The relationship between M3 cholinergic receptor agonist (carbachol) hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen activation or NF-kappaB activation in rats was studied in vitro. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc), and NF-kappaB inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar cells. The results showed that as compared with control group, 10(-3) mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells (P < 0.01) following the treatment with a high concentration of carbachol (10(-3) mol/L) in vitro. The addition of 10(-2) mol/L PDTC didn't result in a significant decrease in the activity of trypsin and the leakage of LDH from pancreatic acinar cells treated with a high concentration of carbachol (10(-3) mol/L) in vitro (P > 0.05). It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-kappaB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.
Carbachol/*pharmacology ; Cholinergic Agonists/pharmacology ; NF-kappa B/*metabolism ; Pancreas/metabolism ; Pancreas/*pathology ; Rats, Wistar ; Receptor, Muscarinic M3/agonists ; Trypsinogen/*metabolism

<b>OBJECTIVEb>To investigate the influence and significance of peroxisome proliferator- activated receptor-gamma (PPAR-gamma) agonist rosiglitazone on the expression of I kappa B kinase-beta(IKK-beta) mRNA and protein induced by LPS in Kupffer cells (KCs) cultured in vitro and to investigate the activity of nuclear factor-kappa B (NF-kappa B) together with the expression of cyclooxygenase-2 (COX-2) in livers of rats with non-alcoholic steatohepatitis (NASH).

<b>METHODSb>(1) KCs from healthy Wistar rats were isolated and purified with IV collagenase digestion and gradient centrifugalization, and then were incubated in the presence or absence of LPS (1 microg/ml) together with two different concentrations of rosiglitazone (10 nmol/L and 50 nmol/L). (2) Thirty-eight healthy Wistar rats were randomly divided into a normal blank control group (10 rats) fed with a normal diet and a NASH model group (28 rats) fed with a fat-rich diet (10% lard + 2% cholesterol + 5% corn oil). After the NASH model was established successfully and confirmed by pathological examination of the livers of 4 rats, 24 rats that continued with the high fat-rich diet, were divided into three groups (8 rats in each group): a control group fed normal saline (NS), a lower dose rosiglitazone group (1 mg.kg(-1).d(-1)) and a higher dose rosiglitazone group (4 mg.kg(-1).d(-1)) for 12 weeks. The mRNA expression of IKK-beta in KCs and COX-2 in livers were measured using reverse transcription-polymerase chain reaction (RT-PCR). The IKK-beta protein in KCs and the NF-kappa B activity of hepatic tissues were determined respectively by Western blot and electrophoretic mobility shift assay (EMSA). The concentration of tumor necrosis factor alpha (TNF alpha) in the supernatant of KCs cultures and serum of the rats was quantified by enzyme linked immunosorbent assay (ELISA).

<b>RESULTSb>LPS significantly increased the expression of IKK-beta mRNA and protein in the KCs and the concentration of TNF alpha in the supernatant of the KCs cultures. The expressions of COX-2 mRNA and protein were more obvious in rats with NASH than those in the normal control group, and the binding activity of NF-kB correlated positively with the expression of COX-2 in the livers and the level of serum TNF alpha of model rats as well. Rosiglitazone blocked the expression of IKK-beta mRNA and protein induced by LPS in KCs, and also inhibited NF-kappa B activation and reduced COX-2 expression in the rats.

<b>CONCLUSIONSb>PPAR-gamma specific agonist rosiglitazone can play an anti-inflammatory role by IKK-beta/I kappa B/NF-kappa B/TNF alpha signal ways, and minimize inflammatory reaction at cellular and molecular levels. This may help to provide a new idea for treating NASH effectively.

Animals ; Cyclooxygenase 2 ; metabolism ; Fatty Liver ; drug therapy ; metabolism ; pathology ; Hypoglycemic Agents ; therapeutic use ; I-kappa B Kinase ; metabolism ; Male ; NF-kappa B ; metabolism ; PPAR gamma ; agonists ; Rats ; Rats, Wistar ; Signal Transduction ; Thiazolidinediones ; therapeutic use ; Tumor Necrosis Factor-alpha ; metabolism

Acute Disease ; Animals ; Blotting, Western ; Disease Models, Animal ; I-kappa B Proteins ; metabolism ; Injections, Intraperitoneal ; Lipopolysaccharides ; Liver ; metabolism ; pathology ; Liver Diseases ; metabolism ; pathology ; Male ; Mice ; NF-kappa B ; metabolism ; NF-kappa B p50 Subunit ; metabolism ; PPAR gamma ; agonists ; Random Allocation ; Signal Transduction ; drug effects ; Thiazolidinediones ; pharmacology

Animals ; Echocardiography ; Female ; Hydroxyl Radical ; metabolism ; Myocardial Contraction ; drug effects ; NF-kappa B ; metabolism ; PPAR gamma ; agonists ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; Superoxides ; metabolism ; Thiazolidinediones ; pharmacology ; Ventricular Function, Left ; drug effects

Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif 10PQENDE15 in STP-A11 reveals that Glu (E)12 residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.
*Transcription, Genetic ; Transcription Factor AP-1/agonists/*metabolism ; TNF Receptor-Associated Factor 6/*metabolism ; Solubility ; STAT3 Transcription Factor/metabolism ; Proto-Oncogene Proteins pp60(c-src)/metabolism ; Protein Binding ; Oncogene Proteins, Viral/*metabolism ; NF-kappa B/agonists/*metabolism ; Ions ; Humans ; Herpesvirus 2, Saimiriine/*metabolism ; Detergents ; Cell Line

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-kappaB and the expression of chemokine MOB-1, MCP-lgenes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-kappaB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10(-3) mol/L, 10(-4) mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10(-3) mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-kappaB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10(-3) mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10(-5) mol/L atropine) or NF-kappaB inhibitor (10(-2) mol/L PDTC) could obviously inhibit the activation of NF-kappaB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-lgenes in pancreatic acinar cells in vitro through the activation of NF-kappaB.
Adaptor Proteins, Signal Transducing ; Carbachol ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; Chemokine CCL2 ; biosynthesis ; genetics ; Chemokines ; biosynthesis ; genetics ; Humans ; NF-kappa B ; biosynthesis ; genetics ; Pancreas, Exocrine ; metabolism ; Pancreatitis ; etiology ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, Muscarinic M3 ; agonists ; physiology ; Signal Transduction

<b>OBJECTIVEb>To observe the effects of peroxisome proliferators-activated receptor-gamma (PPARgamma) on the function of the vital organs in rats with pancreatitis.

<b>METHODSb>Acute pancreatitis (AP) was induced in 30 male SD rats by ductal injection of 4% sodium taurocholate at 1.0 ml/kg. The rats received subsequent intravenously injection of 0.3 mg/kg of PPARgamma ligand (rosiglitazone, n=10), PPARgamma antagonist (GW9662, n=10) followed 10 min later by rosiglitazone administration at 0.3 mg/kg, or left untreated (AP model group, n=10). Another 10 male SD rats receiving no particular treatment served as the control group. The rats were sacrificed 6 h after the operation, and blood samples were collected for measurement of the biochemical indices of the vital organs. The histological changes of the pancreas and portal vein blood endotoxin content were examined.

<b>RESULTSb>The rats in AP group and GW9662 group showed significantly higher level of the biochemical indices for the vital organs, pathological scores of the pancreas and portal vein blood endotoxin content were significantly higher in the control group and roglitazone-treated groups (P<0.05).

<b>CONCLUSIONb>PPARgamma ligand roglitazone can significantly ameliorate multiple organ injuries and effectively protect the functions of the organs in rats with experimental pancreatitis.

Anilides ; pharmacology ; Animals ; Hypoglycemic Agents ; administration & dosage ; therapeutic use ; Injections, Intravenous ; Male ; Multiple Organ Failure ; prevention & control ; NF-kappa B ; metabolism ; PPAR gamma ; agonists ; antagonists & inhibitors ; metabolism ; Pancreatitis, Acute Necrotizing ; drug therapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Thiazolidinediones ; administration & dosage ; therapeutic use

Although the etiology of inflammatory bowel disease is still uncertain, increasing evidence indicates that the excessive activation of NLRP3 inflammasome plays a major role. Norisoboldine (NOR), an alkaloid isolated from Radix Linderae, has previously been demonstrated to inhibit inflammation and IL-1β production. The present study was to examine the effect of NOR on colitis and the underlying mechanism related to NLRP3 inflammasome activation. Our results showed that NOR alleviated colitis symptom in mice induced by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). Moreover, it significantly reduced expressions of cleaved IL-1β, NLRP3 and cleaved Caspase-1 but not ASC in colons of mice. In THP-1 cells, NOR suppressed the expressions of NLRP3, cleaved Caspase-1 and cleaved IL-1β but not ASC induced by lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Furthermore, NOR could activate aryl hydrocarbon receptor (AhR) in THP-1 cells, inducing CYP1A1 mRNA expression, and promoting dissociation of AhR/HSP90 complexes, association of AhR and ARNT, AhR nuclear translocation, XRE reporter activity and binding activity of AhR/ARNT/XRE. Both siAhR and α-naphthoflavone (α-NF) markedly diminished the inhibition of NOR on NLRP3 inflammasome activation. In addition, NOR elevated Nrf2 level and reduced ROS level in LPS- and ATP-stimulated THP-1 cells, which was reversed by either siAhR or α-NF treatment. Finally, correlations between activation of AhR and attenuation of colitis, inhibition of NLRP3 inflammasome activation and up-regulation of Nrf2 level in colons were validated in mice with TNBS-induced colitis. Taken together, NOR ameliorated TNBS-induced colitis in mice through inhibiting NLRP3 inflammasome activation via regulating AhR/Nrf2/ROS signaling pathway.
Alkaloids ; administration & dosage ; Animals ; Colitis ; chemically induced ; drug therapy ; genetics ; immunology ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Inflammasomes ; drug effects ; immunology ; Interleukin-1beta ; genetics ; immunology ; Lindera ; chemistry ; Male ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; genetics ; immunology ; Receptors, Aryl Hydrocarbon ; agonists ; genetics ; metabolism ; Trinitrobenzenesulfonic Acid ; adverse effects

