvette assay. Immune cells in peripheral blood, the levels of reactive oxygen species, and apoptosis of primary lung cells were detected by flow cytometry. The mRNA levels of TLR4, MyD88, IL-1β, and NLRP3 were measured by quantitative real-time polymerase chain reaction. The levels of proteins in apoptosis and the TLR4-MAPKs/NF-κB signaling pathways were detected by Western blot. Hematoxylin-eosin staining showed that CD could improve lung injury; decrease the levels of inflammatory factors, oxidative stress, reactive oxygen species, and cell apoptosis; and regulate the immune system. Moreover, CD could down-regulate the mRNA levels of TLR4, MyD88, NLRP3, and IL-1β in lung, and the protein levels of Keap-1, Cleaved-Caspase-3/Caspase-3, Cleaved-Caspase-9/Caspase-9, TLR4, MyD88, p-ERK/ERK, p-JNK/JNK, p-p38/p38, p-p65/p65, NLRP3, and IL-1β, and up-regulated the levels of Bcl-2/Bax, p-Nrf2/Nrf2, and HO-1. The results suggested that CD could protect mice against LPS-induced acute lung injury by inhibiting oxidative stress, inflammation, and apoptosis via the TLR4-MAPKs/NF-κB signaling pathways.

Infection, particularly prenatal infection leads to an enhanced risk of schizophrenia in the offspring. Interestingly, few data exist on the pathway(s) such as TLR and inflammasome, primarily involved in sensing the microorganisms and inducing downstream inflammatory responses, apoptosis and neuroprogressive changes in the prenatal infection-induced risk of schizophrenia. Herein, we aimed to discern whether prenatal infection-induced maternal immune activation (MIA) causes schizophrenia-like behaviors through activation of TLR and inflammasome pathways in the brain of offspring. Sprague Dawley rats (n=15/group) were injected either with poly (I:C) or LPS or saline at gestational day (GD)-12. Significantly elevated plasma levels of IL-1β, IL-6, TNF-α and IL-17A assessed after 24 hours were observed in poly (I:C) and LPS-treated rats, indicating MIA. The offspring rats of poly (I:C)-and LPS-treated dams displayed increased anxiety-like behaviors, deficits in social behaviors and prepulse inhibition. The hippocampus of offspring rats showed increased expression of TLR3, TLR4, NLRP3, IL-1β, and IL-18 of poly (I:C) and TLR3, TLR4, NLRP3, Cas1, IL-1β, and IL-18 of LPS-treated dams. Notably, the expression of these genes showed a positive correlation with apoptotic and a negative correlation with neuroprotective genes. Furthermore, TLR and inflammasome genes had significant impact on social deficits and impaired prepulse inhibition in offspring rats. The results suggest MIA due to prenatal infection perhaps trigger TLR, inflammasome and apoptotic pathways leading to the induction of schizophrenia-like behaviors in the later stages of life. Prenatal infections seem to drive neuroprogression and subsequently schizophrenia-like symptoms through a common central pathway involving TLR, inflammasome and apoptosis.

Apigenin, as a natural flavonoid present in several plants is characterized with potential anticancer, antioxidant, and anti-inflammatory properties. Recent studies proposed that apigenin affects depression disorder through unknown mechanistic pathways. The effects of apigenin’s anti-depressive properties on streptozocin-mediated depression have been investigated through the evaluation of behavioral tests, oxidative stress, cellular energy homeostasis and inflammatory responses. The results demonstrated anti-depressive properties of apigenin in behavioral test including forced swimming and splash tests and oxidative stress biomarkers such as reduced glutathione, lipid peroxidation, total antioxidant power and coenzyme Q10 levels. Apigenin, also, demonstrated its regulatory potency in cellular energy homeostasis and immune system gene expression through inhibiting Nlrp3 and Tlr4 overexpression. Furthermore, failure in energy production as the key factor in various psychiatric disorders was reversed by apigenin modulating effect on AMPK gene expression. Overall, 20 mg/kg of apigenin was recognized as the dose suitable for minimizing the undesirable adverse effects in the STZ-mediated depression model proposed in this study. Our data suggested that apigenin could be able to adjust behavioral dysfunction, biochemical biomarkers and recovered cellular antioxidant level in depressed animals. The surprising results were achieved by raise in COQ10 level, which could regulate the overexpression of the AMPK gene in stressful conditions. The regulatory effect of apigenin in inflammatory signaling pathways such as Nlrp3, and Tlr4 gene expression was studied at the surface part of the hippocampus.

Early undernutrition has been found to be closely associated with subsequent neurodevelopment. However, studies examining crude growth in terms of body weight/tail length cannot clarify how diets might mediate associations between the gut microbiota and cognitive dysfunction. In the present study, Sprague-Dawley (SD) rats were fed a 7% protein diet and mung bean protein diet (MBPD) for 6 weeks to assess central nervous system functions. Bifidobacterium longum subsp, Alloprevotella, and Lactobacillus were significantly altered after supplementary MBPD. Additionally, tryptophan, tyrosine, and glycine significantly restored in the brain, and the choline system also improved. Moreover, mung bean supplementation also upregulated expression of the brain-derived neurotrophic factor, postsynaptic density 95 protein (PSD95), synaptosome-associated protein 25 (SNAP25), downregulated toll-like receptor 4 (TLR4), and nuclear factor kB (NF-kB). Metabolites in the serum also underwent changes. Together, these results showed that malnutrition perturbed neurodevelopment, while MBPD reversed this trend.

