Reduced Expression of Metallothionein-I/II in Renal Proximal Tubules Is Associated with Advanced Chronic Kidney Disease.

Chronic kidney disease (CKD) is a commonly occurring complex renal syndrome that causes overall mortality in many diseases. The clinical manifestations of CKD include renal tubulointerstitial fibrosis and loss of renal function. Metallothionein-I/II (MT-I/II) is potentially expressed in the liver and kidney, and possesses antioxidant and metal detoxification properties. However, whether MT-I/II expression is associated with the prognosis of nephropathy remains unknown. In this study, we investigated the MT-I/II level in human CKD, using immunohistochemistry. MT-I/II is located on the proximal tubules and is notably reduced in patients with CKD. MT-I/II expression was significantly correlated with the functional and histological grades of CKD. In an aristolochic acid (AAI)-induced nephropathy mouse model, MT-I/II was abundantly increased after AAI injection for 7 days, but decreased subsequently compared to that induced in the acute phase when injected with AAI for 28 days. Furthermore, we found that ammonium pyrrolidinedithiocarbamate (PDTC) restored AAI-induced MT-I/II reduction in HK2 cells. The injection of PDTC ameliorated AAI-induced renal tubulointerstitial fibrosis and reduced the concentrations of blood urea nitrogen and creatinine in mouse sera. Taken together, our results indicate that MT-I/II reduction is associated with advanced CKD, and the retention of renal MT-I/II is a potential therapeutic strategy for CKD.

USP47 promotes apoptosis in rat myocardial cells after ischemia/reperfusion injury via NF-κB activation.

An increasing number of studies have associated nuclear factor-κB (NF-κB) activation to the progression of myocardial infarction (MI). Multiple members of the ubiquitin-specific protease (USP) family regulate the NF-κB signaling pathway. This study attempted to investigate the function and underlying mechanism of USP family members in MI. Monocytes were isolated from peripheral blood samples of MI and healthy control, and the expression of several USP family members and NF-κB was examined. After USP47 knockdown or overexpression, cell apoptosis, related protein levels, and NF-κB promoter activity were measured. We found that USP47 and NF-κB expression was significantly increased in MI, and USP47 was positively correlated with NF-κB. In a rat H9c2 myocardial cell ischemia/reperfusion (I/R) injury model, USP47 expression was increased in a time-dependent manner. USP47 knockdown decreased I/R injury-induced apoptosis, with increased survivin and cytoplasmic NF-κB and decreased cleaved caspase-3 and nuclear NF-κB. USP47-induced apoptosis was potently counteracted by the NF-κB inhibitor, and cytoplasmic NF-κB was increased and nuclear NF-κB was decreased. Luciferase reporter showed that USP47 promotes NF-κB promoter activity. These results suggested that USP47 expression may be correlated with MI progression. USP47 knockdown attenuated myocardial cell I/R injury by inhibiting apoptosis.

Co-delivery of DOX and PDTC by pH-sensitive nanoparticles to overcome multidrug resistance in breast cancer.

Chemotherapeutic drugs have a series of limitations in the conventional clinical treatments, mainly including serious adverse effects and multidrug resistance (MDR). Herein, we developed a pH-sensitive polymeric nanoparticle with using poly(ortho ester urethanes) copolymers for co-delivering doxorubicin (DOX) and pyrrolidinedithiocarbamate (PDTC) to settle these problems. Dual-drug-loaded nanoparticles were nano-sized (˜220 nm) with the spherical morphology and excellent physiological stability. Both drugs both could be quickly released in the mild acidic conditions due to the cleavage of ortho ester bonds. Monolayer cultured cells (2D) and multicellular spheroids (3D) experiments proved that PDTC could reverse multidrug resistance (MDR), improve intracellular drugs accumulation and enhance tumor penetration by down-regulating the expression of P-gp, then resulting in higher DOX-induced cytotoxicity and apoptosis in MCF-7 and MCF-7/ADR cells. Besides, in vivo experiments further demonstrated that co-encapsulated nanoparticles had higher DOX accumulation and superiorer tumor growth inhibition (TGI 82.9%) than free drugs or single-drug-loaded nanoparticles on MCF-7/ADR bearing-mice. Accordingly, the pH-sensitive co-delivery systems possess a promising potential to overcome MDR in cancer therapy.