BACKGROUND/AIMS: Asthma is characterized by airway hyperresponsiveness, inflammation, and remodeling. Peroxisome proliferator-activated receptors have been reported to regulate inflammatory responses in many cells. In this study, we examined the effects of intranasal rosiglitazone on airway remodeling in a chronic asthma model. METHODS: We developed a mouse model of airway remodeling, including smooth muscle thickening, in which ovalbumin (OVA)-sensitized mice were repeatedly exposed to intranasal OVA administration twice per week for 3 months. Mice were treated intranasally with rosiglitazone with or without an antagonist during OVA challenge. We determined airway inflammation and the degree of airway remodeling by smooth muscle actin area and collagen deposition. RESULTS: Mice chronically exposed to OVA developed sustained eosinophilic airway inflammation, compared with control mice. Additionally, the mice developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer. Administration of rosiglitazone intranasally inhibited the eosinophilic inflammation significantly, and, importantly, airway smooth muscle remodeling in mice chronically exposed to OVA. Expression of Toll-like receptor (TLR)-4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) was increased in the OVA group and decreased in the rosiglitazone group. Co-treatment with GW9660 (a rosiglitazone antagonist) and rosiglitazone increased the expression of TLR-4 and NF-kappaB. CONCLUSIONS: These results suggest that intranasal administration of rosiglitazone can prevent not only air way inf lammation but also air way remodeling associated with chronic allergen challenge. This beneficial effect is mediated by inhibition of TLR-4 and NF-kappaB pathways.
Actins/metabolism ; Administration, Inhalation ; Airway Remodeling/*drug effects ; Animals ; Anti-Asthmatic Agents/*administration & dosage ; Asthma/chemically induced/*drug therapy/metabolism/physiopathology ; Chronic Disease ; Collagen/metabolism ; Disease Models, Animal ; Female ; Lung/*drug effects/metabolism/physiopathology ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Ovalbumin ; PPAR gamma/agonists/metabolism ; Pneumonia/chemically induced/physiopathology ; Pulmonary Eosinophilia/chemically induced/prevention & control ; Signal Transduction/drug effects ; Thiazolidinediones/*administration & dosage ; Toll-Like Receptor 4/metabolism

The balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolysis. PAI-1 expression increases in atherosclerotic arteries and vascular smooth muscle cells (VSMCs) are one of major constituents of atheroma. We investigated the impact of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, on the plasminogen activator system of the rat VSMCs. The lysoPC stimulated the protein and gene expressions of PAI-1 but did not affect the protein expression of t-PA. Fibrin overlay zymography revealed that lysoPC increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA. Vitamin E inhibited the lysoPC-induced PAI-1 expression. Further, lysoPC increased the intracellular reactive oxygen species (ROS) formation. Caffeic acid phenethyl ester, an inhibitor of NF-kappaB, blocked this lysoPC effect. Indeed, lysoPC induced the NF-kappaB-mediated transcriptional activity as measured by luciferase reporter assay. In addition, genistein, an inhibitor of protein-tyrosine kinase (PTK), diminished the lysoPC effect, while 7,12-dimethylbenz[a]anthracene, a stimulator of PTK, stimulated PAI-1 production. In conclusion, lysoPC does not affect t-PA expression but induces PAI-1 expression in the VSMC by mediating NF-kappaB and the genistein-sensitive PTK signaling pathways via oxidative stress. Importantly, lysoPC stimulates the enzyme activity of PAI-1 and suppresses that of t-PA.
Animals ; Benz(a)Anthracenes/pharmacology ; Caffeic Acids/pharmacology ; Cells, Cultured ; Genistein/pharmacology ; Lipoproteins, LDL/metabolism ; Lysophosphatidylcholines/*pharmacology ; Muscle, Smooth, Vascular/cytology/*drug effects/metabolism ; NF-kappa B/antagonists & inhibitors/metabolism ; Oxidative Stress/drug effects ; Phenylethyl Alcohol/analogs & derivatives/pharmacology ; Plasminogen Activator Inhibitor 1/agonists/genetics/*metabolism ; Protein Kinase Inhibitors/pharmacology ; Protein-Tyrosine Kinases/antagonists & inhibitors/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Signal Transduction/drug effects ; Tissue Plasminogen Activator/*metabolism ; Transcription, Genetic/drug effects ; Up-Regulation/drug effects ; Vitamin E/pharmacology