Cytoprotective and antioxidant effects of the red alga Alsidium corallinum against hydrogen peroxide-induced toxicity in rat cardiomyocytes.

CONTEXT: Sepsis is the manifestation of the immune and inflammatory responses to infection that may ultimately result in multiorgan failure. Many substances are involved in myocardial dysfunction in sepsis, including hydrogen peroxide. OBJECTIVE: This study evaluates the protective activity of the red alga Alsidium corallinum against hydrogen peroxide (H2O2)-induced toxicity in H9c2 cardiomyocytes. MATERIAL AND METHODS: The biological properties of A. corallinum were firstly investigated. Secondly, the H9c2 cells were pre-treated with alga extract, and then exposed to H2O2. RESULTS: Our results showed richness of the alga in antioxidant compounds, and its biological activities. H2O2 induced a morphological changes and decrease in H9c2 cell viability correlating with an increase in enzymatic and non-enzymatic antioxidants. Pre-treatment with A. corallinum, reduces toxicity and decreased the antioxidants status induced by H2O2. CONCLUSION: These findings indicated for the first time the protective effect of A. corallinum against H2O2-induced toxicity in H9c2 cells.

Limoniastrum guyonianum prevents H2O2-induced oxidative damage in IEC-6 cells by enhancing enzyamtic defense, reducing glutathione depletion and JNK phosphorylation.

Limoniastrum guyonianum is used in several regions of North Africa as a folk medicine. The objective of this study was to determine the in vitro antioxidant activities of L. guyonianum roots and their cytoprotective action on H2O2-challenged rat small intestine epithelial cells (IEC-6 cells). To assess the cytoprotective effect of L. guyonianum extract (LGE), IEC-6 cells were pre-incubated with different LGE concentrations. Then, IEC-6 cultures were exposed to 40µM H2O2 during 4h. Modulation of endogenous antioxidant system including SOD, CAT, MDA, GSH and the expression of possibly involved MAPKs was evaluated. Main results reported that L. guyonianum was rich in polyphenols and exhibited an important antioxidant activity as revealed by different tests (DPPH Assay, IC50=1.6µg/mL; ABTS+ test, IC50=27µg/mL; Fe-reducing power, EC50=44µg/mL). HPLC analysis showed that quercetin, catechin, and isorhamnetin-3-O-rutinoside were major phenolics. The exposure of IEC-6 cells to 40µM H2O2 during 4h resulted in oxidative stress manifested by (i) over 70% cell mortality, (ii) over-activity of CAT (246%), (iii) decrease in GSH level (10.4nmol/mg), (iv) excess in MDA content (18.4nmol/mg), and (v) a trigger of JNK phosphorylation. Pretreatment with LGE, especially at 0.25µg/mL, restored cell viability to 100%, and normal cell morphology in H2O2-chalenged cells. In addition, this extract maintained a high CAT activity, enhanced SOD capacity (120%) and increased GSH level (45.5nmol/mg). Furthermore, reducing cell death seems to be due to dephosphorylated JNK MAPK exerted by L. guyonianum bioactive compounds. In all, L. guyonianum components provided a cross-talk between regulatory pathways, implying their role as cytoprotector against oxidative stress.

Cytoprotective effects of essential oil of Pinus halepensis L. against aspirin-induced toxicity in IEC-6 cells.

CONTEXT: Essential oils from Pinus species have been reported to have various therapeutic properties. OBJECTIVE: This study was undertaken to identify the chemical composition and cytoprotective effects of the essential oil of Pinus halepensis L. against aspirin-induced damage in cells in vitro. MATERIAL AND METHODS: The cytoprotection of the oil against toxicity of aspirin on the small intestine epithelial cells IEC-6 was tested. RESULTS: The obtained results have shown that 35 different compounds were identified. Aspirin induced a decrease in cell viability, and exhibited significant damage to their morphology and an increase in superoxide dismutase (SOD) and catalase (CAT) activities. However, the co-treatment of aspirin with the essential oil of Pinus induced a significant increase in cell viability and a decrease in SOD and CAT activities. CONCLUSION: Overall, these finding suggest that the essential oil of Pinus halepensis L. has potent cytoprotective effect against aspirin-induced toxicity in IEC-6 cells.

