NF-κB P-p65 Research Articles

The roles of Nrf2 and autophagy in modulating inflammation mediated by TLR4 - NFκB in A549 cell exposed to layer house particulate matter 2.5 (PM2.5)

Particulate matter (PM) from layer house has adverse effect on people and chicken respiratory health, which can further influence animal performance and reduce production efficiency. However, little study focus on the respiratory inflammation induced by PM2.5 from layer house and the underlying mechanism also unclear. In this study, human adenocarcinoma alveolar basal epithelial cells (A549 cell) was subjected to the PM2.5 from layer house to evaluate the inflammation reaction caused by PM2.5 and explore the role of Nrf2 and autophagy in regulating the inflammation. Results showed that the viability of A549 cell decreased in a time - and concentration - dependent manner after PM2.5 treatment. TNFα, IL6, and IL8 increased significantly treated with PM2.5 at 12 h. RNA sequencing indicated differentially expressed genes were enriched in immune system process, oxidative stress (OS), endoplasmic reticulum stress (ERS), and autophagy. Further studies showed TLR4 - NFκB p65 signal pathway involved in the inflammation reaction caused by PM2.5. The overexpression of Nrf2 decreased the level of TNFα, IL6, IL8 markedly as well as the level of NFκB p65 and NFκB pp65. OS and ERS were also limited under overactivation of Nrf2 in PM2.5 treated cells. Autophagy induced by PM2.5 promoted the inflammation through increasing the level of NFκB p65 and NFκB pp65. Autophagy deficient strengthened the expression of Nrf2. Collectively, our study revealed Nrf2 prevents inflammation caused by layer house PM2.5 stimulation, however, autophagy exerts a promotive role in TLR4 - NFκB p65 mediating inflammation in A549 cell.

Correlation Between HMGB1 and TLR4 Expression in Sinonasal Mucosa in Patients With Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is one of the most common inflammations in the upper airway. Despite the wide prevalence of CRS, the pathogenesis of this disease is poorly understood. Several components of the innate immune system may play a significant role in CRS, including Toll-like receptor 4 (TLR4), TLR9, and high-mobility group box 1 protein (HMGB1). This study was conducted to determine the expression of TLR4, TLR9, HMGB1, and pNFκ-B p65 in paraffin-embedded blocks of patients with CRS with nasal polyps compared with those of the control group. Twenty-six formalin-fixed, paraffin-embedded samples from patients with confirmed CRS and 26 patients undergoing septoplasty due to anatomic variations and no other inflammatory nasal diseases as the control group were assessed. Expression patterns of HMGB1, TLR9, TLR4, and pNFκ-B p65 genes were examined using real-time quantitative reverse transcription polymerase chain reaction (Real-Time qRT-PCR). Statistical analyses were performed with SPSS and analyzed using unpaired 2-tailed t tests or 1-way analysis of variance. Real-time PCR showed that the expression level of HMGB1 messenger RNA was significantly increased in the tissues of patients with CRS compared with controls (P < .05). The other 3 genes were also upregulated in the patients, but were not significant compared with control. Analysis of the Pearson correlation coefficient (r) revealed a significant positive correlation between HMGB1 and TLR4 (r = 0.79, P < .05) in patients and negative correlation between TLR4 and NfκB in the control group (r = 0.94; P < .05). Both HMGB1 and TLR4 are increased in the paranasal sinus mucosa of patients with CRS. These results suggest a possible contribution of HMGB1 and its internal receptor (TLR4) in the pathophysiology of CRS.

