LPS-induced Phosphorylation Of P38 Research Articles

METTL3 and METTL14 regulate IL-6 expression via RNA m6A modification of zinc transporter SLC39A9 and DNA methylation of IL-6 in periodontal ligament cells

The inflammatory response is a key process in periodontitis. The N6-methyladenosine (m6A) modification has been proven to be involved in various physiological and pathological processes. This study aims to investigate the role and downstream mechanism of N6-adenosine-enzyme subunits methyltransferase (METTL) 3 and 14 in the inflammatory response of periodontal ligament cells (PDLCs). The total m6A content and the expression of METTL3 and METTL14 were upregulated in lipopolysaccharide (LPS)-stimulated PDLCs. Knockdown of METTL3 or METTL14 suppressed the LPS-induced interleukin (IL)-6 expression, as shown by quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Mechanistically, conjoint analysis of m6A sequencing of METTL3-knockdown and METTL14-knockdown PDLCs revealed that the expression of solute carrier family 39 member 9 (SLC39A9) was mediated in a m6A-dependent manner. The suppression of LPS-induced IL-6 by METTL3 or METTL14 knockdown was partially counteracted by SLC39A9 knockdown, which induced downregulation of intracellular zinc via immunofluorescence staining. Amplicon bisulfite sequencing (AmpBS) demonstrated that METTL3/14 knockdown increased the methylation at one position of the IL-6 promoter, while SLC39A9 knockdown decreased it, which was basically consistent with the intracellular zinc concentration and negatively associated with IL-6 expression. Moreover, METTL3 or METTL14 knockdown attenuated the LPS-induced phosphorylation of p38 and JNK mitogen-activated protein kinase (MAPK), which was partially counteracted by SLC39A9 knockdown. These results revealed the “LPS-METTL3/14-SLC39A9-zinc-IL-6” axis and involvement of p38 and JNK MAPK signaling pathway in the inflammatory responses of PDLCs.

Isobavachalcone ameliorates the progression of osteoarthritis by suppressing NF-κB signaling pathway.

Isobavachalcone (IBC), an active component isolated from Psoralea corylifolia L., has been used extensively to treat a wide range of inflammation-associated diseases. However, little is known regarding the potential effect of IBC in the treatment of osteoarthritis (OA). The purpose of this research was to investigate the potential therapeutic effectsof IBC on OA by performingin vitroand in vivo experiments. Meanwhile, the underlying mechanism responsibles for that effect was also explored. Primary rat chondrocytes were isolated from the knee cartilage, and then pretreated with various concentrations of IBC followed by stimulation with or without LPS (1µg/ml) for the indicated times. In vitro, the expression levels of iNOS, COX-2, MMP3, MMP13, ADAMTS5, aggrecan, and collagen II were determined by qRT-PCR, western blot, and immunofluorescence staining. In addition, western blot analysis and immunofluorescence were used to assess alterations to the NF-κB signaling pathway. In vivo, an ACLT-induced rat OA model was established in order to determine the protective effect of IBC. The results showed that IBC treatment inhibited the upregulation of inflammatory factors such as iNOS and COX-2 in response to LPS stimulation. Moreover, IBC significantly suppressed the expression of MMP-3, MMP-13, and ADAMTS5 induced by LPS in a dose-dependent manner. Furthermore, the LPS-induced reduction of collagen II and aggrecan was reversed by IBC. Mechanistically, IBC significantly decreased LPS-induced p65 phosphorylation and IκBα degradation as well as suppressed nuclear translocation of p65 in rat chondrocytes as evidenced by western blot analysis and immunofluorescence staining, indicating that IBC effectively inhibited the LPS-induced activation of the NF-κB pathway. In vivo, IBC treatment prevented cartilage degeneration in the ACLT-induced rat model. In summary, our results suggest that IBC may be able to act as a promising therapeutic drug for treating OA.

Irigenin attenuates lipopolysaccharide-induced acute lung injury by inactivating the mitogen-activated protein kinase (MAPK) signaling pathway.

