Pyrrolidine Dithiocarbamate Treatment Research Articles

PDTC improves cognitive impairment in LPS-induced ARDS by regulating miR-181c/NF-κB axis-mediated neuroinflammation

ABSTRACT Background Cognitive impairment is a severe complication of acute respiratory distress syndrome (ARDS). Emerging studies have revealed the effects of pyrrolidine dithiocarbamate (PDTC) on improving surgery-induced cognitive impairment. The major aim of the study was to investigate whether PDTC protected against ARDS-induced cognitive dysfunction and to identify the underlying mechanisms involved. Methods The rat model of ARDS was established by intratracheal instillation of lipopolysaccharide (LPS), followed by treatment with PDTC. The cognitive function of rats was analyzed by the Morris Water Maze, and pro-inflammatory cytokines were assessed by quantitative real-time PCR, enzyme-linked immunosorbent assay, and western blot assays. A dual-luciferase reporter gene assay was performed to identify the relationship between miR-181c and its target gene, TAK1 binding protein 2 (TAB2). Results The results showed that PDTC improved cognitive impairment and alleviated neuroinflammation in the hippocampus in LPS-induced ARDS model. Furthermore, we demonstrated that miR-181c expression was downregulated in the hippocampus of the ARDS rats, which was restored by PDTC treatment. In vitro studies showed that miR-181c alleviated LPS-induced pro-inflammatory response by inhibiting TAB2, a critical molecule in the nuclear factor (NF)-κB signaling pathway. Conclusion PDTC improves cognitive impairment in LPS-induced ARDS by regulating miR-181c/NF-κB axis-mediated neuroinflammation, providing a potential opportunity for the treatment of this disease.

The Correlation between Lung Ultrasound and Pathology in Rat Model of Monocrotaline-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) is a progressive and complex pulmonary vascular disease with poor prognosis. The aim of this study was to provide a new understanding of the lung pathology of disease and a noninvasive method in monitoring the establishment of animal models for basic and clinical studies of PH, indeed to explore clinical application value of lung ultrasound for patients with PH. Totally 32 male SD rats were randomly divided into control group, MCT (monocrotaline) group, PDTC (pyrrolidine dithiocarbamate) group, and NS (normal saline) group. Rats in the MCT group, PDTC group, and NS group received single intraperitoneal injection of MCT, while the control group received the same dose of NS. Then, PDTC group and NS group received PDTC and NS daily for treatment at the end of the model. Each group received lung ultrasound examination and measurement of pulmonary arterial pressure (PAP). Then, the rats were sacrificed to take the lung specimens to being observed. The ultrasound and pathological results were analyzed with a semiquantitative score. With the pulmonary artery pressure increases, the MCT group had a higher pulmonary ultrasound score and pathological score compared with the control group (p < 0.05). After PDTC treatment, the pulmonary ultrasound score and the pathological score decline (p < 0.05). We investigated both lung ultrasound scores, and the pathological scores were positively correlated with mean pulmonary artery pressure (mPAP) (both r > 0.8, p < 0.0001). Moreover, lung ultrasound scores were positively correlated with pathological scores (r > 0.8, p < 0.0001). We elucidated lung ultrasound evaluation providing more evidence for the management of PH in the rat model. Moreover, lung ultrasound provided a noninvasive method in monitoring the establishment of animal models for basic and clinical studies of PH.

Therapeutic effects of salidroside vs pyrrolidine dithiocarbamate against severe acute pancreatitis in rat.

To evaluate the therapeutic effectiveness of salidroside (Sal) and pyrrolidine dithiocarbamate (PDTC) against severe acute pancreatitis (SAP) in a rat model. Rat models of SAP were established by retrograde infusion of sodium taurocholate solution. SAP rats were randomly divided into 6 groups: SAP 3 h group, SAP 24 h group, low-dose Sal treatment group (Sal L+S), middle-dose Sal treatment group (Sal M+S), high-dose Sal treatment group (Sal H+S) and PDTC treatment group (PDTC+S). The serum amylase, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-10 (IL-10) levels were determined by optical turbidimetry and enzyme-linked immunosorbent assay. The expression of Beclin-1, microtubule-associated protein light chain 3II (LC3 II ), lysosome associated membrane protein 2 (LAMP2), interleukin-1 receptor associated kinase 1 (IRAK1) inhibitor α of nuclear transcription factor-kB (IkBα), nuclear transcription factor-kB 65 (p65) in the pancreas tissues were detected by quantitative real-time polymerase chain reaction and Western blot, while the pIkBα and p-p65 levels were detected by Western blot. Pathological changes of the pancreas and all the other indexes were observed at 3 and 24 h after operation. The serum IL-10 level, IkBα and LAMP2 levels in Sal M+S, Sal H+S and PDTC+S groups were higher than those in SAP 24 h group, while all the other indexes in these three groups were all lower significantly than those in SAP 24 h group. There was no significant difference in all indexes between Sal H+S and PDTC+S groups. High-dose Sal has an effectively therapeutic effect on SAP in rats, which was similar to PDTC.