Background Thalamic pain, a neuropathic pain syndrome, frequently occurs after stroke. This research aimed to investigate the effect of dexmedetomidine (DEX) on thalamic pain. Methods The cellular localization of the TLR4 protein was determined by immunostaining. The expression of Iba1, GFAP and protein associated with the TLR4/NF-κB/ERK1/2 pathway was measured by Western blotting. Continuous pain hypersensitivity was evaluated by behavioural tests. The results were analysed by one-way ANOVA, two-way ANOVA and Tukey’s post hoc test. Results The results demonstrated that DEX obviously alleviated thalamic pain induced by haemorrhage on the ipsilateral side and delayed the development of pain hypersensitivity. Furthermore, the expression levels of Iba1, GFAP and proteins associated with the TLR4/NF-κB/ERK1/2 signalling pathway were greatly increased in mice with thalamic pain, but these effects were reversed by DEX. Conclusion Our findings suggest that DEX alleviates the inflammatory response during thalamic pain through the TLR4/NF-κB/ERK1/2 signalling pathway and might be a potential therapeutic agent for thalamic pain.

Objective. To determine the role of sodium butyrate in intestinal inflammation via regulation of high-mobility group box-1 (HMGB1), we analyzed the potential mechanism in necrotizing enterocolitis (NEC) in a neonatal mouse model. Methods. A NEC model was created with hypoxia and cold exposure and artificial overfeeding. C57BL/6 neonatal mice were randomized into three groups: the control, untreated NEC, and sodium butyrate (150 mM)-pretreated NEC groups. Pathological variations in ileocecal intestinal tissue were observed by HE staining and scored in a double-blind manner. The mRNA expression levels of HMGB1, Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), and inflammatory cytokines in intestinal tissues were determined by quantitative real-time PCR. The protein levels of HMGB1 and associated cytokines in intestinal tissues were evaluated using ELISA. The relative protein expression levels of TLR4 and NF-κB in intestinal tissues were quantified by western blot. Results. Sodium butyrate administration improved the body weight and survival rate of NEC mice; relieved intestinal pathological injury; reduced the intestinal expression of HMGB1, TLR4, NF-κB, interleukin- (IL-) 1β, IL-6, IL-8, and TNF-α; and increased the intestinal expression of IL-10 ( P < 0.05 ). Treatment with butyrate decreased the proportion of opportunistic Clostridium_sensu_stricto_1 and Enterococcus and increased the proportion of beneficial Firmicutes and Lactobacillus in the NEC model. Conclusions. Sodium butyrate intervention relieves intestinal inflammation and partially corrects the disrupted intestinal flora in mice with NEC.

To evaluate the effects of dexmedetomidine (Dex) and oxycodone (Oxy) on neurocognitive and inflammatory response after tourniquet-induced ischemia–reperfusion (I/R) injury. C57/BL6 mice were used to construct the mouse model of tourniquet-induced I/R injury. Mice (n = 48) were randomly divided into sham, I/R, Dex or Oxy group. Morris water maze test was performed to assess the spatial learning and memory function. The expression of NF-κB, TLR4, NR2B, M1 (CD68 and TNF-α) and M2 (CD206 and IL-10) polarization markers in mice hippocampus were detected by western blot or immunofluorescent staining. Spontaneous excitatory post-synaptic currents (sEPSCs) were recorded by electrophysiology. Dex treatment alleviated I/R-induced declines in learning and memory (p

Background: Restoration of blood flow during ischemic stroke leads to Cerebral Ischemia- Reperfusion Injury (CIRI) by activating neuroinflammatory cascades. Pomelo Peel Volatile Oil (PPVO) extracted from Citrus maxima (Burm.) from the genus Rutaceae, comprises some antiinflammatory ingredients, such as limonene and β-myrcene. Objective: The present study aimed to investigate the potential effect of PPVO on alleviating CIRI related to the Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) pathway. Methods: Transient middle cerebral artery occlusion/reperfusion (tMCAO/R) was performed on 65 rats, which were then distributed into five groups (n = 13/group) depending on the intervention they received: Normal Saline (NS) group, normal Glycerin (GL) group, low-dose PPVO (LP, 10mg/kg) group, high-dose PPVO (HP, 30 mg/kg) group, and Sham-operated (SH) group. Neurological Deficit Scores (NDSs) and histological changes were evaluated. Infarct volumes were measured by 2,3,5- Triphenyltetrazolium Chloride (TTC) staining. The expression of TLR4 and neutrophil infiltration were detected by Immunofluorescence (IF) staining. Moreover, the downstream molecules of the TLR4/NF-κB signaling pathway, such as IL-6, IL-1β, TNF-α, p-IκB/IκB, and p-NF-κB p65/NF-κB p65 were analyzed by Western Blot (WB). Results & Discussion: The results showed that PPVO (30 mg/kg) significantly decreased infarct volumes, improved neurological deficits and pathologic changes, inhibited TLR4/NF-κB signaling pathway suppressed neutrophil infiltration, and suppressed pro-inflammatory cytokine release. Conclusion: It can be concluded that PPVO may alleviate neuroinflammation and protect against CIRI via inhibiting the TLR4/NF-κB signaling pathway.

