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Aims Pyroptosis and inflammation are involved in the development of chronic obstructive pulmonary disease (COPD). However, the cigarette smoke-mediated mechanism of COPD remains unclear. In this study, we aimed to investigate the role of nucleotide-binding domain-like receptor protein-3 (NLRP3) inflammasome-mediated pyroptosis in the death of human bronchial epithelial (HBE) cells after cigarette smoke extract (CSE) exposure. Main methods The protein level of NLRP3 in lung tissue was measured after cigarette smoke exposure in vivo. In vitro, HBE cells were treated with CSE. Subsequently, the activity of caspase-1, lactate dehydrogenase (LDH) release, release of interleukin (IL)-1β and NLRP3 expression levels were measured. The involvement of reactive oxygen species (ROS) was also explored. Key findings After exposure to CSE, increased release of LDH, the transcriptional and translational upregulation of NLRP3, the caspase-1 activity levels, and enhanced IL-1β and IL-18 release were observed in 16HBE cells. In addition, NLRP3 was required to activate the caspase-1. Our results suggested that pre-stimulated of 16HBE with a caspase-1 inhibitor, or using NLRP3 siRNA to silence NLRP3 expression, also caused the decrease of IL-1β release and pyroptosis. Significances. CSE induced inflammation and contributed to pyroptosis through the ROS/NLRP3/caspase-1 pathway in 16HBE cells. The NLRP3 inflammasome participates in CSE-induced HBE cell damage and pyroptosis, which could provide new insights into COPD.

Objective To investigate protective efficacies and mechanisms of Cathelicidin-BF (BF-30) peptide on streptozotocin (STZ)-induced diabetic kidney injury. Methods Effects of BF-30 on hydrogen peroxide induced oxidative damage in HK-2 renal cells were assessed by CCK-8 method. Forty STZ-induced diabetic rats with kidney injury were randomly divided into model control group, BF-30 group at different doses (0.1, 0.3 and 0.9 mg/kg). Blood biochemical and kidney related indexes as well adrenal morphological changes, inflammation related markers of diabetic rats were measured. Results Cell viability of HK-2 cells with oxidative damage induced by hydrogen peroxide were significantly improved by BF-30 with 0.8 μg/mL for 56.5% and 1.6 μg/mL for 82.3% compared with control. Moreover, the decreased reactive oxygen species (ROS), and increased intracellular antioxidant enzymes GPX1, SOD2 and GSH were showed in BF-30 treated groups. In addition, co-incubation of BF-30 in HK-2 cells promoted the increase of p-AMPK and LC3, decreased activation of p-mTOR, BAX and Caspase 3. Chronic treatment of BF-30 improved the STZ-induced diabetic characteristics of diabetic kidney disease (DKD) model rats. Further renal histopathological examination revealed 12-week treatment of BF-30 effectively improved the morphology of nephropathy in DKD rats. Moreover, BF-30 also could ameliorate excessive oxidative stress, renal cell apoptosis and fibrosis, thereby protects renal tissues. Conclusion BF-30 exerted protective effects on STZ-induced kidney injury mainly through the inhibiting oxidative stress in kidney tissue, reducing renal fibrosis, increasing autophagy, and reducing the renal cell apoptosis related proteins to decrease the cell damage and protect nephrocytes.

Aims This study evaluated the mechanisms involved in the chemopreventive effects of a mucoadhesive formulation (FITOPROT), containing curcuminoids from Curcuma longa L. (Zingiberaceae) and Bidens pilosa L. (Asteraceae) extract, against 5-FU-induced cellular toxicity using an in vitro oral mucositis model. Main methods Effects of FITOPROT on 5-FU-induced cytotoxicity in HaCaT and SSC-4 cells were evaluated by MTT assay. For mechanistic analyses, HaCaT cells were first pretreated with FITOPROT (0.005%) for 24 h followed by treatment with FITOPROT and simultaneously exposed to 5-FU (10 μg/mL) for additional 24 h. Key findings FITOPROT was able to protect HaCaT cells from 5-FU-triggered cell damage. Moreover, the FITOPROT + 5-FU association showed higher cytotoxic effects on SSC-4 cancer cells. Flow cytometry and/or fluorescence microscopy analysis showed FITOPROT was able to significantly reduce ROS generation and prevent mitochondrial changes in HaCaT cells. In addition, it avoided the release of cytochrome c from mitochondria to the cytoplasm in cells exposed to 5-FU, and restored their proliferative activity via Ki-67 expression. Furthermore, FITOPROT regulated 5-FU-induced oxidative stress via Nrf2 involvement. HaCaT cells pretreated/treated with FITOPROT also showed normal expression of TNF-R1 and NF-κB inflammatory proteins and decreased levels of pro-inflammatory cytokines (TNF, IL-1β, IL-6 and IL-8). Moreover, a high-resolution liquid chromatography-mass spectrometry analysis showed the presence of flavonoids rutin, glucoronylated quercetin and dimethylquercetin rutenoside in FITOPROT. Significance It was showed that FITOPROT, an antioxidant phytochemicals-rich mucoadhesive formulation, exerts chemopreventive effects against 5-FU-triggered toxicity through antioxidant and anti-inflammatory mechanisms and restoration of proliferative capacity in HaCaT cells.

Aims To explore the relationship between renal ischemia/reperfusion injury (RIRI) and the activation of the renal 5-HT degradation system, including 5-HT2A receptor (5-HT2AR), 5-HT synthases and monoamine oxidase-A (MAO-A). Main methods Rat RIRI was induced by removing the right kidney, causing ischemia of the left kidney for 45 min and reperfusion for different times. RIRI model (ischemia for 45 min and reperfusion for 24 h) was pretreated with 5-HT2AR antagonist sarpogrelate hydrochloride (SH) and the 5-HT synthase inhibitor carbidopa. In HK-2 cells, cellular damage was induced by hypoxia (24 h)/reoxygenation (12 h) (H/R) and treated with SH, carbidopa or the MAO-A inhibitor clorgyline. Hematoxylin-eosin, immunohistochemistry, TUNEL and fluorescent probe staining, RT-qPCR, western blotting, ELISA, etc. were used in the tests. Key findings The development of RIRI and the emergence of the RIRI peak were consistent with renal 5-HT degradation system activation. The highest expression regions of the 5-HT degradation system overlapped with those of the most severe lesions in the kidney, which were in proximal renal tubules. Rat RIRI and HK-2 cell damage, including oxidative stress, inflammation and apoptosis, could be almost abolished by synergistic inhibition of SH and carbidopa. Clorgyline also abolished the cellular damage induced by H/R. H/R–induced production of mitochondrial ROS in HK-2 cells was due to MAO-A–catalyzed 5-HT degradation, and 5-HT2AR was involved by mediating the expression of 5-HT synthases and MAO-A. Significance These findings revealed a close association between RIRI and activation of the renal 5-HT degradation system.

