LPS-induced Interleukin-6 Production Research Articles

Corydalis tomentella Franch. Exerts anti-inflammatory and analgesic effects by regulating the calcium signaling pathway

Ethnopharmacological relevanceCorydalis tomentella Franch. is a perennial cespitose plant commonly used to treat stomachaches as a folk medicine. The C. tomentella total alkaloids have good protective effects against acute liver injury and potential anti-hepatoma and anti-Alzheimer's disease activities. Aim of the studyTo establish an effective purification process for total alkaloids from C. tomentella and investigate the mechanism of their anti-inflammatory effects. Materials and methodsCorydalis tomentella were purified using macroporous resin. Then the crude and purified C. tomentella extracts (cCTE and pCTE) were qualitatively analyzed using UPLC-Triple-TOF-MS/MS. The cCTE and pCTE were used to investigate and compare their anti-inflammatory effects on lipopolysaccharide (LPS)-induced RAW264.7 cells. Doses at 100, 200 and 400 mg/kg/d of pCTE were used to study their anti-inflammatory and analgesic activities in mice with xylene-induced ear swelling and acetic acid-induced writhing tests. Content of nitric oxide (NO), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were determined both in RAW264.7 cells and mice. Network pharmacology was used to predict the anti-inflammatory mechanism of C. tomentella, and the key enzymes were validated using qPCR and Western Blot analysis. Concentration of intracellular Ca2+ was detected using flow cytometric analysis. ResultsThe C. tomentella total alkaloid purity increased from 6.29% to 47.34% under optimal purification conditions. A total of 54 alkaloids were identified from CTE. Both cCTE and pCTE could suppress the LPS-induced production of NO, IL-6, IL-1β, and TNF-α in RAW264.7 cells. The pCTE exhibited a more potent anti-inflammatory effect; it also inhibited pain induced by xylene and acetic acid in mice. The calcium signaling pathway is associated with the anti-inflammatory and analgesic activities of C. tomentella. The mRNA expression of nitric oxide synthase (NOS) 2, NOS3 and calmodulin1 (CALM1) was regulated by C. tomentella through the reduction of inflammation-induced Ca2+ influx, and it also exhibited a more pronounced effect than the positive control (L-NG-nitro arginine methyl ester). ConclusionsPurified C. tomentella extract shows anti-inflammatory effect both in vitro and in vivo. It exerts anti-inflammatory and analgesic effects through the calcium signaling pathway by down-regulating NOS2 and CALM1 expression and up-regulating NOS3 expression in LPS-induced RAW264.7 cells, and decreasing intracellular Ca2+ concentration.

Protection against Lipopolysaccharide-Induced Endotoxemia by Terrein Is Mediated by Blocking Interleukin-1β and Interleukin-6 Production.

Terrein is a fungal metabolite and has been known to exert anti-melanogenesis, anti-cancer, and anti-bacterial activities. However, its role in endotoxemia has never been investigated until now. In the present study, we examined the effect of terrein on lipopolysaccharide (LPS)-induced endotoxemia in mice and characterized the potential mechanisms of action. Treatment with terrein increased the survival of mice and decreased the production of inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6) in an LPS-induced endotoxemia model. In addition, terrein suppressed the LPS-induced production of IL-1β and IL-6 in RAW 264.7 cells, a murine macrophage-like cell line, and the mRNA expression of IL-1β and IL-6 was also inhibited by terrein in LPS-stimulated RAW 264.7 cells. Further study demonstrated that terrein blocked LPS-induced phosphorylation of p65 subunit of nuclear factor (NF)/κB and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) was also suppressed by terrein treatment. Collectively, these results suggest that terrein exerts a protective effect again LPS-induced endotoxemia in mice by blocking the production of inflammatory cytokines. Our results also suggest that the anti-inflammatory effect of terrein might be mediated, at least in part, by blocking the activation of NF-κB, JNK, and p38 MAPK signaling pathways.

Comparison of the in vitro Anti-Inflammatory Effect of Cannabidiol to Dexamethasone.

