Down‐regulation of mitogen‐activated protein kinases and nuclear factor‐κB signaling is involved in rapamycin suppression of TLR2‐induced inflammatory response in monocytic THP‐1 cells

Tripalmitoyl‐S‐glycero‐Cys‐(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen‐activated protein kinases (MAPKs) and nuclear factor‐κB (NF‐κB) signal pathway. Rapamycin can suppress TLR‐induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2‐induced inflammatory responses was investigated. It was found that Pam3CSK4‐induced pro‐inflammatory cytokines were significantly down‐regulated at both the mRNA and protein levels in THP‐1 cells pre‐treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3‐kinase/protein kinase‐B (PI3K/AKT) signaling did not suppress the expression of pro‐inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT‐PCR showed that Erk and NF‐κB signal pathways are related to the production of pro‐inflammatory cytokines. Inhibition of Erk or NF‐κB signaling significantly down‐regulated production of pro‐inflammatory cytokines. Additionally, western blot showed that pre‐treatment of THP‐1 cells with rapamycin down‐regulates MAPKs and NF‐κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4‐induced pro‐inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2‐induced inflammatory responses by down‐regulation of Erk and NF‐κB signaling.     

β‐carotene suppresses Porphyromonas gingivalis lipopolysaccharide‐mediated cytokine production in THP‐1 monocytes cultured with high glucose condition

Periodontitis is associated with development of diabetes mellitus. Although lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg), a major pathogen of periodontitis, may lead the progression of diabetes complications, the precise mechanisms are unclear. We, therefore, investigated the effects of β‐carotene on production of Pg LPS‐induced inflammatory cytokines in human monocytes cultured high glucose (HG) condition. THP‐1 cells were cultured under 5.5 mM or 25 mM glucose conditions, and cells were stimulated with Pg LPS. To investigate the productivity of TNF‐α, IL‐6, and MCP‐1, cell supernatants were collected for ELISA. To examine the effects of NF‐kB signals on cytokine production, Bay11‐7082 was used. HG enhanced Pg LPS‐induced production of TNF‐α, IL‐6, and MCP‐1 via NF‐kB signals in THP‐1. β‐carotene suppressed the enhancement of the Pg LPS‐induced cytokine production in THP‐1 via NF‐κB inactivation. Our results suggest that β‐carotene might be a potential anti‐inflammatory nutrient for circulating Pg LPS‐mediated cytokine production in diabetic patients with periodontitis.     

Sodium salicylate modulates inflammatory responses through AMP‐activated protein kinase activation in LPS‐stimulated THP‐1 cells

Sodium salicylate (NaSal) is a nonsteroidal anti‐inflammatory drug. The putative mechanisms for NaSal's pharmacologic actions include the inhibition of cyclooxygenases, platelet‐derived thromboxane A2, and NF‐κB signaling. Recent studies demonstrated that salicylate could activate AMP‐activated protein kinase (AMPK), an energy sensor that maintains the balance between ATP production and consumption. The anti‐inflammatory action of AMPK has been reported to be mediated by promoting mitochondrial biogenesis and fatty acid oxidation. However, the exact signals responsible for salicylate‐mediated inflammation through AMPK are not well‐understood. In the current study, we examined the potential effects of NaSal on inflammation‐like responses of THP‐1 monocytes to lipopolysaccharide (LPS) challenge. THP‐1 cells were stimulated with or without 10 ug/mL LPS for 24 h in the presence or absence of 5 mM NaSal. Apoptosis was measured by flow cytometry using Annexin V/PI staining and by Western blotting for the Bcl‐2 anti‐apoptotic protein. Cell proliferation was detected by EdU incorporation and by Western blot analysis for proliferating cell nuclear antigen (PCNA). Secretion of pro‐inflammatory cytokines (TNF‐α, IL‐1β, IL‐6) was determined by enzyme‐linked immunosorbent assay (ELISA). We observed that the activation of AMPK by NaSal was accompanied by induction of apoptosis, inhibition of cell proliferation, and increasing secretion of TNF‐α and IL‐1β. These effects were reversed by Compound C, an inhibitor of AMPK. In addition, NaSal/AMPK activation inhibited LPS‐induced STAT3 phosphorylation, which was reversed by Compound C treatment. We conclude that AMPK activation is important for NaSal‐mediated inflammation by inducing apoptosis, reducing cell proliferation, inhibiting STAT3 activity, and producing TNF‐α and IL‐1β.     

