BACKGROUND/AIMS: To investigate the expression of Toll-like receptor 4 (TLR4) in the pancreases of rats with acute necrotizing pancreatitis (ANP) and any changes upon treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappaB (NF-kappaB), as well as to determine the relationship between TLR4 and NF-kappaB in ANP pathogenesis. METHODS: A total of 72 SD rats were randomly divided into three groups, namely, the control (sham-operation), ANP, and ANP with PDTC pretreatment groups. The PDTC-pretreated group was intraperitoneally injected with PDTC at a dose of 100 mg/kg 1 hour before the induction of ANP. The expressions of TLR4 and NF-kappaB in pancreatic tissue were evaluated by immunohistochemistry and Western blot analysis. The mRNA levels of cytokines tumor necrosis factor alpha, interleukin (IL)-1beta, and IL-6 were measured by reverse transcription polymerase chain reaction. RESULTS: The expressions of TLR4, NF-kappaB, and cytokine (NF-kappaB target) genes in the pancreatic tissue increased more significantly in the ANP groups than in the sham-operation group at 3, 6, and 12 hours. Pretreatment with PDTC alleviated the inflammatory activation in the pancreas with ANP, causing a significant decrease in the expressions of TLR4, NF-kappaB, and cytokine genes in the pancreatic tissue. CONCLUSIONS: The expressions of TLR4 and NF-kappaB were increased in the pancreases of rats with ANP. PDTC not only inhibits NF-kappaB but also suppresses the expression of TLR4 and downregulates the expression of the related cytokine genes.
Animals ; Antioxidants/*pharmacology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Male ; NF-kappa B/*drug effects/metabolism ; Pancreas/metabolism/pathology ; Pancreatitis, Acute Necrotizing/chemically induced/*drug therapy ; Pyrrolidines/*pharmacology ; RNA, Messenger/metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Thiocarbamates/*pharmacology ; Toll-Like Receptor 4/*drug effects/metabolism ; Tumor Necrosis Factor-alpha/genetics/metabolism

BACKGROUND/AIMS: To investigate the expression of Toll-like receptor 4 (TLR4) in the pancreases of rats with acute necrotizing pancreatitis (ANP) and any changes upon treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappaB (NF-kappaB), as well as to determine the relationship between TLR4 and NF-kappaB in ANP pathogenesis. METHODS: A total of 72 SD rats were randomly divided into three groups, namely, the control (sham-operation), ANP, and ANP with PDTC pretreatment groups. The PDTC-pretreated group was intraperitoneally injected with PDTC at a dose of 100 mg/kg 1 hour before the induction of ANP. The expressions of TLR4 and NF-kappaB in pancreatic tissue were evaluated by immunohistochemistry and Western blot analysis. The mRNA levels of cytokines tumor necrosis factor alpha, interleukin (IL)-1beta, and IL-6 were measured by reverse transcription polymerase chain reaction. RESULTS: The expressions of TLR4, NF-kappaB, and cytokine (NF-kappaB target) genes in the pancreatic tissue increased more significantly in the ANP groups than in the sham-operation group at 3, 6, and 12 hours. Pretreatment with PDTC alleviated the inflammatory activation in the pancreas with ANP, causing a significant decrease in the expressions of TLR4, NF-kappaB, and cytokine genes in the pancreatic tissue. CONCLUSIONS: The expressions of TLR4 and NF-kappaB were increased in the pancreases of rats with ANP. PDTC not only inhibits NF-kappaB but also suppresses the expression of TLR4 and downregulates the expression of the related cytokine genes.
Animals ; Antioxidants/*pharmacology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Male ; NF-kappa B/*drug effects/metabolism ; Pancreas/metabolism/pathology ; Pancreatitis, Acute Necrotizing/chemically induced/*drug therapy ; Pyrrolidines/*pharmacology ; RNA, Messenger/metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Thiocarbamates/*pharmacology ; Toll-Like Receptor 4/*drug effects/metabolism ; Tumor Necrosis Factor-alpha/genetics/metabolism

OBJECTIVETo investigate the effect of paraquat (PQ) on reactive oxygen species (ROS) and neutrophil apoptosis and its possible signal transduction pathways.