BackgroundCannabidiol (CBD) is a non-psychoactive phytocannabinoid constituent of Cannabis sativa with pain-relieving and anti-inflammatory properties. With the emphasis on natural ingredients in cosmetics, CBD has become a new cosmetic ingredient due to its ability to alleviate inflammation. However, in-depth studies that directly compare the effective mechanism and the therapeutic potential of CBD are still needed.PurposeThe aim of the present study was to investigate the anti-inflammatory effect of CBD in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and compare it to dexamethasone (DEX).MethodsRAW264.7 macrophages in the logarithmic growth phase were incubated in the presence or absence of LPS. After that, the production of nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were measured. A luciferase reporter assay for nuclear factor kappa B (NF-κB) was performed, and the phosphorylation levels of the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways were measured.ResultsThe present study indicated that CBD had a similar anti-inflammatory effect to DEX by attenuating the LPS-induced production of NO, IL-6, and TNF-α. However, only CBD attenuated JNK phosphorylation levels, and only DEX attenuated IKK phosphorylation levels.ConclusionThese results suggested that CBD and DEX exhibit similar anti-inflammatory effects on LPS-induced RAW264.7 macrophages mainly through suppressing the MAPK and NF-κB signaling pathways, but with different intracellular mechanisms. These findings suggested that CBD may be considered a natural anti-inflammatory agent for protecting skin from immune disorders.

Anti-inflammatory Effect of Predimenol, A Bioactive Extract from Phaleria macrocarpa, through the Suppression of NF-κB and COX-2.

Inflammation is the response to the reaction of any type of bodily injury by elevating cellular metabolism and releasing soluble mediators. It is also a contributing factor of pain. Predimenol, which has previously been known as DLBS1442, is a bioactive extract from Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae). It can be an alternative treatment for pain relief, especially for long-term use. The objective of this study is to evaluate the anti-inflammatory activities of predimenol through the evaluation of several parameters involved in the inflammatory pathway. Cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-  (TNF-), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), and nuclear factor B (NF-B) were observed after 24 h exposure of predimenol (0-180 µg/mL) to lipopolysaccharides (LPS)-activated RAW 264.7 cell. The inflammatory markers were measured using nitric oxide (NO) assay and enzyme-linked immunosorbent assay (ELISA) for COX-2 inhibitor assay. The gene expressions of TNF-α, IL-1β, IL-2 and IL-6 were quantified using the polymerase chain reaction (PCR) method. Western blotting was applied to detect phosphorylated IB kinase (IKK) protein to confirm the activation of NF-κB. Our study showed a similar mechanism with most non-steroidal anti-inflammatory drugs (NSAIDs). Predimenol consistently downregulated the expression of iNOS and inhibited COX-2 activity. Moreover, predimenol significantly inhibited the LPS-induced production of NO, TNF-α, IL-1β, IL-2 and IL-6. Down-regulation of these markers was suggested due to the reduction of NF-κB transcription level and activation by predimenol. Predimenol exhibits anti-inflammatory activities through NF-kB inactivation-mediated COX-2 suppression, which may suggest that predimenol is a potential analgesic and anti-inflammatory agent.

Slit guidance ligand 2 promotes the inflammatory response of periodontitis through activation of the NF-κB signaling pathway.

Periodontitis is a chronic multifactorial inflammatory disease associated with dental plaque biofilms. Slit guidance ligand 2 (SLIT2) has been shown to guide neuronal migration, regulate the inflammatory response and cancer progression. However, the role of SLIT2 in periodontitis is poorly understood. In this study, we investigated the expression of SLIT2 in the gingiva of periodontitis and its role in periodontitis progression. Gingiva and gingival crevicular fluid (GCF) were collected from healthy people and periodontitis patients. Immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) were used to analyze SLIT2secretion level. Healthy human gingival fibroblasts (hGFs) were isolated and the expression of SLIT2 in lipopolysaccharide (LPS)-treated hGFs was detected. The effect of SLIT2 on inflammation was analyzed using western blot and immunofluorescence. SLIT2knockdown (KD) and overexpression assays in hGFs were performed to investigate the role of SLIT2 in the LPS-induced inflammatory response. Gingival tissues and GCF of periodontitis patients displayed higher expression of SLIT2. Similarly, SLIT2 was upregulated in hGFs in an inflammatory environment (LPS treatment). In addition, SLIT2 treatment increased the expression of the inflammatory mediators interleukin-6 (IL-6) and IL-8 in hGFs. Mechanistically, SLIT2stimulated the activation of nuclear factor-κB (NF-κB) signaling, as well as LPS. Lastly, SLIT2KD impaired LPS-induced IL-6 production in hGFs, while SLIT2 overexpression amplified the inflammatory response. SLIT2may be involved in the aggravation of periodontitis by activating NF-κB signaling in hGFs.