MiR-142a-3p Enhances FlaA N/C Protection Against Radiation-Mediated Intestinal Injury by Modulating the IRAK1/NF-κB Signaling Pathway

Our purpose was to investigate the role of recombinant protein flagellin A N/C (FlaA N/C) protein-mediated pyroptosis inhibition and related miRNA in radiation protection. Mice received 10 Gy irradiation after FlaA N/C pretreatment, IRAK-1/4 Inhibitor I treatment, or pyrrolidine dithiocarbamate treatment. Human intestinal epithelial cells (HIEC) received 10 Gy irradiation after FlaA N/C pretreatment, overexpressed miR-142a-3p with miR-142a-3p mimics, or inhibited miR-142a-3p with miR-142a-3p inhibitor. Mouse & Rat miRNA OneArray determined the change in relevant miRNA after FlaA N/C pretreatment; real-time polymerase chain reaction detected IRAK1 and miR-142a-3p expression; a CCK-8 assay evaluated cell viability; LDH release analyzed cytotoxicity; caspase-1 activity assay, interleukin-1β level, and flow cytometry analyzed pyroptosis in cells; hematoxylin-eosin staining evaluated the damage to intestinal tissue; CO-IP detected the inflammation activation; immunohistochemistry, Western blot analysis, and immunofluorescence analyzed activation of pyroptosis-related proteins and the activation of NF-kB signaling pathways; and luciferase reporter assay and fluorescence in situ hybridization detected the interaction between miR-142a-3p and IRAK1. FlaA N/C reduced radiation-induced pyroptosis in vivo and in vitro, and miR-142a-3p expression increased after FlaA N/C pretreatment. Upregulating the expression of miR-142a-3p inhibited radiation-induced pyroptosis in HIEC, and downregulating the expression of miR-142a-3p led to radiation-induced pyroptosis in HIEC after FlaA N/C pretreatment. IRAK1 was a direct target of miR-142a-3p and played an important role in radiation-induced pyroptosis in HIEC. Inhibiting IRAK1 reduced radiation-mediated pyroptosis in mice intestines. miR-142a-3p downregulated IRAK1 and suppressed the NF-kB pathway. Inhibiting the NF-kB signaling pathway can reduce radiation-mediated pyroptosis in mice intestines. Our findings indicated this new radioprotectant protein regulates miR-142a-3p, effectively inhibiting radiation-induced pyroptosis mediated by the IRAK1/NF-κB signaling pathway in intestinal cells.

The protective effect of NF-κB signaling pathway inhibitor PDTC on mice with chronic atrophic gastritis

Objective To understand the protective effect of NF-κB signaling pathway inhibitor pyrrolidinedithiocarbamate (PDTC) on mice with chronic atrophic gastritis (CAG). Methods Helicobacter pylori (H. pylori) infection combined with high-salt diet was used to construct the CAG mouse model, and 100 or 200 mg/kg/day PDTC was intragastrically treated for 8 weeks. Then, hematoxylin and eosin (HE) and Alcian blue-periodic acid-Schiff (AB-PAS) staining were used to observe the pathology of gastric mucosa, while immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immuno sorbent assay (ELISA) and western blotting were determined to detect the expression of related molecules. Results The nuclear content of NF-κB p65 in the gastric mucosa of the CAG mice was increased accompanying by the structural disorder of the gastric mucosal epithelium, inflammatory cell infiltration, intestinal metaplasia, and increased MUC2 expression, but the symptoms were alleviated after PDTC treatment. In addition, the expressions of TNF-α, IL-1β, IL-6 and COX2 in the gastric mucosa and serum of CAG mice were higher than those control mice, which were reduced in CAG mice treated with either 100 or 200 mg/kg PDTC. Furthermore, 100 mg/kg and 200 mg/kg PDTC treatments reduced the serum PGE2 in CAG mice with the decreased PCNA and Ki-67 expression in gastric mucosa. The therapeutic effect of 200 mg/kg PDTC was significantly better than that of 100 mg/kg PDTC. Conclusion PDTC inhibited inflammation and the excessive proliferation of gastric mucosal epithelial cells, thereby exerting a potential therapeutic effect on CAG.

The ROS scavenger PDTC affects adaptation to treadmill running in mice: distinct effects on murine body mass, resting heart rate and skeletal muscle fiber type composition.