Acute lung injury (ALI) is a serious pulmonary inflammation disease with high mortality. Irigenin, an isoflavone from rhizomes of the Belamcanda chinensis, has been reported to exert anti-inflammatory, anti-oxidative, and anti-apoptotic activities in several diseases. However, it is still unclear whether irigenin can exert a beneficial effect in ALI. A network pharmacology method was utilized to predict the hub targets and potential therapeutic mechanisms of irigenin against ALI. Lipopolysaccharide (LPS) was used to establish the mice model of ALI for evaluating the effects of irigenin. According to the protein-protein interaction (PPI) network, we identified EGFR, HRAS, AKT1, SRC, and HSP90AA1 as the top five significant genes. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment assays showed that irigenin might affect inflammatory response, cytokine production, and cell death by the mitogen-activated protein kinase (MAPK) signaling pathway. In vivo experiment results manifested that irigenin decreased pathological changes, lung Wet/Dry weight ratio, and total protein content in bronchoalveolar lavage fluid (BALF). Irigenin also reduced the production of inflammatory cytokines, including tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-18 (IL-18), and neutrophil infiltration. Additionally, irigenin inhibited pulmonary apoptosis in LPS-treated ALI mice. Moreover, LPS-induced phosphorylation of p38, JNK, and ERK was significantly abated due to the treatment of irigenin. In summary, irigenin ameliorates LPS-induced ALI by suppressing pulmonary inflammation and apoptosis via inactivation of the MAPK signaling pathway. These findings indicated the therapeutic potential of irigenin in ALI.

Apigetrin Abrogates Lipopolysaccharide-Induced Inflammation in L6 Skeletal Muscle Cells through NF-κB/MAPK Signaling Pathways.

Apigetrin is a glycosidic flavonoid derived from Teucrium gnaphalodes that has a wide range of biological activities, including antioxidant, anti-inflammatory, and anticancer. Inflammation is a kind of defense mechanism in the body. Flavonoids are natural phytochemicals that exert anti-inflammatory effects in numerous cells. In the present study, we investigated the anti-inflammatory effect of apigetrin and its underlying mechanism of activity in skeletal muscle cells (L6). The determination of cytotoxicity was performed by MTT assay. We treated L6 cells with apigetrin, and nontoxic concentrations were chosen to perform further experimentation. Apigetrin inhibited the expression of iNOS and COX-2 induced by LPS in a dose-dependent manner. iNOS and COX-2 are inflammatory markers responsible for enhancing the inflammatory response. Apigetrin also inhibited the LPS-induced phosphorylation of p65 and IκB-α. NF-κB signaling regulates the inflammatory process by mediating various proinflammatory genes. Similarly, the MAPK signaling pathway consists of ERK, JNK, and p38, which plays a critical role in the production of cytokines and downstream signaling events leading to inflammation. Apigetrin significantly downregulated the phosphorylation of JNK and p38, but did not affect the phosphorylation of ERK in the LPS-stimulated cells. These findings indicate the correlation between the anti-inflammatory activity of NF-κB and the MAPK signaling pathway. Thus, our overall finding suggests that apigetrin has anti-inflammatory effects and it can be considered for further drug design on L6 skeletal muscle cells.

High glucose enhances lipopolysaccharide‐induced inflammation in cultured BV2 microglial cell line

IntroductionDiabetes mellitus emerges as a global health crisis and is related to the development of neurodegenerative diseases. Microglia, a population of macrophages‐like cells, govern immune defense in the central nervous system. Activated microglia are known to play active roles in the pathogenesis of neurodegenerative diseases.MethodsThis study aimed to investigate the effects of high glucose on low‐dose lipopolysaccharide (LPS)‐induced activations of inflammation‐related signaling molecules in cultured BV2 microglial cells.ResultsCompared to cells cultured in the normal glucose medium (NGM, 5.5 mM), the LPS‐induced activation of NF‐κB lasted longer in cells cultured in high glucose medium (HGM, 25 mM). HGM also enhanced the expression of inducible nitric oxide synthase (iNOS). Among the mitogen‐activated protein kinases, HGM enhanced the LPS‐induced phosphorylation of p38 without affecting the phosphorylation of Erk1/2 or JNK. BV2 cells cultured in HGM expressed higher levels of TLR4 than those cells cultured in NGM.ConclusionHigh glucose aggravated LPS‐induced inflammatory responses of microglia via enhancing the TLR4/p38 pathway and prolonging the activation of NF‐κB/iNOS signaling. Controlling blood glucose levels is advised to manage neuroinflammation and related neurodegenerative diseases.