AKR1B10, One of the Triggers of Cytokine Storm in SARS-CoV2 Severe Acute Respiratory Syndrome.

Preventing the cytokine storm observed in COVID-19 is a crucial goal for reducing the occurrence of severe acute respiratory failure and improving outcomes. Here, we identify Aldo-Keto Reductase 1B10 (AKR1B10) as a key enzyme involved in the expression of pro-inflammatory cytokines. The analysis of transcriptomic data from lung samples of patients who died from COVID-19 demonstrates an increased expression of the gene encoding AKR1B10. Measurements of the AKR1B10 protein in sera from hospitalised COVID-19 patients suggests a significant link between AKR1B10 levels and the severity of the disease. In macrophages and lung cells, the over-expression of AKR1B10 induces the expression of the pro-inflammatory cytokines Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and Tumor Necrosis Factor a (TNFα), supporting the biological plausibility of an AKR1B10 involvement in the COVID-19-related cytokine storm. When macrophages were stressed by lipopolysaccharides (LPS) exposure and treated by Zopolrestat, an AKR1B10 inhibitor, the LPS-induced production of IL-6, IL-1β, and TNFα is significantly reduced, reinforcing the hypothesis that the pro-inflammatory expression of cytokines is AKR1B10-dependant. Finally, we also show that AKR1B10 can be secreted and transferred via extracellular vesicles between different cell types, suggesting that this protein may also contribute to the multi-organ systemic impact of COVID-19. These experiments highlight a relationship between AKR1B10 production and severe forms of COVID-19. Our data indicate that AKR1B10 participates in the activation of cytokines production and suggest that modulation of AKR1B10 activity might be an actionable pharmacological target in COVID-19 management.

Amphiregulin induces interleukin-8 production and cell proliferation in lung epithelial cells through PI3K-Akt/ ERK pathways

Amphiregulin (AR), belongs to the epidermal growth factor (EGF) family, is able to induce a series of pathological and physiological responses by binding and activating epidermal growth factor receptor (EGFR). Interleukin-8 (IL-8) or CXCL8, a pro-inflammatory chemokine, has been suggested to be involved in tumor cell proliferation and inflammatory microenvironment via transactivation of the EGFR. However, whether there is a crosstalk between AR with IL-8 during inflammatory response remain to be fully understood. The current study was designed to investigate the possible mechanism of the interactions between AR and IL-8 production in human lung epithelial cells in vitro. Lung epithelial A549 cells were stimulated with lipopolysaccharide (LPS) to generate ALI model. LPS-induced AR and IL-8 production by A549 cells was measured by real-time PCR, Western Blot, and ELISA. The AR neutralizing antibody, PI3K specific inhibitor LY294002, JNK specific inhibitor SP60012, ERK specific inhibitor PD98089, and p38 inhibitor SB203580 were used to investigate the role of these signal pathways in LPS-induced cell proliferation, AR and IL-8 expression. LPS could induce AR through PI3K/Akt and ERK signal pathways. Furthermore, LPS induced AR promoted the production of IL-8 requires activation of EGFR, PI3K/Akt, and ERK signal pathways. The neutralizing antibody to AR prevented production of IL-8 induced by LPS. Treatment with Erlotinib, PI3K inhibitors, ERK inhibitor significantly inhibited AR-induced IL-8 production and cell proliferation. Our data indicate that a distinct role of EGFR–PI3K–Akt/ERK pathway as a bridge of interaction between AR and IL-8 production, as one of potential mechanisms to regulate inflammation and cell proliferation in human lung epithelial cells.

Abstract 15197: Lipopolysaccharide-Nuclear Factor-kappa B Pathway and Lipoprotein Apheresis Effects in Patients With Familial Hypercholesterolemia and Coronary Artery Disease

