Shp-2／NF-κB Pathway Mediates the Inhibition of Lipoxin A4 onIL-1β-induced Synthesis of IL-6 in Glomerular Mesangial Cells

Objective: To examine whether lipoxin A4 (LXA4) has an antagonistic effect on IL-1β-induced synthesis of IL-6 in glomerular mesangial cells, and to explore the molecular mechanisms of signal pathway in LXA4 actions. Methods: The glomerular mesangial cells of rat were cultured and treated with IL-1β with or without preincubation with LXA4 at different concentrations. The amount of IL-6 in the supernatant of cells was analyzed by enzymelinked immunosorbent assay(ELISA). The expressions of mRNA of IL-6 were determined by RT-PCR. The expressions of Src homology 2( SH2 ) containing protein-tyrosine phosphatase 2(Shp-2) were assessed by immunoprecipitation and immunoblotting. Activities of DNA-binding of nuclear factor-kappa B(NF-κB) were measured by electrophoretic mobility shift assay(EMSA). Results:IL-1β- snulated secretion of protein and expression of mRNA of IL-6 in mesangial cells were inhibited by LXA4 in a dose-dependent manner. LXA4 antagonizes the phosphorylation of Shp-2 and activities of NF-κB induced by IL-1β Conclusion: LXA4 antagonists IL-1β-induced synthesis of IL-6 in glomerular mesangial cellsthrough the mechanism of Shp-2/NF-κB pathway-dependent signal transduction.     

LPS-induced NF-κB activation requires Ca2+ as a mediator in isolated pancreatic acinar cells of rat

Objective To investigate the effect of Ca2+ on lipopolysaccharide (LPS)-induced NF-κB activation in pancreatic acinar cells and the role of NF-κB in LPS-induced acinar cell injury. Methods Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to varying concentrations of LPS (from 1 to 20 mg/L) in the presence or absence of EGTA. At various time points (30 minutes, 1 hour, 2 hours, 4 hours and 10 hours) after treatment with the agents, cell viability was determined by MTT. Nuclear translocation of NF-κB’s subunit p65 was visualized by immunofluorescence staining and nuclei protein was extracted to perform EMSA which was used to assay the activity of NF-κB binding to the DNA sequence containing the recognition site of NF-κB. Results LPS induced cell damage in a time- and concentration-dependent manner while EGTA attenuated LPS-induced cell damage (P<0.05). NF-κB p65 immunofluorescence staining had increased intensity in the cytoplasm and indicated that nuclear translocation occurred within 30 minutes and its zenith was reached at 1 hour after LPS (10 mg/L) treatment. Testing of NF-κB DNA binding activity showed the same alteration phase as p65 immunofluorescence staining. NF-κB activation preceded the pathological alteration of pancreatic acinar cells. The Ca2+ chelator EGTA inhibited LPS-induced NF-κB activation. Conclusions NF-κB activation is an important early event in LPS-induced injury to pancreatic acinar cells. Ca2+ is an important mediator in the process of LPS-induced NF-κB activation.     

Expression of NF-κB in hRPE Cells Stimulated by PDTC and IL-1β

The constitutive expression of nuclear-factor-κB (NF-κB) in human pigment epithelial (hRPE) cells cultivated in vitro and the possible changes when incubated with PDTC and IL-I were investigated. The synchronized hRPE cells in vitro were divided into two groups. In nonPDTC group, hRPE cells were exposed respectively to IL-1β and NS (for detecting the constitutive expressions of NF-κB in hRPE cells) ; In PDTC group, PDTC-pretreated hRPE cells were exposed respectively to IL-1β?Aand NS. (for detecting the constitutive expression of NF-κB in PDTC-pretreated hRPE cells). The expression of NF-κB in hRPE cells in two groups was detected by immunofluorescence stain and flow cytometry. The results showed that the constitutive expression of NF-κB in hRPE cells in vitro was 8.05 %, and increased to 30.26 % by IL-1β. After PDTC pretreatment, the constitutive expression of NF-κB in hRPE cells was decreased to 3.74%, and 3.66 % by IL-l,respectively. It was concluded that the expressions of NF-κB in hRPE cells could be increased significantly by IL-1βand depressed effectively by PDTC. Also, PDTC could significantly inhibit the activation of NF-κB induced by IL-1β.     