Patients with tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) are hypersensitive to Toll-like receptor 9 stimulation.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder characterized by recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNFRSF1A, which encodes tumour necrosis factor receptor 1 (TNF-R1). Our aim was to understand the influence of TRAPS mutations on the response to stimulation of the pattern recognition Toll-like receptor (TLR)-9. Peripheral blood mononuclear cells (PBMCs) and serum were isolated from TRAPS patients and healthy controls: serum levels of 15 proinflammatory cytokines were measured to assess the initial inflammatory status. Interleukin (IL)-1β, IL-6, IL-8, IL-17, IL-22, tumour necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), interferon (IFN)-γ, monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor (TGF)-β were significantly elevated in TRAPS patients' sera, consistent with constitutive inflammation. Stimulation of PBMCs with TLR-9 ligand (ODN2006) triggered significantly greater up-regulation of proinflammatory signalling intermediates [TNF receptor-associated factor (TRAF 3), IL-1 receptor-associated kinase-like 2 (IRAK2), Toll interacting protein (TOLLIP), TRAF6, phosphorylated transforming growth factor-β-activated kinase 1 (pTAK), transforming growth factor-β-activated kinase-binding protein 2 (TAB2), phosphorylated TAK 2 (pTAB2), IFN-regulatory factor 7 (IRF7), receptor interacting protein (RIP), nuclear factor kappa B (NF-κB) p65, phosphorylated NF-κB p65 (pNF-κB p65) and mitogen-activated protein kinase kinase (MEK1/2)] in TRAPS patients' PBMCs. This up-regulation of proinflammatory signalling intermediates and raised serum cytokines occurred despite concurrent anakinra treatment and no overt clinical symptoms at time of sampling. These novel findings further demonstrate the wide-ranging nature of the dysregulation of innate immune responses underlying the pathology of TRAPS and highlights the need for novel pathway-specific therapeutic treatments for this disease.

Zerumbone Protects against Carbon Tetrachloride (CCl4)-Induced Acute Liver Injury in Mice via Inhibiting Oxidative Stress and the Inflammatory Response: Involving the TLR4/NF-κB/COX-2 Pathway.

The natural compound Zerumbone (hereinafter referred to as ZER), a monocyclic sesquiterpenoid, has been reported to possess many pharmacological properties, including antioxidant and anti-inflammatory properties. This study aimed to investigate the underlying mechanism of ZER against acute liver injury (ALI) in CCl4-induced mice models. ICR mice were pretreated intraperitoneally with ZER for five days, then received a CCl4 injection two hours after the last ZER administration and were sacrificed 24 h later. Examination of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and the histopathological analysis confirmed the hepatoprotective effect of ZER. Biochemical assays revealed that ZER pretreatment recovered the activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), restored the glutathione (GSH) reservoir, and reduced the production of malondialdehyde (MDA), all in a dose-dependent manner. Furthermore, administration of ZER in vivo reduced the release amounts of pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) and inhibited the increased protein levels of Toll-like receptor 4 (TLR4), nuclear factor-kappaB (NF-κB) p-p65, and cyclooxygenase (COX-2). Further studies in lipopolysaccharide (LPS)-induced Raw264.7 inflammatory cellular models verified that ZER could inhibit inflammation via inactivating the TLR4/NF-κB/COX-2 pathway. Thus, our study indicated that ZER exhibited a hepatoprotective effect against ALI through its antioxidant and anti-inflammatory activities and the possible mechanism might be mediated by the TLR4/NF-κB/COX-2 pathway. Collectively, our studies indicate ZER could be a potential candidate for chemical liver injury treatment.

Fabrication of resveratrol coated gold nanoparticles and investigation of their effect on diabetic retinopathy in streptozotocin induced diabetic rats

This study includes the fabrication of gold nanoparticles (AuNPs) with the help of a plant polyphenol called Resveratrol through an ecofriendly synthetic process without any use of harmful reductants. In the fabrication of AuNPs, Resveratrol acts as both stabilizing and reducing agent. The prepared AuNPs is tested on streptozotocin (STZ) induced diabetic rats for their amelioration consequence. The images of TEM displayed the development of spherical nanoparticles (NPs) with a median of 20 nm particle size. The STZ injected diabetic rats were administrated orally with calcium dobesilate (CD; 500 mg/kg/day) or AuNPs (200, 300 mg/kg/day) for a period of 3 months. The characteristics displayed by AuNPs were found to be similar with CD in decreasing permeability of blood–retinal barrier in STZ injected diabetic rats. The retinal vessels in the AuNPs administrated diabetic rats were observed to be decreased through the retinal histopathological examination. In the AuNPs administrated diabetic rats, the retinal expression of renal Pigment Epithelium-Derived Factor (PEDF) was observed to be increased and the Vascular Endothelial Growth Factor (VEGF-1), which was increased in diabetic rats was declined on treating with AuNPs. On treating the STZ injected diabetic rats with AuNPs, all the retinal mRNA expressions of VEGF-1, Tumor Necrosis Factor (TNFα), Monocyte Chemotactic Proteins-1 (MCP-1), Intercellular Adhesion Molecule-1 (ICAM-1), and Interleukin (IL)-6, IL-1β were observed to be reduced. Furthermore, AuNPs can reduce phosphorylation of Nuclear Factor Kappa B (NF-κB) p65 and Extracellular signal Regulated Kinase (ERK) 1/2 along with a growth in nuclear translocation of pNF-κB p65 produced by STZ. To conclude, the protective effect of AuNPs on STZ injected diabetic rats could help in redeveloping the balance among the inhibitors and stimulators of angiogenesis. Furthermore, on treating with AuNPs results in inhibiting the signaling pathway of ERK1/2 as well as with amelioration of retinal inflammation through trans repression of NF-κB.

