Butyrylcholinesterase in lipid metabolism: A new outlook.

Cholinesterase enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are traditionally associated with the termination of acetylcholine mediated neural signaling. The fact that these ubiquitous enzymes are also found in tissues not involved in neurotransmission has led to search for alternative functions for these enzymes. Cholinesterases are reported to be involved in many lipid related disease states. Taking into view that lipases and cholinesterases belong to the same enzyme class and by comparing the catalytic sites, we propose a new outlook on the link between BChE and lipid metabolism. The lipogenic substrates of BChE that have recently emerged in contrast to traditional cholinesterase substrates are explained through the hydrolytic capacity of BChE for ghrelin, 4-methyumbelliferyl (4-mu) palmitate, and arachidonoylcholine and through endogenous lipid mediators such as cannabinoids like anandamide and essential fatty acids. The abundance of BChE in brain, intestine, liver, and plasma, tissues with active lipid metabolism, supports the idea that BChE may be involved in lipid hydrolysis. BChE is also regulated by various lipids such as linoleic acid, alpha-linolenic acid or dioctanoylglycerol, whereas AChE is inhibited. The finding that BChE is able to hydrolyze 4-mu palmitate at a pH where lipases are less efficient points to its role as a backup in lipolysis. In diseases such as Alzheimer, in which elevated BChE and impaired lipid levels are observed, the lipolytic activity of BChE might be involved. It is possible to suggest that fatty acids such as 4-mu palmitate, ghrelin, arachidonoylcholine, essential fatty acids, and other related lipid mediators regulate cholinesterases, which could lead to some sort of compensatory mechanism at high lipid concentrations.

12/15-lipoxygenase inhibition attenuates neuroinflammation by suppressing inflammasomes.

Introduction: Lipoxygenases (LOXs) have essential roles in stroke, atherosclerosis, diabetes, and hypertension. 12/15-LOX inhibition was shown to reduce infarct size and brain edema in the acute phase of experimental stroke. However, the significance of 12/15-LOX on neuroinflammation, which has an essential role in the pathophysiology of stroke, has not been clarified yet. Methods: In this study, ischemia/recanalization (I/R) was performed by occluding the proximal middle cerebral artery (pMCAo) in mice. Either the 12/15-LOX inhibitor (ML351, 50 mg/kg) or its solvent (DMSO) was injected i.p. at recanalization after 1 h of occlusion. Mice were sacrificed at 6, 24, and 72-h after ischemia induction. Infarct volumes were calculated on Nissl-stained sections. Neurological deficit scoring was used for functional analysis. Lipid peroxidation was determined by the MDA assay, and the inflammatory cytokines IL-6, TNF-alpha, IL-1beta, IL-10, and TGF-beta were quantified by ELISA. The inflammasome proteins NLRP1 and NLRP3, 12/15-LOX, and caspase-1 were detected with immunofluorescence staining. Results: Infarct volumes, neurological deficit scores, and lipid peroxidation were significantly attenuated in ML351-treated groups at 6, 24, and 72-h. ELISA results revealed that the pro-inflammatory cytokines IL-1beta, IL-6, and TNF-alpha were significantly decreased at 6-h and/or 24-h of I/R, while the anti-inflammatory cytokines IL-10 and TNF-alpha were increased at 24-h or 72-h of ML351 treatment. NLRP1 and NLRP3 immunosignaling were enhanced at three time points after I/R, which were significantly diminished by the ML351 application. Interestingly, NLRP3 immunoreactivity was more pronounced than NLRP1. Hence, we proceeded to study the co-localization of NLRP3 immunoreactivity with 12/15-LOX and caspase-1, which indicated that NLRP3 was co-localized with 12/15-LOX and caspase-1 signaling. Additionally, NLRP3 was found in neurons at all time points but in non-neuronal cells 72 h after I/R. Discussion: These results suggest that 12/15-LOX inhibition suppresses ischemia-induced inflammation in the acute and subacute phases of stroke via suppressing inflammasome activation. Understanding the mechanisms underlying lipid peroxidation and its associated pathways, like inflammasome activation, may have broader implications for the treatment of stroke and other neurological diseases characterized by neuroinflammation.