METHODSCultured neutrophils were treated with different concentrations of PQ for 6-24 h. The apoptosis rate of neutrophils and ROS content were determined by flow cytometry. The exoressions of nuclear factor kappa B (NF-κB) and Caspase 3 were detected by Western blot. These parameters were checked again after NF-κB and Caspase 3 antagonist were applied.

RESULTSPQ could boost ROS generation and depress neutrophil apoptosis significantly. At the same time PQ could enhance the expression of NF-κB and inhibit the expression of Caspase 3. These effects could be reversed by ROS inhibitor diphenyleneiodonium (DPI) and NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC).

CONCLUSIONPQ is a potent inducer of ROS and can inhibit neutrophil apoptosis by activating NF-κB and surpressing Caspase 3 activity.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cells, Cultured ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Neutrophils ; cytology ; drug effects ; Paraquat ; toxicity ; Pyrrolidines ; pharmacology ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Thiocarbamates ; pharmacology

Induced pluripotent stem cells (iPSCs) can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for the extra-embryonic tissues. This iPSC technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large numbers of disease-specific cells for biomedical research. However, the low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limit the potential application of iPSCs. Chemical-induced reprogramming offers a novel approach to generating iPSCs. In this study, a new combination of small-molecule compounds (SMs) (sodium butyrate, A-83-01, CHIR99021, Y-27632) under conditions of transient folate deprivation was used to generate iPSC. It was found that transient folate deprivation combined with SMs was sufficient to permit reprogramming from mouse embryonic fibroblasts (MEFs) in the presence of transcription factors, Oct4 and Klf4, within 25 days, replacing Sox2 and c-Myc, and accelerated the generation of mouse iPSCs. The resulting cell lines resembled mouse embryonic stem (ES) cells with respect to proliferation rate, morphology, pluripotency-associated markers and gene expressions. Deprivation of folic acid, combined with treating MEFs with SMs, can improve the inducing efficiency of iPSCs and reduce their carcinogenicity and the use of exogenous reprogramming factors.
Amides ; pharmacology ; Animals ; Butyric Acid ; pharmacology ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Extraembryonic Membranes ; cytology ; drug effects ; Folic Acid ; pharmacology ; Induced Pluripotent Stem Cells ; cytology ; drug effects ; Kruppel-Like Transcription Factors ; metabolism ; Mice ; Octamer Transcription Factor-3 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Pyrazoles ; pharmacology ; Pyridines ; pharmacology ; Pyrimidines ; pharmacology ; SOXB1 Transcription Factors ; metabolism ; Thiocarbamates ; pharmacology ; Thiosemicarbazones

OBJECTIVETo observe the expressions of α-SMA, integrin α5 and fibronectin (Fn) in acute paraquat poisoned rats and the effect of PDTC. To investigate the mechanism of paraquat-induced pulmonary fibrosis.

METHODSSprague-Dawley rats were randomly divided into three experimental groups: Control group (6 rats), PQ group (36 rats) and PQ+PDTC group (36 rats). On the 1st, 3rd, the 7th, the 14th, the 28th and the 56th day after exposure, the protein expression of α smooth muscle actin (α-SMA) was evaluated by western blot. The mRNA levels of integrin α5 and fibronectin (Fn) were analyzed with real-time quantitative PCR (RT-PCR). Meanwhile, the lung pathological changes were observed and semi-quantified.

RESULTST With the time passing, the expression of α-SMA in PQ group increased gradually compared with control group (P < 0.05 or P < 0.01). The increasing extent was gently on the 3 rd, the 7 th day. While increasing extent was rapidly from the 28 th to the 56 th day. RT-PCR showed PQ significantly increased Fn mRNA level on all time points and increased integrin α5 mRNA level from the 7 rd to 56 th day compared with control group (P < 0.05 or P < 0.01). PDTC treatment significantly deceased α-SMA, Fn, and integrin α5 levels compared with PQ group in corresponding time points (P < 0.05 or P < 0.01) Noteworthy, in PQ+PDTC group, the occurrence of pathological changes were drastically attenuated and pathologic score significantly decreased (P < 0.05 or P < 0.01).

CONCLUSIONSα-SMA, integrin α5 and fibronectin could play an important role in the development of pulmonary fibrosis caused by paraquat poisoning. PDTC, asa strong NF-κB inhibitor, may inhibit NF-κB activity and further significantly decreased expressions of α-SMA, integrin α5 and fibronectin which were important part of ECM, leading to drastically attenuated pulmonary fibrosis. However, the mechanisms of PDTC intervention still remains to be explored.