Introduction: Inflammation may play an important role in atherosclerosis in familial hypercholesterolemia (FH). Lipopolysaccharide (LPS)-nuclear factor-kappa B (NF-κB) pathway is a routine signal process activated in inflammatory status. Hypothesis: We assessed the hypothesis that LPS-NF-κB axis is markedly activated and lipoprotein apheresis has an inhibitory effect on this pathway in patients with FH and coronary artery disease (CAD). Methods: In this matched case-control study a genetically diagnosed FH cohort who presented stable CAD (n=63) was compared with 63 non-FH CAD and 63 non-FH non-CAD controls matched by sex and age. Plasma LPS levels and NF-κB activity were compared among the three groups. In addition, we studied in vitro LPS-induced interleukin-6 production by mononuclear cells from 16 FH cases without previous statin use and compared them with their respective matched control groups. Subsequently, these 16 FH patients underwent lipoprotein apheresis. Blood samples were taken immediately before and regularly after apheresis for measuring LPS and NF-κB. Results: FH plus CAD had higher LPS levels and NF-κB activity than CAD and non-CAD controls (all p <0.01). LPS-induced interleukin-6 production by mononuclear cells of FH plus CAD was also much higher compared with CAD and non-CAD controls (both p <0.01). Moreover, plasma LPS levels ( p <0.001) and NF-κB activity ( p <0.01) were dramatically reduced after apheresis in FH patients. Conclusions: In conclusion, genetically confirmed FH patients with CAD had a marked activation of LPS-NF-κB axis, while lipoprotein apheresis significantly attenuated this key inflammatory pathway, suggesting that inflammation may be an important therapeutic target for FH patients.

CD200R Is Involved in the Anti-inflammatory Effect of Dexmedetomidine in Lipopolysaccharide-Stimulated Microglia.

Dexmedetomidine has been confirmed to have anti-inflammatory effect in numerous diseases. However, the detailed mechanism is still unknown. We explore the hypothesis that CD200R is related to the anti-inflammatory effect of dexmedetomidine. Primary microglia were treated with different concentrations of dexmedetomidine followed with lipopolysaccharide (LPS) for 24h. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) were measured by enzyme-linked immunosorbent assay. Arginase 1 (Arg1), IL-6, and TNF-α were measured by TR-PCR. CD200R was observed by Western blot analysis. We found dexmedetomidine reduced LPS-induced IL-6 and TNF-α production and increase Arg1 in primary microglia. CD200R expression was downregulated by LPS and dexmedetomidine can attenuate the effect of LPS. Our results showed that CD200R may be related to the anti-inflammatory effect of dexmedetomidine.

Noncanonical STAT1 phosphorylation expands its transcriptional activity into promoting LPS-induced IL-6 and IL-12p40 production.

The lipopolysaccharide (LPS)-induced endocytosis of Toll-like receptor 4 (TLR4) is an essential step in the production of interferon-β (IFN-β), which activates the transcription of antiviral response genes by STAT1 phosphorylated at Tyr701 Here, we showed that STAT1 regulated proinflammatory cytokine production downstream of TLR4 endocytosis independently of IFN-β signaling and the key proinflammatory regulator NF-κB. In human macrophages, TLR4 endocytosis activated a noncanonical phosphorylation of STAT1 at Thr749, which subsequently promoted the production of interleukin-6 (IL-6) and IL-12p40 through distinct mechanisms. STAT1 phosphorylated at Thr749 activated the expression of the gene encoding ARID5A, which stabilizes IL6 mRNA. Moreover, STAT1 phosphorylated at Thr749 directly enhanced transcription of the gene encoding IL-12p40 (IL12B). Instead of affecting STAT1 nuclear translocation, phosphorylation of Thr749 facilitated the binding of STAT1 to a noncanonical DNA motif (5'-TTTGANNC-3') in the promoter regions of ARID5A and IL12B The endocytosis of TLR4 induced the formation of a complex between the kinases TBK1 and IKKβ, which mediated the phosphorylation of STAT1 at Thr749 Our data suggest that noncanonical phosphorylation in response to LPS confers STAT1 with distinct DNA binding and gene-regulatory properties that promote both IL12B expression and IL6 mRNA stabilization. Thus, our study provides a potential mechanism for how TLR4 endocytosis might regulate proinflammatory cytokine production.

7-methoxyflavanone alleviates neuroinflammation in lipopolysaccharide-stimulated microglial cells by inhibiting TLR4/MyD88/MAPK signalling and activating the Nrf2/NQO-1 pathway.

