Abstract
Background
Neferine, a bisbenzylisoquinoline alkaloid, isolated from Nelumbo nucifera has a wide range of biological activities. Cobalt chloride (CoCl2) was known to mimic hypoxic condition. In the present study, we assessed the cytoprotective effect of neferine against CoCl2-induced oxidative stress in muscle cells.
Methods
Rhabdomyosarcoma cells were exposed to different concentrations of CoCl2, and the IC50 value was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Lactate dehydrogenase and NO assays were performed in order to determine the cytotoxic effect of CoCl2. Reactive oxygen species generation and cellular antioxidant status were determined for evaluating oxidative stress. For analyzing the effect of neferine on CoCl2-induced apoptosis, propidium iodide staining was performed.
Results
The results of the present study indicate that CoCl2 induces cell death in a dose-dependent manner. Neferine pretreatment at 700 nM concentration offers better cytoprotection in the cells exposed to CoCl2. Lactate dehydrogenase and NO release in the culture medium were restored after neferine pretreatment. CoCl2 triggers time-dependent reactive oxygen species generation in muscle cells. Further, results of propidium iodide staining, mitochondrial membrane potential, and intracellular calcium accumulation confirm that neferine offers protection against CoCl2-induced hypoxic injury. Depleted activities of antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase due to CoCl2 exposure were also reinstated in the group that received neferine pretreatment.
Conclusion
Our study suggests that neferine from N. nucifera offers protection to muscle cells by counteracting the oxidative stress induced by CoCl2.
Keywords: cobalt chloride, hypoxia, neferine, oxidative stress, reactive oxygen species
1. Introduction
Cobalt chloride (CoCl2) has widely been used to mimic hypoxia in cell culture, and it is known to activate hypoxic signaling. Hypoxia is a pathophysiological condition characterized by an increase in reactive oxygen species (ROS) and a change in intracellular redox level. Oxygen flux in the mitochondria of skeletal muscle increases up to 100-fold at high altitude and during physical exercise, which in turn results in an increased generation of free radicals. Physical exercise results in oxidative stress, which is associated with increases in lipid peroxidation (LPO) and protein oxidation.1 High-altitude physiology may be divided into studies of short-term changes that occur with exposure to hypobaric hypoxia (the acute response to hypoxia) and studies of longer-term acclimatization and adaptation. Acute exposure to the ambient atmosphere at extreme altitude (for example, above 8000 m) is rapidly fatal.2 Acclimatization is the set of beneficial processes whereby lowland humans respond to a reduced inspired partial pressure of oxygen. These changes tend to reduce the gradient of oxygen partial pressure from ambient air to tissues (classical oxygen cascade), and are distinct from the pathological changes that lead to altitude illness. Adaptation to high altitude describes changes that have occurred over a number of generations as a result of natural selection in a hypobaric hypoxic environment, and this can be observed in some groups of high-altitude residents.
Cellular response to hypoxia may provide important clues about impaired cellular function and neuronal cell death. ROS have been proposed to act as second messengers in redox-sensitive signal transduction pathways and can damage biomolecules.3 Reactive oxygen intermediates, superoxide radicals (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH), are produced mainly in mitochondria. ROS act as physiological modulators of some mitochondrial functions, but may also damage mitochondria. Oxygen-derived radicals are implicated in LPO events and are critical in injuries after ischemia.4, 5
Physical exercise leads to temporary ischemia in muscles. During strenuous exercise, muscle oxygen consumption increases tremendously to as high as 100–200 times that in normal resting conditions.6 The sudden increase and influx of oxygen cause a calcium overload in cells, leading to an influx of inflammatory cells into reperfused tissue. This will lead to the generation of ROS that are considered responsible for muscle fatigue during exercise.7 Hence, oxidative stress plays a significant role in the initiation and progression of fatigue. Oxidative stress, more specifically LPO, is known to play an important role in the pathophysiology of exercise intolerance at high altitude. Exposure to high altitude appears to decrease the activity and effectiveness of the antioxidant enzyme system. Therefore, supplementation of antioxidants seems to be a necessary step to prevent or decrease the high-altitude-exposure-associated oxidative stress.8