Beneficial effects of aloperine on inflammation and oxidative stress by suppressing necroptosis in lipopolysaccharide-induced acute lung injury mouse model

RationaleAlveolar epithelial cell death, inflammation, and oxidative stress are typical features of acute lung injury (ALI). Aloperine (Alo), an alkaloid isolated from Sophora alopecuroides, has been reported to display various biological effects, such as anti-inflammatory, immunoregulatory, and anti-oxidant properties. In this study, we investigated the effects and mechanisms of Alo in treating a lipopolysaccharide (LPS)-induced ALI in a murine model. MethodsThe effects of Alo in LPS-induced ALI were investigated in C57BL/6 mice. The RIPK1 inhibitor (Nec-1) and the RIPK3 inhibitor (GSK’872) were used to evaluate the relationship of necroptosis, NF-κB activation, and PDC subunits in LPS-treated mouse alveolar epithelial cells (MLE-12). Then the effects of Alo on necroptosis, inflammation, and oxidative stress of LPS-stimulated MLE-12 cells were evaluated. ResultsAlo significantly attenuated histopathological lung injuries and reduced lung wet/dry ratio in LPS-induced ALI mice. Alo also remarkedly reduced total protein and neutrophils recruitment in bronchoalveolar lavage fluid of ALI mice. Meanwhile, Alo ameliorated the LPS-induced necroptosis in the lungs of ALI mice. The RIPK3 inhibitor GSK’872, but not the RIPK1 inhibitor Nec-1, reversed LPS-induced p65 phosphorylation and translocation to the nucleus in MLE-12 cells. GSK’872 also reversed the LPS-induced increase in ROS and binding of RIPK3 and PDC subunits in MLE-12 cells. Moreover, Alo down-regulated the levels of p-RIPK1, p-RIPK3, p-MLKL, p-p65, the translocation of p65 to the nucleus, and reduced the expression of IL-6 and IL-8 in LPS-stimulated MLE-12 cells. Alo also inhibited the binding of RIPK3 and PDC-E1α, PDC-E1β, PDC-E2, and PDC-E3 and the ROS production in LPS-treated MLE-12 cells. ConclusionThe present study validated the beneficial effects of Alo on LPS-induced ALI , suggesting Alo may be a new drug candidate against ALI.

The Anti-Inflammatory Effect of Smilax china L. Extract on LPS-Stimulated THP-1 via Downregulation of MAPK and NF-κB Signaling Pathway.

Background Traditional Chinese medicine Smilax is the rhizome of liliaceous plant Smilax china L., which is used to treat pelvic inflammatory disease and anxieties. Purpose To investigate the mechanism of anti-inflammatory activity of the extract from Smilax china L. (ES). Methods The components of ES were identified by UPLC-QTOF-MS/MS. The anti-inflammatory activities were evaluated in xylene-induced ear oedema and egg white-induced plantar swelling test. Cell viability was examined by CCK-8 assay. The inflammatory mediators, proinflammatory cytokines, and MAPK and NF-κB signals in LPS-stimulated THP-1 cells were determined using ELISA, real-time PCR, and Western blot, respectively. Results 20 compounds of ES were confirmed by comparing with the reference substance. ES displayed more prominent anti-inflammatory activity than the positive control “Jin Gang Teng” capsule in the in vivo acute inflammatory model. ES suppressed the expression of PGE2 and 6-Keot-PGF1α, and the ratio of IC50 (COX-1)/IC50 (COX-2) of ES was 3.15, which indicated that ES could selectively inhibit COX-2. ES dose-dependently (12.5, 25, and 50 mg/L) decreased the production and mRNA levels of proinflammatory cytokines IL-1β, IL-6, and TNF-α. Furthermore, ES significantly decreased LPS-induced phosphorylation of p38, JNK, ERK1/2, and p65, inhibiting the expression of IKKα and the degradation of IκBα. Conclusion The results suggested that ES could selectively inhibit the activity of COX-2, and the anti-inflammatory effect of ES was associated with the inhibition of IL-1β, IL-6, and TNF-α via negative regulation of MAPK and NF-κB signaling pathways in LPS-induced THP-1 cells.

GPER and IGF-1R mediate the anti-inflammatory effect of genistein against lipopolysaccharide (LPS)-induced nigrostriatal injury in rats

Neuroinflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Genistein is an estrogen-like phytoestrogen that can exert biological effects via the crosstalk of estrogen receptor and insulin-like growth factor 1 receptor (IGF-1R). The present study aimed to evaluate the involvement of G protein-coupled estrogen receptor (GPER) and IGF-1R in the anti-inflammatory effects of genistein against lipopolysaccharide (LPS)-induced nigrostriatal injury in ovariectomized rats. Our results showed that genistein treatment could ameliorate the apomorphine-induced rotational behavior in LPS-induced inflammatory PD rat model. Genistein attenuated LPS-induced decrease of the contents of dopamine (DA) and its metabolites in striatum as well as the loss of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the substantia nigra (SN) of the lesioned side, which could be blocked by GPER antagonist G15 or IGF-1R antagonist JB1. Meanwhile, G15 or JB1 could attenuate the anti-inflammatory effects of genistein in LPS-induced microglial activation and production of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, genistein could inhibit the LPS-induced phosphorylation of p38, JNK, ERK and IκB in the lesioned side of SN and these effects could also be blocked by G15 or JB1. Taken together, our data provide the first evidence that genistein can inhibit the increase of microglia and protect dopaminergic neurons at least in part via GPER and IGF-1R signaling pathways in ovariectomized PD rat model.

