Quercetin triggers apoptosis of lipopolysaccharide (LPS)-induced osteoclasts and inhibits bone resorption in RAW264.7 cells.
ABSTRACT Quercetin, a flavonoid present in vegetables, has anti-inflammatory properties and potential inhibitory effects on bone resorption. Up to date, the effect of quercetin on lipopolysaccharide (LPS)-induced osteoclastogenesis has not yet been reported. In the current study, we evaluated the effect of quercetin on LPS-induced osteoclast apoptosis and bone resorption.
RAW264.7 cells were non-treated, treated with LPS alone, or treated with both LPS and quercetin. After treatment, the number of osteoclasts, cell viability, bone resorption and osteoclast apoptosis were measured. The expressions of osteoclast-related genes including tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP9) and cathepsin K (CK) were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of receptor activator of nuclear factor-ĸB (RANK), tumor necrosis factor receptor-associated factor 6 (TRAF6), cyclooxygenase-2 (COX-2), Bax, Bcl-2 and mitogenactivated protein kinases (MAPKs) were measured using Western blotting assays. The MAPK signaling pathway was blocked by pretreatment with MAPK inhibitors.
LPS directly promoted osteoclast differentiation of RAW264.7 cells and upregulated the protein expression of RANK, TRAF6 and COX-2; while quercetin significantly decreased the number of LPS-induced osteoclasts in a dose-dependent manner. None of the treatments increased cytotoxicity in RAW264.7 cells. Quercetin inhibited mRNA expressions of osteoclast-related genes and protein levels of RANK, TRAF6 and COX-2 in LPS-induced mature osteoclasts. Quercetin also induced apoptosis and inhibited bone resorptive activity in LPS-induced mature osteoclasts. Furthermore, quercetin promoted the apoptotic signaling pathway including increasing the phosphorylation of p38-MAPK, c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK), and Bax, while inhibited Bcl-2 expression.
Quercetin could supress LPS-induced osteoclast bone resorption through blocking RANK signaling and inhibiting the expression of osteoclast-related genes. Quercetin also promoted LPS-induced osteoclast apoptosis via activation of the MAPK apoptotic signaling pathway. These findings suggest that quercetin could be of potential use as a therapeutic agent to treat bacteria-induced bone resorption.
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ABSTRACT: Apoptosis is closely associated with the occurrence and development of cardiovascular diseases and is considered as one of the crucial pathological processes of cardiomyopathy, sepsis, ischemia/reperfusion injury, myocardial infarction and heart failure. Hesperetin (HES), a flavanone glycoside found in citrus fruit peels, has been known to exhibit several key biological and pharmacological properties. Previous studies have demonstrated the anti-inflammatory, anti-oxidant and anti-tumor functions of HES. However, with regards to the pro- or anti-apoptotic functions of HES, there are several disagreements within the literature. To examine whether HES has protective effects in cardiac apoptosis, the present study examined the role of HES in lipopolysaccharide (LPS)-stimulated H9C2 cardiomyocytes, aiming to clarify the possible mechanisms underlying its effects. In the present study, HES reduced the percentage of viable apoptotic (VA) cells in a flow cytometry analysis. It had an anti-apoptosis function in LPS-stimulated H9C2 cells. To clarify whether HES alleviated LPS-stimulated apoptosis through the mitochondria-dependent intrinsic apoptotic pathway, certain indicators of this pathway were detected, including members of the caspase family. The data revealed that HES attenuated the activation of capase-3 and caspase-9. These results indicated HES has a mitochondria-dependent anti-apoptosis effect in LPS-stimulated H9C2 cells. To explore the possible mechanisms, the protein expression levels of certain markers in the possible signaling pathway were detected, including JNK and Bcl-2 family. As a result, HES downregulated the protein expression of Bax, upregulated the expression of Bcl-2 and attenuated the phosphorylation level of JNK. Therefore, the anti-apoptosis effects of HES were possibly mediated by the JNK/Bax signaling pathway. In conclusion, HES has a mitochondria-dependent anti-apoptosis effect in LPS-induced H9C2 cells via the JNK/Bax signaling pathway.Molecular Medicine Reports 03/2014; · 1.48 Impact Factor
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ABSTRACT: Background/Aims: Platelets are critically important for primary haemostasis and the major players in thrombotic vascular occlusion. Platelets are activated by agonists, such as thrombin and collagen-related peptide as well as second-wave mediators including thromboxane A2 via different intracellular signaling pathways resulting in degranulation, aggregation and thrombus formation. Platelet activation is paralleled by phosphorylation and activation of p38 MAPK. The limited specificity of hitherto known p38 MAPK inhibitors precluded safe conclusions on the precise role of p38 MAPK in the regulation of platelet function. The present study examined the impact of Skepinone-L, a novel and highly selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), on platelet activation and thrombus formation. Methods: Experiments were performed in freshly isolated human platelets. Protein phosphorylation was quantified by Western blotting, thromboxane B2 synthesis by enzyme immunoassay, ATP release by ChronoLume luciferin assay, cytosolic Ca(2+) concentration by Fura-2 fluorescence-measurements, platelet aggregation by a light transmissions measurement and in vitro thrombus formation by a flow chamber. Results: Skepinone-L (1 μM) virtually abrogated the phosphorylation of platelet p38 MAPK substrate Hsp27 following stimulation with CRP (1 μg/ml), thrombin (5 mU/ml) or thromboxane A2 analogue U-46619 (1 μM). Furthermore, Skepinone-L significantly blunted activation-dependent platelet secretion and aggregation following threshold concentrations of CRP, thrombin and thromboxane A2 analogue U-46619. Skepinone-L did not impair platelet Ca(2+) signaling but prevented agonist-induced thromboxane A2 synthesis through abrogation of p38 MAPK-dependent phosphorylation of platelet cytosolic phospholipase A2 (cPLA2). Skepinone-L further markedly blunted thrombus formation under low (500-s) and high (1700-s) arterial shear rates. Conclusions: The present study discloses a powerful inhibiting effect of p38 MAPK-blocker Skepinone-L on platelet activation and thrombus formation.Cellular Physiology and Biochemistry 06/2013; 31(6):914-924. · 3.55 Impact Factor
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ABSTRACT: Background/Aims: This study was conducted to determine the potential of Ganoderma lucidum polysaccharides (Gl-PS) in protection against lung cancer patient plasma-induced suppression of lymphocytes. Lung cancer is a major cause of disease and loss of life in the United States and worldwide. Cancer cells release immunosuppressive mediators, such as PGE2, TGF-β, IL-10, and VEGF, to inhibit the immune response to escape from immune surveillance. Gl-PS has been shown to counteract this immune inhibition in an animal cell culture model, and thus to facilitate tumor control. The present study explored whether or not such an effect could also be demonstrated in human lung cancer patients. Methods: Immunofluorescence, flow cytometry, MTT, immunocytochemistry, and western blot analysis were used to assess lymphocyte activation with PHA. Results: The plasma of lung cancer patients suppressed proliferation, CD69 expression, and perforin and granzyme B production in lymphocytes upon activation by PHA, effects that were partially of fully reversed by Gl-PS. Conclusion: Lung cancer patient plasma-induced suppression of lymphocyte activation by phytohemagglutinin may be antagonized fully or partially by Gl-PS, an observation suggesting the potential of Gl-PS in cancer therapy. © 2014 S. Karger AG, Basel.Cellular Physiology and Biochemistry 01/2014; 33(2):289-299. · 3.55 Impact Factor