Molecular and Computational Analysis Identify Statins as Selective Inhibitors of Human Butyrylcholinesterase.

Cholinesterase enzyme family consists of acetylcholinesterase (AChE, 3.1.1.7), the major enzyme responsible for hydrolysis of acetylcholine at cholinergic synapses, and butyrylcholinesterase (BChE, 3.1.1.8) a detoxification enzyme of plasma. Statins are cholesterol-lowering medications utilized as protective medicaments in stroke and Alzheimer's disease, which cholinesterases are associated with. Thus, in this study, we characterized the inhibitory effects and mechanisms of common statins, rosuvastatin, atorvastatin, simvastatin and lovastatin, on human erythrocyte AChE and purified serum BChE using in vitro and in silico methods. Kinetic assays identified statins as selective non-competitive inhibitors of human serum BChE. The IC50 and Km values were found as 194.7 ± 55.2 µM and 1.03 ± 0.2 µM for rosuvastatin, 492.5 ± 55.1 µM and 7.2 ± 0.3 µM for atorvastatin, 14.2 ± 0.3 µM and 202.7 ± 23.2 µM for lovastatin, and 17.6 ± 0.1 µM and 207.2 ± 13.2 µM for simvastatin, respectively. The compounds did not display considerable inhibition against AChE. Molecular docking predicted good affinity and strong interactions with the BChE active site for atorvastatin and rosuvastatin. Current study identifies rosuvastatin as the most specific and selective inhibitor of human BChE among the tested statins. As selective inhibitors of BChE statins have the potential to be re-evaluated as medicaments due to their pleiotropic effects.

Novel activity of human BChE: Lipid hydrolysis.

Acetylcholinesterase and butyrylcholinesterase (BChE) typically hydrolyze the neurotransmitter acetylcholine. The multifunctional enzyme BChE is associated with lipid metabolism through an undefined mechanism. Based on lipid-related studies and by comparing the structural similarities between lipases and BChE we postulated that the association of BChE with lipid metabolism could occur through hydrolytic activity. Utilizing purified BChE enzymes from different sources and several lipases as controls, the ability of BChE to hydrolyze 4-methylumbelliferyl (4-mu) palmitate is investigated. Using lectin affinity, inhibition kinetics, and molecular modeling, we demonstrated that purified BChE hydrolyzed 4-mu palmitate at pH 8 as effectively as wheat germ lipase. The affinity Km value of the enzymes for 4-mu palmitate as substrate is found as 10.4 µM, 34.2 µM, 129.8 µM, and 186 µM for wheat germ lipase, purified BChE, pancreatic lipase, and commercial BChE, respectively. Analysis of the inhibitory effect of 4-mu palmitate on BChE using butyrylthiocholine as substrate revealed competitive inhibition with Ki and IC50 values of 448 µM and 987.2 µM, respectively. The binding affinity and interactions of 4-mu palmitate with BChE and pancreatic lipase were predicted by molecular docking. These results suggest that BChE possesses lipolytic activity. The possibility that BChE hydrolyzes not only 4-mu palmitate but also other types of lipids will lead to a new approach to those disease states associated with increased BChE activity/expression.

Inhibitory Action of Omega-3 and Omega-6 Fatty Acids Alpha-Linolenic, Arachidonic and Linoleic acid on Human Erythrocyte Acetylcholinesterase.