Pgc-1α Promotes Phosphorylation, Inflammation, and Apoptosis in H9c2 Cells During the Early Stage of Lipopolysaccharide Induction

Cardiac dysfunction in severe sepsis is associated with increased mortality. However, the molecular mechanisms underlying septic heart dysfunction remain unclear. Expression of peroxisome proliferator-activated receptor-γ coactivator 1α (Pgc-1α), concentrations of inflammatory factors, and activation of the nuclear factor kappa-B (NF-κB) signaling pathway were examined in H9c2 cells after a 24-h lipopolysaccharide (LPS) stimulation period using qPCR, enzyme-linked immunosorbent assays (ELISAs), and western blots (WBs), respectively. Pgc-1α was overexpressed and suppressed in cells using a lentivirus vector and siRNA, respectively. The effects of Pgc-1α dysfunction on the release of inflammatory factors and apoptosis were analyzed. Pgc-1α expression was increased after LPS induction for 0.5 h and returned to the pre-induction level at 2 h. Levels of IL-1β, IL-6, and TNF-α increase after LPS induction for 0.5 h and accumulated in the culture supernatants over time. The WBs revealed the highest Pgc-1α and phospho (p)-p65 protein levels after LPS induction for 0.5 h, followed by a decrease; moreover, the cleaved-caspase-3 level increased after LPS induction for 0.5 h and increased gradually thereafter. A functional analysis of Pgc-1α revealed that overexpression of this protein enhanced LPS-induced inflammatory factors and p-p65 levels and inhibited apoptosis during the early stage after LPS induction (0.5 and 4 h). In contrast, the inhibition of Pgc-1α expression inhibited the LPS expression-associated increases in inflammatory factors and p-p65 and promoted apoptosis. Pgc-1α promoted LPS-induced p65 phosphorylation and inflammatory factor release while inhibiting apoptosis.

Anti-inflammatory Effects of Proanthocyanidin-rich Cranberry Extract through the Suppression of NF-kB Pathway and Histone Acetylase in RAW 264.7 and Mouse Bone Marrow-derived Macrophages

Obesity-mediated chronic inflammation promotes the progression of obesity to metabolic anti-inflammatory effect of cranberries by decreasing plasma inflammatory cytokines. However, its specific mechanisms of action remain unclear. The nuclear factor-κB (NF-κB) pathway in macrophages plays a critical role in regulating the expression of many inflammatory genes, and histone acetylation has been identified as a key epigenetic modification for the NF-κB p65-mediated inflammatory responses. The objective of the study was to investigate if proanthocyanidin (PAC)-rich cranberry extract (CBE) suppresses histone acetylation and NF-κB p65 activation in RAW 264.7 macrophages and mouse bone marrow-derived macrophages (BMDMs). Treatment with 5% and 15% PAC-containing CBEs markedly suppressed the expression of pro-inflammatory mediators (iNos, Cox-2, Tnfα, Mcp-1 and Il-6) in both RAW 264.7 macrophages and BMDMs stimulated with lipopolysaccharides (LPS). CBE significantly reduced LPS-induced phosphorylation of p65 in both cell types without changing total p65 expression levels. Moreover, 15% PAC-CBE increased the expression levels of histone deacetylase 3 (HDAC3) with a concomitant decrease in histone H4 acetylation levels. These results suggest that CBE increases HDAC3 protein expression with the subsequent inhibition of p65 phosphorylation to mediate anti-inflammatory effects in macrophages. Cranberries may serve as a dietary agent to attenuate chronic inflammation in patients with obesity and related complications.

Comparison of PPAR Ligands as Modulators of Resolution of Inflammation, via Their Influence on Cytokines and Oxylipins Release in Astrocytes