Traditional knowledge reveals many medicinal uses of lotus plant. Neferine is a bisbenzylisoquinoline alkaloid extracted (isolated) from the green seed embryo of Nelumbo nucifera Gaertn (lotus), which has a wide range of pharmacological activities. Lotus seeds are in high demand in Ayurvedic medicinal preparations, and are widely used in folk medicines to treat tissue inflammation, cancer, diuretics,9 and skin diseases and as an antidote to poison.10 Embryos of lotus seeds are used in a traditional Chinese drug, called “LianZiXin,” which is used in the treatment of nervous disorders, insomnia, high fevers (with restlessness), and cardiovascular diseases (e.g., hypertension and arrhythmia).11 The major phytochemicals present in lotus seeds are alkaloids (e.g., dauricine, lotusine, nuciferine, pronuciferine, liensinine, isoliensinine, roemerine, nelumbine, and neferine).12, 13 Neferine serves as a free radical scavenger, enhancing superoxide dismutase (SOD) activity and inhibiting LPO due to the hydroxyl group present in its structure.14 Recently, anticancer potential of neferine in HepG2 and A549 cells, and cardioprotective effect against isoproterenol-induced myocardial ischemia have been reported from our laboratory.15, 16, 17
2. Methods
2.1. Chemicals
Dulbecco's modified Eagle's medium, fetal bovine serum, and all other cell culture reagents were obtained from Hi-Media Laboratories, Mumbai, India. Neferine, CoCl2, 3,3′-dihexyloxacarbocyanine iodide (DiOC6), carbonylcyanide-m-chlorophenylhydrazone (CCCP), 2′,7′-dichlorofluorescein diacetate (DCF-DA), and Fura 2/AM were obtained from Sigma-Aldrich (St Louis, MO, USA). Reagents for enzyme assays were obtained from Merck Specialities Chemicals, Mumbai, India.
2.2. Cell culture
Rhabdomyosarcoma (RD) cells were obtained from NCCS (Pune, India) and grown to confluence in 25 cm2 flasks with Dulbecco's modified Eagle's medium and 10% fetal bovine serum (v/v), containing 100 units/mL penicillin, 30 μg/mL streptomycin, and 20 μg/mL gentamycin in a CO2 incubator with 5% CO2. Cells at 80% confluence were used for all the assays. All assays were carried out within 20 passages, between Passage No. 40 and Passage No. 60, to ensure uniformity of cell population and reproducibility.
2.3. Cell proliferation assay
Cell viability was assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, because reduction of tetrazolium salts is widely accepted as a reliable method to examine cell viability/proliferation.18 The cells were seeded at a density of 1 × 104 cells per well, in 200 μL Dulbecco's modified Eagle's medium, and were allowed to attach overnight in a CO2 incubator. Neferine dissolved in 50% (v/v) ethanol (final solvent concentration not exceeding 0.1%) was added to the cells. After different exposure times, 20 μL of MTT (5 mg/mL) was added and incubated at 37 °C for 4 hours after aspirating the medium with neferine and CoCl2. The purple formazan crystals formed were dissolved in 200 μL dimethyl sulfoxide, and the absorbance was read at 570 nm in a microquant plate reader (Bio-Tek Instruments, Winooski, VT, USA).
2.4. Lactate dehydrogenase release and NO assay
Cytotoxicity induced by CoCl2 was evaluated by measuring lactate dehydrogenase (LDH) and NO release by the cells. LDH leakage is a measure of membrane integrity. Nitrite is the stable oxidation product of NO produced by iNOS. The cells were seeded in triplicate in a 24-well plate at a density of 1 × 105 cells/well and were allowed to attach overnight in aCO2 incubator at 37 °C. Cells were treated with appropriate concentrations of neferine and CoCl2 for 24 hours and 48 hours, respectively; the cells were harvested and the supernatant (spent media) was used for the assay.
LDH activity was determined from the linear region of a pyruvate standard graph using regression analysis and expressed as % leakage, as described previously.19 The amount of LDH released was expressed as a percentage. NO assay was carried out by the measurement of nitrite using Greiss reagent in the spent medium.20 The amount of nitrite was expressed as nmoles of nitrite released.