Valsartan ameliorates ageing‐induced aorta degeneration via angiotensin II type 1 receptor‐mediated ERK activity

Angiotensin II (Ang II) plays important roles in ageing‐related disorders through its type 1 receptor (AT₁R). However, the role and underlying mechanisms of AT1R in ageing‐related vascular degeneration are not well understood. In this study, 40 ageing rats were randomly divided into two groups: ageing group which received no treatment (ageing control), and valsartan group which took valsartan (selective AT1R blocker) daily for 6 months. 20 young rats were used as adult control. The aorta structure were analysed by histological staining and electron microscopy. Bcl‐2/Bax expression in aorta was analysed by immunohistochemical staining, RT‐PCR and Western blotting. The expressions of AT₁R, AT₂R and mitogen‐activated protein kinases (MAPKs) were detected. Significant structural degeneration of aorta in the ageing rats was observed, and the degeneration was remarkably ameliorated by long‐term administration of valsartan. With ageing, the expression of AT1R was elevated, the ratio of Bcl‐2/Bax was decreased and meanwhile, an important subgroup of MAPKs, extracellular signal‐regulated kinase (ERK) activity was elevated. However, these changes in ageing rats could be reversed to some extent by valsartan. In vitro experiments observed consistent results as in vivo study. Furthermore, ERK inhibitor could also acquire partial effects as valsartan without affecting AT1R expression. The results indicated that AT1R involved in the ageing‐related degeneration of aorta and AT1R‐mediated ERK activity was an important mechanism underlying the process.     

Down‐regulation of PKM2 enhances anticancer efficiency of THP on bladder cancer

Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels that are correlated with the sensitivity of anticancer chemotherapeutic drugs. THP is one of the major drugs used in non‐muscle‐invasive bladder cancer instillation chemotherapy. However, low response ratio of THP (19.7%) treatment to human genitourinary tumours using collagen gel matrix has been observed. This study aims to investigate the effect of down‐regulation of PKM2 on THP efficiency. Via inhibitor or siRNA, the effects of reduced PKM2 on the efficiency of THP were determined in 2 human and 1 murine bladder cancer cell lines, using MTT, cologenic and fluorescence approaches. Molecular mechanisms of PKM2 on THP sensitization were explored by probing p‐AMPK and p‐STAT3 levels via WB. Syngeneic orthotopic bladder tumour model was applied to evaluate this efficiency in vivo, analysed by Kaplan‐Meier survival curves, body and bladder weights plus immunohistochemistric tumour biomarkers. PKM2 was overexpressed in bladder cancer cells and tissues, and down‐regulation of PKM2 enhanced the sensitivity of THP in vitro. Activation of AMPK is essential for THP to exert anti‐bladder cancer activities. On the other hand, down‐regulating PKM2 activates AMPK and inhibits STAT3, correlated with THP sensitivity. Compared with THP alone (400 μmol L^?1, 50 μL), the combination with metformin (60 mmol L^?1, 50 μL) stopped growth of bladder cancer completely in vivo (combination group VS normal group P = .078). Down‐regulating the expression of PKM2 enhances the anticancer efficiency of THP. This study provides a new insight for improving the chemotherapeutic effect of THP.     