MicroRNA-146a-5p Downregulates the Expression of P-Glycoprotein in Rats with Lithium-Pilocarpine-Induced Status Epilepticus.

Increasing evidence supports that the efflux transporters, especially P-glycoprotein (P-gp), have vital roles on drug resistance in epilepsy. Overexpression of P-gp in the brain could reduce the anti-epileptic drugs (AEDs) concentration in the epileptogenic zone, resulting in drug resistance. Studies have demonstrated that recurrent seizures induce the expression of P-gp and status epilepticus (SE) could upregulate the expression of P-gp, resulting in drug resistance. MicroRNAs (miRNAs), as endogenous regulators, represent small regulatory RNA molecules that have been shown to act as negative regulators of gene expression in different biological processes. We investigated the impact of miR-146a-5p on the expression of P-gp in status epilepticus rat model. The expression of miR-146a-5p in rat cortex and hippocampus was measured by quantitative RT-PCR at 2 weeks after induction of SE. Meanwhile, we detected the expression of P-gp in the brain of SE rats using Western blotting and immunohistochemistry. Upregulation of miR-146a-5p and overexpression of P-gp were evident at 2 weeks after SE. Moreover, the expression of P-gp was downregulated by injection of miR-146a mimic into the hippocampus. We also detected the expression of interleukin-1 receptor-associated protein kinases-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) and nuclear factor-kappaB (NF-κB) p65 using Western blotting and immunohistochemistry, which indicated the expression of IRAK1, TRAF6 and NF-κB p-p65/p65 increased in the brain of SE rats, and overexpression of miR-146a-5p could downregulate the expression of IRAK1, TRAF6, NF-κB p-p65/p65 and P-gp. Our study indicated that miR-146a-5p may decrease the expression of P-gp in status epilepticus rats via NF-κB signaling pathway.

Combination of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis Shows a Stronger Anti-Inflammatory Effect than Individual Strains in HT-29 Cells.

Inflammatory bowel disease (IBD) is an emerging health problem associated with the dysregulation of the intestinal immune system and microbiome. Probiotics are able to reduce inflammatory responses in intestinal epithelial cells (IECs). However, entire signaling pathways and the interaction between different probiotics have not been well-documented. This study was designed to investigate the anti-inflammatory effects and mechanisms of single and combined probiotics. HT-29 cells were induced by lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α, treated with Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis or their combination and analyzed for inflammation-related molecules. Both L. acidophilus and B. animalis subsp. lactis reduced interleukin (IL)-8 secretion and the expressions of phosphorylated p65 nuclear factor-kappa B (p-p65 NF-κB), phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK), vascular cell adhesion molecule-1 (VCAM-1) and cyclooxygenase-2 (COX-2), while they increased toll-like receptor 2 (TLR2) expression. L. acidophilus did not decrease intercellular adhesion molecule-1 (ICAM-1) but enhanced the inhibitory efficacy of B. animalis subsp. lactis. Combined probiotics showed the best anti-inflammatory activity. These results suggest that L. acidophilus and B. animalis subsp. lactis may exert a potent anti-inflammatory effect through modulating TLR2-mediated NF-κB and MAPK signaling pathways in inflammatory IECs. Both strains, especially their combination, may be novel adjuvants for IBD therapy.