Silica (SiO2) nanoparticles (NPs) were synthesized by laser ablation method and were characterized by TEM and DLS techniques. Afterwards, their inhibition activity against carbonic anhydrase (CA) isoforms (CA I and CA II) was explored by experimental and theoretical analysis. Also, the protective effect of SiO2 NPs against H2O2-induced oxidative stress in alveolar epithelial cells (A549) were assessed by measurement of MTT, ROS level, CAT and SOD activity and GSH content. Finally, the NPs were screened for their antimicrobial activity using the MICs method against the Klebsiella pneumoniae. The result showed that the synthesized NPs have a size of around 40 nm. The inhibition activity by comparing IC50 values with acetazolamide as a positive control revealed that SiO2 NPs in comparison with acetazolamide served as potent inhibitors against CA isoforms which was also confirmed by docking studies. The cellular assays indicated that the SiO2 NPs with a concentration of 20 μg/mL stimulated a significant antioxidant activity against H2O2-induced oxidative cell damage through activation of CAT and SOD, an increase in the GSH content and reducing the level of ROS. The synthesize NPs also showed a good inhibition effect against Klebsiella pneumoniae as compared to Sulfamethoxazole as a positive control. In conclusion, this data may provide some useful information on the development of some platforms for pneumonia treatment and management.

Aims The magnesium ion (Mg2 +) fulfils several important functions for living organisms. We investigated whether there is a protective effect of MgSO4 on 6-OHDA-induced neurotoxicity in SH-SY5Y cells, and gained insight into the effects of cellular mRNA and protein expression of the magnesium transporters SLC41A1, NIPA1, MagT1 and CNNM2 on 6-OHDA-induced neurotoxicity. Main methods The effect of MgSO4 on cell viability in 6-OHDA-treated SH-SY5Y cells was measured using a CCK-8 kit. The mRNA and protein expression of SLC41A1, NIPA1, MagT1, and CNNM2 were detected using reverse transcription–qPCR and Western blot. Key findings The results showed that SH-SY5Y cells treated with 25– 50 μM 6-OHDA for 24 h significantly decreased cell viability, while if pre-incubated with 0.125– 1 mM MgSO4 for 1 h before adding 6-OHDA it partially prevented the cell damage. There was a significant decrease in cellular mRNA and protein expression of SLC41A1, NIPA1, MagT1 and CNNM2 in 6-OHDA treated SH-SY5Y cells, and MgSO4 can reverse its decline. Significance Our results suggest that MgSO4 may protect SH-SY5Y cells against 6-OHDA-induced cell injury and that gene expression of SLC41A1, NIPA1, MagT1, and CNNM2 might be involved in dopaminergic neurons.

Aims Sodium propionate (SP) has been reported to possess an anti-inflammatory and anti-apoptotic potential by inhibiting certain signaling pathways and helps in reducing the pathological damages of the mammary gland. However, the effects of sodium propionate on attenuating Lipopolysaccharide (LPS)-induced inflammatory condition and cell damage in bovine mammary epithelial cells (bMECs) are not comprehensively studied yet. Therefore, the aim of the current investigation was to evaluate the protective effects of sodium propionate on LPS-induced inflammatory conditions and to clarify the possible underlying molecular mechanism in bMECs. Main methods The effects of increasing doses of SP on LPS-induced inflammation, oxidative stress and apoptosis was studied in vitro. Furthermore, the underlying protective mechanisms of SP on LPS-stimulated bMECs was investigated under different experimental conditions. Key findings The results reveled that increased inflammatory cytokines, chemokines and those of tight junction's mRNA expression was significantly attenuated dose-dependently by propionate. Biochemical analysis revealed that propionate pretreatment modulated the LPS-induced intercellular reactive oxygen species (ROS) accumulation, oxidative and antioxidant factors and apoptosis rate. Furthermore, we investigated that the LPS activated nuclear factor-kB (NF-kB), caspase/Bax apoptotic pathways and Histone deacetylases (HDAC) was significantly attenuated by propionate in bMECs. Significance Our results suggest that sodium propionate is a potent agent for ameliorating LPS-mediated cellular disruption and limiting detrimental inflammatory responses, partly via maintaining blood milk barrier integrity, inhibiting HDAC activity and NF-kB signaling pathway.

Aim Diabetes is a high-risk factor for neurocognitive dysfunction. Diabetic acute hyperglycaemia accompanied by high osmotic pressure can induce immune cell dysfunction, but its mechanism of action in brain microglia remains unclear. This study aimed to evaluate the role of the mechanically sensitive ion channel Piezo1 in the dysfunction of microglia in acute hyperglycaemia. Materials and methods To construct an in vitro acute hyperglycaemia model using the BV2 microglial cell line, Piezo1 in microglia was inhibited by GsMTx4 and siRNA, and the changes in microglial function were further evaluated. Key findings High concentrations of glucose upregulated the expression of Piezo1, led to weakened cell proliferation and migration, and reduced the immune response to inflammatory stimulating factors (Aβ and LPS). Additionally, LPS upregulated Piezo1 in BV2 microglial cultures in vitro. The activation of Piezo1 channels increased the intracellular Ca2+ concentration and reduced the phosphorylation of JNK1 and mTOR. Inhibiting Piezo1 did not affect cell viability at average glucose concentrations but improved acute HCG-induced cell damage and increased the phosphorylation of JNK1 and mTOR, suggesting that the latter modification may be a potential downstream mechanism of Piezo1. Significance Piezo1 is necessary for microglial damage in acute hyperglycaemia and may become a promising target to treat hyperglycaemic brain injury.

Aim Apoptosis of endothelial cells (ECs) is a crucial factor in blood-spinal cord barrier (BSCB) disruption post spinal cord injury (SCI). Insulin-like growth factor-1 (IGF-1) is a protective cytokine that plays an important role in multiple diseases, whereas the distinct role in SCI-induced remains critical questions to address. Here we designed to explore the role and underlying mechanism of IGF-1 in endothelial damage after SCI. Main methods In the current study, we established mouse microvascular endothelial cells (MVECs) injury model via LPS and cDNA of IGF-1 was transfected into MVECs. In vivo SCI mice, overexpression of IGF-1 (SCI-IGF-1) and its corresponding empty vehicle (SCI-NC) were conducted using lentivirus, then apoptosis degree, component of tight junction, and inflammatory damage were evaluated. Key findings IGF-1 treatment in MVECs displayed a milder apoptosis and cell damage under LPS insult. IGF-1 increased the level of PI3K/AKT pathway, which impeded the procedure of apoptosis. Blocking of PI3K/AKT pathway markedly neutralized the effect of IGF-1 treatment. Transfection of excess IGF-1 into SCI mice significantly corrected microenvironment of neural tissue repair, reduced area of injured core and improved functional recovery with greater activation of PI3K/AKT pathway. Significance The results above argue that the promising roles played by IGF-1 is potentially vital for developing effective future therapies in SCI.

Aims Diosmin is a citrus flavonoid with broad biological activities. Moreover, the administration of biologically active compounds during the myocardial reperfusion potentiates the cardioprotection and survival rate. Hence, this study aimed to evaluate whether the reperfusion with diosmin elicits protection to ischemic hearts and to investigate the mechanisms involved in these effects. Main methods Male Wistar rats were subjected to myocardial ischemia-reperfusion (I/R) injury in the presence or absence of diosmin (0.1 μM). When appropriated, a non-selective nitric oxide synthase (NOS) inhibitor was used. Key findings Here, we show that the severe impairment of contractile function induced by I/R was fully recovered by the administration of diosmin. Our results also showed that reperfusion with diosmin markedly reduced the incidence of cardiac arrhythmias and enhanced coronary microvascular function. Moreover, the rise of end-products of lipid peroxidation, biomarkers of cell damage, and infarct size were dramatically attenuated with diosmin reperfusion. However, all cardioprotective effects mediated by diosmin were fully abolished by inhibition of NOS, including the contractile function, arrhythmias index, coronary flow, and infarct size. Significance Together, our data show that inclusion of diosmin during myocardial reperfusion potentiates the cardioprotection through a NOS-dependent mechanism.