Essential involvement of cross‐talk between IFN‐γ and TNF‐α in CXCL10 production in human THP‐1 monocytes

Interferon (IFN)‐γ‐induced protein 10 (IP‐10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN‐γ depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN‐γ and TNF‐α have not been adequately elucidated in human monocytes. In this study, we showed that TNF‐α had more potential than IFN‐γ to induce CXCL10 production in THP‐1 monocytes. Furthermore, IFN‐γ synergistically enhanced the production of CXCL10 in parallel with the activation of NF‐κB in TNF‐α‐stimulated THP‐1 cells. Blockage of STAT1 or NF‐κB suppressed CXCL10 production. JAKs inhibitors suppressed IFN‐γ plus TNF‐α‐induced production of CXCL10 in parallel with activation of STAT1 and NF‐κB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF‐κB, but not that of STAT1. IFN‐γ‐induced phosphorylation of JAK1 and JAK2, whereas TNF‐α induced phosphorylation of ERK1/2. Interestingly, IFN‐γ alone had no effect on phosphorylation and degradation of IκB‐α, whereas it significantly promoted TNF‐α‐induced phosphorylation and degradation of IκB‐α. These results suggest that TNF‐α induces CXCL10 production by activating NF‐κB through ERK and that IFN‐γ induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF‐α‐induced NF‐κB may be the primary pathway contributing to CXCL10 production in THP‐1 cells. IFN‐γ potentiates TNF‐α‐induced CXCL10 production in THP‐1 cells by increasing the activation of STAT1 and NF‐κB through JAK1 and JAK2. J. Cell. Physiol. 220: 690–697, 2009. © 2009 Wiley‐Liss, Inc.     

S‐allyl cysteine,an active ingredient of garlic,attenuates acute liver dysfunction induced by lipopolysaccharide/ d‐galactosamine in mouse: Underlying mechanisms

In the present study, beneficial effect of S‐allyl cysteine (SAC) was evaluated in the lipopolysaccharide/d ‐galactosamine (LPS/d ‐Gal) model of acute liver injury (ALI). To mimic ALI, LPS and d ‐Gal (50 μg/kg and 400 mg/kg, respectively) were intraperitoneally administered and animals received SAC per os (25 or 100 mg/kg/d) for 3 days till 1 hour before LPS/d ‐Gal injection. Pretreatment of LPS/d ‐Gal group with SAC‐lowered activities of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase and partially reversed inappropriate alterations of hepatic oxidative stress‐ and inflammation‐related biomarkers including liver reactive oxygen species, malondialdehyde, and hepatic activity of the defensive enzyme superoxide dismutase, ferric reducing antioxidant power (FRAP), toll‐like receptor‐4 (TLR4), cyclooxygenase 2, NLR family pyrin domain containing 3 (NLRP3), caspase 1, nuclear factor κB (NF‐κB), interleukin 1β (IL‐1β), IL‐6, tumor necrosis factor‐α, and myeloperoxidase activity. Additionally, SAC was capable to ameliorate apoptotic biomarkers including caspase 3 and DNA fragmentation. In summary, SAC can protect liver against LPS/d ‐Gal by attenuation of neutrophil infiltration, oxidative stress, inflammation, apoptosis, and pyroptosis which is partly linked to its suppression of TLR4/NF‐κB/NLRP3 signaling.     

TLR4‐dependent induction of vascular adhesion molecule‐1 in rheumatoid arthritis synovial fibroblasts: Roles of cytosolic phospholipase A2α/cyclooxygenase‐2