Inhibition of Nf-ҝb prevents trauma-induced heterotopic ossification in rat model.

PURPOSE: To investigate the pathogenesis and find a better prophylactic method of acquired heterotopic ossification (HO). MATERIALS AND METHODS: In the first part, we designed the brain-traumatic/burn/tenotomy rat model and testified its efficacy as HO model. 44 rats were randomly divided into experimental group and control group. After operation, the bilateral tendons of 2 rats were collected at the 2nd, 3rd, 4th, 6th, 8th, and 10th weeks to determine the expression levels of p65. Additionally, the remaining rats were exposed to X-Ray examination at the 10th week. In the second part, 124 rats were randomly divided into four groups based on the administration dosage of Ammonium pyrrolidinedithiocarbamate (PDTC). Then, three rats of each group were euthanized every week in the first seven weeks to collect tendon to detect the expression levels of p65 by qRT-PCR and Western Blot. The remaining rats were exposed to X-Ray examination at the 10th week to assess the size of HO before being euthanized for HE staining. RESULTS: The success rate of Brain-traumatic/Burn/Tenotomy model was 100%. Pharmacologic inhibition of Nf-ҝb signaling pathway by PDTC could significantly reduce the expression levels of p53 and the size of HO, and the reduction was most significant in the 0.6mg dosage group. CONCLUSIONS: Brain-traumatic/Burn/Tenotomy model was highly reliable HO model. Inhibition of Nf-ҝb signaling pathway by PDTC could significantly reduce HO formation, and the most effective concentration was 6 mg/ml for local injection.

Regulation of il-1 and TNF-α on metabolic enzyme cyp2e1 in immunological liver injury in rats / 中国药理学通报

Aim To investigate the regulation and mechanism of inflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) on the drug metabolism enzyme cytochrome P450 2E1 (CYP2E1) during immune-mediated liver injury. Methods By injection of Bacillus Calmette-Guerin(BCG, 125 mg·kg-1) prepared rat model of immunological liver damage. To detection of CYP2E1 probe drug chlorzoxazone in blood concentration changes of rats over time reflects its metabolic activity by high-performance liquid chromatography (HPLC) assay. The changes of IL-1β and TNF-α in liver tissue of rats were detected by ELISA. The expression of CYP2E1 protein was determined by Western blot analysis. Results After 14 days of BCG injection of rats, a large number of mononuclear cells and lymphocytes infiltrated around the liver parenchyma and the portal area, resulting in a large number of granuloma masses with different sizes and diffuse distribution, IL-1β and TNF-α high expression (P < 0.01), CYP2E1 metabolic activity and protein expression were significantly decreased (P < 0.05). The protein content of CYP2E1 was significantly negatively correlated with IL-1β (r=0.222, P=0.027). Activation of PDTC-mediated nuclear factor-kappa B (NF-κB) inhibited the high expression of IL-1β and TNF-α in liver tissue and slowed down the down-regulation of CYP2E1 metabolism and protein expression. Conclusions NF-κB may be involved in the regulation of CYP2E1 by regulating the inflammatory cytokines IL-1β and TNF-α.

Pyrrolidinedithiocarbamate attenuates bleomycin-induced pulmonary fibrosis in rats: Modulation of oxidative stress, fibrosis, and inflammatory parameters.

OBJECTIVE: The current study aimed to investigate the modulatory effects of pyrrolidinedithiocarbamate (PDTC; 100 mg/kg) on bleomycin-induced pulmonary fibrosis (5 mg/kg; intratracheal) in rats. MATERIALS AND METHODS: Rats were randomly assigned to three groups: normal control, bleomycin control, and PDTC-treated groups. Lung injury was evaluated through histological examination, immunohistochemical detection of inducible nitric oxide synthase (iNOS) in lung tissue and evaluating the total and differential leucocytes count in bronchoalveolar lavage fluid. Lung tissue was used for biochemical assessment of lung content of hydroxyproline, transforming growth factor beta-1 (TGF-ß1), tumor necrosis factor-alpha (TNF-α) as well as analysis of lipid peroxides, reduced glutathione (GSH), and total nitrite contents. RESULTS: PDTC attenuated bleomycin-induced pulmonary fibrosis as evidenced by histological observations, decreased iNOS expression and prevention of bleomycin-induced altered total and differential leukocytes count. Additionally, PDTC caused a significant decrease in lung contents of hydroxyproline, TGF-ß1, TNF-α, lipid peroxides, and total nitrite coupled with increase in lung GSH content as compared to bleomycin control group. CONCLUSION: PDTC attenuated bleomycin-induced pulmonary fibrosis in rats via its anti-inflammatory, antioxidant, and antifibrotic activities.