Regular exercise induces a broad spectrum of adaptation reactions in a variety of tissues and organs. However, the respective mechanisms are incompletely understood. In the context of their analysis, animal model systems, specifically rodent treadmill running protocols, play an important role. However, few researchers have studied different aspects of adaptation, such as cardiorespiratory and skeletal muscle training effects, within one set of experiments. Here, we analyzed physiological adaptation to 10weeks of regular, moderate-intensity, uphill treadmill running in mice, a widely used model for endurance exercise training. To study the effects of reactive oxygen species (ROS), which have been suggested to be major regulators of training adaptation, a subgroup of mice was treated with the ROS scavenger PDTC (pyrrolidine dithiocarbamate). We found that mass gain in mice that exercised under PDTC treatment lagged behind that of all other experimental groups. In addition, both exercise and PDTC significantly and additively decreased resting heart rate. Furthermore, there was a trend towards an enhanced proportion of type 2A skeletal muscle fibers and differential expression of metabolism-associated genes, indicating metabolic and functional adaptation of skeletal muscle fibers. By contrast, there were no effects on grip strength and relative mass of individual muscles, suggesting that our protocol of uphill running did not increase skeletal muscle hypertrophy and strength. Taken together, our data suggest that a standard protocol of moderate-intensity uphill running induces adaptation reactions at multiple levels, part of which might be modulated by ROS, but does not enhance skeletal muscle hypertrophy and force.

Upregulation of miR-335-3p by NF-κB Transcriptional Regulation Contributes to the Induction of Pulmonary Arterial Hypertension via APJ during Hypoxia.

Pulmonary arterial hypertension (PAH) is a cardiopulmonary disease that can lead to heart failure and eventually death. MicroRNAs (miRs) play essential roles during PAH progression; however, their exact mechanism of action remains unclear. Apelin is a small bioactive peptide with a key protective function in the pathogenesis of PAH mediated by binding to the APJ gene. The aim of the present study was to investigate the role of miR-335-3p in chronic normobaric hypoxia (CNH)-induced PAH in mice and the potential underlying regulatory mechanism. Adult male C57BL/6 mice were exposed to normoxia (~21% O2) or CNH (~10% O2, 23 h/d) for 5 weeks. MiR-335-3p was significantly increased in lung tissue of CNH-induced PAH mice. Blocking miR-335-3p attenuated CNH-induced PAH and alleviated pulmonary vascular remodeling. Bioinformatics analysis and luciferase reporter assay indicated that nuclear factor-kappa beta (NF-κB) acted as a transcriptional regulator upstream of miR-335-3p. Pyrrolidine dithiocarbamate treatment reversed the CNH-induced increase in miR-335-3p expression and diminished CNH-induced PAH. Moreover, p50-/- mice were resistant to CNH-induced PAH. Finally, APJ was identified as a direct targeting gene downstream of miR-335-3p, and pharmacological activation of APJ by its ligand apelin-13 reduced CNH-induced PAH and improved pulmonary vascular remodeling. Our results indicate that NF-κB-mediated transcriptional upregulation of miR-335-3p contributes to the inhibition of APJ and induction of PAH during hypoxia; hence, miR-335-3p could be a potential therapeutic target for hypoxic PAH.

Protective effect of pyrrolidine dithiocarbamate on isoniazid/rifampicin‑induced liver injury in rats.

Isoniazid (INH) and rifampicin (RIF) continue to be first line anti‑tuberculosis (TB) drugs. However, the use of these drugs is associated with hepatotoxicity. Nuclear factor‑κB (NF‑κB) plays a crucial role in regulating immunity and inflammation. It has been reported that pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF‑κB, exerts a hepatoprotective effect on acute and chronic liver damage. The aim of the present study was to explore the INH/RIF‑induced protective effects and mechanisms of PDTC on liver injury. Rats were intragastrically administered INH (50mg/kg/day) and RIF (50mg/kg/day) daily for 28days. PDTC (50mg/kg/day) was intraperitoneally injected 2h after the co‑administration of INH and RIF to compare liver biochemical indicators in the serum, histopathological damage, NF‑κB activity, oxidative stress, hepatic mRNA expression of tumor necrosis factor (TNF)‑α, bile salt export pump (BSEP), and protein expression of BSEP. It was found that the inhibition of NF‑κB activation by PDTC treatment markedly alleviated liver biochemical and histological injury, decreased oxidative stress and mRNA levels of TNF‑α, and prevented decreases in BSEP mRNA and protein expression induced by the co‑administration of INH and RIF. Collectively, the present data suggested that INH/RIF‑induced liver injury is dependent on the activation of NF‑κB. PDTC exerted a therapeutic effect on INH/RIF‑induced liver injury by increasing BSEP expression, and exhibiting antioxidant and anti‑inflammatory activities.