Background and purpose Early brain injury is an essential pathological process after subarachnoid hemorrhage (SAH), with many cell death modalities. Ferroptosis is a newly discovered regulated cell death caused by the iron-dependent accumulation of lipid peroxidation, which can be prevented by glutathione peroxidase 4 (GPX4). Our study aimed to investigate the role of GPX4 in neuronal cell death after experimental SAH. Methods In vivo experimental SAH was induced by injecting autologous arterial blood into the prechiasmatic cistern in male Sprague-Dawley rats. Meanwhile, the in vitro SAH model was performed with primary rat cortical neurons cultured in medium containing hemoglobin (Hb). Adenovirus was used to overexpress GPX4 before experimental SAH. GPX4 expression was detected by western blot and immunofluorescence experiments. Malondialdehyde (MDA) was measured to evaluate the level of lipid peroxidation. Nissl staining was employed to assess cell death in vivo, whereas lactate dehydrogenase (LDH) release was used to evaluate cell damage in vitro. The brain water content and neurological deficits were evaluated to determine brain injury. Results Endogenous GPX4 was mainly expressed in neurons, and its expression decreased at 24 h after experimental SAH. Overexpression of GPX4 significantly reduced lipid peroxidation and cell death in the experimental SAH models both in vivo and in vitro. Moreover, overexpression of GPX4 ameliorated brain edema and neurological deficits at 24 h after SAH. Conclusions The decrease of GPX4 expression potentially plays an important role in ferroptosis during early brain injury after SAH. Overexpression of GPX4 has a neuroprotective effect after SAH.

Stem cell therapy is widely employed in treating osteoarthritis (OA), and bone marrow-derived mesenchymal stem cells (BMSCs) has gradually become the most attractive new method for treating OA due to the benefit for cartilage tissue repair. However, the apoptosis in the neural stem cell transplantation severely decreases repairing efficacy. Icariin has been reported to exert multiple effects on BMSCs, including its proliferation, osteogenic, and chondrogenic differentiation. However, its effects on the injury induced by oxygen, glucose and serum deprivation (OGD) remains unknown. We prospectively investigated the role of ICA on rabbit BMSCs under conditions of OGD. Firstly, BMSCs were cultured under conditions of OGD, ICA relieved OGD-induced cell damage by promoting cell proliferation and suppressing apoptosis. Secondly, Markers of endoplasmic reticulum stress (ERs), ER stress IRE-1 pathway, and autophagy were both inhibited by ICA via inhibition of phosphor-extracellular regulated protein kinases (p-ERKs), p-P38, p-c-Jun N-terminal kinase (p-JNK) or si-MAPK. Finally, decrease of ERs marker levels enhanced protective effect of ICA against OGD-induced injury by limiting apoptosis and autophagy. Moreover, an autophagy inhibitor (3-methyladenine: 3-MA) contributed to a synergistic effect in conjunction with ICA, in promoting cell proliferation, suggesting that ICA exerts anti-ERs and anti-autophagy effects in OGD-treated BMSCs. Therefore, ICA protected rabbit BMSCs from OGD-induced apoptosis through inhibitory regulation of ERs-mediated autophagy related to the MAPK signaling pathway, which provided insights for a potential therapeutic strategy in OA.

Aims This study was designed to reveal the role and underlying mechanism of excessive autophagy mediated by ERS via the PERK-eIF2α pathway in the apoptosis and formation of CaOx kidney stones. Main methods Ethylene glycol (EG) was used to establish a rat model of CaOx kidney stones, and 100 mg/kg of ERS inhibitor 4-phenylbutyric acid (4-PBA) or 60 mg/kg of autophagy inhibitor chloroquine (CQ) was administered daily to the rats. Four weeks after administration, we collected blood and kidney tissues to analyze the occurrence of ERS and autophagy, apoptosis, renal function, renal tubular crystal deposition, and kidney damage, respectively. Key findings We observed that both 4-PBA and CQ treatment significantly inhibited the excessive autophagy and reduced apoptosis as well as decreasing p-PERK and p-eIF2α expressions. Meanwhile, the proportion of kidney weight, contents of creatinine and blood urea nitrogen, excretion of neutrophil gelatinase-associated lipocalin and kidney injury molecule 1, and renal tubular deposition were markedly down-regulated. Significance The findings in this study suggested that ERS induced excessive autophagy via the PERK-eIF2α pathway, regulating cell damage and apoptosis. ERS-mediated inhibition of excessive autophagy effectively protected kidney function and prevented the apoptosis and formation of kidney stones.

Peripheral diabetic neuropathy (DN) manifests in nearly 60% of diabetic patients, being pain its most debilitating symptom. Although electrophysiological and morphological aspects are well described, little is known about its development and progression, undermining effective therapies. Hyperglycemia and insulin signaling impairment are considered the triggering events of oxidative stress observed in the dying nerves, however there are still many gaps in the knowledge of intracellular plastic changes it generates. Aims In this study we aimed to evaluate the early transcriptome changes in DN when the first symptoms of the disease start to appear. Main methods Next-Generation Sequencing of messenger RNA (RNA-Seq) of L4 and L5 dorsal root ganglia (DRG) four weeks post-diabetes induction in a rat model for type 1 diabetes. Key findings RNA sequencing found 66 transcripts differentially expressed between diabetic and control groups, related mainly to the following biological processes: inflammation, hyperalgesia/analgesia, cell growth and cell survival. Given their roles, the differentially expressed genes suggest an attempt to switch to a survival/regenerative program. Significance Our results show that changes in the transcriptome profile start to appear early in the course of DN and might be related to secure cell homeostasis. Hence, the present data may indicate how DRG cells are responding to hyperglycemia in its early stages and which mechanisms first fail to respond, further leading to cell damage and cell death. Early screening of cell alterations in DN might lead to more concrete targets for pharmaceutical interventions, which could more efficiently delay cell damage.

Exposure of macrophages to lipopolysaccharide (LPS) induces oxidative and inflammatory stresses, which cause cell damage. Antioxidant and anti-inflammatory properties have been attributed to the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), commonly used in free radical analysis, but these aspects of DMPO have been little explored. In this study, we sought to establish the anti-inflammatory activity of DMPO, presumably by removing free radicals which otherwise help activate inflammatory response and damage cells.RAW 264.7 macrophages were treated with LPS and/or DMPO for different time points, cell damage, production of inflammatory mediators, inducible nitric oxide synthase (iNOS) expression, NF-κB p65 activation, phosphorylation of MAPKs and Akt, and intracellular reactive oxygen species (ROS) were determined.After cells were treated with LPS and/or DMPO for 24 h, DMPO reduced the LPS-induced inflammatory response as indicated by downregulated iNOS expression and production of inflammatory mediators. Accordingly, DMPO protected cells from LPS-induced cytotoxicity. In order to understand the mechanistic basis of these DMPO effects, the NF-κB p65 activation and the phosphorylation of MAPKs and Akt were examined. We found, by assaying cells treated with LPS and/or DMPO for 15-60 min, that DMPO inhibited the phosphorylation of MAPKs, Akt, and IκBα, and reduced the NF-κB p65 translocation. Furthermore, we demonstrated that DMPO inhibited LPS-induced ROS production.DMPO showed the anti-inflammatory activity and attenuated LPS-induced cell damage, most likely by reducing ROS production and thus preventing the subsequent inflammatory activation and damage.