Potential protective effects of alpha-pinene against cytotoxicity caused by aspirin in the IEC-6 cells.

Alpha-pinene is a key compound of the essential oils extracted from many species of coniferous trees. It is known for its biological activities. The aim of the present study was to determine the preventive effect of alpha-pinene on aspirin-induced toxicity in vitro, using IEC-6 cells, and to investigate its antioxidant activities. The antioxidant activities were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). The cytotoxicity and oxidative stress were detected by cell viability, antioxidant enzyme activity, malondialdehyde (MDA) and GSH production, and the activation of MAPK pathways. The results indicated that alpha-pinene revealed an important antioxidant activity. It was evaluated by DPPH test (EC50=310±10µg/mL) and FRAP test (EC50=238±18.92µg/mL). The co-exposure of alpha-pinene with aspirin on cells significantly increased the survival of cells and the level of GSH, and decreased the levels of MDA and total SOD and the activity of Mn-SOD. In addition, the activation of p38 and JNK was blocked by alpha-pinene. Therefore, these findings suggest that alpha-pinene can protect IEC-6 cells against aspirin-induced oxidative stress.

Tamarix gallica phenolics protect IEC-6 cells against H2O2 induced stress by restricting oxidative injuries and MAPKs signaling pathways.

Polyphenolic compounds gained interest in the pharmaceutical research area due to their beneficial properties. Herein, antioxidant and cytoprotective capacities of T. gallica extract on H2O2-challenged rat small intestine epithelial cells were investigated. To set stress conditions, IEC-6 cultures were challenged with numerous H2O2 doses and durations. Then, 40µM H2O2 during 4h were selected to assess the cytoprotective effect of different T. gallica extract concentrations. Oxidative parameters, measured through CAT and SOD activities as well as MDA quantification were assessed. In addition, the expression of possibly involved MAPKs was also valued. Main results reported that T. gallica was rich in polyphenols and exhibited an important antioxidant activity (DPPH Assay, IC50=6µgmL-1; ABTS+ test, IC50=50µgmL-1; Fe-reducing power, EC50=100µgmL-1). The exposure of IEC-6 cultures to 40µM H2O2 during 4h caused oxidative stress manifested by (i) over 70% cell mortality, (ii) over-activity of CAT (246%), (iii) excess in MDA content (18.4nmolmg-1) and (iiii) a trigger of JNK phosphorylation. Pretreatment with T. gallica extract, especially when used at 0.25µgmL-1, restored cell viability to 122%, and normal cell morphology in H2O2-chalenged cells. In addition, this extract normalized CAT activity and MDA content (100% and 14.7nmolmg-1, respectively) to their basal levels as compared to control cells. Furthermore, stopping cell death seems to be due to dephosphorylated JNK MAPK exerted by T. gallica bioactive compounds. In all, T. gallica components provided a cross-talk between regulatory pathways leading to an efficient cytoprotection against harmful oxidative stimulus.

Protective effects of essential oil of Citrus limon against aspirin-induced toxicity in IEC-6 cells.

Aspirin, one of the widely used nonsteroidal anti-inflammatory drugs, is the most highly consumed pharmaceutical product in the world. However, it has several side effects in cells. This study was designed to investigate the antioxidative activity and cytoprotective effects of essential oil of Citrus limon (EOC) extracted from leaves against aspirin-induced damages in the rat small intestine epithelial cells (IEC-6). Biochemical indicators were used to assess cytotoxicity and oxidative damages caused by aspirin treatment on IEC-6. Our results showed that the chemical characterization of EOC identified 25 compounds representing 98.19% of the total oil. The major compounds from this oil were z-citral (53.21%), neryl acetate (13.06%), geranyl acetate (10.33%), and limonene (4.23%). Aspirin induced a decrease in cell viability as well as an increase in superoxide dismutase (SOD) and catalase (CAT) activities. Contrariwise, the co-exposure of cells to aspirin and EOC alleviated every above syndrome by an increase in cell survival and decrease in SOD and CAT activities. In conclusion, the essential oil of C. limon has a potent cytoprotective effect against aspirin-induced toxicity in IEC-6 cells.