Pyrrolidine Dithiocarbamate Attenuates Cardiocyte Apoptosis and Ameliorates Heart Failure Following Coronary Microembolization in Rats

Background:Nuclear factor-kB is highly activated in cardiovascular disorders. However, few articles have targeted at the role of nuclear factor-kB inhibitor in heart failure.Aims:To evaluate the effects of nuclear factor-kB inhibitor pyrrolidine dithiocarbamate on cardiocyte apoptosis and cardiac function in a rat heart failure model.Study Design:Animal experiment.Methods:A stable and reproducible rat heart failure model (n=64) was prepared by injecting homologous microthrombotic particles into the left ventricle of Sprague–Dawley rats while obstructing the ascending aorta to produce coronary microembolization. Rats with heart failure were randomized into untreated (HFu) and pyrrolidine dithiocarbamate-treated (HFp) groups; the latter received an intraperitoneal injection of pyrrolidine dithiocarbamate (100 mg/kg/day) 1 h prior to surgery as well as on postoperative days 1-7. The sham group comprised 32 Sprague–Dawley rats. Eight rats from each group were sacrificed on days 1, 3, 7, and 14 postoperatively. Masson’s trichrome staining was used to determine the micro-fibrotic area to indicate the severity of myocardial loss. Terminal transferase uridine triphosphate nick end labeling staining was used to detect apoptotic cardiomyocytes. Echocardiography and hemodynamics were performed to evaluate left ventricular function.Results:Rats with heart failure exhibited pathological changes evidenced by patchy myocardial fibrosis, remarkably elevated severity of myocardial loss, and persistently reduced left ventricular function. At the end of the study, compared with the HFu group, myocardial infarct size was reduced by 28% (p=0.001), cardiocyte apoptosis was suppressed (7.17%±1.47% vs 2.83%±0.75%, p<0.001), cardiac function parameters such as left ventricular ejection fraction (80%±4% vs 61%±6%), left ventricular + dP/dt max (4828±289 vs 2918±76 mmHg.s−1), left ventricular - dP/dt max (4398±269 vs 2481±365 mmHg.s−1), and left ventricular systolic pressure (126±13 vs 100±10 mmHg) were significantly increased, and left ventricular end-diastolic pressure was reduced (18±2 vs 13±1 mmHg) (p<0.001, for all) in the HFu group.Conclusion:Our rat model can adequately mimic heart failure via coronary vessel embolization. Moreover, pyrrolidine dithiocarbamate treatment can reduce cardiocyte apoptosis and improve cardiac function, which may be beneficial for patients with heart failure secondary to myocardial infarction.

CXCL16/CXCR6 is involved in LPS-induced acute lung injury via P38 signalling.

Although several chemokines play key roles in the pathogenesis of acute lung injury (ALI), the roles of chemokine (C‐X‐C motif) ligand 16 (CXCL16) and its receptor C‐X‐C chemokine receptor type 6 (CXCR6) in ALI pathogenesis remain to be elucidated. The mRNA and protein expression of CXCL16 and CXCR6 was detected after lipopolysaccharide (LPS) stimulation with or without treatment with the nuclear factor‐κB (NF‐κB) inhibitor pyrrolidine dithiocarbamate (PDTC). Lung injury induced by LPS was evaluated in CXCR6 knockout mice. CXCL16 level was elevated in the serum of ALI patients (n = 20) compared with healthy controls (n = 30). CXCL16 treatment (50, 100, and 200 ng/mL) in 16HBE cells significantly decreased the epithelial barrier integrity and E‐cadherin expression, and increased CXCR6 expression, reactive oxygen species (ROS) production, and p38 phosphorylation. Knockdown of CXCR6 or treatment with the p38 inhibitor SB203580 abolished the effects of CXCL16. Moreover, treatment of 16HBE cells with LPS (5, 10, 20 and 50 μg/mL) significantly increased CXCL16 release as well as the mRNA and protein levels of CXCL16 and CXCR6. The effects of LPS treatment (20 μg/mL) were abolished by treatment with PDTC. The results of the luciferase assay further demonstrated that PDTC treatment markedly inhibited the activity of the CXCL16 promoter. In conclusion, CXCL16, whose transcription was enhanced by LPS, may be involved in ROS production, epithelial barrier dysfunction and E‐cadherin down‐regulation via p38 signalling, thus contributing to the pathogenesis of ALI. Importantly, CXCR6 knockout or inhibition of p38 signalling may protect mice from LPS‐induced lung injury by decreasing E‐cadherin expression.