Aims With the advancement of small intestinal (double balloon and capsule) endoscopy technology, incidence of small intestinal lesion caused by nonsteroidal anti-inflammatory drugs (NSAIDs) has been known to be high. However, therapy for small intestinal mucosal lesion has not yet been developed. Previous studies have shown that heat shock proteins (HSPs) are involved in cytoprotection mediated by their function as a molecular chaperone. In this study, we examined the effect of HSP60 or HSP70 overexpression on hydrogen peroxide-induced (H 2 O 2 ) or indomethacin-induced cell damage in the small intestinal epithelial cells. Main methods cDNA of human HSP60 or HSP70 was transfected to rat small intestinal (IEC-6) cells, and HSP60- or HSP70-overexpressing cells were cloned. IEC-6 cells transfected with vector only were used as control cells. These cells were treated with H 2 O 2 (0–0.14 mM) or indomethacin (0–2.5 mM). The cell viability was determined by MTT-assay. Cell necrosis was evaluated by LDH-release assay. Further, apoptosis was evaluated by caspases-3/7 activity and TUNEL assay. Key findings Cell viability after H 2 O 2 or indomethacin treatment was significantly higher in HSP60-overexpressing cells compared with that in control cells and HSP60-overexpressing cells. Apoptotic cells were also reduced in HSP60-overexpressing. Conclusion : These results indicate that HSP60 plays an important role in protecting small intestinal mucosal cells from H 2 O 2 -induced or indomethacin-induced cell injury. HSP70-overexpressing cells did not show anti-apoptotic ability. Significance These findings possibly suggest that function of each HSP is different in the small intestine. Therefore, for the therapy of small intestinal mucosal lesion, HSP60-induction therapy could be a new therapeutic strategy.

There is evidence that using lidocaine-treated cellular culture produces cell damage. However, there are no studies in vivo demonstrating the potential injurious effect of lidocaine on the central nervous system. Therefore, the aim of our study was to investigate if lidocaine is involved in neuronal damage in the CA3 hippocampus and amygdala regions when using a single subconvulsive or a convulsive lidocaine dose. Two-month-old male Wistar rats (57) were used. The animals were randomly assigned to one of three groups. Group I received 0.9% saline ip (n = 9), group II received a single lidocaine dose of 60 mg/kg (n = 18), and group III received 90 mg/kg ip (n = 12). At day 2, 7, and 10 after the dosing, three to six rats per group were sacrificed. The brains of the rats were removed and were embedded in paraffin. Coronal cuts of 7 μm were made. Each brain section was stained with cresyl-eosin. We evaluated the number of normal and abnormal neurons in the hippocampal CA3 (pyramidal) and basolateral amygdala (large and medium neurons) regions in a 10,000 μm2 section. To explore an association between lidocaine-induced seizure and neuronal damage, diazepam was used (10 mg/kg ig) as an anticonvulsant two hours before a 90 mg/kg dose of lidocaine. Lidocaine causes a morphological neuronal alteration in the CA3 hippocampal region and the basolateral amygdala and possibly an inhibition–excitation imbalance. Diazepam prevents lidocaine-induced seizures, but not neuronal damage in brain structures. Interaction of lidocaine with the membrane components produces disrupted Ca+ 2 homeostasis and causes neuronal damage. Moreover, it is possible that lidocaine or its metabolites could actively participate in the neuronal damage observed.

This paper evaluates the effects of testosterone (0.5 mg/kg subcutaneously (s.c.) for 8 days) on oxidative stress and cell damage induced by 3-nitropropionic acid (20 mg/kg intraperitoneally (i.p.) for 4 days) in ovariectomized rats. Gonadectomy triggered oxidative damage and cell loss, evaluated by the detection of caspase-3, whereas 3-nitropropionic acid increased the levels of oxidative stress induced by ovariectomy and prompted cell damage characterized by enhanced levels of lactate dehydrogenase. These changes were blocked by testosterone administration. Our results support the following conclusions: i) ovariectomy triggers oxidative and cell damage via caspase-3 in the striatum; ii) 3-nitropropionic acid exacerbates oxidative stress induced by ovariectomy and leads to cell damage characterized by increased levels of lactate dehydrogenase; iii) testosterone administration decreases oxidative stress and cell damage. Additionally, these data support the hypothesis that testosterone might play an important role in the onset and development of neurodegenerative diseases.

Aims Age-related macular degeneration (AMD) is one of the most common diseases leading to blindness in elderly people. The progression of AMD may be prevented through anti-inflammation and antioxidation in retinal pigment epithelium (RPE) cells. Lycopene, a carotenoid, has been shown to possess both antioxidative and anti-inflammatory properties. This research was conducted to detail the mechanisms of these effects of lycopene-treated RPE cells. Main methods We exposed ARPE-19 cells to TNFα after pretreatment with lycopene, and measured monocyte adhesion, ICAM-1 expression, NF-κB nuclear translocation, and transcriptional activity. Cell viability was assayed with Alamar Blue. The cell redox state was tested by glutathione (GSH) and reactive oxygen species (ROS) levels. The importance of the Nrf2 pathway was tested in nuclear translocation, promoter reporter assay, and siRNA. Key findings Lycopene could reduce TNF-α-induced monocyte adhesion and H2O2– induced cell damage in RPE cells. Furthermore, lycopene inhibits ICAM-1 expression and abolishes NF-κB activation for up to 12 h in TNFα-treated RPE cells. Lycopene upregulates Nrf2 levels in nuclear extracts and increases the transactivity of antioxidant response elements. The use of Nrf2 siRNA blocks the inhibitory effect of lycopene in TNF-α-induced ICAM-1 expression and NF-κB activation. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the de novo synthesis of GSH. We found that lycopene increases intracellular GSH levels and GCL expression. Following lycopene treatment, TNF-α-induced ROS production was abolished. Significance The Nrf2-regulated antioxidant property plays a pivotal role in the anti-inflammatory mechanism underlying the inhibition of NF-κB activation in lycopene-treated ARPE-19 cells.

We previously reported that the Smilacis chinae rhizome inhibits amyloid beta protein (25-35) (Abeta (25-35))-induced neurotoxicity in cultured rat cortical neurons. Here, we isolated catechin and epicatechin from S. chinae rhizome and also studied their neuroprotective effects on Abeta (25-35)-induced neurotoxicity in cultured rat cortical neurons. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced neuronal cell death at a concentration of 10 microM, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Catechin and epicatechin also inhibited glutamate release into medium induced by 10 microM Abeta (25-35), which was measured by HPLC, generation of reactive oxygen species (ROS) and activation of caspase-3. These results suggest that catechin and epicatechin prevent Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release, generation of ROS and caspase-3 activity. Furthermore, these effects of catechin and epicatechin may be associated with the neuroprotective effect of the S. chinae rhizome.