Atorvastatin reduces long pentraxin 3 expression in vascular cells by inhibiting protein geranylgeranylation.

BACKGROUND: The long pentraxin PTX3 is an acute-phase multi-functional protein that might play both positive and detrimental effects under different pathophysiological conditions. We previously showed that statins down-regulate the release of PTX3 in human endothelial cells (ECs). The present study investigated the mechanism mediating this effect, its occurrence in other cells involved in atherogenesis, and whether it takes place in experimental atherosclerosis. METHODS AND RESULTS: We found that atorvastatin (1-5 µmol/L) decreased the production and release of PTX3 in human ECs through a post-transcriptional effect. Co-incubation with mevalonate or geranylgeranyl pyrophosphate prevented this effect. Direct blockade of geranylgeranyl transferase I by GGTI-286, treatment with the Rac inhibitor NSC23766 or silencing of the geranylgeranylated GTPase Rac2 by siRNA closely mimicked the action of atorvastatin. In contrast, inactivation of other geranylgeranylated proteins such as RhoA, RhoB, and RhoC or Rac1 did not affect PTX3 release. In addition, we found that atorvastatin also decreased PTX3 secretion in aortic SMCs through a mechanism likely dependent on protein geranylgeranylation, while no effect was observed in monocytes. Finally, we found that atherosclerotic lesions from cholesterol-fed rabbits treated with atorvastatin (2.5 mg/kg/day for 8 weeks) showed less immunoreactive PTX3 than lesions from control animals. CONCLUSIONS: Results suggest that statins may interfere with PTX3 expression in vascular cells via inhibition of protein geranylgeranylation. Since PTX3 is increasingly regarded as an important mediator of the inflammatory response underlying atherosclerosis and its complications, these results highlight the need for further studies of the role of PTX3 and its potential pharmacological modulation in cardiovascular disease.

Development of a new green non-dispersive ionic liquid microextraction method in a narrow glass column for determination of cadmium prior to couple with graphite furnace atomic absorption spectrometry.

Easy and innovative non-dispersive ionic liquid based microextraction (NDILME) has been developed for preconcentration of trace level of cadmium (Cd) in aqueous real surface water samples prior to couple with graphite furnace atomic absorption spectrometry (GFAAS). A 200 cm long narrow glass column containing aqueous solution of standard/sample was used to increase phase transfer ratio by providing more contact area between two medium (aqueous and extractive), which drastically improve the recoveries of labile hydrophobic chelate of Cd ammonium pyrrolidinedithiocarbamate (APDC), into ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate [C4mim][PF6]. Different aspect of the desire method have been investigated and optimized. Under the optimized key experimental variables, limit of detection (LOD) and enhancement factor (EF) were achieved to be 0.5 ng L(-1) and 150, respectively. Reliability of the model method was checked by relative standard deviation (%RSD), which was found to be <5%. Validity and accuracy of the developed method was checked by analysis of certified reference water samples (SLRS-4 Riverine water) using standard addition method. Application of the model method was productively performed by analysis of Cd in real surface water samples (tap and sea).

Effect of PDTC-induced inhibition of NF-κB expression on cerebrovascular remodeling following subarachnoid / 第二军医大学学报

Objective: To observe the expression of NF-κB in the basilar artery of rabbit model after subarachnoid hemorrhage (SAH) and its effect on cerebrovascular smooth muscle proliferation. Methods: Sixteen male New Zealand rabbits were evenly randomized into two groups, ammonium pyrrolidinedithiocarbamate (PDTC) group and SAH group. Double injection of fresh autologous arterial blood into the cisterna magna was used to establish animal models of cerebral vasospasm. In PDTC group PDTC was injected each day into the cisterna magna in the model animals. The animals were sacrificed 7 days after model establishment; the smooth muscle thickness of basilar artery was observed; and expression of NF-κB, proliferating cell nuclear antigen (PCNA) in the basilar artery was examined by immunohistochemistry and Western blotting analysis. Results: Compared with SAH group, PDTC group had significantly decreased NF-κB and PCNA protein expression and significantly reduced smooth muscle thickness (P<0. 05). Conclusion: PDTC can reduce the proliferation of smooth muscle through inhibiting the activation of NF-κB after SAH in rabbits.