Biological properties of citral and its potential protective effects against cytotoxicity caused by aspirin in the IEC-6 cells.

Citral, 3,7-dimethyl-2,6-octadienal, is a key component of several essential oils extracted from lemon-scented herbal plants. The present study was designed to investigate the antioxidant activities of citral and assess its possible protective effects against aspirin-induced toxicity in vitro. We used IEC-6 cells (rat small intestine epithelial cells). The antioxidant activities were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH), ß-carotene/linoleic acid and Ferric reducing antioxidant power (FRAP). Cytotoxicity was evaluated by cell viability, anti-oxidant enzyme activities, malondialdehyde (MDA) production and by the expression of MAPKs (Mitogen-Activated Protein Kinases) pathways. According to results, citral showed an important antioxidant activity. It inhibited the oxidation of linoleic acid, a moderate DPPH was found and it showed a Ferric reducing antioxidant potential with an EC50 value of 125±28.86µg/mL. Then, the co-treatment of aspirin with citral significantly decreased the aspirin-induced cell death, and the MDA level. It modulated the superoxide dismutase (SOD) and glutathione (GSH) activities. Also, the activation of MAPKs was attenuated by citral. These findings suggest that citral can protect IEC-6 cells against aspirin-induced oxidative stress that may help to discover new chemicals out of natural antioxidant substances.

The diaphragm is better protected from oxidative stress than hindlimb skeletal muscle during CLP-induced sepsis.

OBJECTIVES: The aim of this study was to determine whether non-lethal sepsis induced by cecal ligation and puncture (CLP) modulates oxidative damage and enzymatic antioxidant defenses in diaphragm and hindlimb skeletal muscles (soleus and Extensor Digitorus Longus (EDL)). METHODS: Female Wistar rats were divided into four experimental groups: (1) control animals, (2) animals sacrificed 2 hours or (3) 7 days after CLP, and (4) sham-operated animals. At the end of the experimental procedure, EDL, soleus, and diaphragm muscles were harvested and 4-hydroxynonenal (HNE)-protein adducts and protein carbonyl contents were examined in relation to superoxide dismutase and catalase expression and activities. RESULTS: We observed that both non-respiratory oxidative (i.e. soleus) and glycolytic skeletal muscles (i.e. EDL) are more susceptible to sepsis-induced oxidative stress than diaphragm, as attested by an increase in 4-HNE protein adducts and carbonylated proteins after 2 hours of CLP only in soleus and EDL. DISCUSSION: These differences could be explained by higher basal enzymatic antioxidant activities in diaphragm compared to hindlimb skeletal muscles. Together, these results demonstrate that diaphragm is better protected from oxidative stress than hindlimb skeletal muscles during CLP-induced sepsis.

The protective effect of Citrus limon essential oil on hepatotoxicity and nephrotoxicity induced by aspirin in rats.

Citrus limon is a member of the large Rutaceae family characterized by its therapeutic proprieties and has been widely used in traditional medicine to treat various diseases. This study investigates the protective effect of Citrus limon essential oil against a high dose of aspirin-induced acute liver and kidney damage in female Wistar albino rats. Twenty-eight adult female Wistar rats were divided into 4 groups of 7 each: (1) a control group; (2) a group of rats which was kept untreated for 56days then treated with aspirin (A) (600mg/kg) for 4 days; (3) a group fed with essential oil of Citrus limon for 56days then (A) for 4 days; and (4) a group of rats receiving essential oil of Citrus limon for 56 days, then given NaCl for 4 days. Estimations of biochemical parameters in blood were determined. Lipid peroxidation levels (TBARS), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidas (GPx) activities in liver and kidney was determined. A histopathological study was done. Under our experimental conditions, aspirin induced an increase of serum biochemical parameters and it resulted in an oxidative stress in both liver and kidney. This was evidenced by significant increase in TBARS in liver and kidney by 108% and 55%, respectively, compared to control. On the other hand, a decrease in the activities of SOD by 78% and 53%, CAT by 53% and 78%, and GPx by 78% and 51% in liver and kidney, respectively. Administration of EOC to rats attenuated the induced an effect of the high dose of aspirin induced in the afore mentioned serum biochemical parameters. In conclusion, our data suggest that treatment with essential oil of Citrus limon prevented the liver and kidney damage induced by aspirin.