Role and mechanism of rosiglitazone on the impairment of insulin secretion induced by free fatty acids on isolated rat islets

Background Prolonged exposure of pancreatic β-cells to fatty acids increases basal insulin secretion but inhibits glucose-stimulated insulin secretion. Rosiglitazone is a new antidiabetic agent of the thiazolidinediones. However, the relationship between thiazolidinediones and insulin secretion is highly controversial. The aim of this study is to explore the effect and mechanism of rosiglitazone on insulin secretion of islets under chronic exposure to free fatty acids （FFA）. Methods Pancreatic islets were isolated from the pancreata of male Sprague-Dawley rats by the collagenase digestion and by the dextran gradient centrifugation method. The purified islets were cultured in the presence or absence of rosiglitazone and palmitate for 48 hours. The insulin secretion was measured by radioimmunoassay. The mRNA level of peroxisome proliferator-activated receptor y, uncoupling protein 2 0dCP-2） and insulin were determined by real-time polymerase chain reaction （PCR）. The cell cytotoxicity assay was measured by cell counting kit-8. Results Islets exposed to elevated palmitate for 48 hours showed an increased basal and a decreased glucose-stimulated insulin secretion （P〈0.01）. The mRNA level of UCP-2 was increased by 3.7 fold in the 0.5 mmol/L concentration of palmitate. When islets were cultured with palmitate （0.5 mmol/L） in the presence of rosiglitazone （1.0 pmol/L）, both basal and glucose-stimulated insulin secretion reversed to a pattern of control islets （P〈0.05, P〈0.0 1）. The addition of rosiglitazone in the culture medium decreased the mRNA level of UCP-2 by 2.2 fold, having a statistically significant difference （P〈0.05） as compared with islets cultured with palmitate alone. The cell viability was not affected. Conclusion The protective effects of rosiglitazone on insulin secretion of isolated pancreatic islets under chronic exposure to palmitate might be mediated through the downregulation of UCP-2 expression.     

Resveratrol Induces Apoptosis in Pancreatic cancer cells

Background  Pancreatic cancer is one of the most lethal human cancers with a very low survival rate of 5 years. Conventional cancer treatments including surgery, radiation, chemotherapy or combinations of these show little effect on this disease. Several proteins have been proved critical to the development and the progression of pancreatic cancer. The aim of this study was to investigate the effect of resveratrol on apoptosis in pancreatic cancer cells.

Methods  Several pancreatic cancer cell lines were screened by resveratrol, and its toxicity was tested by normal pancreatic cells. Western blotting was then performed to analyze the molecular mechanism of resveratrol induced apoptosis of pancreatic cancer cell lines.

Results  In the screened pancreatic cancer cell lines, capan-2 and colo357 showed high sensitivity to resveratrol induced apoptosis. Resveratrol exhibited insignificant toxicity to normal pancreatic cells. In resveratrol sensitive cells, capan-2 and colo357, the activation of caspase-3 was detected and showed significant caspase-3 activation upon resveratrol treatment; p53 and p21 were also detected up-regulated upon resveratrol treatment.

Conclusion  Resveratrol provides a promising anti-tumor stratagy to fight against pancreatic cancer.