Actins ; metabolism ; Animals ; Disease Models, Animal ; Fibronectins ; metabolism ; Integrin alpha5 ; metabolism ; Male ; Paraquat ; poisoning ; Pulmonary Fibrosis ; chemically induced ; metabolism ; Pyrrolidines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Thiocarbamates ; pharmacology

OBJECTIVETo investigate the effect of PDTC (inhibitor of NF-κb) on apoptosis of human gastric cancer cell line SGC-7901 induced by tumor necrosis factor α (TNF-α) and explore the related mechanisms.

METHODSAfter the treatment with different concentrations of PDTC, TNF-α or PDTC combined with TNF-α on gastric cancer cell line SGC-7901, the growth inhibition of SGC-7901 was measured by MTT assay. Hoechst was used to assess SGC-7901 cell apoptosis. The protein expressions of survivin and caspase-3 were detected by Western blot assay.

RESULTSThe growth inhibition rate of SGC-7901 induced by PDTC (15, 30, 60, 100 μmol/L) was (12.14±0.91)%, (20.00±1.11)%, (37.63±1.01)% and (41.46±1.07)%. Different concentrations of PDTC all inhibited the growth of SGC-7901 significantly (all P<0.01), The growth inhibition rate of SGC-7901 induced by 25 mg/L TNF-α was (2.38±0.67)%, which could not significantly inhibit the growth of SGC-7901 [control (1.50±0.81)%], while TNF-α of 50, 100, 150 mg/L could inhibit the growth of SGC-7901 significantly [(4.53±0.85)%, (4.43±0.70)% and (4.74±1.07)%, all P<0.05]. PDTC (15 μmol/L) combined with TNF-α (25, 50, 100, 150 mg/L) significantly increased the cell growth inhibition rate compared with TNF-α alone or PDTC 15 μmol/L alone (all P<0.01). Hoechst assay showed that 100 mg/L TNF-α, 15 μmol/L PDTC and combination of above two all induced cell apoptosis (P<0.01), and the combination group had significantly higher percentage of cell apoptosis (P<0.01). Survivin protein was significantly down-regulated in combination group as compared with single TNF-α (100 mg/L) group, but was not significant down-regulated as compared with single PDTC (15 μmol/L) group. Caspase-3 protein expression was significantly increased in combination group as compared with other two groups.

CONCLUSIONPDTC can enhance the cell apoptosis induced by TNF-α, which may be associated with the blocking of TNF-α-activated NF-κB signaling pathway by PDTC, the down-regulation of survivin expression, and up-regulation of caspase-3 expression.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Gene Expression Regulation, Neoplastic ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; NF-kappa B ; antagonists & inhibitors ; Proline ; analogs & derivatives ; pharmacology ; Signal Transduction ; Stomach Neoplasms ; metabolism ; pathology ; Thiocarbamates ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo study possible immunobiological potential of Osmunda japonica Thunb.

METHODSImmunomodulatory effects of ethanol extracts prepared from rhizomes of O. japonica and phenolic compounds isolated from the extracts were investigated under the in vitro conditions using the rat peritoneal cells (2×10(6)/mL; 24 h culture). Biosynthesis of nitric oxide (NO) was assayed by Griess reagent, production of prostaglandin E2 (PGE2) and secretion of cytokines were determined by enzyme-linked immunoabsorbent assay.

RESULTSThe extracts activated dose dependently, with the onset at 2.5-5 μmol/L concentrations, the high output NO production, and secretion of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Mild enhancement of NO was produced by the aldehyde-type phenolics 4-hydroxybenzaldehyde and 3,4-hydroxybenzaldehyde. In contrasts, the acetone-type phenolics 4-hydroxybenzalacetone and 3,4-hydroxybenzalacetone inhibited production of immune mediators including cytokines (TNF-α, IL-1β, IL-6), NO, and PGE2. The 3,4-hydroxybenzalacetone was more effective than 4-hydroxybenzaldehyde. The IC50s estimates ranged within the interval of 5-10 μmol/L. No signs of cytotoxicity were observed up to the 50 μmol/L concentration of the compounds.