Protective Effects of Pinus halepensis L. Essential Oil on Aspirin-induced Acute Liver and Kidney Damage in Female Wistar Albino Rats.

Aromatic and medicinal plants are sources of natural antioxidants thanks to their secondary metabolites. Administration of Pinus halepensis L. (Pinaceae family) in previous studies was found to alleviate deleterious effects of aspirin-induced damage on liver and kidney. The present study, carried out on female rats, evaluates the effects of P. halepensis L. essential oil (EOP) on aspirin (A)-induced damage to liver and kidney. The animals used in this study were rats (n=28) divided into 4 groups of 7 each: (1) a control group (C); (2) a group given NaCl for 56 days then treated with (A) (600 mg/kg) for 4 days (A); (3) a group fed with (EOP) for 56 days then (A) for 4 days; and a group fed with only (EOP) for 56 days and given NaCl for 4 days. Estimations of biochemical parameters in blood were determined using kit methods (Spinreact). Lipid peroxidation levels (TBARS), superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) activities were determined. Histopathological study was done by immersing pieces of both organs in a fixative solution followed by paraffin embeddeding and hematoxylin-eosin staining. Under our experimental conditions, Aspirin at dose 600 mg/kg body weight induced an increase of serum biochemical parameters as well as an oxidative stress in both organs. An increase occurred in TBARS by 108% and 55%, a decrease in SOD by 78% and 53%, CAT by 53% and 78%, and GPx by 78% and 51% in liver and kidney, respectively, compared to control. Administration of EOP given to rats enabled correction in these parameters. It could be concluded that the treatment with P. halepensis L. essential oil inhibited aspirin-induced liver and kidney damage.

Cytoprotective and antioxidant effects of the edible halophyte Sarcocornia perennis L. (swampfire) against lead-induced toxicity in renal cells.

Lead (Pb) exposure is considered as a risk factor responsible for renal impairment in humans. On the other hand, the halophyte Sarcocornia perennis is a fresh vegetable crop suitable for leafy vegetable production. This study was designed to evaluate the in vitro protective activity of S. perennis against lead-induced damages in HEK293 kidney cells. Morphological and biochemical indicators were used to assess cytotoxicity and oxidative damages caused by Pb treatment on the cells. Our results showed that lead induced (1) a decrease in cell viability (MTT), (2) cell distortion and cohesion loss, (3) superoxide anion production and lipid peroxidation. Conversely, addition of S. perennis extract to the lead-containing medium alleviated every above syndrome. Thus, cell survival was increased and the production of reactive oxygen species caused by Pb treatment was inhibited. Taken together, our study revealed that S. perennis has potent cytoprotective effect against Pb-induced toxicity in HEK 293 cell. Such action would proceed through the decrease in ROS levels and resulting oxidative stress, which suggests a potential interest of this halophyte in the treatment of oxidative-stress related diseases.

Effect of okadaic acid on cultured clam heart cells: involvement of MAPkinase pathways.