Neuroinflammation is a key process of many neurodegenerative diseases and other brain disturbances, and astrocytes play an essential role in neuroinflammation. Therefore, the regulation of astrocyte responses for inflammatory stimuli, using small molecules, is a potential therapeutic strategy. We investigated the potency of peroxisome proliferator-activated receptor (PPAR) ligands to modulate the stimulating effect of lipopolysaccharide (LPS) in the primary rat astrocytes on (1) polyunsaturated fatty acid (PUFAs) derivative (oxylipins) synthesis; (2) cytokines TNFα and interleukin-10 (IL-10) release; (3) p38, JNK, ERK mitogen-activated protein kinase (MAPKs) phosphorylation. Astrocytes were exposed to LPS alone or in combination with the PPAR ligands: PPARα (fenofibrate, GW6471); PPARβ (GW501516, GSK0660); PPARγ (rosiglitazone, GW9662). We detected 28 oxylipins with mass spectrometry (UPLC-MS/MS), classified according to their metabolic pathways: cyclooxygenase (COX), cytochrome P450 monooxygenases (CYP), lipoxygenase (LOX) and PUFAs: arachidonic (AA), docosahexaenoic (DHA), eicosapentaenoic (EPA). All tested PPAR ligands decrease COX-derived oxylipins; both PPARβ ligands possessed the strongest effect. The PPARβ agonist, GW501516 is a strong inducer of pro-resolution substances, derivatives of DHA: 4-HDoHE, 11-HDoHE, 17-HDoHE. All tested PPAR ligands decreased the release of the proinflammatory cytokine, TNFα. The PPARβ agonist GW501516 and the PPARγ agonist, rosiglitazone induced the IL-10 release of the anti-inflammatory cytokine, IL-10; the cytokine index, (IL-10/TNFα) was more for GW501516. The PPARβ ligands, GW501516 and GSK0660, are also the strongest inhibitors of LPS-induced phosphorylation of p38, JNK, ERK MAPKs. Overall, our data revealed that the PPARβ ligands are a potential pro-resolution and anti-inflammatory drug for targeting glia-mediated neuroinflammation.

Anti‑inflammatory effects of leaf and branch extracts of honeyberry (Lonicera caerulea) on lipopolysaccharide‑stimulated RAW264.7 cells through ATF3 and Nrf2/HO‑1 activation.

Honeyberry (Lonicera caerulea) has long been used as a traditional medicine in China, Japan and northern Russia. Functional studies of honeyberry have mainly focused on the fruits, which have been reported to exert various pharmacological activities, including anti-inflammatory activity, with limited or no studies on the other parts of the plant, such as the leaves and branches. In the present study, the anti-inflammatory effects of extracts of the leaves (HBL), branches (HBB) and fruit (HBF) of honeyberry plant were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. HBL and HBB significantly inhibited the production of pro-inflammatory mediators in LPS-stimulated RAW264.7 cells, and the inhibitory effects of HBL and HBB were stronger than those of HBF. HBL and HBB blocked the nuclear accumulation of p65 independently of IκB-α. HBL did not inhibit the phosphorylation of ERK1/2 or p38; however, HBB effectively inhibited the phosphorylation of p38 but not ERK1/2. HBL and HBB increased the expression of heme oxygenase-1 (HO-1) protein by inducing the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) through the activation of the reactive oxygen species (ROS)/p38 pathway; the reduction in inducible nitric oxide synthase (iNOS) and interleukin-1β (IL-1β) expression by HBL and HBB was inhibited by HO-1 knockdown. In addition, HBL and HBB increased the expression of activating transcription factor-3 (ATF3), and the reduction in iNOS and IL-1β expression by HBL and HBB was inhibited by ATF3 knockdown. Collectively, HBL and HBB inhibited LPS-induced nuclear factor-κB activation by blocking the nuclear accumulation of p65, increasing HO-1 expression through activation of the ROS/p38/Nrf2 pathway, and increasing ATF3 expression. Furthermore, HBB inhibited LPS-induced p38 phosphorylation. These findings suggest that HBL and HBB may have great potential as natural products for the development of anti-inflammatory drugs.

Anti-Inflammatory Activity of Oligomeric Proanthocyanidins Via Inhibition of NF-κB and MAPK in LPS-Stimulated MAC-T Cells.

Oligomeric proanthocyanidins (OPCs), classified as condensed tannins, have significant antioxidation, anti-inflammation and anti-cancer effects. This study was performed to investigate the anti-inflammatory effects of OPCs and the mechanism underlying these effects in lipopolysaccharide (LPS)-stimulated bovine mammary epithelial cells (MAC-T). Real-time PCR and ELISA assays indicated that OPC treatment at 1, 3 and 5 μg/ml significantly reduced the mRNA and protein, respectively, of oxidant indicators cyclooxygenase-2 (COX-2) (p < 0.05) and inducible nitric oxide synthase (iNOS) (p < 0.01) as well as inflammation cytokines interleukin (IL)-6 (p < 0.01), IL-1β (p < 0.01) and tumor necrosis factor-α (TNF-α) (p < 0.05) in LPS-induced MAC-T cells. Moreover, OPCs downregulated LPSinduced phosphorylation of p65 and inhibitor of nuclear factor kappa B (NF-κB) (IκB) in the NF-κB signaling pathway (p < 0.01), and they inhibited p65 translocation from the cytoplasm to the nucleus as revealed by immunofluorescence test and western blot. Additionally, OPCs decreased phosphorylation of p38, extracellular signal regulated kinase and c-jun NH2-terminal kinase in the MAPK signaling pathway (p < 0.01). In conclusion, the anti-inflammatory and antioxidant activities of OPCs involve NF-κB and MAPK signaling pathways, thus inhibiting expression of pro-inflammatory factors and oxidation indicators. These findings provide novel experimental evidence for the further practical application of OPCs in prevention and treatment of bovine mastitis.