519-P: Acute Kidney Injury Sensitivity in Diabetic Murine Model

Background: We recently reported that diabetes can upregulate two discrete isoforms of the MMP-2 (full length and N-terminal truncated, FL-MMP-2, NTT-MMP-2) in renal tubules and especially, inducible NTT-MMP-2 could be related to renal tubular damage. Also, pyrrolidinedithiocarbamate (PDTC), NF-Kb inhibitor, can suppress NTT-MMP-2 selectively. This study was conducted to analyze the effects of acute kidney injury on diabetic animal model and test the effect of PDTC in acute kidney injury with diabetes. Methods: We performed unilateral ischemia-reperfusion injury (uIRI) in streptozotocin-induced diabetic animals as a model of acute kidney injury and followed for 48 hours and six weeks. Mice were randomized into four groups as follows; control (A), non-DM+uIRI (B), DM+uIRI (C), DM+uIRI+PDTC (D). Results: In a 48-hour experiment, tubular necrosis was more severe in diabetic IR kidney compared with nondiabetic IR kidney. Outer medulla in group C was becoming devastated and led to the extensive coagulative necrosis. Those findings were diminished by PDTC. Peritubular capillary density was increased by PDTC and cortical macrophage density was decreased by PDTC compared to group B and C. The nitrotyrosin was lesser expressed by PDTC compared to group B and C. In a six-week experiment, IRI caused the kidney to be stiffen and shrink both in group B and C and PDTC can ameliorate these gross changes compared to group C. Peritubular capillary density was increased by PDTC compared to group C. Cortical macrophage density was higher than group B and D and PDTC ameliorate the recruitment of macrophage in renal cortex. The expression of cleaved caspase-3/caspase 3 in group C was higher than group B and D. Conclusion: Ischemia-reperfusion injury caused more intense renal damage in diabetic murine model and could lead to chronic renal change related to inflammation and apoptotic pathway. These changes may be mitigated by PDTC treatment associated with anti-oxidative mechanism and inhibition of NTT-MMP-2. Disclosure S. Song: None. S. Kim: None. I. Kim: None. Funding National Research Foundation of Korea (2016R1A2B4008243)

Overexpression of glutathione peroxidase-1 attenuates cocaine-induced reproductive dysfunction in male mice by inhibiting nuclear factor κB

Since reproductive toxicity is associated with oxidative stress, nuclear factor κB (NFκB), a redox-sensitive transcription factor, may be involved in the reproductive dysfunction induced by the abusive drug, such as cocaine. In the present study, we investigated whether NFκB mediates cocaine-induced reproductive dysfunction in male mice, and whether glutathione peroxidase (GPx)-1, a well-known enzymatic antioxidant, modulates NFκB activity to affect this reproductive dysfunction. Cocaine treatment significantly increased nuclear translocation of NFκB and its DNA binding activity in the testis of mice. Treatment with cocaine resulted in a significant increase in sperm abnormality, and in significant decreases in the sperm viability and sperm level. Furthermore, cocaine significantly reduced hypothalamic gonadotropin-releasing-hormone expression and plasma testosterone level. These alterations were more pronounced in the GPx-1 knockout (GPx-1 KO) than wild type (WT) mice, and they were less pronounced in GPx-1 overexpressing transgenic (GPx-1 TG) than in non-transgenic (non-TG) mice. Pyrrolidine dithiocarbamate (PDTC), an NFκB inhibitor, was more effective in attenuating cocaine-induced reproductive toxicity in GPx-1 KO than in WT mice. Although PDTC treatment was also significantly protective against the reproductive toxicity in non-TG mice, PDTC did not show additional positive effects against the protective potential mediated by GPx-1 overexpression in mice. Therefore, our results suggest that GPx-1 gene is a protective factor in response to reproductive dysfunction induced by cocaine in male mice, and that NFκB is a critical mediator of protective activity of GPx-1 gene in our experimental conditions.

Activation of NF-κB signaling pathway during HCG-induced VEGF expression in luteal cells.

Vascular endothelial growth factor (VEGF) plays an essential role in luteal angiogenesis, the present study therefore utilized luteal cells cultured in vitro to further investigate the activation and contribution of nuclear factor (NF)-κB to VEGF expression induced by human chorionic gonadotrophin (HCG). The present results showed HCG induced VEGF expression as well as hypoxia-inducible factor (HIF)-1α mRNA and protein expressions, which was blocked by NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Further analysis found that these increases of VEGF and HIF-1α mRNA induced by HCG were also blocked by NF-κB siRNA transfection, which was consistent with PDTC treatment. However, HIF-1α siRNA treatment significantly decreased HCG induced-VEGF expression with no effect on NF-κB mRNA expression. Furthermore, combination of HIF-1α siRNA and PDTC treatment did not further decrease VEGF mRNA expression, and the result of chromatin immunoprecipitation indicated NF-κB may regulate HIF-1α transcription through binding with its promoter. Taken together, the present results clearly demonstrated that NF-κB was activated to regulate VEGF expression by increasing HIF-1α transcription in luteal cells treated with HCG. Therefore, the present study provided a new and important mechanism of luteal angiogenesis during the formation of corpus luteum in mammals.