Okadaic acid (OA) is one of the main diarrhetic shellfish poisoning toxins and a potent inhibitor of protein phosphatases 1 and 2A. The downstream signal transduction pathways following the protein phosphatase inhibition are still unknown and the results of most of the previous studies are often conflicting. The aim of the present study was to evaluate the effects of OA on heart clam cells and to analyse its possible mechanisms of action by investigating the signal transduction pathways involved in OA cytotoxicity. We showed that OA at 1 µM after 24 h of treatment induces disorganization of the actin cytoskeleton, rounding and detachment of fibroblastic cells. Moreover, treatment of heart cells revealed a sequential activation of MAPK proteins depending on the OA concentration. We suggest that the duration of p38 and JNK activation is a critical factor in determining cell apoptosis in clam cardiomyocytes. In the opposite, ERK activation could be involved in cell survival. The cell death induced by OA is a MAPK modulated pathway, mediated by caspase 3-dependent mechanism. OA was found to induce no significant effect on spontaneous beating rate or inward L-type calcium current in clam cardiomyocytes, suggesting that PP1 was not inhibited even by the highest dose of OA.

New antibacterial and cytotoxic activities of falcarindiol isolated in Crithmum maritimum L. leaf extract.

Bioassay-guided fractionation of a chloroformic extract obtained from Crithmum maritimum leaves led to the chemical isolation of falcarindiol, a polyacetylene widely distributed within the Apiaceae family. Structure of this compound was confirmed by NMR and activity was tracked using a screening microplate bioassay. Falcarindiol strongly inhibited the growth of Micrococcus luteus and Bacillus cereus, with a minimum inhibitory concentration (MIC) value of 50 microg ml(-1). Moreover, this compound showed cytotoxicity against IEC-6 cells with an IC50 value of 20 microM after 48 h of exposition. These results suggest that Crithmum maritimum could be potentially used in food manufactures and cosmetology as preservative agents and biopesticides, or in medicine as new antibiotics, confirming the interest in studying halophytic species as sources of bioactive compounds.

Effects of chronic sepsis on contractile properties of fast twitch muscle in an experimental model of critical illness neuromyopathy in the rat.

OBJECTIVE: Critical illness polyneuromyopathy has been extensively studied in various animal models regarding electrophysiological aspects or molecular mechanisms involved in its physiopathology; however, little data are available on its main clinical feature, that is, muscular weakness. We have studied the effects of chronic sepsis in rats with special consideration to contractile and neuromuscular blockade properties in relation with the level of messenger RNA (mRNA) coding for ryanodine and acetylcholine receptors. DESIGN: This was an experimental animal study. SETTING: This study was conducted at a university laboratory. SUBJECTS: Subjects consisted of Wistar rats. INTERVENTIONS: Chronic sepsis was achieved by cecal ligation and needle perforation. Ten days after surgery, fast twitch extensor digitorum longus was excised for extraction and assays of mRNA coding for ryanodine and acetylcholine receptor subunits and contralateral muscle was tested in vivo on a mechanical bench. A fatigability index was measured and neuromuscular blockade properties using atracurium were evaluated. MEASUREMENTS AND MAIN RESULTS: A decrease in active force developed by extensor digitorum longus associated with an increase in passive force is induced by chronic sepsis. Maximal force at optimal length during twitch contraction was significantly reduced (0.25 +/- 0.09 N vs. 0.17 +/- 0.06 N); contraction and relaxation speeds were higher as shown by the decrease of respective time constants (3.75 +/- 0.01 msec vs. 2.70 +/- 0.0 msec, 10.76 +/- 0.03 msec vs. 7.62 +/- 0.03 msec) in the control group compared with the septic group. Fatigability index was significantly lower (23 +/- 0.11% vs. 59 +/- 0.19%) in septic rats. These rats also showed quicker blockade and shorter recovery after atracurium administration. Sepsis induced a significant increase of the expression of ryanodine receptor (RyR) RyR1 along with a steady expression of RyR3 mRNA, leading to a 5.6-fold increase of RyR1/RyR3 ratio with a steadiness of mRNA corresponding to acetylcholine-receptors. CONCLUSIONS: Chronic inflammation and sepsis induced a decrease in contractile performances of extensor digitorum longus along with accelerated kinetics of atracurium possibly induced by modified expression of RyR1 receptors and not acetylcholine-receptors.

Effects of chronic sepsis on the voltage-gated sodium channel in isolated rat muscle fibers.