The effects of dihydroxyphenyl lactic acid on alleviating blood-brain barrier injury following subarachnoid hemorrhage in rats

BackgroundIn this study, the protective effects of 3,4-dihydroxyphenyl lactic acid (DLA) on blood-brain barrier (BBB) injury following subarachnoid hemorrhage (SAH) has been explored. MethodsMale Sprague-Dawley rats (weight 300–350 g) were used to establish the SAH model using the endovascular perforation method. The animals were randomly divided into four groups: sham (n = 40), SAH (n = 46), SAH + vehicle (n = 44), and SAH + DLA (n = 40) treatment groups. At 1 h after SAH, either DLA (10 mg/kg) or normal saline (vehicle) was administered by femoral vein injection. The effects of DLA on mortality, neurological function, brain water content, and BBB were observed. Additionally, immunohistochemistry and western blot techniques were applied to investigate the mechanism of action of DLA. ResultsWe found that the administration of DLA (10 mg/kg) following SAH could improve neurological functions, reduce brain water content, and maintain BBB integrity. The expression of pro-inflammatory and pro-apoptotic factors such as toll-like receptor 4 (TLR4), NF-κB (p-p65), tumor necrosis factor-α, p-p38 MAPK, p-p53, and caspase-3 were significantly increased after SAH. These same factors were markedly attenuated following treatment with DLA. ConclusionsThese findings showed that DLA can alleviate BBB injury following SAH through its anti-inflammatory and anti-apoptotic effects via suppression of TLR4 and its downstream NF-κB and p38 MAPK pathways.

NLRC5 negatively regulates LTA-induced inflammation via TLR2/NF-κB and participates in TLR2-mediated allergic airway inflammation.

NLRC5, the largest member of the Nod-like receptor (NLR) family, has been reported to play a pivotal role in regulating inflammatory responses. Recent evidence suggests that NLRC5 participates in Toll-like receptor (TLR) signaling pathways and negatively modulates nuclear factor-κB (NF-κB) activation. In this study, we investigated the interaction between NLRC5 and TLR2 in the NF-κB inflammatory signaling pathway and the involvement of NLRC5 in TLR2-mediated allergic airway inflammation. We knocked down TLR2 and NLRC5, respectively in the RAW264.7 macrophage cell line by small interfering RNA (siRNA) and then stimulated the knockdown cells with lipoteichoic acid (LTA). In comparison with the negative siRNA group, the level of NLRC5 expression was lower in the TLR2 siRNA group, with a reduction in the NF-κB-related inflammatory response. Conversely, in the NLRC5 knockdown cells, after LTA-treated the level of TLR2 expression did not change but the expression levels of both NF-κB pp65 and NLRP3 increased remarkably. Thus, we hypothesize that NLRC5 participates in the LTA-induced inflammatory signaling pathway and regulates the inflammation via TLR2/NF-κB. Similarly, in subsequent in vivo experiments, we demonstrated that the expression level of NLRC5 was significantly increased in the ovalbumin-induced allergic airway inflammation. However, this effect disappeared in TLR2-deficient (TLR2 -/- ) mice and was accompanied by reduced levels of NF-κB expression and airway inflammation. In conclusion, NLRC5 negatively regulates LTA-induced inflammatory response via a TLR2/NF-κB pathway in macrophages and also participates in TLR2-mediated allergic airway inflammation.

Thymol reduces acetic acid-induced inflammatory response through inhibition of NF-kB signaling pathway in rat colon tissue.

The aim of the present study was to evaluate the anti-inflammatory effect of thymol in acetic acid-induced rat colitis through inhibiting the NF-κB signaling pathway. Colitis was induced by intra-rectal administration of 2mL of diluted acetic acid (4%) solution using a flexible plastic rubber catheter in Wistar rats. Colitis was induced on the first day and all treatments were applied 5days after the induction of colitis. Thymol was dissolved in 0.2% tween 80 in saline and administered orally at doses of 10, 30, and 100mg/kg per day. Macroscopic and histopathologic investigations were done. The expression of myeloperoxidase (MPO) and tumor necrosis factor-α (TNF-α) was determined by immunohistochemistry (IHC) assay. The protein expression level of pNF-κB p65 was measured by the Western blot technique. Treatment with thymol reduced mucosal and histological damages compared to the acetic acid group. Our results showed that thymol markedly inhibited the production of MPO and TNF-α in the colon tissue of the acetic acid-induced group. In addition, thymol decreased acetic acid-induced up-regulation of pNFκB p65 protein. The results of our study suggest that thymol exerts an anti-inflammatory effect in acetic acid-induced rat colitis by inhibiting the NF-κB signaling pathway and downregulating TNF-α and MPO expressions.