Acetylcholinesterase (AChE, E.C. 3.1.1.7) termed as the true cholinesterase functions to end cholinergic transmission at synapses. Due to its diverse expression in non-neural tissues such as erythrocytes and bones along with its various molecular forms, researchers seek a non-classical role for this protein. Here, the inhibitory action of unsaturated 18 carbon fatty acids linoleic acid and alpha-linolenic acid and 20 carbon fatty acid arachidonic acid on AChE were investigated. Enzyme activity was measured in kinetic assay method according to Ellman assay utilizing acetylthiocholine. Analysis of the activity data revealed that among the fatty acids examined the IC50 values differed according to the length of the fatty acid and the number of the double bonds. Arachidonic acid, a 20-carbon fatty acid with 4 unsaturated bonds (20:4 n-6, cis 5,8,11,14) displayed an IC50 value of 2.78 µM and Ki value of 396.35 µM. Linoleic acid, an essential 18-carbon fatty acid (18:2 n-6, cis 9,12) had an IC50 value of 7.95 µM and Ki value of 8027.55 µM. The IC50 value of alpha-linolenic acid, 18-carbon fatty acid (18:3 n-3, cis-9,12,15) was found as 179.11 µM. Analysis of the data fit the inhibition mechanism for linoleic, alpha-linolenic and arachidonic acid as mixed-type; non-competitive. Molecular docking complied with these results yielding the best score for arachidonic acid. The alkenyl chain of the fatty acids predictably reached to the catalytic site while the carboxylate strongly interacted with the peripheric anionic site.

Relation between orexin A and epileptic seizures.

INTRODUCTION: One of the unknown mechanisms in epilepsy pathogenesis is the involvement of the hypothalamic neuropeptide orexin. Although the relationship between orexin and sleep has been revealed, its effect in epilepsy has not been fully clarified. In this study, we aimed to show the relationship between orexin A and the seizures that occur during sleep and wakefulness. MATERIAL AND METHODS: This study included 40 patients with drug-resistant focal epilepsy and 37 healthy controls. Night basal orexin (NBO) and morning basal orexin (MBO) levels were measured using enzyme-linked immunosorbent assay in patients and controls. Serum samples were collected from patients after epileptic seizures during sleep and wakefulness. RESULTS: In both patients and controls, MBO levels (median: 1039 pg/mL, interquartile ranges [IQR] (899-1078)) were higher than NBO levels (median 989 pq/mL, IQR (893-1078) (p = 0.02). Basal orexin levels were lower in patients than in controls (p < 0.001). However, while the duration of seizures was shortened in awake seizures, the level of orexin increased (p = 0.007). Additionally, orexin levels after nocturnal seizure were higher in patients who had an ictal electroencephalography onset in the left hemisphere or a lesion in the left temporal lobe (p = 0.02; p = 0.01, respectively). There was no relationship between postictal somnolence and orexin levels. Although there was no significant difference, the level of post-seizure orexin increased compared to the basal values, especially in seizures during sleep. DISCUSSION: The increase in serum orexin levels, especially after seizures, suggests that orexin may be associated with the epileptogenic effect. In further studies, determination of orexin from cerebrospinal fluid (CSF) and correlation of CSF and serum orexin levels may provide more useful information regarding the relationship between orexin and epilepsy.

Melatonin is effective in attenuating cisplatin-induced neurotoxicity.

Cisplatin (Cis) is a chemotherapeutic agent that has many side effects. Neurotoxicity is one of the most important of these side effects. Oxidative stress and neuroinflammation are the best-known mechanisms in the pathogenesis of neurotoxicity development. In this study, we aimed to determine whether melatonin (Mel), with antioxidant and anti-inflammatory effects, is effective in preventing Cis-induced neurotoxicity. Forty-eight male Sprague-Dawley rats were divided into six groups (n = 8) as follows: control (0.9% NaCl), vehicle (5% ethanol), Cis (6 mg/kg), Cis (6 mg/kg) + vehicle (5% ethanol), Mel (20 mg/kg), and Cis (6 mg/kg) + Mel (20 mg/kg) groups. Cis was administered as a single dose on the 3rd day of the experiment while Mel was given for 5 days. All administrations were performed via intraperitoneal injection. After injections, T-maze, rotarod, and hot plate tests were performed to evaluate cognitive, motor, and sensory functions, respectively. Following sacrification oxidative stress markers, cholinergic function, and proinflammatory cytokines were studied from brain homogenates. Cis impaired cognitive function and motor performance in the Cis and Cis+Vehicle groups. The drug also increased oxidative stress in the brain. Mel significantly improved brain oxidant/antioxidant status and also decreased the overproduction of proinflammatory cytokines (superoxide dismutase activities in Cis+Vehicle and Cis+Mel groups: 104.55 ± 9.50 µU/mg protein vs. 150.13 ± 4.70 µU/mg protein, respectively, p < 0.05; tumor necrosis factor-α levels in Cis and Cis+Mel groups: 40 pg/ml vs. 20 pg/ml, respectively, p < 0.05). It seems that Mel can improve Cis neurotoxicity. For a more firm conclusion, further studies using Mel at different doses with larger groups should be performed.

cAMP/PKA-CREB-BDNF signaling pathway in hippocampus of rats subjected to chemically-induced phenylketonuria.