Peri‑implantitis, which is characterized by peri‑implant mucositis and alveolar bone resorption, significantly shortens the service life of dental implants. Melatonin is well-known for its anti-inflammatory and osteoprotective activities. Nevertheless, the effects and mechanisms of melatonin to prevent peri‑implantitis remain unknown. In this study, the lipopolysaccharide-induced peri‑implantitis model was established after the titanium implants were osseointegrated, and the rats received daily administrations of melatonin. The gingival fibroblasts and osteoclasts/osteoblasts were also co-cultured to simulate the inflammatory environment in vitro. We found that prophylactic administration of melatonin decreased proinflammatory cytokine levels and osteoclast numbers, attenuated alveolar bone resorption, and reduced the incidence of peri‑implantitis in vivo. Furthermore, melatonin suppressed osteoclastic formation and function in the inflammatory co-culture environment, while melatonin promoted osteoblastic differentiation and function in the in vitro model. Mechanistically, melatonin reduced TLR4 protein levels, and inhibited activation of NF-κB to downregulate the levels of TNF, IL-1β, and IL-6. These data showed that melatonin was a potent agent to prevent peri‑implantitis through inhibiting TLR4/NF-κB signaling. Our findings provide a novel strategy to prevent peri‑implantitis, and expand the applications of melatonin. Statement of significance Dental implants have become the first choice for restoring partial and full edentulism, but its service life is seriously affected by peri‑implantitis. Exploration of novel and effective approaches to prevent peri‑implantitis is an important and urgent need. In the present study, we have reported for the first time that prophylactic administration of melatonin delayed the occurrence and reduced the incidence of peri‑implantitis by decreasing proinflammatory cytokine levels, inhibiting osteoclastogenesis, and promoting osteogenesis. The study is expected to have an important significance on the prevention of peri‑implantitis.

With the progress made in the widespread application of interventional radiology procedures, there has been an increasing number of patients who suffer from cardiovascular diseases and go through imaging and interventional treatment with iodine contrast medium (ICM) year by year. In turn, there has been an increasing amount of concern over acute kidney injury (AKI) brought about by ICM. As evidenced by numerous studies, the initiation of inflammatory response plays a critical role in the development of ICM-induced AKI. Correspondingly, the strategy of targeting renal inflammatory response and cytokine release could provide an effective solution to mitigating the ICM-induced AKI. Moreover, Ginsenoside Rb1 (GRb1) constitutes one of the major active components of ginseng and features a wide range of vital biological functions. Judging from the research findings, GRb1 could impose antioxidant and anti-inflammatory effects on cardiovascular diseases, in addition to lung, liver and kidney diseases. However, reports on whether GRb1 could impose a protective effect against contrast-induced nephropathy (CIN) are absent. In this study, we have examined the therapeutic effects imposed by GRb1 as well as the potential molecular mechanism by establishing an in vivo and in vitro model of CIN. In addition, we have set up a mouse model of CIN through sequential intravenous injection of indomethacin, N(ω)-nitro-Larginine methyl ester (L-NAME), and iopromide. To further enhance the bioavailability of GRb1, we have encapsulated GRb1 with polyethylene glycol (PEG)/poly lactic-co-glycolic acid (PLGA) nanocarriers to generate GRb1 nanoparticles (NPs) conducting the in vivo experiments. During the in vitro experiments, we have adopted GRb1 to treat NRK-52E cells or cells transfected with the high mobility group box 1 gene (HMGB1) overexpression plasmid. As shown by the in vivo experimental results, GRb1 NPs could evidently improve the renal dysfunction in CIN, diminish the extent of apoptosis of tubular epithelial cells, and reduce the expression of high mobility group box 1 (HMGB1) and cytokines (tumor necrosis factor (TNF-α), interleukin (IL) 6 and IL-1β). In addition, GRb1 NPs are found to be capable of preventing the activation of Toll-like receptor 4 (TLR4)/NF-κB signaling pathway triggered by contrast medium. The in vitro experimental results have exactly confirmed the findings of the in vivo experiments. In the meantime, through the observation of the in vitro assays, overexpression of HMGB1 can partially counteract the beneficial effects imposed by GRb1. Judging from our research data, GRb1 could impose a protective effect against CIN by inhibiting inflammatory response via HMGB1/TLR4/NF-κB pathway, whereas HMGB1 constitutes a critical molecular target of GRb1.