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

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 study the role of oxiracetam on traumatic brain injury in rats.

METHODSThirty Wistar rats were randomly divided into 3 groups: sham operation group, model group and treatment group. Feeney method were used to establish traumatic brain injury (TBI) model in rats in model and treatment group, and rats in sham group were only broached without hydraumatic fitted. Rats in treatment group were successive administration for 21 days with oxiracetam (100 mg/kg, ig). Neurologic impairment scores were undertook after operation of 1 d, 4 d, 7 d, 14 d and 21 d, and Morris water maze test were proceeded during 15 to 19 days after operation. Average escape latency, searching time in target quadrant and number of crossing target platform in rats were recorded.

RESULTSNeurologic impairment scores of rats in treatment group were significantly less than those of model group after operation of 7, 14 and 21 d (P < 0.05). Average escape latency of model group were significantly higher than those of sham operation group and treatment group (P < 0.05, P < 0.01). Searching time in target quadrant and number of crossing target platform of model group were lower than those of sham operation and treatment group (P < 0.05)).

CONCLUSIONOxiracetam could decrease neural injury and increase ability of learning, memory and space cognition in traumatic brain injury rats.

Animals ; Brain Injuries ; drug therapy ; psychology ; Male ; Maze Learning ; drug effects ; Pyrrolidines ; pharmacology ; therapeutic use ; Rats ; Rats, Wistar

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

This study was purposed to investigate the relationship between activation of nuclear factor-κB (NF-κB) and multidrug resistance in K562/AO₂ cells and its mechanism. Human erythroleukemic cell line K562 and its adriamycin-resistant counterpart K562/AO₂ cells were used in the study. After inhibiting the activation of NF-κB with noncytotoxic concentration of antioxidant pyrrolidine dithiocarbamate (PDTC) in vitro, the multiple of drug resistance of K562/AO₂ cells was assessed by MTT assay. RT-PCR and flow cytometry method were used to detect the relative expression of mdr-1 mRNA and P-gp, respectively. The results showed that (1) multidrug resistance of K562/AO₂ cells to ADM was 59 times higher than that of K562 cells. When being pretreated with 0.2 μmol/L PDTC which is noncytotoxic to cells, the IC₅₀ of ADM in K562/AO₂ cells was sharply decreased with relative reverse efficiency of 93.03%, which was more higher than that of classic modifying agents Verapamil (Ver); (2) NF-κB activity of K562/AO₂ cells was significantly higher than that of K562 cells (p < 0.01). When being treated with PDTC, the activation of NF-κB was sharply decreased in K562/AO₂ cells; with 0.2 μmol/L PDTC for 24 hours it decreased to the lowest, nearly to the K562 cell level (p > 0.05); (3) the relative expression of both mdr-1 mRNA and P-gp in K562/AO₂ cells was more higher; the expressions of mdr-1 mRNA and P-gp both were inhibited in K562/AO₂ cell group treated with PDTC for 48 hours. It is concluded that the PDTC used as an inhibitor of NF-κB activity can partially reverse the multidrug resistance of K562/AO₂ cells, which mechanism can be associated with the down-regulation of mdr-1 mRNA and P-gp.
ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans ; K562 Cells ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Protease Inhibitors ; pharmacology ; Pyrrolidines ; pharmacology ; Thiocarbamates ; pharmacology

This investigation was made with special reference to the effect of the combined use of nuclear factor-kappaB (NF-kappaB) inhibitor Pyrrolidine dithiocarbamate(PDTC) and Paclitaxel on the expression of Matrix metalloproteinases (MMP-9) and its inhibitor TIMP-1, and on the proliferation and invasion of human breast cancer cell line MCF-7. The MCF-7 cells were treated with PDTC and Paclitaxel. The effect on proliferation was evaluated by MTT assay. The cell cycle was analyzed by flow cytometry. Western blot was used to determine the change of NF-kappaB p65, MMP-9 and TIMP-1 expression in MCF-7 cells after treatment. RT-PCR was used to detect NF-kappaB p65 mRNA expression. The invasion ability of MCF-7 cells was tested by the invasion, migration and cell adhesion assay. The cell growth was significantly slowed down and the cell cycle was arrested at G0/G1 phase after the combined treatment. The expression of NF-kappaB p65 and MMP-9 was down-regulated and the invasion ability of MCF-7 cells was decreased after the combined treatment. In conclusion,PDTC combined with paclitaxel effectively inhibited cell proliferation, induced cell cycle arrest, and decreased cell invasion ability of breast cancer MCF-7 cells. The mechanism may be associated with the inhibiting effect of PDTC on the NF-kappaB-related gene expression.
Antineoplastic Agents, Phytogenic ; pharmacology ; Breast Neoplasms ; pathology ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; drug effects ; Drug Synergism ; Female ; Humans ; NF-kappa B ; antagonists & inhibitors ; Neoplasm Invasiveness ; Paclitaxel ; pharmacology ; Pyrrolidines ; pharmacology ; Thiocarbamates ; pharmacology