2.5. Estimation of ROS
Relative changes in intracellular ROS in RD cells were monitored using a fluorescent probe, 2′,7′-dichlorofluoresceindiacetate (DCF-DA). The treatment groups were maintained as described for LDH leakage, and the time course for ROS generation by CoCl2 was estimated. Cells were incubated with 10 μM of DCF-DA and incubated for 30 minutes, followed by incubation with CoCl2 for different time periods (15 minutes, 30 minutes, and 1 hour). One treatment group with 50 μM H2O2 was included to serve as a positive control. In the neferine pretreatment samples, the cells were treated with 700 nM neferine for 24 hours and then exposed to DCF-DA and CoCl2 for 30 minutes each. The cells were then harvested, centrifuged, washed, and resuspended in phosphate-buffered saline (PBS), and read in a Hitachi spectrofluorimeter (excitation 480 nm, emission 520 nm). The estimations were carried out thrice in triplicate, using the same number of cells per treatment group to ensure reproducibility. The values were expressed as % relative fluorescence compared to the control.
2.6. Lipid peroxidation
Malonaldehyde, an indicator of LPO, which is formed during the breakdown of polyunsaturated fatty acids, serves as a convenient index for determining the extent of peroxidation reaction. LPO content in different treatment groups was estimated according to Ohkawa et al.21 All experiments were carried out thrice in triplicate to ensure reproducibility, and the LPO content was expressed as nmoles of Thiobarbituric acid (TBA) reactants.
2.7. Cellular antioxidant status
After completing the treatment schedule, cell extracts were prepared by sonication in 50 mM Tris, 5 mM EDTA, 10 μg/mL phenyl methyl sulfonyl fluoride, pH 7.6. The cell debris was removed by centrifugation at 1800 g for 5 minutes at 4 °C, and the protein content of the supernatant was determined by the Lowry method and used for antioxidant assays. Both reagent blanks and enzyme blanks were measured for all assays. All assays were carried out in the linear range and expressed as specific activities of enzymes.
The intracellular concentration of reduced glutathione (GSH) was estimated by the fluorimetric assay, as described by Hissin and Hilf.22 Cells from the different treatment groups were treated with 25% ortho-phosphoric acid followed by sonication. The values obtained were calculated based on the linear region of a GSH standard graph using regression analysis. All experiments were carried out thrice in triplicate to ensure reproducibility, and the amount of GSH was expressed as nmoles of GSH/mg protein.
SOD activity was measured following the procedure described previously by Marklund and Marklund,23 and expressed as the amount of enzyme required for 50% inhibition of pyrogallol auto-oxidation. Catalase was determined following the method described by Aebi.24 Catalase activity in the samples was calculated based on the extinction coefficient of hydrogen peroxide and expressed as specific activity. Glutathione S-transferase (GST) was determined by the method of Habig et al.25 The enzyme activity was calculated based on the extinction coefficient of Glutathione-1-chloro-2,4-dinitrobenzene (GS-CDNB), and expressed as specific activity. The activity of glutathione peroxidase (GPx) was determined following the method described previously by Rotruck et al.26 The amount of GSH consumed was calculated from a standard graph, using linear regression. The enzyme activity was expressed as units/mg protein.
2.8. Protein carbonyl content
Protein carbonyl content is a cellular marker of oxidative stress. The cells were harvested and washed with PBS, and the levels of protein carbonyl content were estimated by the method previously described by Levine et al.27
2.9. Intracellular calcium measurement
RD cells at a density of 1 × 105 cells/well in a 24-well plate were treated with neferine and CoCl2 for 24 hours and 48 hours, respectively. After treatment, the cells were washed twice with buffer A (5 mM KCl, 2 mM CaCl2, 0.5 mM KH2PO4, 137 mM NaCl, 4 mM NaHCO3, and 0.2 mM Na2HPO4). The cells were loaded with 4 μM of Fura 2/AM in buffer A for 45 minutes. Fluorescence emission (500 nm) was monitored, with the excitation wavelength cycling between 340 nm and 380 nm, using a fluorescence spectrophotometer. The ratio of intensities at 340 nm and 380 nm excitation (F340/F380) was used as an index of [Ca2+]i.
2.10. Measurement of mitochondrial transmembrane potential (ΔΨM)
Mitochondrial energization was determined as the retention of the dye DiOC6 in normal cells and its exclusion by cells with mitochondrial membrane damage. Cells were treated with neferine as described in the LDH leakage experiment. After the treatment period, the medium was flicked off and the cells were washed twice with PBS. Then the cells were treated with 50 nM DiOC6 at 37 °C for 30 minutes and washed twice with PBS. The cells were collected and resuspended in 2 mL PBS, and fluorescence intensity was measured in a fluorescent spectrophotometer using excitation/emission as 488 nm/500 nm. CCCP (50 μM) was used as a positive control for maximum ΔψM disruption. CCCP was incubated with cells for 15 minutes prior to the addition of DiOC6.