Brain angiotensin regulates iron homeostasis in dopaminergic neurons and microglial cells.

Dysfunction of iron homeostasis has been shown to be involved in ageing, Parkinson's disease and other neurodegenerative diseases. Increased levels of labile iron result in increased reactive oxygen species and oxidative stress. Angiotensin II, via type-1 receptors, exacerbates oxidative stress, the microglial inflammatory response and progression of dopaminergic degeneration. Angiotensin activates the NADPH-oxidase complex, which produces superoxide. However, it is not known whether angiotensin affects iron homeostasis. In the present study, administration of angiotensin to primary mesencephalic cultures, the dopaminergic cell line MES23.5 and to young adult rats, significantly increased levels of transferrin receptors, divalent metal transporter-1 and ferroportin, which suggests an increase in iron uptake and export. In primary neuron-glia cultures and young rats, angiotensin did not induce significant changes in levels of ferritin or labile iron, both of which increased in neurons in the absence of glia (neuron-enriched cultures, dopaminergic cell line) and in the N9 microglial cell line. In aged rats, which are known to display high levels of angiotensin activity, ferritin levels and iron deposits in microglial cells were enhanced. Angiotensin-induced changes were inhibited by angiotensin type-1 receptor antagonists, NADPH-oxidase inhibitors, antioxidants and NF-kB inhibitors. The results demonstrate that angiotensin, via type-1 receptors, modulates iron homeostasis in dopaminergic neurons and microglial cells, and that glial cells play a major role in efficient regulation of iron homeostasis in dopaminergic neurons.

Ionic liquid-linked dual magnetic microextraction of lead(II) from environmental samples prior to its micro-sampling flame atomic absorption spectrometric determination.

A novel and rapid microextraction approach termed as ionic liquid-linked dual magnetic microextraction (IL-DMME), was developed for the atomic absorption spectrometric determination of lead. The developed method based on a combination of dispersive liquid-liquid microextraction (DLLME) and dispersive micro solid-phase extraction (D-µ-SPE). In the first DLLME step, 1-butyl-3-methylimidazolium hexafluorophosphate [C4mim][PF6], was selected to extract the lead-pyrrolidine-dithiocarbamate (Pb-PDC) complex from sample solution by the assistance of vortex agitator. After the first step, fifty milligrams of Fe3O4 magnetic nanoparticles (MNPs) were added to extraction of the ionic liquid and Pb-PDC complex in aqueous solution. The effective factors in proposed IL-DMME procedure, including volume of 1-butyl-3-methylimidazolium hexafluorophosphate, amount of Fe3O4 magnetic nanoparticles, vortex time, amount of ammonium pyrrolidinedithiocarbamate, sample volume and matrix effect were optimized in details. Under the optimal conditions, the method present has low detection limit (0.57 µg L(-1)), high preconcentration factor (160) and good repeatability (<7.5%, n=10). The accuracy of the developed method was evaluated by the analysis of the certified reference materials and addition-recovery tests. The method was successfully applied to the determination of lead in water, plant and hair samples.

Evaluation of lead levels in biological samples of mentally retarded children in different stages using advanced extraction method.

In present study the lead (Pb) levels has been assessed by analyzing the scalp hair and blood samples of mentally retarded/intellectual disabled (MR/ID) children of both genders, age ranged 3-8 years. For comparative purpose, healthy age matched children were also selected. The cloud point extraction of Pb from digested biological samples was carried out by complexed with ammonium pyrrolidinedithiocarbamate. The complexed analyte was subsequently isolated from the aqueous matrix in the micelles of a non-ionic surfactant (Triton X-114). Dilution of the surfactant-rich phase with acidified ethanol was performed after phase separation, and the Pb content was measured by flame atomic absorption spectrometer. Factors affecting the cloud point extraction were evaluated and optimized. The proposed procedure allowed the determination of lead in certified standard and real samples with detection limits of 0.834µgL(-) and enhancement factor 55. The results were compared with those of healthy children have same age, socioeconomic status and residential areas.

Speciation of chromium in environmental samples by dual electromembrane extraction system followed by high performance liquid chromatography.