Lipopolysaccharide (LPS)/Toll‐like receptor 4 (TLR4)‐mediated signaling pathways have caught the attention of strategies designed for rheumatoid arthritis (RA). In this study, we identified that cPLA₂α acted as a modulator of LPS‐induced VCAM‐1 expression and THP‐1 (human acute monocytic leukemia cell line) adherence. Treatment of RA synovial fibroblasts (RASFs) with LPS, a TLR4 agonist, promoted the VCAM‐1 expression and THP‐1 adherence which were decreased by pretreatment with a selective cytosolic phospholipase A₂ (cPLA₂) inhibitor (AACOCF₃), implying the involvement of cPLA₂α in these responses. This notion was further confirmed by knockdown of cPLA₂α expression by transfection with cPLA₂α small interfering RNA (siRNA) leading to a decrease in VCAM‐1 expression and THP‐1 adherence induced by LPS. Subsequently, the LPS‐stimulated cPLA₂α phosphorylation was attenuated by pretreatment with a MEK1/2 inhibitor (U0126), suggesting that LPS‐stimulated cPLA₂α phosphorylation and activity are mediated through an ERK‐dependent mechanism. Moreover, COX‐2‐derived PGE₂ production appeared to involve in LPS‐induced VCAM‐1 expression which was attenuated by pretreatment with selective COX‐2 inhibitors (NS‐398 and celecoxib), transfection with COX‐2 siRNA, or PGE₂ receptor antagonists. In addition, pretreatment with ecosapentaenoic acid (EPA), a substrate competitor of arachidonic acid (AA), also blocked LPS‐induced VCAM‐1 mRNA and protein expression, and THP‐1 adherence. Collectively, these results suggest that LPS‐induced VCAM‐1 expression and adhesion of THP‐1 cells are mediated through the TLR4/ERK/cPLA₂α phosphorylation and COX‐2 expression/PGE₂ synthesis in RASFs. J. Cell. Physiol. 223: 480–491, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of mechanical vibration on cell morphology,proliferation, apoptosis,and cytokine expression/secretion in osteocyte‐like MLO‐Y4 cells exposed to high glucose

Diabetic patients exhibit significant bone deterioration. Our recent findings demonstrate that mechanical vibration is capable of resisting diabetic bone loss, whereas the relevant mechanism remains unclear. We herein examined the effects of mechanical vibration on the activities and functions of osteocytes (the most abundant and well‐recognized mechanosensitive cells in the bone) exposed to high glucose (HG). The osteocytic MLO‐Y4 cells were incubated with 50 mM HG for 24 h, and then stimulated with 1 h/day mechanical vibration (0.5 g, 45 Hz) for 3 days. We found that mechanical vibration significantly increased the proliferation and viability of MLO‐Y4 cells under the HG environment via the MTT, BrdU, and Cell Viability Analyzer assays. The apoptosis detection showed that HG‐induced apoptosis in MLO‐Y4 cells was inhibited by mechanical vibration. Moreover, increased cellular area, microfilament density, and anisotropy in HG‐incubated MLO‐Y4 cells were observed after mechanical vibration via the F‐actin fluorescence staining. The real‐time polymerase chain reaction and western blotting results demonstrated that mechanical vibration significantly upregulated the gene and protein expression of Wnt3a, β‐catenin, and osteoprotegerin (OPG) and decreased the sclerostin, DKK1, and receptor activator for nuclear factor‐κB ligand (RANKL) expression in osteocytes exposed to HG. The enzyme‐linked immunosorbent assay assays showed that mechanical vibration promoted the secretion of prostaglandin E₂ and OPG, and inhibited the secretion of tumor necrosis factor‐α and RANKL in the supernatant of HG‐treated MLO‐Y4 cells. Together, this study demonstrates that mechanical vibration improves osteocytic architecture and viability, and regulates cytokine expression and secretion in the HG environment, and implies the potential great contribution of the modulation of osteocytic activities in resisting diabetic osteopenia/osteoporosis by mechanical vibration.     

Z‐FA.FMK activates duodenal epithelial cell proliferation through oxidative stress,NF‐κB and IL‐1β in d‐GalN/TNF‐α‐administered mice

This study was designed to evaluate the effect of Z‐FA.FMK (benzyloxycarbonyl‐l ‐phenylalanyl‐alanine‐fluoromethylketone), a pharmacological inhibitor of cathepsin B, on the proliferation of duodenal mucosal epithelial cells and the cellular system that controls this mechanism in these cells in vivo. For this investigation, BALB/c male mice were divided into four groups. The first group received physiological saline, the second group was administered Z‐FA.FMK, the third group received d ‐GalN (d ‐galactosamine) and TNF‐α (tumour necrosis factor‐α) and the fourth group was given both d ‐GalN/TNF‐α and Z‐FA.FMK. When d ‐GalN/TNF‐α was administered alone, we observed an increase in IL‐1β‐positive and active NF‐κB‐positive duodenal epithelial cells, a decrease in PCNA (proliferative cell nuclear antigen)‐positive duodenal epithelial cells and an increase in degenerative changes in duodenum. On the other hand, Z‐FA.FMK pretreatment inhibited all of these changes. Furthermore, lipid peroxidation, protein carbonyl and collagen levels were increased, glutathione level and superoxide dismutase activity were decreased, while there was no change in catalase activity by d ‐GalN/TNF‐α injection. On the contrary, the Z‐FA.FMK pretreatment before d ‐GalN/TNF‐α blocked these effects. Based on these findings, we suggest that Z‐FA.FMK might act as a proliferative mediator which is controlled by IL‐1β through NF‐κB and oxidative stress in duodenal epithelial cells of d ‐GalN/TNF‐α‐administered mice.     