2.11. Apoptotic cell detection by propidium iodide
After treatment, the cells were collected, washed with PBS, fixed overnight with 70% ethanol at 4 °C, and incubated with propidium iodide at room temperature for 30 minutes; nuclear morphology was examined using a fluorescent microscope.
2.12. Statistical analysis
All experiments were performed three times in triplicates. Results were expressed as mean ± the standard error of the mean. Multiple comparisons were done using one-way analysis of variance followed by Tukey's multiple comparison test, using SPSS 16 software (SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant when p < 0.05.
3. Results
In RD cells, different concentrations of CoCl2 and neferine were tested, and the effective dosage was fixed by calculating the cell viability using MTT assay. CoCl2 induces cell death in RD cells in time- and dose-dependent manner. From this, IC50 of CoCl2 for 48 hours was found to be 200 μM, and pretreatment with neferine for 24 hours at 700 nM provides better protection (Fig. 1). All experiments were carried out using this dosage and time point.
Fig. 1.
Protective effect of neferine on CoCl2-induced cytotoxicity assessed by MTT assay. RD cells were treated with CoCl2 (0 μM, 100 μM, 150 μM, 200 μM, and 250 μM) for 48 hours and 24 hours in the presence and absence of neferine pretreatment (600 nM, 700 nM, and 800 nM), respectively. Cell viability was assessed by MTT assay. Results shown are mean ± SEM of three separate experiments performed in triplicate.
#p > 0.05, not significant compared to control.
* p < 0.01, compared to control.
+p > 0.05 not significant compared to the CoCl2-treated group (one-way ANOVA followed by Tukey's multiple comparison).
$ p < 0.01, compared to the CoCl2-treated group.
ANOVA, analysis of variance; CoCl2, cobalt chloride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nef, neferine; RD, rhabdomyosarcoma; SEM, standard error of the mean.
Light microscopic images revealed the morphological changes in the CoCl2-treated cells. The control and neferine groups showed normal morphological characters of RD cells, while morphological changes such as cell shrinkage, rounding up, and detachment of the cells from the surface were observed in the CoCl2-treated group. These morphological changes were reduced in the group that received neferine pretreatment (Fig. 2).
Fig. 2.
Effect of neferine on CoCl2-induced morphological changes. Results shown are representative images of three separate experiments carried out in the following groups: (A) control, (B) neferine, (C) CoCl2, and (D) neferine + CoCl2 (100×).
CoCl2, cobalt chloride.
Nitrite is the stable oxidation product of NO produced by iNOS. The effect of CoCl2-induced hypoxia on nitrite production was determined by NO assay. Treatment of RD cells with CoCl2 resulted in a statistically significant increase in nitrite production when compared to the control. Cells that received neferine treatment, followed by CoCl2, showed lower nitrite production in a statistically significant manner compared to control (Fig. 3A). LDH leakage was a measure of membrane integrity. CoCl2 treatment of cells resulted in a statistically significant increase in LDH leakage compared to the control. Whereas pretreatment with neferine resulted in a significant increase in the LDH level compared to the CoCl2-treated group. Pretreatment with neferine did not show a statistically significant difference from the control (Fig. 3B). CoCl2 treatment resulted in significantly elevated levels of protein carbonyl content compared to the control, which may be attributed to oxidative stress. Pretreatment of cells with neferine resulted in a significant decrease in protein carbonyl content levels compared to CoCl2-treated cells. Cells treated with neferine alone did not show significant changes (Fig. 3C).
Fig. 3.
Effects of neferine on nitrite release, LDH leakage, and protein carbonyl content. (A) Nitrite release was measured using Griess reagent. RD cells were treated with neferine for 24 hours and CoCl2 for 48 hours for neferine and CoCl2-alone groups, respectively; for pretreatment with neferine, the cells were treated with neferine for 24 hours followed by CoCl2 exposure for 48 hours. (B) LDH leakage was measured to assess membrane integrity. Results are expressed as % LDH release. (C) Protein carbonyl content was determined using dinitrophenyl hydrazine (DNPH). Results shown are mean ± SEM of three separate experiments performed in triplicate.