Phenylketonuria (PKU) is an inborn error disease in phenylalanine metabolism resulting from defects in the stages of converting phenylalanine to tyrosine. Although the pathophysiology of PKU is not elucidated yet, the toxic effect of phenylalanine on the brain causes severe mental retardation. In relation to learning and memory, the hippocampal PKA / CREB / BDNF pathway may play a role in learning deficits in PKU patients. This study aimed to investigate PKA/CREB/BDNF pathway in hippocampus of chemically induced PKU rats with regard to gender. Sprague-Dawley rat pups were randomized into two groups of both genders. To chemically induce PKU, animals received subcutaneous administration of phenylalanine (5.2 mmol / g) plus p-chlorophenylalanine, phenylalanine hydroxylase inhibitor (0.9 mmol / g); control animals received 0.9% NaCl. Injections started on the 6th day and continued until the 21st day after which locomotor activity, learning and memory were tested. In male PKU rats, locomotor activity was reduced. There were no differences in learning and memory performances of male and female PKU rats. In PKU rats, pCREB / CREB levels in males was unchanged while it decreased in females. Elevated PKA activity, BDNF levels and decreased pCREB/CREB ratio found in female PKU rats were not replicated in PKU males in which BDNF is decreased. Our results display that in this disease model a gender specific differential activation of cAMP/PKA-CREB-BDNF signaling pathway in hippocampus occurs investigation of which can help us to a better understanding of disease pathophysiology.

Composite nanofibers incorporating alpha lipoic acid and atorvastatin provide neuroprotection after peripheral nerve injury in rats.

Despite the new treatment strategies within the last 30 years, peripheral nerve injury (PNI) is still a worldwide clinical problem. The incidence rate of PNIs is 1 in 1000 individuals per year. In this study, we designed a composite nanoplatform for dual therapy in peripheral nerve injury and investigated the in-vivo efficacy in rat sciatic nerve crush injury model. Alpha-lipoic acid (ALA) was loaded into poly lactic-co-glycolic acid (PLGA) electrospun nanofibers which would release the drug in a faster manner and atorvastatin (ATR) loaded chitosan (CH) nanoparticles were embedded into PLGA nanofibers to provide sustained release. Sciatic nerve crush was generated via Yasargil aneurism clip with a holding force of 50 g/cm2. Nanofiber formulations were administered to the injured nerve immediately after trauma. Functional recovery of operated rat hind limb was evaluated using the sciatic functional index (SFI), extensor postural thrust (EPT), withdrawal reflex latency (WRL) and Basso, Beattie, and Bresnahan (BBB) test up to one month in the post-operative period at different time intervals. In addition to functional recovery assessments, ultrastructural and biochemical analyses were carried out on regenerated nerve fibers. L-929 mouse fibroblast cell line and B35 neuroblastoma cell line were used to investigate the cytotoxicity of nanofibers before in-vivo experiments. The neuroprotection potential of these novel nanocomposite fiber formulations has been demonstrated after local implantation of composite nanofiber sheets incorporating ALA and ATR, which contributed to the recovery of the motor and sensory function and nerve regeneration in a rat sciatic nerve crush injury model.

Effect of Asiatic Acid on the Treatment of Spinal Cord Injury: An Experimental Study in Rats.