Neuroprotective potential of 7-methoxyflavanone (7MF) and its underlying mechanism was investigated. Inhibitory effects of 7MF on microglial activation and neuroinflammation were evaluated by employment of lipopolysaccharide (LPS)-induced BV2 microglial cells. Changes in expression of genes and proteins of interest were investigated by RT-qPCR analysis and Western blot analysis. Inhibitory effects of 7MF on microglial overactivation were verified in LPS-treated C57BL/6J mice using ionized calcium-binding adaptor molecule-1 (Iba1) in the brain and interleukin-6 (IL-6) in serum as indicators. In BV2 cells, pretreatment with 7MF antagonized LPS-induced production of inflammatory factors IL-6, tumour necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1). Mechanistic studies revealed reduced expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor-88 (MyD88), phosphorylated forms of c-Jun N-terminal kinase (p-JNK) and extracellular signal-regulated kinases 1/2 (p-ERK) but increased nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and cellular expression of NAD(P)H quinone dehydrogenase-1 (NQO-1) by 7MF. In LPS-treated mice, pretreatment with 7MF reduced the brain level of Iba1 and serum level of IL-6. 7-methoxyflavanone inhibited LPS-stimulated TLR4/MyD88/MAPK signalling and activated Nrf2-mediated transcription of antioxidant protein NQO-1, showing antineuroinflammatory effect, so it is a potential neuroprotective agent.

Mast Cell Degranulation Decreases Lipopolysaccharide-Induced Aortic Gene Expression and Systemic Levels of Interleukin-6 In Vivo.

Mast cells play an important role in immunomodulation and in the maintenance of vascular integrity. Interleukin-6 (IL-6) is one of the key biomarkers and therapeutic target in systemic vasculitis. The objective of the current study is to describe the role of mast cells in arterial IL-6 homeostasis. Eight- to ten-week-old male C57BL/6 (wild-type) mice were injected with either (a) saline, (b) compound 48/80 (a systemic mast cell degranulating agent), (c) lipopolysaccharide (LPS), or (d) a combination of C48/80 and LPS. Twenty-four hours after the injections, mice were sacrificed and serum samples and aortic tissues were analyzed for determining inflammatory response and cytokine expression profile. The results revealed that induction of mast cell degranulation significantly lowers serum IL-6 levels and aortic expression of IL-6 in LPS-treated mice. Significantly higher aortic expression of toll-like receptor-2 (TLR-2) and TNF-α was seen in the LPS and LPS+C48/80 groups of mice compared to controls. Aortic expression of TLR-4 was significantly decreased in LPS+C48/80 compared to C48/80 alone. LPS+C48/80-treated mice presented with a 3-fold higher aortic expression of suppressor of cytokine signaling (SOCS-1) compared to saline-injected groups. The inhibition of LPS-induced increase in serum IL-6 levels by mast cell degranulation was not seen in H1R knockout mice which suggests that mast cell-derived histamine acting through H1R may participate in the regulatory process. To examine whether the mast cell-mediated downregulation of LPS-induced IL-6 production is transient or cumulative in nature, wild-type mice were injected serially over a period of 10 days (5 injections) and serum cytokine levels were quantified. We found no significant differences in serum IL-6 levels between any of the groups. While mice injected with C48/80 or LPS had higher IL-10 compared to vehicle-injected mice, there was no difference between C48/80- and LPS+C48/80-injected mice. In conclusion, in an in vivo setting, mast cells appear to partially and transiently regulate systemic IL-6 homeostasis. This effect may be regulated through increased systemic IL-10 and/or aortic overexpression of SOCS-1.

Circulating versus lipopolysaccharide-induced inflammatory markers as correlates of subthreshold depressive symptoms in older adults

Objectives: Circulating cytokines have been associated with depression, but their detection has limitations, which may be overcome by direct detection of intracellular cytokines (ICCs) after lipopolysaccharide (LPS) stimulation in vitro. This study compared circulating versus LPS-induced inflammatory markers as correlates of subthreshold depressive symptoms.Methods: Secondary data analysis of a cross-sectional insomnia study in healthy community-dwelling older adults was conducted. In 117 participants (≥55 years), plasma tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP) and in vitro LPS-induced monocyte production of IL-6 and TNF-α were assayed. Depressive symptoms were assessed using the clinician-rated Inventory of Depressive Symptomatology (IDS-C). Multivariate linear regression was conducted to test the associations between inflammatory markers and subthreshold depressive symptoms in the entire sample as well as in subgroups stratified into higher and lower inflammation levels.Results: LPS-induced TNF-α (adjusted β = 0.28, p = .04), IL-6 (0.29, p = .03) and TNF-α + IL-6 (0.43, p = .001) significantly positively correlated with subthreshold depressive symptoms only in higher inflammation subgroups. No circulating biomarkers positively correlated in any subgroups. In the entire sample, no biomarkers were significantly associated with subthreshold depressive symptoms.Conclusions: LPS-induced cytokines may be more sensitive correlates of subthreshold depressive symptoms than circulating cytokines, particularly in older adults with higher systemic inflammation.Clinical Trials Registry: ClinicalTrials.gov NCT00280020.