Angiotensin II‐induced redox‐sensitive SGLT1 and 2 expression promotes high glucose‐induced endothelial cell senescence

High glucose (HG)‐induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co‐transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG‐induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence‐associated beta‐galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT‐PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2‐NBD‐glucose. HG increased ECs senescence markers and oxidative stress, down‐regulated eNOS expression and NO formation, and induced the expression of VCAM‐1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX‐4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H₂O₂ in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG‐induced ECs senescence and SGLT1 and 2 expression. Thus, HG‐induced ECs ageing is driven by the local angiotensin system via the redox‐sensitive up‐regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.     

Hypoglycemic Effects of a Polysaccharide from Tetrastigma hemsleyanum Diels & Gilg in Alloxan‐Induced Diabetic Mice

Tetrastigma hemsleyanum Diels & Gilg , a well‐known traditional Chinese medicine, possesses antitumor and anti‐inflammatory activity, etc. However, the anti‐diabetic effect has not been determined. In our present study, a water‐soluble polysaccharide, named THP with molecular weight of 93 307 Da, was isolated from T. hemsleyanum by DEAE‐52 ion‐exchange and Sephadex G‐100 chromatography. It contains rhamnose, arabinose, mannose, glucose, and galactose in the molar ratio of 0.07:0.14:0.38:0.21:0.31. Then anti‐diabetic effects of THP were examined by treating alloxan‐induced diabetic mice with different doses (100, 200, and 300 mg/kg) of THP orally. The results showed that THP could decrease the blood glucose, TC, TG, LDL‐C levels, increase the body weight, HDL‐C, insulin levels, and enhance the activities of antioxidant enzyme system in alloxan‐induced diabetic mice. Furthermore, the histopathological examination of pancreas, liver, and kidney indicated that THP could protect and reverse β‐cells in diabetic mice with low damage to liver and kidney, which suggests that THP may stimulate pancreatic release of insulin and can be an effectively potential candidate for diabetes mellitus.     

Critical role of Toll‐like receptor 2 in Bacteroides fragilis‐mediated immune responses in murine peritoneal mesothelial cells

In this study, the role of Toll‐like receptor 2 (TLR2) in immune responses of murine peritoneal mesothelial cells against Bacteroides fragilis was investigated. Enzyme linked immunosorbent assay was used to measure cytokines and chemokines. Activation of nuclear factor κB (NF‐κB‐α) and mitogen‐activated protein kinases (MAP kinases) was investigated by western blot analysis. B. fragilis induced production of interleukin‐6, chemokine (C‐X‐C motif) ligand 1 (CXCL1) and chemokine (C‐C motif) ligand 2 (CCL2) in wild type peritoneal mesothelial cells; this was impaired in TLR2‐deficient cells. In addition, in response to B. fragilis, phosphorylation of inhibitory NF‐κB‐α and c‐Jun N‐terminal kinase mitogen‐activated protein kinase (MAPK) was induced in wild type mesothelial cells, but not in TLR2‐deficient cells,. Inhibitor assay revealed that NF‐κB and MAPKs are essential for B. fragilis‐induced production of CXCL1 and CCL2 in mesothelial cells. These findings suggest that TLR2 mediates immune responses in peritoneal mesothelial cells in response to B. fragilis.