Colominic acid (CA), produced by Escherichia coli K1, is a polymer of sialic acid linked through α (2→8) glycosidic linkages. Although there are several studies on the biological activities of chemically sulfated CA, the activity of CA has been incompletely understood. In the present study, we investigated the effects of CA, prepared as an α2,8-linked homopolymer of N -acetylneuraminic acid, on the proliferation and monolayer maintenance of bovine aortic endothelial cells in culture. The results indicate that CA potently inhibits the proliferation of sparse endothelial cells without nonspecific cell damage. The inhibitory effect of CA was markedly stronger than those of sodium spirulan and calcium spirulan, known polysaccharides that inhibit endothelial cell proliferation. On the other hand, in dense endothelial cells, CA induced nonspecific cell damage and markedly injured the monolayer. These results indicate that CA has two distinct effects on vascular endothelial cells: one is the inhibition of proliferation when the cell density is low, and the other is the nonspecific cytotoxicity when the cell density is high. Interestingly, these cell density-dependent effects of CA could be prevented by sulfation of the CA chains. Therefore, it is concluded that CA not only inhibits the proliferation of sparse endothelial cells without nonspecific cell damage but also injures dense cells in a monolayer by nonspecific cytotoxicity, which can be prevented by sulfation of the polysaccharide.

We have evaluated the effect of a creatine supplementation protocol upon inflammatory and muscle soreness markers: creatine kinase (CK), lactate dehydrogenase (LDH), prostaglandin E2) (PGE2) and tumor necrosis factor-alpha (TNF-alpha) after running 30km. Runners with previously experience in running marathons, with their personal best between 2.5-3h were supplemented for 5 days prior to the 30km race with 4 doses of 5g of creatine and 15g of maltodextrine per day while the control group received the same amount of maltodextrine. Pre-race blood samples were collected immediately before running the 30km, and 24h after the end of the test (the post-race samples). After the test, athletes from the control group presented an increase in plasma CK (4.4-fold), LDH (43%), PGE2 6.6-fold) and TNF-alpha (2.34-fold) concentrations, indicating a high level of cell injury and inflammation. Creatine supplementation attenuated the changes observed for CK (by 19%), PGE2 and TNF-alpha (by 60.9% and 33.7%, respectively, p

It has been suggested that the neurodegeneration that occurs with Alzheimer's disease (AD) may result from apoptosis, a process of programmed cell death. Neuronal injury, induced by abnormal aggregates of beta-amyloid peptide, has been identified as an apoptotic trigger. In the present study, brain tissue samples were obtained from the insular cortex (INS) and midfrontal gyrus (MFG) of Alzheimer subjects and age-matched, nondemented controls. Tissue sections from all samples were alternately stained by an in situ TUNEL assay to identify 3' termini DNA strand breaks characteristic of apoptosis or immunohistochemically for beta-amyloid deposition in senile plaques. The incidence of DNA fragmentation detected in pyramidal neurons was relatively infrequent overall, but was significantly higher in AD compared to controls. AD subjects consistently exhibited a dense accumulation of plaques, with a twofold greater concentration in MFG as INS. There was no significant difference in pyramidal cell number regardless of subject or brain region. Taken together, our results indicate that the TUNEL assay may be revealing cell damage rather than cell loss. Our finding of a moderate correlation between the incidence of TUNEL-positive cells and plaque density implicates beta-amyloid as one of multiple factors provoking cell injury in AD. A notable contribution of this study is the identification of distinctive neuropathologies co-occurring in two brain regions interconnected with each other and with limbic and cortical areas typically damaged during AD.

Bacterial endotoxin lipopolysaccharide (LPS) treatment of neuron-rich cells and glia-rich cells exhibited significant cell damage 12 hr after incubation, although no severe or significant cell damage induced by LPS appeared in neuron-glia co-cultured cells. Moreover, severe and significant time-dependent cell damage was induced by a larger dose treatment (10 mM) of glutamate (Glu), and this damage was seen in neuron-rich cells, neuron-glia co-cultured cells, and glia-rich cells. Examining extracellular tumor necrosis factor alpha (TNFalpha) induced by either LPS or Glu treatment, the levels of extracellular TNFalpha induced by LPS were significantly higher than those induced by Glu. These significant increases of TNFalpha were measured within 2 hr after LPS treatment in neuron-glia co-cultured cells and glia-rich cells, although no significant changes were detected in the neuron-rich cells. With Glu treatment, a significant increase in TNFalpha levels was detected after 6 hr of Glu treatment only in glia-rich cells. Our results indicate that cerebral TNFalpha is mainly produced in glia cells and that its production is dependently regulated by each stimulant. In addition, the production of TNFalpha is not directly related to the trigger of cell injury.

Different in vitro free radical generating systems were used to assess the antioxidative activity of aqueous extracts of the five herbal components of Wu-zi-yan-zong-wan, a traditional Chinese medicinal formula with a long history of use for tonic effects. Fructus Rubi [Rubus chingii (Rosaceae) fruits] was found to be the most potent. It was further investigated using the primary rat hepatocyte system. tert-Butyl hydroperoxide (t-BHP) was used to induce oxidative stress. Being a short chain analog of lipid hydroperoxide, t-BHP is metabolized into free radical intermediates by the cytochrome P450 system in hepatocytes, which in turn, initiate lipid peroxidation, glutathione depletion and cell damage. Pre-treatment of hepatocytes with Fructus Rubi extract (50 microg/ml to 200 microg/ml) for 24 h significantly reversed t-BHP-induced cell viability loss, lactate dehydrogenase leakage and the associated glutathione depletion and lipid peroxidation. The amount of reactive oxygen species formed was also decreased as visualized by the fluorescence probe 2',7'-dichlorofluorescin diacetate. These results suggested that Fructus Rubi was useful in protecting against t-BHP-induced oxidative damage and may also be capable of attenuating cytotoxicity of other oxidants.

Oxidative stress plays a key role in several ailments including neurodegenerative conditions. The aim of the study was to demonstrate the effect of rosmarinic acid (RA) in preventing oxidative stress related death of neuronal cell lines.In the present study, we demonstrated direct neuroprotective effect of RA using H2O2-induced oxidative challenge in N2A mouse neuroblastoma cells. The mechanism of neutralization of H2O2-induced toxicity by RA was evaluated using MTT, lactate dehydrogenase, mitochondrial membrane potential (MMP), intracellular ROS, and comet assays. Up-regulation of brain neuronal markers at molecular level was performed by RT-PCR.Results presented in the paper indicate that H2O2-induced cytotoxicity in N2A cells was suppressed by treatment with RA. Moreover, RA is very effective in attenuating the disruption of lactate dehydrogenase, mitochondrial membrane potential and intracellular ROS. Pretreatment with RA significantly prevents genotoxicity (3.7-fold, p0.01) and promotes the up-regulation of tyrosine hydroxylase (TH) (4.5-fold, p0.01), and brain-derived neurotrophic factor (BDNF) genes (5.4-fold, p0.01) against H2O2-induced cytotoxicity in N2A cells.Our results revealed that N2A cells are suitable cellular models to evaluate neuroprotective effects of RA, and suggest that RA may potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress.