This study proposes the dual electromembrane extraction followed by high performance liquid chromatography for selective separation-preconcentration of Cr(VI) and Cr(III) in different environmental samples. The method was based on the electrokinetic migration of chromium species toward the electrodes with opposite charge into the two different hollow fibers. The extractant was then complexed with ammonium pyrrolidinedithiocarbamate for HPLC analysis. The effects of analytical parameters including pH, type of organic solvent, sample volume, stirring rate, time of extraction and applied voltage were investigated. The results showed that Cr(III) and Cr(VI) could be simultaneously extracted into the two different hollow fibers. Under optimized conditions, the analytes were quantified by HPLC instrument, with acceptable linearity ranging from 20 to 500 µg L(-1) (R(2) values≥0.9979), and repeatability (RSD) ranging between 9.8% and 13.7% (n=5). Also, preconcentration factors of 21.8-33 that corresponded to recoveries ranging from 31.1% to 47.2% were achieved for Cr(III) and Cr(VI), respectively. The estimated detection limits (S/N ratio of 3:1) were less than 5.4 µg L(-1). Finally, the proposed method was successfully applied to determine Cr(III) and Cr(VI) species in some real water samples.

Minimal NF-κB activity in neurons.

Nuclear factor-kappa B (NF-κB) is a ubiquitous transcription factor that regulates immune and cell-survival signaling pathways. NF-κB has been reported to be present in neurons wherein it reportedly responds to immune and toxic stimuli, glutamate, and synaptic activity. However, because the brain contains many cell types, assays specifically measuring neuronal NF-κB activity are difficult to perform and interpret. To address this, we compared NF-κB activity in cultures of primary neocortical neurons, mixed brain cells, and liver cells, employing Western blot of NF-κB subunits, electrophoretic mobility shift assay (EMSA) of nuclear κB DNA binding, reporter assay of κB DNA binding, immunofluorescence of the NF-κB subunit protein p65, quantitative real-time polymerase chain reaction (PCR) of NF-κB-regulated gene expression, and enzyme-linked immunosorbent assay (ELISA) of produced proteins. Assay of p65 showed its constitutive presence in cytoplasm and nucleus of neurons at levels significantly lower than in mixed brain or liver cells. EMSA and reporter assays showed that constitutive NF-κB activity was nearly absent in neurons. Induced activity was minimal--many fold lower than in other cell types, as measured by phosphorylation and degradation of the inhibitor IκBα, nuclear accumulation of p65, binding to κB DNA consensus sites, NF-κB reporting, or induction of NF-κB-responsive genes. The most efficacious activating stimuli for neurons were the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-beta (IL-ß). Neuronal NF-κB was not responsive to glutamate in most assays, and it was also unresponsive to hydrogen peroxide, lipopolysaccharide, norepinephrine, ATP, phorbol ester, and nerve growth factor. The chemokine gene transcripts CCL2, CXCL1, and CXCL10 were strongly induced via NF-κB activation by TNFα in neurons, but many candidate responsive genes were not, including the neuroprotective genes SOD2 and Bcl-xL. Importantly, the level of induced neuronal NF-κB activity in response to TNFα or any other stimulus was lower than the level of constitutive activity in non-neuronal cells, calling into question the functional significance of neuronal NF-κB activity.

Interactions sorafenib of and pyrrolidine dithiocarbamate in pancreatic cancer cells / 中国癌症杂志

Background and purpose: Sorafenib, a multiple targeted agent, can inhibit proliferation and induce apoptosis of diverse tumor cell in vitro. It has extensive biological activities, but the pancreatic cancer effect of monotherapy is poor. This may be related to nuclear factor-kappa B (NF-κB) pathway activation in cancer. Therefore, it is necessary to combine with Pyrrolidinedithiocarbamate (PDTC, a NF-κB activation inhibitor) to enhance curative effect. To investigate the influences of cell proliferation, cell cycle and expression of NF-κB via their acting on human pancreatic cancer PANC-1, and explore their possible mechanism. Methods:The experiment groups were divided into sorafenib group with different concentrations (1.5, 3.0, 6.0, 12.0 μmol/L), PDTC group with different concentrations (10.0, 25.0, 50.0, 100.0 μmol/L) and combination group with low concentration (3.0 μmol/L sorafenib+25.0 μmol/L PDTC). The proliferative activity of PANC-1 of each group was measured by MTT assay at different time points of 24