     

Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Proliferation in Human Leukemia U937 Cells

This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor(VEGF) and subsequent proliferation of human leukemia U937 cells,and explored the mechanisms involved.Human leukemia U937 cells were treated with resveratrol of different concentrations(12.5-200 μmol/L) for different time lengths(12-48 h).The proliferation of the U937 leukemic cells was determined by MTT assay.Apoptosis was observed by Annexin-Ⅴ-FIFC/PI double staining and flow cytometry(FCM).Cells cycle was analyzed by PI staining and FCM.The content of VEGF was determined by ELISA.Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations.The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose-and time-dependent manner.Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells.Resveratrol inhibited the secretion of VEGF in U937 cells.Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner.It was concluded that resveratrol could down-regulate the secretion of VEGF,induce apoptosis and suppress the proliferation of U937 cells.     

Interleukin-13 inhibits cytokines synthesis by blocking nuclear factor-κB and c-Jun N-terminal kinase in human mesangial cells

Objective： Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin （IL） -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells （HMCs）. Methods： The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay （RPA）. Activity of nuclear factor-kappa B （NF-κB） and activa- tor protein-1 （AP-1） was examined by electrophoretic mobility shift assay （EMSA）. NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase （JNK） activity were assayed by immunoblot. Results： Recombinant IL-13 inhibited tumor necrosis factor-α （TNF-u）, IL-1α, IL-1β, monocyte chemoattractant protein-1 （MCP-1）, IL-8, and transforming growth factor-β1 （TGF-β1） mRNA expressions in a dose-dependent manner. Lipopoly- sacchorides （LPS） dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner. Conclusion： IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.     

Oxymatrine improves TNBS-induced colitis in rats by inhibiting the expression of NF-κB p65

Inflammatory bowel disease is thought to be regulated by the balance between Th1 and Th2 cytokines secreted by T cells, and NF-κB p65 also plays a predominant role in the intestinal inflammation. We evaluated the potency of oxymatrine, one of active components of Sophora Root, in inhibiting the immune responses and inflammation in 2,4,6-trinitrobenzene sulfonic acid （TNBS）-induced colitis. The inflammation was markedly ameliorated in the oxymatrine-treated rats. The level of IL-2 was increased and that of IL-10 was decreased in colon tissue in the rat model, which was reversed by the treatment of oxymatrine. Moreover, the elevated expression of NF-κB p65 in colon tissue in the model was also improved by oxymatrine treatment. Our results suggest that oxymatrine might be beneficial for the abnormal immune responses and inflammation by regulating the unbalance of Th1 and Th2 cytokines secretion and inhibiting the expression of NF-κB p65 in colon tissue.     

Oxymatrine Improves TNBS-induced Colitis in Rats by Inhibiting the Expression of NF-κB p65

Inflammatory bowel disease is thought to be regulated by the balance between Th1 and Th2 cytokines secreted by T cells, and NF-κB p65 also plays a predominant role in the intestinal inflammation. We evaluated the potency of oxymatrine, one of active components of Sophora Root, in inhibiting the immune responses and inflammation in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. The inflammation was markedly ameliorated in the oxymatrine-treated rats. The level of IL-2 was increased and that of IL-10 was decreased in colon tissue in the rat model, which was reversed by the treatment of oxymatrine. Moreover, the elevated expression of NF-κB p65 in colon tissue in the model was also improved by oxymatrine treatment. Our results suggest that oxymatrine might be beneficial for the abnormal immune responses and inflammation by regulating the unbalance of Th1 and Th2 cytokines secretion and inhibiting the expression of NF-κB p65 in colon tissue.     

Nuclear factor-Y mediates pancreatic β-cell compensation by repressing reactive oxygen species-induced apoptosis under metabolic stress

Background: Pancreatic β-cells elevate insulin production and secretion through a compensatory mechanism to override insulin resistance under metabolic stress conditions. Deficits in β-cell compensatory capacity result in hyperglycemia and type 2 diabetes (T2D). However, the mechanism in the regulation of β-cell compensative capacity remains elusive. Nuclear factor-Y (NF-Y) is critical for pancreatic islets’ homeostasis under physiological conditions, but its role in β-cell compensatory response...     