#p > 0.05, not significant compared to control (one-way ANOVA followed by Tukey's multiple comparison).
*p < 0.01, compared to control.
+p < 0.01, compared to the only CoCl2-treated group.
ANOVA, analysis of variance; CoCl2, cobalt chloride; LDH, lactate dehydrogenase; RD, rhabdomyosarcoma; SEM, standard error of the mean.
ROS are generated during mitochondrial respiration. We tested whether ROS generation contributed to the effect of CoCl2-induced hypoxia on RD cell death. CoCl2 induced ROS generation in a time-dependent manner. In CoCl2-treated cells, a significant increase in ROS generation in muscle cells was observed at 15 minutes, which reached a maximum at 30 minutes. RD cells pretreated with neferine showed diminished ROS generation when exposed to CoCl2 (Fig. 4).
Fig. 4.
CoCl2-induced ROS generation in a time-dependent manner. ROS production was assessed by the spectrofluorimetric method using 2,7-dichlorodihydrofluorescein diacetate (DCF-DA). Results shown are mean ± SEM of three separate experiments performed in triplicate.
* p < 0.01, compared to control.
#p > 0.05, not significant compared to control (one-way ANOVA followed by Tukey's multiple comparison).
+p < 0.01, compared to the only CoCl2-treated group.
ANOVA, analysis of variance; CoCl2, cobalt chloride; ROS, reactive oxygen species; SEM, standard error of the mean.
The role of antioxidants in cytotoxicity induced by CoCl2 was also investigated. Reduced GSH is a major antioxidant and protects cells from oxidative stress by scavenging peroxides in the cytosol and mitochondria; hence, measurement of the levels of GSH helps in finding the severity of oxidative damage induced by CoCl2. There was considerable reduction in the levels of GSH in the group exposed to CoCl2 compared with the control group. In neferine-pretreated group, the GSH level was significantly increased compared to CoCl2-treated cells (Fig. 5A). LPO was one of the main manifestations of oxidative damage in cells. Lipid peroxide content measured as Thiobarbituric Acid Reactive Substances (TBARS) in RD cells was significantly increased in the CoCl2-treated cells. Cells that received neferine pretreatment showed significantly reduced LPO content compared with the CoCl2-treated cells (Fig. 5B).
Fig. 5.
Effects of neferine on Glutathione and lipid peroxidation. (A) GSH levels were estimated using ortho-phthalaldehyde (OPT) spectrofluorimetrically. (B) A significant increase in lipid peroxidation, measured as thiobarbituric acid reactive substance (TBARS) formation, was observed in the CoCl2-treated group, which was reduced by neferine pretreatment. Results shown are mean ± SEM of three separate experiments performed in triplicate.
# p > 0.05, not significant compared to control (one-way ANOVA followed by Tukey's multiple comparison).
*p < 0.01, compared to control.
+p < 0.01, compared to the only CoCl2-treated group.
ANOVA, analysis of variance; CoCl2, cobalt chloride; GSH, reduced glutathione; SEM, standard error of the mean.
In order to evaluate the effect of CoCl2 and neferine on the activities of antioxidant enzymes, catalase, SOD, GPx, and GST assays were carried out in the various treatment groups. Catalase, SOD, and GPx are enzymes primarily involved in combating cellular oxidative stress; in cells subjected to CoCl2 treatment, all the antioxidant enzyme activities were significantly decreased. Pretreatment with neferine significantly increased activities of catalase, SOD, GPx, and GST (Table 1).
Table 1.
Effect of neferine on CoCl2-induced alterations in antioxidant system
| Parameters | Group I | Group II | Group III | Group IV |
|---|---|---|---|---|
| SOD (U/mg of protein) | 6937.3 ± 14.32 | 6893.6 ± 31.35* | 5548.832 ± 23.17† | 6137.3 ± 16.95‡ |
| CAT (U/mg of protein) | 298.23 ± 5.36 | 293.72 ± 2.31* | 159.31 ± 8.81† | 256.65 ± 6.41‡ |
| GST (U/mg of protein) | 42.291 ± 2.35 | 41.872 ± 4.81* | 24.791 ± 3.76† | 30.382 ± 3.29‡ |
| GPx (U/mg of protein) | 397.058 ± 8.82 | 391.176 ± 10.58* | 296.361 ± 6.97† | 358.207 ± 8.46‡ |
Results shown are mean ± SEM of three separate experiments performed in triplicate.
p > 0.05 compared to control (one-way ANOVA followed by Tukey's multiple comparison).
p < 0.01, compared to control.
p < 0.01, compared to the only CoCl2-treated group.