OBJECTIVE: Physiopathology of critical illness polyneuromyopathy was investigated in several animal-based models. Electrophysiologic approach was achieved in denervated and corticosteroid-induced myopathy; other models based on sepsis or inflammatory factors (zymosan, cytokines) were also used but did not consider voltage-gated sodium channel implication in neuromuscular weakness. We have studied electrophysiologic effects of chronic sepsis on an intact neuromuscular rat model with special consideration to the subtypes of sodium channels involved. DESIGN: Experimental animal study. SETTING: University laboratory. SUBJECTS: Wistar rats. INTERVENTIONS: Chronic sepsis was achieved by a technique of cecal ligature and needle perforation. Ten days after surgery, the rats were killed. Fast-twitch flexor digitorum brevis was excised and dissociated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline supplemented with 3.0 mg/mL collagenase. Fast sodium currents were recorded by a macropatch clamp technique at room temperature (22+/-2 degrees C) in a cell-attached configuration. MEASUREMENTS AND MAIN RESULTS: A decrease in maximal sodium current and in conductance was evidenced without modification of the sodium Nernst potential. A shift of the voltage inactivation curve toward more negative potentials could explain the observed decrease in excitability. In parallel, we observed an up-regulation of NaV 1.5-type sodium channels. CONCLUSIONS: Chronic inflammation and sepsis induced modifications of sodium channel properties that could contribute to muscular inexcitability. This inexcitability can be elicited by a modification of properties or type of voltage-gated sodium channels. Our results lead us to explain this inexcitability by an up-regulation of NaV 1.5 sodium channel.

Effects of immobilizing a single muscle on the morphology and the activation of its muscle fibers.

A single muscle of Wistar female rats, either soleus or peroneus longus, was immobilized by fixing its cut distal tendon to the bone during 8 weeks. We observed a transitory weight loss in both muscles; the mean fiber cross-sectional area (CSA) showed a reduction at day 30, followed by an increase at day 60. The time course of the activation of the immobilized muscle was evaluated by recording the chronic electromyographic (EMG) activity during short periods (1 min every other day) of treadmill locomotion. During immobilization, the integrated EMG amplitude of the soleus increased, reaching a maximum at 4 weeks, but remained close to control values during 8 weeks for the peroneus. The median frequency (MF) of the power density spectrum of the soleus EMG was not statistically different between immobilized and control muscles, while MF of the immobilized peroneus EMG was permanently higher than that of control muscles. This suggests two different modes of adaptation in motor unit command, depending on the muscle profile, which could be concomitant with the restoration of muscle fibers CSA after 8 weeks.

Characterization of the voltage-activated currents in cultured atrial myocytes isolated from the heart of the common oyster Crassostrea gigas.

Using the macro-patch clamp technique, we show that cardiac myocytes isolated from the heart of the oyster Crassostrea gigas possess several types of voltage-activated ionic currents. (1) A classical non-inactivating potassium current of the IK type that is inhibited by tetraethyl ammonium and shows an outward rectification and a slow activation. (2) A potassium current of the IA type that shows rapid activation and inactivation, and is blocked by 4-amino pyridine or preliminary depolarisation. (3) A potassium calcium-dependent current that is inhibited by charybdotoxin, activated by strong depolarisations and shows a large conductance. (4) A calcium inward current of the L-type that is inhibited by verapamil, cobalt and high concentrations of cadmium. This current is identified in most cells, but a T-type calcium current and classical fast sodium current are only identified in few cells, and only after a strong hyperpolarizing pulse. This suggests that these channels are normally inactivated in cultured cells and are not involved in the spontaneous activity of these cells. When they exist, the fast sodium channel is blocked by tetrodotoxin. The L-type calcium conductance is increased by serotonin. The identification in cultured oyster atrial cells of classical ionic currents, which are observed in most vertebrate species but only in a few species of molluscs, demonstrates that these cells are an interesting model. Moreover the viability and the electrophysiological properties of these cells are not significantly modified by freezing and thawing, thus increasing their usefulness in various bioassays.