Progestin and AdipoQ Receptor 3 Upregulates Fibronectin and Intercellular Adhesion Molecule-1 in Glomerular Mesangial Cells via Activating NF-κB Signaling Pathway Under High Glucose Conditions.

Progestin and adipoQ receptor 3 (PAQR3), is a Golgi-anchored membrane protein containing seven transmembrane helices. It has been demonstrated that PAQR3 mediates insulin resistance, glucose and lipid metabolism, and inflammation. In addition, kidney inflammatory fibrosis is an important pathological feature of diabetic nephropathy (DN). Therefore, we aimed to investigate the role of PAQR3 in diabetic kidney fibrosis as well as inflammation in DN. The effect of PAQR3 on NF-κB signaling pathway, expressions of fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in glomerular mesangial cells (GMCs) cultured by high glucose (HG) were examined. Diabetic mouse and rat models were induced by streptozotocin (STZ). GMCs were treated with HG and transfected with PAQR3 plasmids or small-interfering RNA targeting PAQR3 or NF-κB. The protein levels of FN and ICAM-1 were examined by Western blotting, and the transcriptional activity and DNA binding activity of NF-κB were measured by dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). The interaction between PAQR3 and IKKβ (inhibitor of nuclear factor κB kinase β) was analyzed by co-immunoprecipitation. PAQR3 was increased in both STZ-induced diabetic models and HG-treated GMCs. PAQR3 overexpression further increased HG-induced FN and ICAM-1 upregulation. In contrast, silencing of PAQR3 suppressed the expressions of FN and ICAM-1. PAQR3 overexpression promoted the nuclear accumulation, DNA binding activity, and transcriptional activity of NF-κB. Mechanically, PAQR3 directly interacted with IKKβ. The upregulation effect of PAQR3 overexpression on the expressions of FN and ICAM-1 was abolished by the treatment of NF-κB siRNA or PDTC (ammonium pyrrolidinedithiocarbamate) in HG-treated GMCs. PAQR3 promotes the expressions of FN and ICAM-1 via activating NF-κB signaling pathway. Mechanistically, PAQR3 activates NF-κB signaling pathway to mediate kidney inflammatory fibrosis through direct interaction with IKKβ in DN.

Effects of 10weeks of regular running exercise with and without parallel PDTC treatment on expression of genes encoding sarcomere-associated proteins in murine skeletal muscle.

Physical exercise can induce various adaptation reactions in skeletal muscle tissue, such as sarcomere remodeling. The latter involves degradation of damaged sarcomere components, as well as de novo protein synthesis and sarcomere assembly. These processes are controlled by specific protease systems in parallel with molecular chaperones that assist in folding of newly synthesized polypeptide chains and their incorporation into sarcomeres. Since acute exercise induces oxidative stress and inflammation, leading to activation of the transcription factor NFκB (nuclear factor kappa B), we speculated that this transcription factor might also play a role in the regulation of long-term adaptation to regular exercise. Thus, we studied skeletal muscle adaptation to running exercise in a murine model system, with and without parallel treatment with the NFκB-inhibitory, anti-oxidant and anti-inflammatory drug pyrrolidine dithiocarbamate (PDTC). In control mice, 10weeks of uphill (15° incline) treadmill running for 60min thrice a week at a final speed of 14m/min had differential, but only minor effects on many genes encoding molecular chaperones for sarcomere proteins, and/or factors involved in the degradation of the latter. Furthermore, there were marked differences between individual muscles. PDTC treatment modulated gene expression patterns as well, both in sedentary and exercising mice; however, most of these effects were also modest and there was little effect of PDTC treatment on exercise-induced changes in gene expression. Taken together, our data suggest that moderate-intensity treadmill running, with or without parallel PDTC treatment, had little effect on the expression of genes encoding sarcomere components and sarcomere-associated factors in murine skeletal muscle tissue.

Effect of pyrrolidine dithiocarbamate on oxidative stress and mitochondrial function of lung tissue in mice with acute lung injury