CONCLUSIONPhenolic compounds contained in medicinal herb Osmunda japonica possess distinct immunomodulatory activity.

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Dinoprostone ; biosynthesis ; Female ; Ferns ; chemistry ; Immunologic Factors ; pharmacology ; Interferon-gamma ; pharmacology ; Lipopolysaccharides ; pharmacology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Peritoneum ; cytology ; drug effects ; Phenols ; chemistry ; isolation & purification ; pharmacology ; Plant Extracts ; chemistry ; isolation & purification ; pharmacology ; Polymyxin B ; pharmacology ; Proline ; analogs & derivatives ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Thiocarbamates ; pharmacology

OBJECTIVETo observe the changes in the expression of connective tissue growth factor (CTGF), type I collagen (Col I), and type III collagen (Col III) among the rats with acute paraquat (PQ) poisoning and the intervention effect of pyrrolidine dithiocarbamate (PDTC) on their expression, and to investigate the mechanism of PQ-induced pulmonary fibrosis and the intervention effect of PDTC on the disease.

METHODSSprague-Dawley rats were randomly divided into control group (n = 6), PQ group (n = 36), and PQ + PDTC group (n = 36). The PQ group and PQ + PDTC group were given a single dose of saline-diluted PQ (80 mg/kg) by gavage; 2 h later, the PQ + PDTC group was intraperitoneally injected with a single dose of PDTC (100 mg/kg), and the PQ group was intraperitoneally injected with the same amount of saline. The control group was given saline (1 ml/kg) by gavage and was intraperitoneally injected with the same amount of saline 2h later. At 1, 3, 7, 14, 25, and 56 days after operation, the protein expression of CTGF was evaluated by Western blot; the mRNA expression of CTGF, Col I, and Col III was analyzed by real-time quantitative PCR; the content of hydroxyproline in lung tissue was measured, and the pathological changes of lung tissue of the poisoned rats were observed.

RESULTSThe protein expression of CTGF in the PQ group increased as the time went on, slowly from the 3rd to the 14th day and rapidly from the 28th to the 56th day, significantly higher than that in the control group at each time point (P < 0.05 or P < 0.01). The mRNA expression of CTGF in the PQ group began to rise markedly on the 1st day, increased rapidly from the 3rd to the 14th day, and remained at a relatively high level from the 28th to the 56th day, significantly higher than that in the control group at each time point (P < 0.01). The mRNA expression of Col I in the PQ group changed little on the 1st and 3rd day, increased slightly on the 7th day, and increased greatly from the 14th to the 56th day, significantly higher than that in the control group from the 7th to the 56th day (P < 0.05 or P < 0.01). The mRNA expression of Col III in the PQ group began to rise on the 1st day, reached the peak level on the 7th day, and then declined, significantly higher than that in the control group at each time point (P < 0.05 or P < 0.01). Masson staining showed that fibroblasts proliferated from the 14th to the 28th day, and collagen fibers increased gradually. Compared with the PQ group, the PQ + PDTC group showed significantly decreased protein expression of CTGF as well as mRNA expression of CTGF, Col I, and Col III (P < 0.05 or P < 0.01).

CONCLUSIONIn PQ-induced pulmonary fibrosis, the expression of CTGF keeps rising, and the collagen secretion and matrix synthesis are increased probably by upregulating the transcriptional levels of Col I and Col III; CTGF plays an important role in PQ-induced pulmonary fibrosis. PDTC can inhibit the expression of CTGF, thus reducing the lung injury in rats with PQ poisoning.

Animals ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Connective Tissue Growth Factor ; metabolism ; Male ; Paraquat ; poisoning ; Proline ; analogs & derivatives ; pharmacology ; Pulmonary Fibrosis ; chemically induced ; metabolism ; Rats ; Rats, Sprague-Dawley ; Thiocarbamates ; pharmacology