P650Lipopolysaccharide-nuclear factor-kappa B pathway and lipoprotein apheresis effects in patients with familial hypercholesterolemia and coronary artery disease

Abstract Abstract Background Inflammation may play an important role in atherosclerosis in familial hypercholesterolemia (FH). Lipopolysaccharide (LPS)-nuclear factor-kappa B (NF-κB) pathway is a routine signal process activated in inflammatory status. Purpose This study aimed to examine the LPS-NF-κB axis status and the impact of lipoprotein apheresis (LA) on this pathway in patients with FH and coronary artery disease (CAD). Methods In this matched case-control study a genetically diagnosed FH cohort who presented stable CAD (n=63) was compared with 63 non-FH CAD and 63 non-FH non-CAD controls matched by sex and age. Plasma LPS levels and NF-κB activity were compared among the three groups. In addition, we studied in vitro LPS-induced interleukin-6 (IL-6) production by mononuclear cells from 16 FH cases without previous statin use and compared them with their respective matched control groups. Subsequently, these 16 FH patients underwent LA. Blood samples were taken immediately before and regularly after LA for measuring LPS and NF-κB. Results FH plus CAD had higher LPS levels and NF-κB activity than CAD and non-CAD controls (all p values <0.01). LPS-induced IL-6 production by mononuclear cells of FH plus CAD was also much higher compared with CAD and non-CAD controls (both p values <0.01). Moreover, plasma LPS levels (p<0.001) and NF-κB activity (p<0.01) were dramatically reduced after apheresis in FH patients. Conclusion Genetically confirmed FH patients with CAD had a marked activation of LPS-NF-κB axis, while LA significantly attenuated this key inflammatory pathway, suggesting that inflammation may be an important therapeutic target for FH patients.

Quercetin Inhibits Inflammatory Response Induced by LPS from Porphyromonas gingivalis in Human Gingival Fibroblasts via Suppressing NF-κB Signaling Pathway.

Quercetin, a natural flavonol existing in many food resources, has been reported to be an effective antimicrobial and anti-inflammatory agent for restricting the inflammation in periodontitis. In this study, we aimed to investigate the anti-inflammatory effects of quercetin on Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide- (LPS-) stimulated human gingival fibroblasts (HGFs). HGFs were pretreated with quercetin prior to LPS stimulation. Cell viability was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), along with chemokine interleukin-8 (IL-8), were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA levels of IL-1β, IL-6, IL-8, TNF-α, IκBα, p65 subunit of nuclear factor-kappa B (NF-κB), peroxisome proliferator-activated receptor-γ (PPAR-γ), liver X receptor α (LXRα), and Toll-like receptor 4 (TLR4) were measured by real-time quantitative PCR (RT-qPCR). The protein levels of IκBα, p-IκBα, p65, p-p65, PPAR-γ, LXRα, and TLR4 were characterized by Western blotting. Our results demonstrated that quercetin inhibited the LPS-induced production of IL-1β, IL-6, IL-8, and TNF-α in a dose-dependent manner. It also suppressed LPS-induced NF-κB activation mediated by TLR4. Moreover, the anti-inflammatory effects of quercetin were reversed by the PPAR-γ antagonist of GW9662. In conclusion, these results suggested that quercetin attenuated the production of IL-1β, IL-6, IL-8, and TNF-α in P. gingivalis LPS-treated HGFs by activating PPAR-γ which subsequently suppressed the activation of NF-κB.