To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on oxidative stress and mitochondrial function of lung tissue in mice with acute lung injury (ALI) induced by lipopolysaccharide (LPS). Forty female Balb/c mice were randomly divided into normal saline (NS) control group, LPS model group, PDTC group, and PDTC+LPS group, with 10 mice in each group. The model of mice with ALI was reproduced by intraperitoneal injection of 15 mg/kg LPS. PDTC was administered intraperitoneally with 50 mg/kg PDTC 1 hour before LPS treatment in the PDTC+LPS group. The mice in NS control group was given intraperitoneal injection of 0.1 mL NS only, and those in PDTC group was given intraperitoneal injection of 50 mg/kg PDTC only. The mice were sacrificed at 24 hours after model reproduction, and the lung tissues were harvested. The total antioxidant capacity (T-AOC) of lung tissue was measured by spectrophotometric kits. The content of malondialdehyde (MDA) was determined by thiobarbituric acid reactive substances assay. The protein expressions of superoxide dismutases (SOD1, SOD2) and catalase (CAT) in lung tissue were determined by Western Blot. Mitochondria from mouse lungs were isolated, and adenosine triphosphate (ATP) synthesis was measured with a luciferase/luciferin-based approach. The mitochondrial membrane potential (ΔΨm) was estimated by using Rhodamine. The mRNA expressions of mitochondrial uncoupling proteins (UCPs) were determined by reverse transcription-polymerase chain reaction (RT-PCR). LPS stimulation could significantly increase oxidative stress in lung tissue of mice and lead to mitochondrial dysfunction. The results showed that the protein expressions of T-AOC and SOD1 were decreased, the level of MDA was increased, the ATP synthesis was decreased in the mitochondrial, the ΔΨm was decreased, and the mRNA expression of UCP2 was decreased. However, there was no significant change in the expressions of SOD2, CAT in lung tissue and UCP1, UCP3 in the mitochondria. Pretreatment with PDTC could obviously alleviate the increase in LPS-induced oxidative stress in lung tissue and mitigate mitochondrial dysfunction. Compared with the LPS model group, T-AOC in lung tissue of PDTC+LPS group was significantly increased (U/g: 0.35±0.08 vs. 0.31±0.07), the level of MDA was significantly decreased (μmol/mg: 13.29±1.13 vs. 17.54±1.72), the protein expression of SOD1 was significantly upregulated (SOD1 protein: 1.13±0.11 vs. 0.71±0.09), ATP synthesis was significantly increased in the mitochondrial (μmol/mg: 49.23±5.42 vs. 36.92±2.21), ΔΨm was significantly increased (mV: 226.03±11.69 vs. 194.86±7.79), and the mRNA expression of UCP2 was significantly increased (2-ΔΔCt: 0.88±0.06 vs. 0.73±0.04). The differences were statistically significant (all P < 0.05). In lung tissue of normal mice, PDTC treatment also had the effect of anti-oxidizing, reducing oxidative stress and promoting ATP synthesis in the mitochondrial. Compared with the NS control group, the level of T-AOC (U/g: 0.49±0.09 vs. 0.43±0.06) and the protein expressions of SOD2 and CAT (SOD2 protein: 1.33±0.08 vs. 1.00±0.11, CAT protein: 1.39±0.08 vs. 1.00±0.11), and ATP synthesis in the mitochondrial of PDTC group was significantly increased (μmol/mg: 61.53±4.92 vs. 53.33±3.20), MDA was significantly decreased (μmol/mg: 10.27±1.25 vs. 12.27±1.36), with statistical differences, but had no effect on the protein expression of SOD1 in lung tissue and ΔΨm and UCPs mRNA expressions in mitochondrion. LPS can induce ALI in mice, increased oxidative stress in lung tissue, and induce mitochondrial dysfunction by inhibiting ATP synthesis. PDTC pretreatment has anti-oxidative effect on LPS-induced ALI, and can mitigate mitochondrial dysfunction.

Modulation of inflammation by toll-like receptor 4/nuclear factor-kappa B in diarrhea-predominant irritable bowel syndrome.

In order to investigate the function of toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signal pathways in the pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D), IBS-D animal models were established in wistar rats challenged with acute and chronic stresses (29 days). Wistar rats without stress-challenged were used as controls. IBS-D models were randomly divided into two groups: one was treated with normal saline, another group was treated with TLR4/NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) (50mg/kg/week) for continuous four times. Our results demonstrate that continuous stresses can induce the characteristic symptoms of IBS-D, including high wet stool rate and intestinal flora imbalance. Further examinations of colon tissues show that the protein expression levels of TLR4 and NF-κB in IBS-D groups are higher than that in control group. The secretory levels of interleukin (IL-8), tumor necrosis factor α (TNFα), and myeloid differentiation factor 88 (MyD88) are significantly increased in IBS-D group. Administration with PDTC effectively downregulates levels of these inflammatory factors. In contrast, interleukin-10 (IL-10) is in an opposite alteration with lower levels in IBS-D groups and the PDTC treatment increases it to the levels as in control group. Moreover, inhibition of the TLR4/NF-κB by PDTC improves the microstructure of intestinal mucosa mainly by increasing the height of villi. Our results suggest that TLR4/NF-κB signal pathway plays an important role in the modulation of inflammatory responses in IBS-D, which might be a therapeutic target for the IBS-D. All of these findings also provide the evidence concerning an inherent linkage between the axis of stress/NF-κB/inflammation and IBS-D.