ANOVA, analysis of variance; CAT 1 U, the amount of catalase enzyme that consumes 1 nmole H2O2/min; CoCl2, cobalt chloride; GPx 1 U, the amount of glutathione peroxidase enzyme that converts 1 μmole glutathione to GSSG in the presence of H2O2/min; Group I, control group; Group II, neferine group; Group III, CoCl2 group; Group IV, neferine + CoCl2 group; GST 1 U, the amount of glutathione S-transferase enzyme that conjugates 1 μmole 1-chloro-2,4-dinitrobenzene (CDNB)/min; NS, not significant; SEM, standard error of the mean; SOD 1 U, the amount of superoxide dismutase enzyme required for 50% inhibition of pyrogallol auto-oxidation.
CoCl2-induced apoptosis was determined by propidium iodide staining. Propidium iodide staining revealed chromatin condensation and formation of apoptotic bodies in CoCl2-treated cells, confirming the apoptosis event. Whereas cells pretreated with neferine showed apparently normal architecture with no sign of apoptosis (Fig. 6).
Fig. 6.
Protective effect of neferine on the formation of hypoxia-induced apoptotic bodies in muscle cells. PI fluorescent staining was used. Results shown are representative images of three separate experiments for (A) control, (B) neferine, (C–E) CoCl2, (F,G) neferine + CoCl2 (400×), and (H) percentage of apoptotic cells. A total of 200 cells per treatment group were screened and analyzed for the PI staining.
# p > 0.05, not significant compared to control (one-way ANOVA followed by Tukey's multiple comparison).
*p < 0.01, compared to control.
+p < 0.01, compared to the CoCl2-treated group.
ANOVA, analysis of variance; CoCl2, cobalt chloride; PI, propidium iodide.
Intracellular calcium was measured using Fura 2/AM and expressed as relative percentage. Intracellular calcium accumulation was higher in those cells that received CoCl2 treatment. By contrast, neferine-pretreated cells showed significantly decreased calcium accumulation compared to CoCl2-treated cells (Fig. 7A). When RD cells were exposed to CoCl2, the ΔΨM was dissipated rapidly, as revealed by decreases in the fluorescence measured using a fluorescent spectrophotometer. Whereas in the neferine-pretreated cells, the ΔΨM was significantly higher compared to that in the CoCl2-treated cells (Fig. 7B).
Fig. 7.
Effect of neferine on intracellular calcium accumulation and mitochondrial membrane potential on CoCl2-treated cells. Results shown are mean ± SEM of three separate experiments performed in triplicate. (A) CoCl2 induced intracellular calcium accumulation in RD cells was estimated using Fura-2/AM. Results were expressed as relative % of Fura-2AM fluorescence. (B) Cells were treated with neferine (500 nM) followed by CoCl2. 50 μM CCCP was used as positive control. Results were expressed as relative % of DiOC6 fluorescence.
# p > 0.05, not significant compared to control (one-way ANOVA followed by Tukey's multiple comparison).
*p < 0.01, compared to control.
+p < 0.01, compared to the only CoCl2-treated group.
ANOVA, analysis of variance; CCCP, carbonylcyanide-m-chlorophenylhydrazone; CoCl2, cobalt chloride; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; SEM, standard error of the mean.
4. Discussion
Cobalt chloride mimics hypoxic/ischemic conditions, including ROS production, in muscle cells at high altitude. CoCl2-induced cell death in RD cells may serve as a simple and convenient in vitro model of hypoxia-induced cytotoxicity in muscle cells. It is a well-known fact that hypoxia leads to the generation of reactive oxygen and nitrogen species, which leads to oxidative stress resulting in the oxidation of biological molecules.28 Substantial weight loss, in particular, a considerable reduction in muscle mass, was one of the remarkable features of chronic exposure to high altitude.29 Radák et al30 revealed that physical training at high altitude increases the reactive carbonyl derivatives in amino acids of skeletal muscle.