Homocysteine-induced Enhanced Expression of Tissue Factor in Human Vascular Smooth Muscle Cells

The homocysteine (Hcy)-induced tissue factor (TF) expression in human vascular smooth muscle cells (VSMCs) and the effect of Hcy on the activity of nuclear factor-kappaB (NF-кB) and the expression of inducible nitric oxide synthase (iNOS) were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC (NF-кB inhibitor). Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-кB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF pro- tein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-кB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-ΚB, subsequently mediate TF gene expression and protein synthesis. NF-кB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.     

Effect of resveratrol on platelet aggregation in vivo and in vitro

Objective Low or moderate consumption of red wine has a greater benefit than the consumpti on of other beverages in the prevention of atherosclerosis and coronary heart di sease and this is increasingly attributed to the polyphenol compounds in red win e, such as resveratrol. In the present study, we investigated the effect of res veratrol on platelet aggregation in vitro and in vivo. Methods Platelet aggregation in rabbits and normal subjects was measured using Born’s me thod. Results Resveratrol, at 10-1000 μmol/L, significantly inhibited platelet aggregation i n vitro induced by collagen, thrombin, and ADP in healthy subjects. The inhibit ory effect was concentration- dependent. Hypercholesterolemia induced by high- cholesterol diet enhanced ADP- induced platelet aggregation. Resveratrol 4 mg ·kg(-1)·d(-1)inhibited ADP- induced platelet aggregation in vivo de spite no changes in serum lipid levels. Conclusions Resveratrol inhibits platelet aggregation both in vitro and in vivo. This may b e one of the mechanisms by which resveratrol prevents atherosclerosis.     

Triptolide-induced apoptosis by inactivating nuclear factor-kappa B apoptotic pathway in multiple myeloma <Emphasis Type="Italic">in vitro</Emphasis>

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro,as well as the roles of nuclear factor-kappa B(NF-κB) and IκBα was investigated.The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay.Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay.The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy.The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells.Triptolide at nM range induced proliferation inhibition in a dose-and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells.Besides,we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol.These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.     

Perfluorocarbon attenuates lipopolysaccharide-mediated inflammatory responses of alveolar epithelial cells in vitro

Background  Toll-like receptor-4 (TLR-4) is integrally involved in lipopolysaccharide (LPS) signaling and has a requisite role in the activation of nuclear factor-κB (NF-κB). The exact mechanisms that lend perfluorocarbon (PFC) liquids a cytoprotective effect have yet to be elucidated. Therefore we examined in an in vitro model the cytoprotective effect of PFC on LPS-stimulated alveolar epithelial cellls (AECs). 

Methods  AECs (A549 cells, human lung adenocarcinoma cell line) were divided into four groups: control, PFC, LPS and LPS + PFC (coculture group) groups. Intercellular adhesion molecule-1 (ICAM-1) was detected by ELISA, tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) were detected by radioimmunological methods. The expression of TLR-4 mRNA and protein was detected by real time PCR and Western blotting, respectively. The activation of NF-κB was detected by Western blotting (proteins of I-κBα and NF-κB p65). 

Results  ICAM-1, TNF-α and IL-8 were significantly increased in LPS-stimulated AECs groups. The expression of TLR-4 mRNA and protein in LPS-stimulated groups was markedly increased. Meanwhile, NF-κB was activated as indicated by the significant degradation of IκB-α and the significant release of NF-κB P65 and its subsequent translocation into the nucleus. There were no significant effects of PFC alone on any of the factors studied while the coculture group showed significant downregulation of the secretion of ICAM-1, TNF-α and IL-8, the expression of TLR-4 mRNA and the activity of NF-κB. 

Conclusions  Taken together, our results demonstrate that LPS can induce AEC-related inflammatory injury via the activation of TLR-4 and subsequent activation of NF-κB. PFC is able to protect AECs from LPS-induced inflammatory injury by blocking the initiation of the LPS signaling pathway, which is indicated by the significant decrease of TLR-4 expression and NF-κB activation.