Recent epidemiologic studies clearly showed that early intensive glucose control has a legacy effect for preventing diabetic macrovascular complications. However, the cellular and molecular processes by which high glucose leads to macrovascular complications are poorly understood. Vascular smooth muscle cell (VSMC) dysfunction due to high glucose is a characteristic of diabetic vascular complications. Activation of nuclear factor-kappaB (NF-kappaB) may play a key role in the regulation of inflammation and proliferation of VSMCs. We examined whether VSMC proliferation and plasminogen activator inhibitor-1 (PAI-1) expression induced by high glucose were mediated by NF-kappaB activation. Also, we determined whether selective inhibition of NF-kappaB would inhibit proliferation and PAI-1 expression in VSMCs. VSMCs of the aorta of male SD rats were treated with various concentrations of glucose (5.6, 11.1, 16.7, and 22.2 mM) with or without an inhibitor of NF-kappaB or expression of a recombinant adenovirus vector encoding an IkappaB-alpha mutant (Ad-IkappaBalphaM). VSMC proliferation was examined using an MTT assay. PAI-1 expression was assayed by real-time PCR and PAI-1 protein in the media was measured by ELISA. NF-kappaB activation was determined by immunohistochemical staining, NF-kappaB reporter assay, and immunoblotting. We found that glucose stimulated VSMC proliferation and PAI-1 expression in a dose-dependent manner up to 22.2 mM. High glucose (22.2 mM) alone induced an increase in NF-kappaB activity. Treatment with inhibitors of NF-kappaB such as MG132, PDTC or expression of Ad-IkappaB-alphaM in VSMCs prevented VSMC proliferation and PAI-1 expression induced by high glucose. In conclusion, inhibition of NF-kappaB activity prevented high glucose-induced VSMC proliferation and PAI-1 expression.
Animals ; Aorta/cytology ; Cardiovascular Diseases/prevention & control ; Cell Proliferation/*drug effects ; Cells, Cultured ; Diabetes Complications/prevention & control ; Gene Expression Regulation/drug effects ; Glucose/immunology/*metabolism ; Leupeptins/pharmacology ; Male ; Muscle, Smooth, Vascular/*cytology ; Myocytes, Smooth Muscle/cytology/*drug effects/immunology/metabolism ; NF-kappa B/*antagonists & inhibitors/immunology ; Plasminogen Activator Inhibitor 1/*genetics ; Proline/analogs & derivatives/pharmacology ; Rats ; Rats, Sprague-Dawley ; Thiocarbamates/pharmacology

OBJECTIVETo investigate the relationship between activation of nuclear factor-K-gene binding (NF-κB) and apoptosis induced by matrine(MT) in transplanted tumor of human hepatocellular carcinoma in nude mouse.

METHODSTumors were established by injection of hepatocellular carcinoma cell line HepG2 into the back of nude mice. The mice were divided randomly into four groups: Control group, MT group (35 mg/kg), PDTC group (120 mg/kg) and Combination group: PDTC + MT group (120 mg/kg + 35 mg/kg), the reagents were injected peritoneally. The tumor growth curve of nude mice bearing transplanted tumor were observed and the inhibition ratios were evaluated. Apoptosis of carcinoma cells was analyzed by TUNEL. The DNA-binding activity of NF-κB was determined by electrophoretic mobility shift assay (EMSA). Expression of bcl-2 and bax in carcinoma tissue were detected by immunohistochemical method. NF-κB mRNA, bcl-2 mRNA and bax mRNA in carcinoma tissue were detected by RT-PCR.

RESULTSPyrrolidine dithiocarbamate (PDTC) could enhance the inhibition of matrine on carcinoma proliferation (P < 0.05). The apoptosis and activation of NF-κB in carcinoma cells could be induced by matrine. PDTC significantly suppressed NF-κB activation induced by matrine in carcinoma cells from 93.64 ± 2.95 to 65.78 ± 5.65 (F = 124.754, P < 0.01). Meanwhile, PDTC increased the apoptosis induced by matrine from 55.9% ± 2.8% to 74.3% ± 4.8% (P < 0.05).A positive correlation observed between the expressions of NF-κB and of bcl-2 (Pearson correlation coefficient = 0.983, P < 0.01).

CONCLUSIONSMatrine could induce apoptosis and activation of NF-κB in transplanted tumor. PDTC could increase apoptosis in hepatocellular carcinoma cells might be due to the suppression of NF-κB activation and the enhancement of bcl-2 expression.

Alkaloids ; pharmacology ; Animals ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; NF-kappa B ; metabolism ; Neoplasm Transplantation ; Pyrrolidines ; pharmacology ; Quinolizines ; pharmacology ; Thiocarbamates ; pharmacology