Nucleolin Mediates LPS-induced Expression of Inflammatory Mediators and Activation of Signaling Pathways

SummaryIn this study, we investigated the effects of nucleolin on lipopolysaccharide (LPS)-induced activation of MAPK and NF-KappaB (NF-κB) signaling pathways and secretion of TNF-α, IL-1β and HMGB1 in THP-1 monocytes. Immunofluorescence assay and Western blotting were used to identify the nucleolin expression in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Inactivation of nucleolin was induced by neutralizing antibody against nucleolin. THP-1 monocytes were pretreated with anti-nucleolin antibody for 1 h prior to LPS challenge. The irrelevant IgG group was used as control. Secretion of inflammatory mediators (TNF-α, IL-1β and HMGB1) and activation of MAPK and NF-κB/I-κB signaling pathways were examined to assess the effects of nucleolin on LPS-mediated inflammatory response. Nucleolin existed in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Pretreatment of anti-nucleolin antibody significantly inhibited the LPS-induced secretion of TNF-α, IL-1β and HMGB1. P38, JNK, ERK and NF-κB subunit p65 inhibitors could significantly inhibit the secretion of IL-1β, TNF-α and HMGB1 induced by LPS. Moreover, the phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65) was significantly increased after LPS challenge. In contrast, pretreatment of anti-nucleolin antibody could significantly inhibit the LPS-induced phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). However, the irrelevant IgG, as a negative control, had no effect on LPS-induced secretion of TNF-α and IL-1β and phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). We demonstrated that nucleolin mediated the LPS-induced activation of MAPK and NF-κB signaling pathways, and regulated the secretion of inflammatory mediators (TNF-α, IL-1β and HMGB1).

Kmeria duperreana (Pierre) Dandy Extract Suppresses LPS-Induced iNOS and NO via Regulation of NF-κB Pathways and p38 in Murin Macrophage RAW 264.7 Cells.

Development of anti-inflammatory products remains in high demand due to the incidence of inflammatory diseases, including diabetes, cardiovascular disease, and neurodegenerative diseases. In this study, we examined the potential anti-inflammatory activity of the nutraceutical, Kmeria duperreana (Pierre) Dandy extract (KDE). We evaluated the ability of KDE to inhibit lipopolysaccharide (LPS)-induced inflammatory markers, including nitric oxide (NO), nuclear factor kappa-B, and mitogen-activated protein kinases, in RAW 264.7 cells. KDE suppressed LPS-induced nitrite production and inducible NO synthase (iNOS) expression in RAW 264.7 cells, but has no effect on cyclooxygenase-2 expression. KDE also suppressed LPS-induced phosphorylation of p65, IκB kinase, and p38 in RAW 264.7 cells. Through Western blot assays and immunofluorescence results, we showed that KDE suppresses LPS-induced p65 translocation from cytosol to the nucleus in RAW 264.7 cells. Moreover, KDE suppressed mRNA expression of LPS-induced interleukin (IL)-1β in RAW 264.7 cells, but had no effect on mRNA expression of IL-6 or tumor necrosis factor-a. These results demonstrate that KDE may be a promising anti-inflammatory nutraceutical. KDE may act by suppressing iNOS expression and subsequent NO production by inhibiting phosphorylation of p65 and p38 and suppressing translocation of p65 from the cytosol to the nucleus.