AIM: Spinal cord injury (SCI) is a devastating condition of the central nervous system. There is no proven therapeutic agent for the treatment of this complex disorder. Asiatic acid (AA) has been used as an anti-inflammatory and anti-oxidant agent in Eastern countries for many years. The aim of this study was to investigate the effectiveness of AA on the treatment of traumatic SCI in rats. MATERIAL AND METHODS: Thirty-two adult male Sprague-Dawley rats were divided into 4 groups as laminectomy, laminectomy+trauma, vehicle, and AA treatment groups. SCI was created by the modified Allen"s weight-drop technique. After the injury, the levels of pro-inflammatory cytokines (IL-6, IL1-Β, TNF-α) and lipid peroxidation products (MDA) were measured. Tarlov functional recovery scores were also determined for each rat. The One-way ANOVA test was used for the analysis of difference between 4 experimental groups and the groups were compared individually by Tukey-LSD post hoc analysis test (p=0.001). RESULTS: AA administration just after SCI attenuated the levels of lipid peroxidation products (MDA) and pro-inflammatory cytokines (TNF-α, IL1Β). It also increased the Tarlov functional recovery scores of the rats. CONCLUSION: AA administration could attenuate a number of deleterious reactions after traumatic SCI. Further studies are needed to elucidate the pathways of neuroprotective effects of AA after spinal trauma.

The effect of HDL-bound and free PON1 on copper-induced LDL oxidation.

Oxidative modification of LDL plays an important role in the development of atherosclerosis. High-density lipoprotein (HDL) confers protection against atherosclerosis and the antioxidative properties of paraoxonase 1 (PON1) has been suggested to contribute to this effect of HDL. The PON1 exist in two major polymorphic forms (Q and R), which regulate the concentration and activity of the enzyme and alter its ability to prevent lipid oxidation. However, the association of Q192R polymorphism with PON1's capacity to protect against LDL lipoperoxidation is controversial. The aim of this study was to evaluate the effects of the purified PON1 Q192R and the partially purified HDL-bound PON1 Q192R isoenzymes (HDL-PON1 Q192R) on LDL oxidation, with respect to their arylesterase/homocysteine thiolactonase (HTLase) activities. Cupric ion-induced LDL oxidation was reduced up to 48% by purified PON1 Q192, but only 33% by an equivalent activity of PON1 R192. HDL-PON1 Q192 isoenzyme caused a 65% reduction, whereas HDL-PON1 R192 isoenzyme caused only 46% reduction in copper ion-induced LDL oxidation. These findings reflect the fact that PON1 Q and PON1 R allozymes may have different protective characteristics against LDL oxidation. The protection against LDL oxidation provided by HDL-PON1 Q192R isoenzymes is more prominent than the purified soluble enzymes. Inhibition of the Ca(+2)-dependent PON1 Q192R arylesterase/HTLase by the metal chelator EDTA, did not alter PON1's ability to inhibit LDL oxidation. These studies indicate that the active site involvement of the purified enzyme is not similar to the HDL-bound one, in terms of both PON1 arylesterase/HTLase activity and the protection of LDL from copper ion-induced oxidation. Moreover, PON1's ability to protect LDL from oxidation does not seem to require calcium.

Butyrylcholinesterase expression is regulated by fatty acids in HepG2 cells.

Butyrylcholinesterase (BChE) is mostly associated with the detoxification of xenobiotics. In this study to analyze the involvement of BChE in lipid metabolism, linoleic acid (LA) and α-linolenic acid (ALA) were applied to HepG2 cells along with expression of wild type human BChE. After 48 h of these treatments WST-1 cell proliferation assay, FACS analysis, RT-PCR, Oil Red O staining and activity assays were performed. Application of high concentrations of LA to HepG2 cells without BChE transfection lead to detachment of the cells. The IC50 value LA was found as 149.3 µM whereas the IC50 value for ALA could not be calculated. Hence, in order to display minimal effects on cell viability, 5 µM was chosen as appropriate concentration for LA and ALA application to HepG2 cells. Transfection of wild-type BChE plasmid to HepG2 cells yielded increased BChE expression. Application of 5 µM ALA after BChE transfection to HepG2 cells resulted in increased expression of BChE. Although with this low concentration the number of apoptotic cells was decreased with ALA treatments, LA application did not cause a similar result with the same dose. Moreover ghost cell like property was observed in LA-treated cells. Application of ALA, on the other hand, led to an overall increase in cell numbers, BChE expression and activity. Our results indicate that BChE expression might be regulated by ALA in HepG2 cells.