Neuroimmune and epigenetic involvement in adolescent binge ethanol-induced loss of basal forebrain cholinergic neurons: Restoration with voluntary exercise.

Binge drinking and alcohol abuse are common during adolescence and cause lasting pathology. Preclinical rodent studies using the adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2‐day on/2‐day off from postnatal day [P]25 to P55) model of human adolescent binge drinking report decreased basal forebrain cholinergic (ie, ChAT+) neurons that persist into adulthood (ie, P56‐P220). Recent studies link AIE‐induced neuroimmune activation to cholinergic pathology, but the underlying molecular mechanisms contributing to the persistent loss of basal forebrain ChAT+ neurons are unknown. We report here that the AIE‐induced loss of cholinergic neuron markers (ie, ChAT, TrkA, and p75NTR), cholinergic neuron shrinkage, and increased expression of the neuroimmune marker pNF‐κB p65 are restored by exercise exposure from P56 to P95 after AIE. Our data reveal that persistently reduced expression of cholinergic neuron markers following AIE is because of the loss of the cholinergic neuron phenotype most likely through an epigenetic mechanism involving DNA methylation and histone 3 lysine 9 dimethylation (H3K9me2). Adolescent intermittent ethanol caused a persistent increase in adult H3K9me2 and DNA methylation at promoter regions of Chat and H3K9me2 of Trka, which was restored by wheel running. Exercise also restored the AIE‐induced reversal learning deficits on the Morris water maze. Together, these data suggest that AIE‐induced adult neuroimmune signaling and cognitive deficits are linked to suppression of Chat and Trka gene expression through epigenetic mechanisms that can be restored by exercise. Exercise restoration of the persistent AIE‐induced phenotypic loss of cholinergic neurons via epigenetic modifications is novel mechanism of neuroplasticity.

Galectin-3 prospects as a therapeutic agent for multiple sclerosis.

Galectin-3 prospects as a therapeutic agent for multiple sclerosis.

Pyropheophorbide-α methyl ester-mediated photodynamic therapy induces apoptosis and inhibits LPS-induced inflammation in RAW264.7 macrophages.

This study aimed to determine the effect of pyropheophorbide-α methyl ester (MPPa)-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and inflammation of murine macrophage RAW264.7 cells. Uptake and subcellular localization of MPPa was detected by flow cytometry and confocal fluorescence microscope. Cell viability was assessed by CCK-8; ROS levels were assessed by DCFH-DA. Cell apoptosis was measured by flow cytometry and Hoechst 33342 staining, whereas mitochondrial membrane potential was detected by JC-1 staining. Secretion of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) was determined using ELISA kits. Caspase-3, cleaved caspase-3, procaspase-9, cleaved caspase-9, PARP, cleaved PARP, Bcl-2, Bax, NF-κB p-p65, p-IKKα/β, and p-IκBα were measured by western blotting. Nuclear factor κB (NF-κB)-p65 nuclear translocation was observed by immunofluorescence. MPPa -PDT influenced cell viability in a light dose-dependent manner. It induced ROS formation and RAW264.7 cell apoptosis. It also increased the expression of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, decreased the expression of Bcl-2. While TNF-α, IL-1β, and IL-6 increased in LPS group (model of inflammation), it deceased in LPS-MPPa-PDT group. NF-κB p-p65, p-IKKα/β, and p-IκBα had higher expression in LPS group while that reduced in LPS-MPPa-PDT group. Simultaneously, MPPa-PDT inhibited nuclear translocation of NF-κB-p65 caused by LPS. MPPa-PDT can induce apoptosis and attenuate inflammation in mouse RAW264.7 macrophages, thereby suggesting a promising therapy for atherosclerosis.

Anti-Inflammatory Effects of Aster incisus through the Inhibition of NF-κB, MAPK, and Akt Pathways in LPS-Stimulated RAW 264.7 Macrophages.