Morin alleviates LPS-induced mastitis by inhibiting the PI3K/AKT, MAPK, NF-κB and NLRP3 signaling pathway and protecting the integrity of blood-milk barrier

Mastitis is a common veterinary clinical disease that restricts the development of dairy farming around the world. Morin, extracted from Mulberry Tree and other herbs, has been reported to possess the function of anti-bacteria, anti-oxidant, and anti-inflammatory. However, whether morin could protect lipopolysaccharide (LPS)-induced mouse mastitis in vivo has not well known. This study firstly aims to evaluate the effects of morin on LPS-induced mouse mastitis in vivo, and then try to illustrate the mechanism involved in the process. Before injected with LPS, mice were intraperitoneally pre-injected with different concentrations of morin, and mice of the control and LPS group were injected with the same amount of saline. Pathologic changes of mammary gland were determined by histopathological examination. Myeloperoxidase (MPO) activities of mammary gland were determined by the MPO kits. The mRNA expressions of inflammatory cytokines including TNF-α, IL-1β and IL-6, and those of chemokine factors CCL2 and CXCL2, and those of tight junctions occludin claudin-3 were examined by qRT-PCR analysis. The activities of IκB, p65, ERK, P38, AKT, PI3K, NLPR3, claudin-1, claudin-3 and occludin were determined by western blotting. The results showed that morin alleviated LPS-induced edema, destructed structures and infiltrated inflammatory cells of mammary gland. Morin administration significantly decreased LPS-induced TNF-α, IL-1β, IL-6, CCL2 and CXCL2 mRNA expressions. Furthermore, western blot analysis also showed that morin significantly reduced LPS-induced phosphorylation of p65, IκB, p38 and ERK, and enhanced LPS-induced phosphorylation of AKT and PI3K. It was also found that LPS-decreased claudin-3 and occludin expressions were also inhibited by morin treatment. In summary, above results suggest that morin indeed protect LPS-induced mouse mastitis in vivo, and the mechanism was through inhibiting the PI3K/AKT, MAPK, NF-κB and NLRP3 signaling pathways and protecting the integrity of blood-milk barrier by regulating the tight junction proteins expressions.

(3β,16α)-3,16-Dihydroxypregn-5-en-20-one from the Twigs of Euonymus alatus (Thunb.) Sieb. Exerts Anti-Inflammatory Effects in LPS-Stimulated RAW-264.7 Macrophages.

To discover new pharmacologically active natural products, here, we performed the phytochemical analysis of a Korean medicinal plant. Euonymus alatus (Thunb.) Sieb. is a traditional medicinal plant that has been used as a remedy for various diseases in Asian countries. In particular, the cork cambium on the twigs of E. alatus has been used to treat dysmenorrhea, tumors, diabetes, and wound. Phytochemical analysis of the methanolic extract of E. alatus twigs led to the isolation of a sterol, which was identified as (3β,16α)-3,16-dihydroxypregn-5-en-20-one (1) by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry. The stereochemistry of 1 was established with nuclear Overhauser effect spectroscopy (NOESY) analysis and comparison of electronic circular dichroism (ECD) data. To the best of our knowledge, the isolation of compound 1 from nature is first reported here, as well as the complete and revised NMR data assignment of 1. In lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages, compound 1 significantly inhibited nitric oxide (NO) production at an IC50 value of 12.54 ± 0.05 μM as well as the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, the pre-treatment with compound 1 attenuated the LPS-induced phosphorylation of nuclear factor kappa B (NF-κB) p65 through the inhibition of the phosphorylation of IκB kinase alpha (IKKα), IKKβ, and inhibitor of kappa B alpha (IκBα). Compound 1 also inhibited the LPS-induced phosphorylation of p38, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Taken together, compound 1 may serve as an anti-inflammatory constituent of E. alatus twigs and its anti-inflammatory property is thought to be associated with the inhibition of NO production via suppression of iNOS and COX-2 expression through inhibition of IKKα/β, I-κBα and NF-κB p65 activation and downregulation of p38, JNK, and ERK mitogen-activated protein kinase signal pathways in RAW 264.7 macrophages. These findings also provide experimental evidence that compound 1 identified from E. alatus twigs could be a candidate for an anti-inflammatory agent.

LDK378 inhibits the recruitment of myeloid-derived suppressor cells to spleen via the p38-GRK2-CCR2 pathway in mice with sepsis.

Myeloid-derived suppressor cells (MDSCs) are functionally immunosuppressive cells that are persistently increased in abundance and associated with adverse clinical outcomes in sepsis. Here, we investigated the therapeutic potential of an anaplastic lymphoma kinase inhibitor, LDK378, in cecal ligation and puncture (CLP)-induced polymicrobial sepsis and examined its effects on the recruitment of MDSCs. LDK378 significantly improved the survival of CLP-induced polymicrobial septic mice, which was paralleled by reduced organ injury, decreased release of inflammatory cytokines and decreased recruitment of MDSCs to the spleen. Importantly, LDK378 inhibited the migration of MDSCs to the spleen by blocking the CLP-mediated upregulation of CC chemokine receptor 2 (CCR2), a chemokine receptor critical for the recruitment of MDSCs. Mechanistically, LDK378 treatment blocked the CLP-induced CCR2 upregulation of MDSCs via partially inhibiting the phosphorylation of p38 and G-protein-coupled receptor kinase-2 (GRK2) in bone marrow MDSCs of septic mice. Furthermore, invitro experiments also showed that lipopolysaccharide (LPS)-induced migration of MDSCs was similarly owing to the activation of GRK2 and upregulation of CCR2 by LPS, whereas the treatment with LDK378 partially blocked the LPS-induced phosphorylation of p38 and GRK2 and decreased the expression of CCR2 on the cell surface, therefore leading to the suppression of MDSC migration. Together, these findings unravel a novel function of LDK378 in the host response to infection and suggest that LDK378 could be a potential therapeutic agent for sepsis.