Lipopolysaccharide-Nuclear Factor-Kappa B Pathway and Lipoprotein Apheresis Effects in Patients with Familial Hypercholesterolemia and Coronary Artery Disease

Background: Inflammation may play an important role in atherosclerosis in familial hypercholesterolemia (FH). Lipopolysaccharide (LPS)-nuclear factor-kappa B (NF-κB) pathway is a routine signal process activated in inflammatory status. However, the LPS-NF-κB axis status and the impact of lipoprotein apheresis (LA) on this pathway in patients with FH and coronary artery disease (CAD) has not been clarified. Methods: A genetically diagnosed FH cohort who presented stable CAD (n=63) was compared with 63 non-FH CAD and 63 non-FH non-CAD controls matched by sex and age. Plasma LPS levels and NF-κB activity were compared among them. In vitro LPS-induced interleukin-6 (IL-6) production by mononuclear cells from 16 FH cases without previous statin use was measured and compared with their respective matched control groups. Subsequently, these 16 FH patients underwent LA. Blood samples were regularly taken for measuring LPS and NF-κB. Findings: FH plus CAD had higher LPS levels and NF-κB activity than CAD and non-CAD controls (all p<0.01). LPS-induced IL-6 production by mononuclear cells of FH plus CAD was also much higher compared with control groups (both p<0.01). Moreover, plasma LPS levels (p<0.001) and NF-κB activity (p<0.01) were dramatically reduced after apheresis in FH patients. Interpretation: Genetically confirmed FH patients with CAD had a marked activation of LPS-NF-κB axis, while LA significantly attenuated this key inflammatory pathway, suggesting that inflammation may be an important therapeutic target for FH patients. Funding Statement: This work was partially supported by the Capital Health Development Fund [201614035] and CAMS Major Collaborative Innovation Project [2016-I2M-1-011] awarded to Dr. J.J.L. Declaration of Interests: R.D.S. has received honoraria related to consulting, speaker and/or research activities from Amgen, Akcea, Astra Zeneca, Biolab, Esperion, Kowa, Novo-Nordisk, Merck and Sanofi/Regeneron. Ethics Approval Statement: The study complied with the Declaration of Helsinki and Title 45, US Code of Federal Regulations, Part 46, Protection of Human Subjects, Revised November 13, 2001, effective December 13, 2001, and was approved by the hospital’s ethical review board (Fu Wai Hospital & National Center for Cardiovascular Diseases, Beijing, China). Each participant provided written, informed consent before enrollment.

Sensitivity to glucocorticosteroids and heterogeneity of in vitro cell response in patients with chronic obstructive pulmonary disease

Inhaled corticosteroids are widely used for the treatment of chronic obstructive pulmonary disease (COPD), but their efficacy significantly varies between patients. The aim of the study was to establish approaches to reveal steroid-sensitive and steroid-resistant patients with COPD using the blood and lung cells. Methods. Forty five patients with COPD undergoing bronchoscopy were recruited for the study of cytokine secretion by alveolar macrophages under the influence of glucocorticoids. Alveolar macrophages isolated from bronchoalveolar lavage fluid were cultured with lipopolysaccharide (LPS) and different concentrations of dexamethasone (0.01 – 1000 nM) for 24 h. Then, supernatants were removed and analyzed for concentrations of interleukin 6 (IL-6), IL-8 and tumor necrosis factor α (TNF-α). Binding of the glucocorticoid with its receptors was investigated in 24 patients with COPD, 20 healthy smokers and 20 healthy non-smokers. Blood cells were cultured with fluorescein isothiocyanate (FITC)-labelled dexamethasone and monoclonal antibodies against surface antigens of lymphocyte and monocyte populations. Fluorescence intensity of FITC-labelled dexamethasone was analyzed in blood cells using flow cytometry. Results. Dexamethasone significantly inhibited IL-6, IL-8, and TNF-α production in alveolar macrophages in a dose dependent manner. The maximal inhibition of cytokine production was observed at dexamethasone concentration of 100 nM, and the maximal cell response variability was found at 10 nM. IL-8 was less sensitive to the corticosteroid compared to IL-6 and TNF-α. Dexamethasone at any concentration failed to reach &gt;50% inhibition of LPS-induced production of IL-8, IL-6 and TNF-α in alveolar macrophages of 40.0%; 11.1% and 8.9% of COPD patients, respectively. The fluorescence intensity of FITC-labelled dexamethasone in blood lymphocytes and monocytes was lower in smokers with COPD compared to healthy smokers and healthy non-smokers. The binding of dexamethasone with its receptors in the blood cells was higher in healthy non-smokers compared to healthy smokers. Conclusion. In vitro response of alveolar macrophages to glucocorticoids in COPD patients is characterized by significant inter-individual variability. The weak corticosteroid-related inhibition of IL-8 production can contribute to neutrophilic inflammation in COPD. The capacity of glucocorticoid receptors to bind with their ligands in blood lymphocytes and monocytes is decreased in COPD patients.