Excessive Activation of TLR4/NF-\u03baB Interactively Suppresses the Canonical Wnt/\u03b2-catenin Pathway and Induces SANFH in SD Rats

Nuclear factor-kappa B (NF-κB) interactively affects the Wnt/β-catenin pathway and is closely related to different diseases. However, such crosstalk effect in steroid-associated necrosis of femoral head (SANFH) has not been fully explored and evaluated. In this study, early-stage SANFH was induced by two doses of lipopolysaccharide (LPS, 2 mg/kg/day) and three doses of methylprednisolone (MPS, 40 mg/kg/day). LPS and pyrrolidine dithiocarbamate (PDTC) were administered to activate the TLR4/NF-κB pathway and selectively block the activation of NF-κB, respectively. Results showed that PDTC treatment significantly reduced NF-κB expression, diminished inflammation, and effectively decreased bone resorption processes (osteoclastogenesis, adipogenesis, and apoptosis), which were evidently reinforced after osteonecrosis induction. Moreover, PDTC remarkably increased the interfered Wnt/β-catenin pathway and elevated bone formation processes (osteogenesis and angiogenesis). Ultimately, PDTC treatment effectively reduced the incidence of SANFH. Therefore, the excessive activation of TLR4/NF-κB may interactively suppress the Wnt/β-catenin pathway and induce SANFH. Hence, we propose NF-κB-targeted treatment as a novel therapeutic strategy for SANFH.

Effects of NF-κB inhibitor on nuclear factor-κB and intercellular adhesion moceule-1 in lung injury associated with radiation of rats

Objective: To investigate the mechanism of lung injury caused by radiation-induced lung injury by observing the change of nuclear factor (NF-κB) and intercellular adhesion moceule-1 (ICAM-1) in rats and the effects of pyrrolidine dithiocarbamate (PDTC) . Methods: 80 SD female rats were randomly classified into 4 groups: control group, radiation group, PDTC treatment group and PDTC group.The radiation induced pulmonary injury model was preformed by using 6 MV X-rays to deliver 8 Gy per day for 5 consecutive days with 40 Gy in total to the thorax of each animal.PDTC was given from 3 d before radiation to 30 d after the first radiation. Rats in control group and PDTC group received the same dose of saline. Animals were sacrificed at 8 week and 24 week after radiation, respectively. The lungs were removed and processed for HE and Masson staining, hydroxyproline content measurement, and real-time quantitative reverse transcription-polymerase chain (RT-PCR) ICAM-1mRNA and NF-κB p65mRNA were detected, Statistical analysis were carried out. Results: Compared with radiation group, there was an obvious amelioration in pathological injury of lung tissue in PDTC treatment group. The lung coefficient and the content of Hyp in PDTC treatment group were significantly lower than those in radiation group (t=3.651, 5.293, 2.348 and 4.126, respectively, allP<0.05) , while slightly higher than those in control group. The levels of ICAM-1mRNA and NF-κB p65mRNA were significantly higher in radiation group than that in PDTC treatment group (allP<0.05) , There were no significant differences in these indicators between control group and PDTC group (P>0.05) . Conclusion: The expression of ICAM-1mRNA and NF-κB p65mRNA is increased in rats with radiation-induced lung injury. Suggest that ICAM-1 and NF-κB are a key factor lead to radiation-induced lung injury. PDTC may inhibits NF-κB activity and further significantly deceases expression of ICAM-1, leading to significantly attenuated pulmonary inflammation and fibrosis, which provides a new therapeutic target for the prevention and treatment of radiation-induced lung injury.

Pyrrolidine dithiocarbamate sensitizes U251 brain glioma cells to temozolomide via downregulation of MGMT and BCL-XL.

The current study investigated the effect of pyrrolidine dithiocarbamate (PDTC) on the proliferation, apoptosis, cell cycle and sensitivity to temozolomide (TMZ) of the U251 glioma cell line. Proliferation, apoptosis and cell cycle analysis of U251 cells following treatment with PDTC and TMZ was determined by an MTT assay and flow cytometry, respectively. The mRNA and protein expression levels of O-6-methylguanine-DNA methyltransferase (MGMT), B-cell lymphoma extra-large (BCL-XL) and survivin were further determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting analysis. The results revealed that treatment with TMZ, PDTC and TMZ + PDTC significantly inhibited cell proliferation, induced apoptosis and contributed to cell cycle arrest in U251 cells. A combination of PDTC and TMZ induced the highest rates of proliferation inhibition and apoptosis. PDTC treatment markedly reduced the expression levels of MGMT, BCL-XL and survivin. The expression levels of MGMT and BCL-XL, were significantly upregulated by TMZ but not by combination treatment of TMZ and PDTC. The results of the present study suggest that treatment with PDTC inhibits cell proliferation, induces apoptosis and cell cycle arrest, and enhances sensitivity to TMZ in U251 cells, which is partly induced by downregulation of MGMT and BCL-XL.