Anti-Inflammatory Activity of Adenosine 5'-Trisphosphate in Lipopolysaccharide-Stimulated Human Umbilical Vein Endothelial Cells Through Negative Regulation of Toll-Like Receptor MyD88 Signaling.

Activation of TLR4-MyD88-NF-κB signaling by lipopolysaccharide (LPS) evokes a proinflammatory immune response, and plays a pivotal role in initiation and progression of atherosclerosis (AS). ATP (adenosine 5'-trisphosphate), a powerful extracellular signal transduction molecule, functions to regulate immune inflammatory responses depending on the type of P2 receptors and cell lines. In this study, we first performed RT-PCR to detect the mRNA expression of monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-1β induced by different concentrations of LPS in human umbilical vein endothelial cells (HUVECs). Protein level of TLR4 signaling including TLR4, myeloid differentiation factor (MyD88), and CD14 induced by LPS (1 μg/mL) at different times (0, 10, 30, 60, 120 min) was analyzed by Western blot. Then, RT-PCR was performed to detect the effect of different concentrations of ATP on mRNA expression of IL-1β and MCP-1 induced by LPS (1 μg/mL) and the TLR4 signaling pathway. Western blot was performed to detect the effect of low concentrations of ATP on phosphorylation of p65 induced by 1 μg/mL LPS. Finally, we used P2Y receptor blocker Suramin to verify whether the role of ATP on LPS-induced inflammatory cytokine expression was through P2Y receptors. The results showed that LPS upregulated the expression of MCP-1, IL-8, and IL-1β in a dose-dependent manner accompanied by the activation of TLR4-MyD88 signaling in HUVECs. Only low concentration ATP (1, 10 μM) inhibited LPS-induced mRNA expression of IL-1β and MCP-1. ATP at low concentrations also downregulated the mRNA expression of TLR4, CD14, and MyD88 and inhibited LPS-induced phosphorylation of p65. Furthermore, Suramin, a nonspecific P2Y receptor antagonist, did not attenuate the inhibition of ATP on LPS-induced IL-1β and MCP-1 expression. Taking this together, low concentration ATP inhibited LPS-induced inflammation in HUVECs by negatively regulating TLR4-MyD88 signaling, and P2Y receptors were not involved in this process, which might provide new ideas for prevention and treatment of inflammatory diseases such as AS.

MRNA Expression Profile of SFKs and Involvement of SFKs in the Regulation of LPS-Induced Erk1/2 Signaling in PBMCs of Active BD Patients.

Behcet's Disease (BD) is a multisystemic inflammatory disorder affecting large vessels, lungs joints, gastrointestinal and neurological systems. The pathogenesis of BD remains poorly understood. Identifying the key signaling pathway is crucial for a complete understanding of the pathogenesis of BD. The aim of this study was to determine mRNA expression level of Src family kinases (SFKs) members and their involvement in lipopolysaccharide (LPS)-induced mitogen-activated protein kinases (MAPKs) regulation in peripheral blood mononuclear cells (PBMCs) of active BD patients. Twenty- five active BD patients and twenty-five healthy controls were included in the study. PBMCs were isolated from total blood by density gradient centrifugation. The mRNA expression levels of SFKs members were measured by real-time quantitative PCR (RT-qPCR). The effect of SFKs activity on LPS-induced activation MAPKs (Erk1/2, p38 and JNK) was examined by Western blot. The mRNA expression levels of Hck, Src, Lyn, Yes and Fyn were found to be slightly decreased in active BD patients compared to the control subjects, but a slight change in mRNA level of SFKs members did not impact on protein levels and protein activity. LPS-induced Erk1/2 phosphorylation was significantly increased in the absence of SFKs activity in active BD patients. However, inhibition of SFKs activity had no effect on LPS-induced phosphorylation of p38 and JNK in both controls and active BD patients. SFKs downregulate LPS-induced Erk1/2 phosphorylation in PBMCs of active BD patients.