Comparative Biochemical and Motor Function Analysis of Alpha Lipoic Acid and N-Acetyl Cysteine Treatment on Rats with Experimental Spinal Cord Injury.

AIM: Spinal Cord Injury (SCI) is a devastating health problem both for the patient and the clinician. Numerous treatment modalities have been studied to reverse the effects of spinal cord injury. Herein is reported the effects and the comparison of Alpha Lipoic Acid and N-Acetyl Cysteine on rats with SCI. MATERIAL AND METHODS: 38 adult male Sprague-Dawley rats were randomly divided into 5 groups: only laminectomy, laminectomy and trauma, laminectomy trauma and Alpha Lipoic Acid 100 mg/kg IP administration, laminectomy trauma and N-Acetyl Cysteine 300 mg/kg IP administration, and vehicle group (PEG). The trauma model was the Modified Allen Weight drop method. After the procedure, the rats' motor function was evaluated using the modified Tarlov Scale and consequently they were sacrificed and the spinal cord tissue was analyzed biochemically for inflammation markers. RESULTS: Both Alpha Lipoic Acid and N-Acetyl Cysteine administration after the injury significantly improved the results. There was no statistically significant difference in between the agents. CONCLUSION: Although these agents both proven to be effective in ameliorating the effects of SCI, there was not enough evidence in this research to conclude the benefit of one agent over the other.

Effects of tadalafil-Type-V phosphodiesterase enzyme inhibitor-On rats with spinal trauma.

In this research, the effect of tadalafil, a selective inhibitor of cyclic guanosine monophosphate-specific phosphodiesterase type 5, on rats with spinal trauma was evaluated. The evaluation consisted of neurological examination and biochemical parameters. Twenty healthy male Wistar albino rats were used in this study. They were separated into three groups: tadalafil-receiving (TD) group (n=7), laminectomy and trauma (LT) group (n=7), and just laminectomy group (n=6). The TD group received daily dose of tadalafil (10 mg/kg) for a week along with bait and water. Each rat's spinal cord was dissected with utter caution. The spinal cord was traumatized by Allen's weight-drop method. Using a standard apparatus, 5 g of weight was dropped from a height of 10 cm on the spinal cords of the TD and LT (laminectomy+trauma) group. No extra maneuvers were conducted on the laminectomy group. A day later, the rat's functional neurological status was examined followed by re-exploration of the spinal cord for sampling 1 cm of tissue. The Tarlov scale was used to evaluate the functional neurological status. The modified Tarlov scale was rated to be significantly higher in the TD group than that in the LT group. For the biochemical parameters, malondialdehyde (MDA) and cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) involved in the inflammatory process were examined. MDA--an indicator of lipid peroxidation--was found to be significantly lower in the TD group compared with that in the LT group. TNF-α and IL-6 levels were also found to be lower in the TD group compared with those in the LT group. Shortly, this research showed that the use of TD group in spinal trauma resulted in better neurological outcome and significant improvement in biochemical parameters.

Effects of conjugated linoleic acid supplementation and exercise on post-heparin lipoprotein lipase, butyrylcholinesterase, blood lipid profile and glucose metabolism in young men.

This study was designed to investigate the effects of conjugated linoleic acid (CLA) supplementation and endurance exercise training-induced changes on post-heparin lipoprotein lipase (PH-LPL) and butyrylcholinesterase (BChE) activities along with leptin, insulin and lipid levels in plasma by a randomized double blind experiment. Eighteen sedentary male volunteers were randomly divided into CLA and Placebo (PLC) supplementation groups. Both groups underwent daily supplementation of either 3g CLA or 3g placebo for 30 days, respectively, and performed exercise on a bicycle ergometer 3 times per week for 30-40 min at 50% VO2 peak workload. For plasma glucose, insulin and leptin levels and BChE activity fasting blood was used. For PH-LPL measurements, blood was collected 15 min after 50 IU/kg iv heparin injection. In all groups, there is a statistically significant decrease in BChE (p = 0.03, p = 0.02) and leptin (p = 0.002), insulin and HOMA-IR levels (p = 0.02). Exercise with or without CLA supplementation decreased insulin levels and increased insulin sensitivity. PH-LPL activity was increased significantly in both groups, displaying increased fatty acid mobilization. We conclude that though CLA supplementation and exercise can affect these parameters, CLA is not more effective than exercise alone. Hence, a prolonged supplementation regime may be more effective. Taken together in our small study group, our findings display that BChE is a potential marker for synthetic function of liver, fat metabolism, an obesity marker, a function long overlooked.