Primary Cultures from Rat Dorsal Root Ganglia: Responses of Neurons and Glial Cells to Somatosensory or Inflammatory Stimulation

Primary cultures of rat dorsal root ganglia (DRG) consist of neurons, satellite glial cells and a moderate number of macrophages. Measurements of increased intracellular calcium [Ca2+]i induced by stimuli, have revealed that about 70% of DRG neurons are capsaicin-responsive nociceptors, while 10% responded to cooling and or menthol (putative cold sensors). Cultivation of DRG in the presence of a moderate dose of lipopolysaccharide (LPS, 1 µg/ml) enhanced capsaicin-induced Ca2+ signals. We therefore investigated further properties of DRG primary cultures stimulated with 10 µg/ml LPS for a short period. Exposure to LPS for 2 h resulted in pronounced release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) into the supernatants of DRG cultures, increased expression of both cytokines in the DRG cells and increased TNF immunoreactivity predominantly in macrophages. We further observed an accumulation of the inflammatory transcription factors NF-IL6 and STAT3 in the nuclei of LPS-exposed DRG neurons and macrophages. In the presence of the cytotoxic agent cisplatin (5 or 10 µg/ml), the number of macrophages was decreased significantly, the growth of satellite glial cells was markedly suppressed, but the vitality and stimulus-induced Ca2+ signals of DRG neurons were not impaired. Under these conditions the LPS-induced production and expression of TNF-α and IL-6 were blunted. Our data suggest a potential role for macrophages and satellite glial cells in the initiation of inflammatory processes that develop in sensory ganglia upon injury or exposure to pathogens.

Lipopolysaccharide-induced CCN1 production enhances interleukin-6 secretion in bronchial epithelial cells

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a clinical complication caused by primary or secondary lung injury, as well as by systemic inflammation. Researches regarding molecular pathophysiology of ALI/ARDS are immerging with an ultimate aim towards developing prognostic molecular biomarkers and molecule-based therapy. However, the molecular mechanisms concerning ALI/ARDS are still not completely understood. The purpose of the present study was to identify a crucial role of CCN1 in inflammatory microenvironment during ALI/ARDS and focus on a potential communication between CCN1 and interleukin-6 (IL-6) in the airway epithelial cells. Our data illustrated that the expression levels of CCN1 and IL-6 in bronchoalveolar lavage fluid (BALF) in a lipopolysaccharide (LPS)-induced ALI mouse model were significantly elevated and the pulmonary expression of CCN1 was restricted to bronchial epithelial cells. Interestingly, both endogenous and exogenous CCN1 stimulated IL-6 production in vitro. Furthermore, LPS-induced IL-6 production in a bronchial epithelial cell line was blocked by CCN siRNA whereas CCN1 induced by LPS was sensitive to PI3K inhibition. Together, our data indicate a linear signal pathway, LPS-CCN1-IL-6, existing in bronchial epithelial cells after LPS exposure. This finding may represent an additional mechanism and a novel target for development of therapy and biomarker on ALI/ARDS.

P32 is Required for Appropriate Interleukin-6 Production Upon LPS Stimulation and Protects Mice from Endotoxin Shock

Sepsis is a major cause of morbidity and mortality in seriously ill patients and mitochondrial dysfunction is associated with poor outcomes in septic patients. Although interleukin-6 (IL-6) is a good prognostic marker for sepsis, the relationship between mitochondrial dysfunction and IL-6 remains poorly understood. We identified p32/C1QBP/HABP1 as a regulator of IL-6 production in response to lipopolysaccharide (LPS). LPS induced IL-6 overproduction in p32 deficient mouse embryonic fibroblasts (MEFs) through NF-κB independent but activating transcription factor (ATF) 4 dependent pathways. Short hairpin RNA-based knockdown of ATF4 in p32 deficient MEFs markedly inhibited LPS-induced IL-6 production. Furthermore, MEFs treated with chloramphenicol, an inhibitor of mitochondrial translation, produced excessive IL-6 via ATF4 pathways. Using a LPS-induced endotoxin shock model, mice with p32 ablation in myeloid cells showed increased lethality and overproduction of IL-6. Thus, this study provides a molecular link how mitochondrial dysfunction leads to IL-6 overproduction and poor prognosis of sepsis.