Aster incisus is a common flower found in almost all regions of South Korea. In the current study, we investigated the potential antioxidant and anti-inflammatory properties of the Aster incisus methanol extract in LPS-stimulated RAW 264.7 cells. We analyzed the phytochemicals contained in the extract by GC-MS. GC-MS results showed that the Aster incisus extract contains 9 known compounds. Later on, DPPH assay, WST-1 assay, nitric oxide (NO) assay, Western blot, and RT-PCR were conducted to investigate the anti-inflammatory effects of the extract. Our WST-1 assay results revealed that Aster incisus did not affect the viability of all tested cell lines up to a concentration of 200 μg/ml; therefore, lower concentrations (50 μg/ml and 150 μg/ml) were used for further assays. Aster incisus scavenged DPPH and inhibited the production of NO. Aster incisus also reduced significantly the production of inflammation-related enzymes (iNOS, Cox-2) and cytokines (TNFα, IL-1β, and IL-6) and the gene expression of the proinflammatory cytokines. Additionally, further Western blot results indicated that Aster incisus inhibited the expression of p-PI3K, p-IκBα, p-p65 NF-κB, p-ERK1/2, p-SAPK/JNK, and p-Akt. Our results demonstrated that Aster incisus suppressed the expression of the inflammation mediators through the regulation of NF-κB, MAPK, and Akt pathways.

Adolescent binge ethanol-induced loss of basal forebrain cholinergic neurons and neuroimmune activation are prevented by exercise and indomethacin.

Basal forebrain cholinergic neurons mature in adolescence coinciding with development of adult cognitive function. Preclinical studies using the rodent model of adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-days on/2-days off from postnatal day [P]25 to P55) reveal persistent increases of brain neuroimmune genes that are associated with cognitive dysfunction. Adolescent intermittent ethanol exposure also reduces basal forebrain expression of choline acetyltransferase (ChAT), an enzyme critical for acetylcholine synthesis in cholinergic neurons similar to findings in the post-mortem human alcoholic basal forebrain. We report here that AIE decreases basal forebrain ChAT+IR neurons in both adult female and male Wistar rats following early or late adolescent ethanol exposure. In addition, we find reductions in ChAT+IR somal size as well as the expression of the high-affinity nerve growth factor (NGF) receptor tropomyosin receptor kinase A (TrkA) and the low-affinity NGF receptor p75NTR, both of which are expressed on cholinergic neurons. The decrease in cholinergic neuron marker expression was accompanied by increased phosphorylation of NF-κB p65 (pNF-κB p65) consistent with increased neuroimmune signaling. Voluntary wheel running from P24 to P80 prevented AIE-induced cholinergic neuron shrinkage and loss of cholinergic neuron markers (i.e., ChAT, TrkA, and p75NTR) as well as the increase of pNF-κB p65 in the adult basal forebrain. Administration of the anti-inflammatory drug indomethacin (4.0 mg/kg, i.p prior to each ethanol exposure) during AIE also prevented the loss of basal forebrain cholinergic markers and the concomitant increase of pNF-κB p65. In contrast, treatment with the proinflammatory immune activator lipopolysaccharide (1.0 mg/kg, i.p. on P70) caused a loss of cholinergic neuron markers that was paralleled by increased pNF-κB p65 in the basal forebrain. These novel findings are consistent with AIE causing lasting activation of the neuroimmune system that contributes to the persistent loss of basal forebrain cholinergic neurons in adulthood.

Toll-like receptor 4 shRNA attenuates lipopolysaccharide-induced epithelial-mesenchymal transition of intrahepatic biliary epithelial cells in rats