Plasma 3-nitrotyrosine estimates the reperfusion-induced cerebrovascular stress, whereas matrix metalloproteinases mainly reflect plasma activity: a study in patients treated with thrombolysis or endovascular recanalization.

Cerebral reperfusion injury may account for complications of thrombolysis and endovascular recanalization. Experimental studies have shown that brain matrix metalloproteinase (MMP) activity increases during reperfusion and is correlated with oxidative/nitrative stress. Increased plasma MMP levels have been reported in stroke, but no information is available for reperfusion-induced plasma MMP and 3-nitrotyrosine (3-NT, a marker of oxidative/nitrative stress) changes immediately after recanalization. We obtained plasma from 29 patients undergoing endovascular recanalization, 12 patients treated with thrombolysis, and six control patients having diagnostic angiogram before and 1,3, and 24 h after treatment to investigate the effect of cerebral reperfusion on plasma MMP gelatinolytic activity and 3-NT level. Hypoperfusion was shown distal to the stenotic artery in endovascular treatment patients. Presence of an occluded artery and recanalization was documented in thrombolysis patients. A significant increase was detected in plasma 3-NT levels 3 and 24 h after stenting/angioplasty. Plasma MMP-9 gelatinolytic activity rose more than 50% of the pre-treatment level in 12 of 29 patients. However, this was not statistically significant and not correlated with any of the clinical or radiological correlates of reperfusion injury (e.g., hyperperfusion and hemorrhage). After thrombolysis, a significant increase in plasma MMP-9 gelatinolytic activity at 3 and 24 h and the cleaved form of MMP-9 were detected. 3-NT levels increased by 44% and 62% at 3 and 24 h, which did not achieve statistical significance, but was highly correlated with admission NIH Stroke Scale (r = 0.930 p < 0.001). No change was detected in MMP-2 in all groups. In conclusion, these data suggest that the increased plasma MMP-9 levels is not a direct measure of MMP-9 activity in the reperfused brain but rather a consequence of tissue plasminogen activator infusion, whereas plasma 3-NT levels appear to originate from the reperfused brain vasculature. The changes in 3-NT levels may therefore be useful to monitor oxygen/nitrogen radical formation during reperfusion with serial measurements.

Transport of a caspase inhibitor across the blood-brain barrier by chitosan nanoparticles.

The current treatment of neurological and psychiatric diseases is far beyond being satisfactory. In addition to highly complex disease mechanisms, the blood-brain barrier (BBB) also remains as a challenge by limiting the delivery of the majority of currently available therapeutics to the central nervous system. Several approaches taking advantage of molecular and physicochemical characteristics of the BBB have been developed recently to improve drug delivery to the brain. Here, we introduce a nanomedicine that can efficiently transport BBB-impermeable peptides to the brain. This nanomedicine is made of chitosan nanoparticles into which considerable amounts of a peptide can be incorporated. The nanoparticle surface is modified with polyethylene glycol to enhance the plasma residence time by preventing their capture by the reticuloendothelial system. Monoclonal antibodies against the transferrin receptor (TfR), which is highly expressed on the brain capillary endothelium, are conjugated to nanoparticles via biotin-streptavidin bonds. The activation of TfR by the nanoparticle-antibody complex induces transcytosis and thus delivers the loaded drug to the brain. Penetration of nanoparticles to the brain can be illustrated in vivo by intravital microscopy as well as ex vivo by fluorescence or electron microscopy. N-Benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK)-loaded nanoparticles rapidly release their contents within brain parenchyma, inhibit ischemia-induced caspase-3 activity, and thereby provide neuroprotection.