Estrogen can enhance or reduce lymphocyte functions in vitro depending on dose and exposure duration. The purpose of this study was to determine the effect of in vivo 17 beta-estradiol (E2) on apoptosis and necrosis in lymphoid tissue of female C567BL/6 mice. Animals were ovariectomized (OVX), ovariectomized and 17 beta-estradiol supplemented (OVX + E2; 71 micrograms E2 per day for 14 days), sham ovariectomized (SHAM), or unhandled controls (CONTROL). Thymus and spleen were removed aseptically, cells dispersed into single cell suspensions in RPMI-1640, and measures of cell damage performed: an annexin V flow cytometric assay for markers of apoptosis and an enzyme-linked immunoassay for measures of DNA fragmentation and necrosis. OVX + E2 mice had 620 +/- 72 pg/ml 17 beta-estradiol in serum in contrast to OVX mice which had 7.6 +/- 5 pg/ml, the SHAM mice which had 2.8 +/- 1 pg/ml of serum E2, and the CONTROL mice which had 3.9 +/- 0.8 pg/ml of serum E2 (p < 0.001). There was a significantly lower percentage of viable thymocytes in OVX + E2 mice compared to the other treatment conditions (p < 0.001, respectively). There was also a significantly higher percentage of annexin V positive thymocytes in OVX + E2 mice (p < 0.005). Measures of DNA fragmentation by ELISA were higher in splenocytes from OVX + E2 mice than in the OVX, SHAM or CONTROL mice (p < 0.005). These results suggest that supraphysiological levels of estrogen in vivo induce damage in lymphoid cells; however, the impact of estrogen associated lymphoid tissue damage on specific immune functions remains to be determined.

Aims Melatonin possesses various pharmacological effects including neuroprotective effects against brain ischemia. Post-ischemic increases in matrix metalloproteinase-9 (MMP-9) expression and activity mainly contribute to neuronal damage by degradation of the extracellular matrix. This study was designed to examine whether melatonin has a neuroprotective effect and an influence on MMP-9 in transient global brain ischemia. Main methods Mice were subjected to 20 min of global brain ischemia and sacrificed 72 h later. Melatonin (30 mg/kg) was administered 30 min before and 2 h after ischemia as well as once daily until sacrifice. Key findings Hippocampal pyramidal cell damage after ischemia was significantly decreased by melatonin. As observed by zymography, melatonin inhibited the increase of MMP-9 activity after ischemia. In the brain sections, the increased gelatinase activity was mainly observed in the hippocampus after ischemia and melatonin also reduced gelatinase activity. The laminin and NeuN expression levels were reduced in the hippocampal CA1 and CA2 regions after ischemia, and melatonin reduced laminin degradation and neuronal loss. A TUNEL assay demonstrated that there were TUNEL-positive cells in the hippocampus and the number of TUNEL-positive cells was significantly decreased by melatonin. There was no difference in the ischemia-induced hippocampal neuronal damage between the vehicle- and melatonin-treated groups of MMP-9 knock-out mice. Significance These data demonstrate that melatonin suppressed the occurrence of neuronal injury, which might be partly due to its inhibitory effects on MMP-9 in addition to its anti-oxidative effects. MMP-9 may be an important key target of melatonin in neuroprotection against global ischemia.

Aims The effects of bisphenol A (BPA) on NCTC Clone 1469, non-parenchymal hepatocytes, were examined to clarify the molecular basis of BPA-induced liver injury. Main methods We analyzed the expression of C/EBP homologous protein ( CHOP ) mRNA, Bcl2 mRNA, caspase12, and glucose-regulated protein 78 kDa (GRP78)/Ig heavy chain-binding protein (BiP), to determine whether endoplasmic reticulum (ER) stress was involved in the 100 μM BPA-induced cell death. To examine the features of damaged hepatocytes, we analyzed the morphological changes in BPA-treated NCTC Clone 1469 by transmission electron microscopy. In addition, we analyzed the intracellular reactive oxygen species (ROS) level in BPA-treated NCTC Clone 1469 by the 2′, 7′-dichlorofluorescein diacetate (DCFDA) method. Key findings Increases in the expression of CHOP mRNA, caspase-12, and GRP78/BiP in NCTC Clone 1469-treated with 100 μM BPA were detected (CHOP, 1.42 fold; caspase-12, 1.33 fold; GRP78/Bip, 1.36 fold). These observations suggested that BPA induced ER stress-associated apoptosis. A morphological analysis revealed remarkable elongation of the rough ER, supporting the finding of ER stress. Intracellular ROS production was increased in NCTC Clone 1469-treated with BPA, and N -acetyl- l -cysteine (NAC) prevented the cell damage induced by BPA. However, these effects of BPA were not inhibited by estrogen receptor inhibitors. Significance We found that BPA induced ER stress-associated apoptosis in non-parenchymal hepatocytes. The ER stress is due to ROS production and is independent of estrogen receptors.

The effect of ischemia on atrial natriuretic peptide (ANP) release from heart ventricles was studied by exposing the perfused hearts of Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats to global ischemia after excision of the atria. Ischemia for 2, 5 and 20 min caused an increase of 0.3 +/- 1.1, 12.4 +/- 5.5 and 11.4 +/- 4.2 ng/g dry weight in ANP release of the WKY ventricles, respectively. ANP release increased 3.4 +/- 2.8 ng/g dry weight after 5 minutes' ischemia from the SHR ventricles. The increase was not caused by cell damage, as only processed form of the peptide was detected in the perfusates. The increase in ANP release in the WKY ventricles correlated positively with the tissue lactate/pyruvate ratio (r = 0.85) and adenosine (r = 0.99), and negatively with the phosphorylation potential (r = -0.70). The results indicate that ventricular ischemia increases ANP release, probably due to changes in myocardial energy metabolism.

Aims High blood glucose may auto-oxidize and generate free radicals, which are proposed to induce apoptosis in cardiac cells. The aim of the present study was to investigate the cell damage induced by glucose/glucose oxidase-dependent oxidative stress and the protective effect of N-acetylcysteine (NAC) on H9c2 cardiac muscle cells. Main methods H9c2 cells were exposed to 33 mM glucose (G) + 1.6 milliunits (mU) of glucose oxidase (GO) and termed G/GO. Cell apoptosis, generation of reactive oxygen species (ROS-super oxide anion and hydrogen peroxide) and reactive nitrogen species (RNS-peroxinitrite), and the change in mitochondrial membrane potential (ΔΨm) was studied using flow cytometry and confocal microscopy, and cytochrome c release was measured using confocal microscopy. The expression of Bcl-2, Bax and the activation of procaspase-9 was studied by western blot. Key findings Exposure of H9c2 cells to G/GO resulted in a significant increase in cellular apoptosis (P

Aims This study was performed to evaluate the therapeutic efficacy of nanocapsulated flavonoidal quercetin (QC) in combating arsenic-induced reactive oxygen species (ROS)-mediated oxidative damage in hepatocytes and brain cells in a rat model. Main methods Hepatic and neuronal cell damage in rats was made by a single injection (sc) of sodium arsenite (NaAsO2, 13 mg/kg b. wt. in 0.5 ml of physiological saline). A single dose of 500 µl of quercetin suspension (QC) (QC 8.98 µmol/kg) or 500 µl of nanocapsulated QC (NPQC) (QC 8.98 µmol/kg) was given orally to rats at 90 min prior to the arsenite injection. Key findings Inorganic arsenic depositions (182 ± 15.6 and 110 ± 12.8 ng/g protein) were found in hepatic and neuronal mitochondrial membranes. Antioxidant levels in hepatic and neuronal cells were reduced significantly by arsenic. NPQC prevented the arsenite-induced reduction in antioxidant levels in the liver and brain. Arsenic induced a substantial decrease in liver and brain cell membrane microviscosities, and NPQC treatment resulted in a unique protection against the loss. A significant correlation between mitochondrial arsenic and its conjugated diene level was observed both in liver and brain cells for all experimental rats. Significance Arsenic-specific antidotes are used against arsenic-induced toxicity. However, the target site is poorly recognized and therefore achieving an active concentration of drug molecules can be a challenge. Thus, our objective was to formulate NPQC and to investigate its therapeutic potential in an oral route against arsenite-induced hepatic and neuronal cell damage in a rat model.