Background and aimIntrahepatic biliary epithelial cells (IBECs) of the bile duct in liver tissue of patients with hepatolithiasis promoted the development of diseases through epithelial–mesenchymal transition (EMT). This study investigated whether lipopolysaccharide (LPS), a cell-wall constituent of gram-negative bacteria, could induce EMT of IBECs and toll-like receptor 4 (TLR4) had a regulatory role via activating the nuclear factor-κB (NF-κB)/Snail signaling pathway during this process in vivo. MethodsTLR4 short hairpin RNA (shRNA) adenovirus or negative control shRNA (NC shRNA) adenovirus (1 × 109 plaque-forming unit (PFU), respectively) was injected into the caudal vein of rats. After 96 h, 1 mg/kg LPS was infused retrogradely into the common bile duct for 48 h per rat. The effects of TLR4 shRNA on LPS-induced EMT were determined by evaluating the histopathological changes in IBECs using hematoxylin and eosin staining and the changes in the levels of EMT markers, TLR4, NF-κB p65, pNF-κB p65, and Snail using real-time polymerase chain reaction and Western blot analysis. ResultsCompared with normal saline treatment, a loss of epithelial cell markers (E-cadherin and cytokeratin 7) and a gain of mesenchymal cell markers (N-cadherin and matrix metalloproteinase 2) were revealed. The levels of TLR4, NF-κB phosphorylation, and Snail significantly increased after LPS treatment, whereas pretreatment with TLR4 shRNA inhibited the LPS-induced EMT by downregulating the NF-κB/Snail signaling pathway. ConclusionsLPS induced the EMT of IBECs by activating TLR4. The RNAi-mediated knockdown of TLR4 suppressed EMT occurrence via downregulating the NF-κB/Snail signaling pathway, implicating TLR4 as a new target for human hepatolithiasis.

Effects of honokiol on particulate matter 2.5-induced lung injury in asthmatic mice and its mechanisms

To explore the therapeutic effect of honokiol on particulate matter 2.5 (PM2.5)-induced lung injury in asthmatic mice and the possible mechanisms. Methods: A total of 32 BALB/C mice were randomly divided into four groups: a normal saline group, a model group, a PM2.5 group and a honokiol group (n=8 in each group). The asthma mouse model was established by ovalbumin treatment. The mice were treated with physiological saline, ovalbumin, PM2.5 and honokiol, respectively. Lung tissues and serum were collected. The pathological changes of lung tissues were evaluated. The levels of inflammatory cytokines in bronchoalveolar lavage fluid (BALF) and serum were measured and the expressions of Toll like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), retinoid-related orphan receptor gamma-t (RORγt) and forkhead box protein 3 (Foxp3) in lung tissues were detected. Results: 1) The lung tissues of mice in the asthma group showed obvious pathological changes and inflammatory state, suggesting that the asthma model was established successfully. PM2.5 could aggravate the pathological condition of inflammatory injury in lung tissues in asthmatic mice. 2) Compared to the PM2.5 group, the pathological symptoms in the lung tissues were alleviated in the honokiol group and the percentage of inflammatory cells in BALF and the levels of inflammatory cytokines in BALF and serum were significantly reduced (all P<0.05). 3) Compared to the PM2.5 group, the expressions of TLR4, NF-κB (p-p65) and RORγt in lung tissues were significantly decreased, while the expression of Foxp3 was increased; the ratio of RORγt/Foxp3 was also decreased in the honokiol group (all P<0.05). Conclusion: Honokiol can resist lung injury induced by PM2.5 in asthmatic mice. These effects are through inhibiting TLR4-NF-κB pathway-mediated inflammatory response or regulating the balance of Th17/Treg cells.

Protective effect of chlorogenic acid on lipopolysaccharide-induced inflammatory response in dairy mammary epithelial cells

Mastitis is a major disease of dairy cattle. Given the recent emergence of antibiotics resistance to mastitis, new intramammary treatments are urgently required. In the present study, we investigated whether lipopolysaccharide (LPS) could induce the increase in the proinflammatory cytokines in bovine mammary epithelial cells (MECs), and whether a natural antimicrobial compound Chlorogenic acid (CGA) could attenuate the inflammatory responses induced by LPS and thus could be a potential therapeutic compound for bovine mastitis. Our results indicated that LPS could induce the expression of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukine (IL)-1β and IL-6, and the activation of NF-κB p65 and p-p65 in primary bovine MECs. Furthermore, CGA significantly inhibited not only the protein expression of NF-κB p65 and p-p65 but also the mRNA expression of TNF-α, IL-1β and IL-6 after LPS treatment in primary bovine MECs. These results suggested that CGA had anti-inflammatory role by inhibiting NF-κB activation. In conclusion, CGA could be possibly used as a potential therapeutic compound for bovine mastitis.