Rosuvastatin induces apoptosis in cultured human papillary thyroid cancer cells.

Statins show antiproliferative activity in various cancer cells. The aim of this study was to evaluate the effects of rosuvastatin treatment on papillary thyroid carcinoma. The papillary thyroid carcinoma (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines were treated with rosuvastatin at 12.5, 18.5, 25, 50, 100, and 200 µM concentrations. After 48 and 72 h of rosuvastatin treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Ki-67 immunolabeling, FACS analysis, electron microscopy, caspase-3, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis were performed. Decreased cell viability and G1 phase arrest were detected in papillary thyroid cell line treated with rosuvastatin. Positive immunoreactivity of Ki-67 and dose-dependent increase in S phase on Nthy-ori 3-1 cells were also detected. B-CPAP cells showed intense vacuolisation and autophagosomes with low concentrations and 48 h incubations, while Nthy-ori 3-1 cells showed these changes at higher concentrations. A decrease in the percentage of cells showing autophagy was determined with increasing concentrations of rosuvastatin in B-CPAP cells. Rosuvastatin treatment also caused a dose- and time-dependent increase in caspase-3 activity and apoptotic index by TUNEL assay in B-CPAP cells compared with the Nthy-ori 3-1 cells. Apoptotic cells with nuclear condensation and fragmentation were observed in B-CPAP cell line. Rosuvastatin induced autophagic changes in B-CPAP papillary thyroid cancer cells in lower doses and caused a shift from autophagy to apoptosis. Rosuvastatin may be an alternative treatment for refractory papillary thyroid cancer. Further in vivo studies are necessary to clarify the effects of rosuvastatin in papillary thyroid carcinoma and the clinical implications of rosuvastatin treatment.

Counter-regulation of cholinesterases: differential activation of PKC and ERK signaling in retinal cells through BChE knockdown.

The ubiquitous cholinesterase (ChE) enzymes, functioning in the termination of acetylcholine mediated neural transmission, are also reported to have additional functions. Through application of siRNAs against butyrylcholinesterase (BChE) in R28 cells, a retinal cell line with pluripotent properties, a counter-regulation between ChEs was revealed. BChE knock down resulted in an up-regulation of not only acetylcholinesterase (AChE), but also altered the signaling status of PKC and ERK. Knockdown of BChE modified ERK signaling most notably through ERK1/2 proteins, together with the transcription activator P90RSK1 and c-fos. Stimulation of the R28 cell line by forskolin revealed that ChEs are involved in an intricate cross talk between different signaling pathways. Forskolin-stimulated R28 cells displayed a robust cholinergic response, as detected by both electrophysiology and ChE expression, and changed the activation status of PKC/ERK signaling pathways. The findings in R28 cells show that ChE expressions are inversely co-regulated and act through the transcription factors c-fos and P90RSK1. Since R28 cells have the capacity to differentiate into different cell types through stimulation of signaling pathways, ChEs are likely to be associated with cell fate determination, rather than just terminating cholinergic responses.

A quadruped study on chitosan microspheres containing atorvastatin calcium: preparation, characterization, quantification and in-vivo application.

Atorvastatin is commonly used as a cholesterol lowering agent in patients. Recently, the neuroprotective effects of atorvastatin became the focus of many research studies. In this study, we have formulated chitosan microspheres containing atorvastatin calcium. In-vitro characterization of chitosan microspheres and quantification of atorvastatin calcium from formulations were also evaluated. The neuroprotective efficiency of atorvastatin calcium was investigated by an experimental spinal cord injury model. Atorvastatin calcium microspheres were implanted at the laminectomy area (1 mg/kg) immediately after trauma. Twenty-four hours after injury, motor functions of animals were scored according to modified Tarlov Scale. In spinal cord tissues tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and lipid peroxidation levels were quantified and ultrastructural changes have been investigated. The results of all parameters indicate that microspheres containing atorvastatin calcium were capable of improving functional outcome, attenuating the expression of TNF-alpha, IL-1beta and IL-6; lowering lipid peroxidation levels and maintaining the preservation of the cellular uniformity.