OBJECTIVES: To evaluate the protective effect of Bufei Yishen Formula (BYF) against cigarette smoke extract (CSE)-induced injuries in human bronchial epithelial BEAS-2B cells and explore the underlying mechanism. METHODS: BEAS-2B cells exposed to CSE were treated with normal rat serum, BYF-medicated rat serum at low or high doses, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), PDTC combined with high-dose BYF-medicated serum, or S-carbomethyloysteine (S-CMC, as the positive control). CCK-8 assay was used to determine the optimal concentration and treatment time of CSE, BYF-medicated serum and S-CMC. The treated cells were examined for inflammatory factor levels in the supernatant and cellular expressions of MUC5AC and MUC5B using ELISA, cell ultrastructural changes with transmission electron microscopy, and cell apoptosis rate using flow cytometry. The expression levels of TLR4/NF‑κB pathway-associated mRNAs and proteins were determined by qRT-PCR and Western blotting. RESULTS: CSE exposure significantly increased secretions of IL-1β, IL-6 and TNF-α, mRNA and protein expressions of MUC5AC and MUC5B, and early and total apoptosis rates in BEAS-2B cells, where the presence of apoptotic bodies was detected. CSE also significantly enhanced the mRNA and protein expressions of TLR4, I-κB, and NF-κB and reduced mRNA and protein expressions of AQP5. Treatments of the CSE-exposed cells with BYF-medicated serum, PDTC and S-CMC all significantly lowered inflammatory factor levels, MUC5AC and MUC5B expressions, and early and total cell apoptosis rates, and partly reversed the changes in cellular ultrastructure and mRNA and protein expressions of the TLR4/NF-κB pathway, and the effects were the most conspicuous following the combined treatment with high-dose BYF-medicated serum and PDTC. CONCLUSIONS BYF can inhibit cell apoptosis, inflammation and mucus hypersecretion in CSE-induced BEAS-2B cells by inhibiting the TLR4/NF-κB signaling pathway.
Humans ; Epithelial Cells/cytology* ; Drugs, Chinese Herbal/pharmacology* ; NF-kappa B/metabolism* ; Bronchi/cytology* ; Smoke/adverse effects* ; Apoptosis/drug effects* ; Mucin 5AC/metabolism* ; Cell Line ; Toll-Like Receptor 4/metabolism* ; Mucin-5B/metabolism* ; Signal Transduction/drug effects* ; Nicotiana ; Rats ; Thiocarbamates/pharmacology* ; Animals

Tetanic stimulation of the sciatic nerve (TSS) triggers long-term potentiation in the dorsal horn of the spinal cord and long-lasting pain hypersensitivity. CX3CL1-CX3CR1 signaling is an important pathway in neuronal-microglial activation. Nuclear factor κB (NF-κB) is a key signal transduction molecule that regulates neuroinflammation and neuropathic pain. Here, we set out to determine whether and how NF-κB and CX3CR1 are involved in the mechanism underlying the pathological changes induced by TSS. After unilateral TSS, significant bilateral mechanical allodynia was induced, as assessed by the von Frey test. The expression of phosphorylated NF-κB (pNF-κB) and CX3CR1 was significantly up-regulated in the bilateral dorsal horn. Immunofluorescence staining demonstrated that pNF-κB and NeuN co-existed, implying that the NF-κB pathway is predominantly activated in neurons following TSS. Administration of either the NF-κB inhibitor ammonium pyrrolidine dithiocarbamate or a CX3CR1-neutralizing antibody blocked the development and maintenance of neuropathic pain. In addition, blockade of NF-κB down-regulated the expression of CX3CL1-CX3CR1 signaling, and conversely the CX3CR1-neutralizing antibody also down-regulated pNF-κB. These findings suggest an involvement of NF-κB and the CX3CR1 signaling network in the development and maintenance of TSS-induced mechanical allodynia. Our work suggests the potential clinical application of NF-κB inhibitors or CX3CR1-neutralizing antibodies in treating pathological pain.
Animals ; Antibodies ; therapeutic use ; Antioxidants ; therapeutic use ; CX3C Chemokine Receptor 1 ; immunology ; metabolism ; Cytokines ; metabolism ; Disease Models, Animal ; Enzyme Inhibitors ; therapeutic use ; Ganglia, Spinal ; drug effects ; metabolism ; Hyperalgesia ; etiology ; metabolism ; Nerve Tissue Proteins ; metabolism ; Pain Threshold ; physiology ; Physical Stimulation ; adverse effects ; Proline ; analogs & derivatives ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; physiology ; Signal Transduction ; physiology ; Spinal Cord ; drug effects ; metabolism ; Thiocarbamates ; therapeutic use ; Up-Regulation ; drug effects ; physiology

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

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

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 establish a method for determining 2, 4-D butylate in serum by gas chromatography (GC)and to provide a basis for the diagnosis and treatment of clinical poisoning.

METHODSSerum 2, 4-D butylate level was determined by the following steps: mixing serum (0.5 ml)with trichloromethane (2.0 ml), adequately shaking for extraction, standing for 5 min, centrifuging at 4 000 rpm for 10 min, blow-drying the trichloromethane layer with nitrogen, adding ethanol (50 µl)to a certain volume, adding the sample (1.0 µl), and performing GC with a hydrogen flame ionization detector.

RESULTSSerum 2, 4-D butylate level showed a linear relationship within 5∼40 µg/ml, with a regression equation of y = 1 831.6.4x-899.4 (r = 0.999 2); the minimum detectable concentration was 1.0 µg/ml. The recovery rate was 88.7%∼103.0% (relative standard deviation (RSD) 3.8%∼5.0%). The intra-day RSD and inter-day RSD were 3.87-4.92% and 3.33%∼5.34%, respectively.

CONCLUSIONThis determination method is simple, efficient, and accurate and provides a good means for rapid diagnosis and treatment of 2, 4-D butylate poisoning.

Chromatography, Gas ; methods ; Humans ; Serum ; chemistry ; Thiocarbamates ; blood

Adult ; Delirium ; chemically induced ; Female ; Humans ; Thiocarbamates ; poisoning

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

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