Artesunate ameliorates high glucose-induced rat glomerular mesangial cell injury by suppressing the TLR4/NF-κB/NLRP3 inflammasome pathway

Inflammatory response is important for the development and progression of diabetic nephropathy (DN). Artesunate (ART), an antimalarial drug, possesses anti-inflammatory effect and exhibits protective effect on chronic kidney diseases. However, the effect of ART on DN is unknown. The aim of the present study was to evaluate the effect and the molecular mechanism of ART on DN in an in vitro model. The rat mesangial cell line, HBZY-1, was induced by high glucose (HG; 30 mM d-glucose) in the presence or absence of ART (15 and 30 μg/ml) and incubated for 24 h. We found that HG induced the proliferation of HBZY-1 cells, while treatment with ART inhibited the cell proliferation. Treatment with ART inhibited HG-induced inflammatory cytokines production and expression of extracellular matrix (ECM). Besides, HG induced reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and inhibited the superoxide dismutase (SOD) activity of HBZY-1 cells, and the effects were attenuated by ART treatment. ART decreased HG-induced the expression levels of toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MyD88), nuclear factor κB (NF-κB) p-p65, and nod-like receptor protein 3 (NLRP3). Inhibition of the TLR4/NF-κB pathway suppressed NLRP3 inflammasome in HBZY-1 cells. In conclusion, ART exhibited protective effect on HG-induced HBZY-1 cells by inhibiting the inflammatory response, oxidative stress and ECM accumulation. The TLR4/NF-κB/NLRP3 inflammasome pathway was involved in the protective effect of ART. The results suggested that ART might be a potential therapy agent for the DN treatment.

Mycophenolic Acid Synergizing with Lipopolysaccharide to Induce Interleukin-1β Release via Activation of Caspase-1.

Background:The previous study showed that mycophenolic acid (MPA) synergizing with lipopolysaccharide (LPS) promoted interleukin (IL)-1β release, but the mechanism is unclear. This study aimed to investigate the mechanism of MPA synergizing with LPS to induce IL-1β release.Methods:Undiluted human blood cells, THP-1 human myeloid leukemia mononuclear cells (THP-1) cells, or monocytes were stimulated with LPS and treated with or without MPA, and the supernatant IL-1β was detected by enzyme-linked immunosorbent assay. The mRNA levels of IL-1β were detected by real-time quantitative polymerase chain reaction. The intracellular protein levels of nuclear factor kappa B (NF-κB) phospho-p65 (p-p65), precursor interleukin-1β (pro-IL-1β), NOD-like receptor pyrin domain containing-3 (NLRP3), and cysteine aspartic acid-specific protease-1 (caspase-1) p20 in THP-1 cell were measured by Western blot.Results:The MPA alone failed to induce IL-1β, whereas MPA synergized with LPS to increase IL-1β in a dose-dependent manner (685.00 ± 20.00 pg/ml in LPS + 5 μmol/L MPA group, P = 0.035; 742.00 ± 31.58 pg/ml in LPS + 25 μmol/L MPA group, P = 0.017; 1000.00 ± 65.59 pg/ml in LPS + 75 μmol/L MPA group, P = 0.024; versus 408.00 ± 35.50 pg/ml in LPS group). MPA alone has no effect on the IL-1β mRNA expression, LPS induced the expression of IL-1β mRNA 2761 fold, and LPS + MPA increased the IL-1β expression 3018 fold, which had the same effect with LPS group (P = 0.834). MPA did not affect the intracellular NF-κB p-p65 and pro-IL-1β protein levels but activated NLRP3 inflammasome. Ac-YVAD-cmk blocked the activation of caspase-1 and subsequently attenuated IL-1β secretion (181.00 ± 45.24 pg/ml in LPS + MPA + YVAD group vs. 588.00 ± 41.99 pg/ml in LPS + MPA group, P = 0.014).Conclusions:Taken together, MPA synergized with LPS to induce IL-1β release via the activation of caspase-1, rather than the enhanced production of pro-IL-1β. These findings suggested that patients immunosuppressed with mycophenolate mofetil may have overly activated caspase-1 during infection, which might contribute to a more sensitive host defense response to invading germs.