Influenza is a major threat to millions of people worldwide. Vaccines and antiviral agents are two main options available to reduce the impact of the influenza virus, while anti-influenza agents are the most effective means to prevent the transmission of the highly contagious virus and to treat the epidemics of disease. At present, four anti-influenza agents have been approved by the FDA for the treatment of influenza, including two M2 protein ion channel inhibitors-amantadine and rimantadine and two neuraminidase inhibitors-zanamivir and oseltamivir. Arbidol hydrochloride, launched in Russia, is a potent inhibitor of influenza virus, too. Neuraminidase inhibitors could be classified generally by structure into six different kinds: sialic acid derivatives, benzoic acid derivatives, cyclohexene derivatives, cyclopentane derivatives, pyrrolidine derivatives and natural products. In this paper, recent progress in the research of the action mechanisms and structure-activity relationships of these anti-influenza virus agents were reviewed.
Amantadine ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Antiviral Agents ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Cyclopentanes ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Guanidines ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Humans ; Indoles ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Influenza, Human ; drug therapy ; Neuraminidase ; antagonists & inhibitors ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Orthomyxoviridae ; drug effects ; Oseltamivir ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Pyrrolidines ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Rimantadine ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Structure-Activity Relationship ; Viral Matrix Proteins ; antagonists & inhibitors ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Zanamivir ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use

OBJECTIVETo develop a technology for production of recombinant SAG1 of Toxoplasma gondii(T.g) in batches.

METHODSTwelve healthy mongrel dogs undergoing CPB were randomly allocated into control group (group C, n=6) and PDTC pretreatment group (group P, n=6). In group P, the dogs received intravenous injection of PDTC at 30 mg/kg before CPB, while in group C, normal saline was given instead. The myocardial tissues were obtained before CPB, 60 min after aortic cross-clamping (AC) and 60 min after declamping (DC) for determining the myocardial contents of adenine nucleotides (ATP, ADP, AMP, TAN, EC) and malondialdehyde (MDA) and evaluating the total anti-oxidation capacity (T-AOC) and mitochondrial swelling degree (MSD). The heart rate (HR), mean arterial pressure (MAP) and cardiac output (CO) were monitored before CPB, 30 min and 60 min after DC.

RESULTSIn both groups, the myocardial contents of ATP, TAN, EC and T-AOC decreased while MDA content and MSD increased after AC as compared to the values before CPB (P<0.01). In group C, ATP, TAN, EC and T-AOC decreased while MDA content and MSD increased after DC as compared to the values before CPB (P<0.01). At 60 min after DC, the dogs in group P showed no significant variation in the contents of ATP, TAN, EC, MDA, T-AOC or MSD (P>0.05). ATP, TAN, EC and T-AOC were significantly lowered while MDA and MSD increased at 60 min after AC and after DC in group P in comparison with the measurements in group C (P<0.01). HR, MAP and CO of group P recovered rapidly at 30 min and 60 min after DC as compared with those in group C (P<0.01).

CONCLUSIONCPB can induce serious energy exhaustion and delay in the recovery of energy metabolism. PDTC pretreatment can substantially ameliorate myocardial energy depletion and protect the myocardial mitochondria to attenuate myocardial ischemia/reperfusion injury.

Animals ; Antioxidants ; pharmacology ; Cardiopulmonary Bypass ; Dogs ; Energy Metabolism ; drug effects ; Female ; Male ; Myocardial Reperfusion Injury ; metabolism ; prevention & control ; Myocardium ; metabolism ; Postoperative Complications ; metabolism ; prevention & control ; Preoperative Care ; methods ; Pyrrolidines ; pharmacology ; Random Allocation ; Thiocarbamates ; pharmacology