The present study was designed to assess the preventive effect of licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid against mitochondrial damage and cell death in lung epithelial cells exposed to 3-morpholinosydnonime, a donor of nitric oxide and superoxide. Treatment of lung epithelial cells with 3-morpholinosydnonime resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. Treatment of glycyrrhizin and 18beta-glycyrrhetinic acid attenuated the 3-morpholinosydnonime-induced mitochondrial damage, formation of reactive oxygen species and GSH depletion and revealed a maximal inhibitory effect at 10 and 1 muM, respectively; beyond these concentrations the inhibitory effect declined. Melatonin, carboxy-PTIO, rutin and uric acid reduced the 3-morpholinosydnonime-induced cell death. The results show that glycyrrhizin and 18beta-glycyrrhetinic acid seem to prevent the toxic effect of 3-morpholinosydnonime against lung epithelial cells by suppressing the mitochondrial permeability transition that leads to the release of cytochrome c and activation of caspase-3. The preventive effect may be ascribed to the inhibitory action on the formation of reactive oxygen species and depletion of GSH. The findings suggest that licorice compounds seem to prevent the nitrogen species-mediated lung cell damage.

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.

Curcumin is a natural antioxidant isolated from the medicinal plant Curcuma longa Linn. We previously reported that manganese complexes of curcumin (Cp-Mn) and diacetylcurcumin (DiAc-Cp-Mn) exhibited potent superoxide dismutase (SOD)-like activity in an in vitro assay. Nitric oxide (NO) is a free radial playing a multifaceted role in the brain and its excessive production is known to induce neurotoxicity. Here, we examined the in vivo effect of Cp-Mn and DiAc-Cp-Mn on NO levels enhanced by kainic acid (KA) and L-arginine (L-Arg) in the hippocampi of awake rats using a microdialysis technique. Injection of KA (10 mg/kg, i.p.) and L-Arg (1000 mg/kg, i.p.) significantly increased the concentration of NO and Cp-Mn and DiAc-Cp-Mn (50 mg/kg, i.p.) significantly reversed the effects of KA and L-Arg without affecting the basal NO concentration. Following KA-induced seizures, severe neuronal cell damage was observed in the CA1 and CA3 subfields of hippocampal 3 days after KA administration. Pretreatment with Cp-Mn and DiAc-Cp-Mn (50 mg/kg, i.p.) significantly attenuated KA-induced neuronal cell death in both CA1 and CA3 regions of rat hippocampus compared with vehicle control, and Cp-Mn and DiAc-Cp-Mn showed more potent neuroprotective effect than their parent compounds, curcumin and diacetylcurcumin. These results suggest that Cp-Mn and DiAc-Cp-Mn protect against KA-induced neuronal cell death by suppression of KA-induced increase in NO levels probably by their NO scavenging activity and antioxidative activity. Cp-Mn and DiAc-Cp-Mn have an advantage to be neuroprotective agents in the treatment of acute brain pathologies associated with NO-induced neurotoxicity and oxidative stress-induced neuronal damage such as epilepsy, stroke and traumatic brain injury.

The labdane diterpene solidagenone 1 and its semisynthetic and biotransformation derivatives 2 – 10 were assessed for gastroprotective and ulcer-healing effect using human epithelial gastric cells (AGS) and human lung fibroblasts (MRC-5). The ability of the compounds to protect the AGS cells against the damage induced by sodium taurocholate (NaT), to stimulate the cellular reduced glutathione (GSH), prostaglandin E 2 content, enhance AGS and MRC-5 cell proliferation and to scavenge superoxide anion in vitro was studied. The cytotoxicity of the compounds was assessed towards MRC-5 fibroblasts and AGS cells. A significant reduction of cell damage after NaT incubation was observed when the AGS cells were pretreated with compounds 2 and 6 . Treatment with compounds 4 – 6 , 8 and 9 significantly stimulated the GSH content in AGS cell cultures. None of the studied compounds was active as a superoxide anion scavenger. In AGS cells treated with compounds 1 – 10 , only compound 5 was able to increase prostaglandin content. Concerning the proliferation assays, a significant stimulating effect was observed for compounds 2 , 8 , 9 on AGS cells and for 5 , 7 – 9 on MRC-5 fibroblasts. Regarding cytotoxicity, solidagenone showed higher toxicity while compounds 4 and 7 were the less toxic. Our results showed that most of the studied compounds act in vitro as gastroprotectors increasing the cellular GSH content. Additionally, some derivatives exhibited in vitro ulcer-healing effect stimulating the cell proliferation.

The mood stabilizing drug lithium is a highly effective treatment for bipolar disorder. Previous studies in our laboratory found that chronic treatment with the mood stabilizing drug valproate in rat brain increased the expression of endoplasmic reticulum (ER) stress proteins GRP78, GRP94 and calreticulin. We report here that in primary cultured rat cerebral cortical cells, expression of GRP78, GRP94 and calreticulin are increased not only by valproate, but also by lithium after chronic treatment for 1 week at therapeutically relevant concentrations. However, two other mood stabilizing drugs carbamazepine and lamotrigine had no effect on expression of GRP78, GRP94 or calreticulin. Chronic treatment with lithium for 1 week increased both mRNA and protein levels of ER stress proteins. In contrast to a classic GRP78 inducer thapsigargin, an inhibitor of the ER Ca 2+ -ATPase, chronic treatment with lithium or valproate for 1 week modestly increased GRP78 expression in neuronal cells, had no effect on basal intracellular free Ca 2+ concentration and does not induce cell death. These results indicate that lithium and valproate may increase expression of GRP78, GRP94 and calreticulin in primary cultured rat cerebral cortical cells without causing cell damage. These results also suggest that the mechanism of GRP78 increase induced by lithium and valproate may be different from that of thapsigargin.

Although information on energy metabolism during hypoxemic-ischemic states is abundant, data on perinatal asphyxia (PA) are limited. As results from hypoxia-ischemia cannot be directly extrapolated to PA, a clinical entity characterized by acidosis, hypoxemia and hypercapnia, we decided to use a rat model of graded PA during delivery. Cesarean section was performed at the 21st day of gestation and the pups, still in the uterus horns, were asphyxiated from 0 to 20 minutes. In this model survival decreases with the length of asphyxia. Early changes of energy-rich phosphates in brain, heart and kidney were determined by HPLC. ATP and phosphocreatine gradually decreased with the length of asphyxia, with highest ATP depletion rate occurring in the kidney. ATP: brain 1.39 +/- 0.71 (0 min) to 0.06 microM/g wwt (20 min); heart 4.73 +/- 0.34 (0 min) to 1.08 +/- 0.47 (20 min); kidney 1.62 +/- 0.11 (0 min) to 0.02 +/- 0.02 (20 min). Phosphocreatine: brain 1.65 +/- 0.68 (0 min) to 0.51 +/- 0.45 microM/g (20 min); heart 6.98 +/- 0.38 (0 min) to 6.17 +/- 1.07 (20 min); kidney 8.23 +/- 0.86 (0 min) to 3.76 +/- 0.54 (20 min). We present data on energy derangement in a rat model of PA, closely resembling the clinical situation, showing that energy depletion precedes cell damage and death.

The calcium paradox stands for the cell damage that occurs when isolated hearts are perfused with a Ca(2+)-free solution followed by perfusion with a Ca(2+)-containing solution. Although it is generally accepted that a massive Ca2+ influx during the Ca(2+)-repletion phase is responsible for the cell damage, there is no consensus about what makes the heart susceptible to the calcium paradox during the Ca(2+)-depletion phase. It has been suggested that the extent of the calcium paradox is primarily determined by accumulation of Na+ during Ca2+ depletion and a subsequent accumulation of Ca2+ via reverse Na(+)-Ca2+ exchange during Ca2+ repletion. According to another theory, weakening of intercalated disc junctions during Ca2+ depletion and contracture-mediated disruption of the cell membrane during Ca2+ repletion are responsible for the calcium paradox. In the present study we further investigated the possible role of Na+ in the development of the calcium paradox. During Ca2+ depletion, lidocaine was used to inhibit Na+ entry through the Na+ channels. Isolated rat hearts were perfused with Krebs Henseleit buffer (KH) containing 1.4 mM Ca2+ for 15 min, followed by 10 min of Ca(2+)-free perfusion and 10 min of reperfusion with Ca2+. In the treated group 0.1 mM lidocaine was present throughout the experiment. At the end of each experiment, Ca2+ cytochemistry was performed and the intracellular Ca2+ content was analyzed by laser microprobe mass analysis (LAMMA). The results show that during Ca2+ depletion, the intracellular Ca2+ content did not change significantly. Ca2+ repletion, however, gave rise to a full calcium paradox irrespective of the presence of lidocaine: massive cell damage and Ca2+ accumulation in the mitochondria. The results provide further evidence that intracellular Na+ accumulation during Ca2+ depletion is not involved in the occurrence of the calcium paradox.

Primary cultures of newborn rat myocardial cells were treated in various extracellular calcium concentrations (0, 1.35, 2.7, 4.05, and 5.4 mM) with three different drugs; namely, ouabain, sulmazole, and chlorpromazine. Lactate dehydrogenase (LDH) release was used as an indicator of damage. The results showed 10−3M ouabain caused apparent damage of the cells and the damage was increased by an increased extracellular calcium concentration. Sulmazole (10−3M) caused damage of the cells in the absence of calcium; but it did not cause damage of the cells in the presence of calcium; it protected the cells from damagedcaused by high calcium concentrations (4.05 and 5.4 mM). Chlorpromazine (1.6 × 10−4M) caused severe damage of the cells. The various calcium concentrations had no influence on the degree of the damage. Correlation coefficients showed that correlations between the calcium concentrations and the cell damage caused by ouabain, sulmazole and chlorpromazine were positive correlation, negative correlation, and no correlation, respectively. It is suggested that influx of extracellular calcium is not a final common pathway of drug-induced myocardial cell injury, although it plays an important role in cell injury.

Publisher Summary This chapter elaborates a study analyzing mechanisms of exocytosis. Exocytosis is a method of discharge of secretory products in which there is selective release of granule-bound material. Other forms of secretion may cause nonselective release of cellular constituents. In the adrenal medulla, for instance, elevated pH causes the release not only of catecholamines but also of a cytoplasmic marker, lactic dehydrogenase (LDH). This is probably indicative of cell damage. Reserpine can release catecholamines without discharge of other soluble granule constituents. This represents another mechanism of catecholamine release and is also different from exocytosis. Evidence that exocytosis occurs in the adrenal medulla in response to the physiological transmitter, acetylcholine, and other drugs have been reviewed. This chapter discusses some of the factors believed to be involved in exocytisis in the adrenal medulla. The chapter highlights that inhibitors of ATP synthesis increase the spontaneous release of catecholamines from the adrenal gland, while inhibiting the evoked release. These findings have been interpreted along with the evidence that ATP activates calcium uptake by cell organelles and that ATP can release catecholamines from isolated chromaffin granules.

Mitochondria are known to develop a series of abnormalities as a result of ischemia. The inability of mitochondria to resume normal function following reperfusion has been implicated as an important factor in irreversible cell damage. However, the mechanism of mitochondrial injury after ischemic brain insult is poorly understood. In this paper a hypothesis is proposed which concentrates on the interrelated roles of phosphate, calcium, and electron transport on ischemic brain cell injury.

Oxidative stress and covalent binding have been proposed as possible mechanisms involved in the cytotoxic effects of the parkinsonism-causing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the toxicity induced by MPTP in isolated rat hepatocytes seems to be relatively independent of oxygen radical-induced oxidative stress. Here we demonstrate that MPTP cytotoxicity is not potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, nor prevented by the antioxidant N,N′-diphenyl-p-pphenylenediamine (DPPD) or the iron-chelating agent desferrioxamine. Moreover, preincubation of hepatocytes with diethylmaleate to lower the level of intracellular reduced glutathione (to 20% of the initial value) did not affect either the rate or extent of MPTP cytotoxicity. Thus, nucleophilic soluble thiols do not seem to play a protective role against MPTP-induced cell damage, in contrast to what one would have expected if covalent protein binding and oxidative stress were involved as toxic mechanisms. On the other hand, MPTP cytotoxicity was potentiated by pretreatment of hepatocytes with cytochrome P-450 inhibitors (e.g., SKF 525A and metyrapone) and a more rapid depletion of ATP was observed in these experimental conditions. We conclude that mitochondrial damage and subsequent ATP depletion are likely to play a critical role in the toxicity of MPTP to isolated hepatocytes and that the metabolism of MPTP via the cytochrome P-450 monooxygenase system can be considered to be a detoxifying pathway.

Data reviewed here indicate that acute and chronic ethanol ingestion induce a decrease in the concentration of GSH and an increase in lipoperoxidation in the liver both in experimental animals and in man, changes that are closely interrelated GSH depletion is suggested to be due to an oxidation in the liver tissue and to a translocation into the extrahepatic medium as free glutathione and/or as conjugates with ethanol-derived acetaldehyde. As a result, the hepatic GSH/GSSG ratio is drastically reduced. Lipoperoxidation seems to be related to the metabolism of ethanol and acetaldehyde by secondary pathways that are known to generate oxygen-related free radicals. Being lipoperoxidation a process associated with cell damage and death, its stimulation by ethanol ingestion could play a role in the production of alcoholic liver damage in man. The involvement of several contributory factors in the development of a high lipoperoxidative index in the liver in this situation is discussed.

The effects of vinpocetine on hippocampal cell damage and local cerebral blood flow (LCBF) were measured in a rat model of forebrain ischemia (2-vessel occlusion and hypotension). Duration of ischemia was 10 min. LCBF was determined after 2 min of recirculation using the 14C-iodoantipyrine technique. Hippocampal cell loss was quantified histologically 7 days post-ischemia. Intraperitoneal application of vinpocetine (10 mg/kg) 15 min prior to ischemia significantly reduced neuronal cell loss in hippocampal CA 1 sector from 60% to 28%. The drug led to a marked increase in blood flow in cortical areas, whereas LCBF remained unchanged in hippocampus and all other structures measured. It is suggested that the protective effect of vinpocetine does not depend on increased postischemic blood flow.