From the MTT result it was evident that CoCl2 induces cell death in a dose- and time-dependent manner. The concentration at which neferine (700 nM) offers better protection was found to be very low. To evaluate the CoCl2-induced hypoxic cell death, we performed cytotoxicity assays such as LDH and NO assays. Measurement of LDH enzyme activity serves as a cellular injury marker in the biological system; hence, measurement of LDH enzyme activity may serve as an indicator of the cell injury during hypoxia. Significantly increased LDH release in hypoxia-exposed hepatocytes was previously observed by Frank et al.31 Neferine with a strong antioxidative property protects cells against the cellular injury triggered by CoCl2, which is evident from a significant decrease in the LDH in the medium of neferine-pretreated cells. Biomolecules such as DNA and protein are damaged by the NO formed by iNOS; hence, measurement of NO content in the culture medium is an indirect method of measuring the expression of iNOS, which serves as an indicator of the pathological and stress conditions. CoCl2 induced a significant increase in NO production in the muscle cells (RD). NO levels in the neferine-pretreated cells were found to be significantly decreased compared to that in cells treated with CoCl2 alone. Recently, it has been reported by Lalitha et al15 that neferine possesses strong antioxidant property and maintains the membrane integrity of cardiac cells in isoproterenol-treated rats. Wang et al13 have reported that neferine restores serum marker enzyme levels to normal in ischemic liver. Our results are in line with the above reports.
Increased intracellular ROS have been reported to contribute to CoCl2-induced cell death.32 ROS also induce apoptotic cell death associated with the loss of mitochondrial membrane potential.33 Oxygen free radicals are highly reactive species that promote damage to lipids, DNA, carbohydrates, and proteins, and induce production of several immune/inflammatory proteins, leading to cell death.34 It has also been shown that antioxidants can scavenge ROS and protects cells from injury.35 In our study, neferine with its antioxidant activity could effectively scavenge the ROS generated during CoCl2 treatment.
Pathogenesis of hypoxic damage was reported to be initiated by LPO, protein oxidation, and irreversible protein modification, ultimately leading to cell membrane damage.36 Reports of Sevanian and Hochstein37 demonstrated that LPO was initiated by free radicals, causing alteration of membrane integrity, fluidity, and permeability. Oxidative damage to DNA and lipids in muscle tissue was reported in many patients with chronic fatigue syndrome.38, 39 Results of the present study showed significantly increased levels of TBA reactive substances in the CoCl2-treated group compared to that in the control group. Generation of free radicals and activation of LPO could result in irreversible membrane damage in muscle cells. GSH is the main nonenzymatic antioxidant that protects cells from oxidative injury, by preventing the accumulation of oxygen radicals in the cells.40 Neferine, due to its strong radical scavenging activity, restored the cellular GSH content in the CoCl2-treated RD cells. A previous study by Joanny et al41 showed that ascorbic acid restored the increased TBARS content and decreased GSH levels in the blood of the individuals subjected to the hypoxic condition, which proves that antioxidant intake will reduce hypoxia-mediated oxidative damage.
The cellular antioxidant defense system plays a role in protecting cells from ROS generated during metabolic process. Aerobic cells develop enzymatic and nonenzymatic antioxidant systems to regulate the effects of reactive oxygen and nitrogen species. The enzymatic system contains mitochondrial, cytosolic, and extracellular SOD to convert reactive superoxide to less powerful hydrogen peroxide. GPx and catalase decompose hydrogen peroxide to water.8 The CoCl2-treated cells were subjected to a continuous flux of superoxide anions and hydrogen peroxide, resulting in oxidative stress in the cells. Exposure to extreme conditions such as hypoxia results in a decline in the activities of cellular antioxidant enzymes such as SOD, catalase, GPx, and GST, and antioxidant supplementation will help in improving the cellular antioxidant status.42 Lalitha et al15 reported that neferine offered cardioprotection against isoproterenol-induced oxidative stress due to the strong antioxidant nature of neferine. In the present study, pretreatment with neferine improved the cellular antioxidant status in CoCl2-treated cells, which matches with the earlier findings.
Membrane damage and subsequent mitochondrial dysfunction by ROS can lead to modifications in mitochondrial DNA.38 Maintenance of the ΔψM is fundamental to the normal performance and survival of cells that have a high energy requirement. Pathological conditions, such as oxidative stress, adenosine triphosphate depletion, Ca2+ overload of mitochondrial matrix, and some physiological processes, can trigger the opening of a nonspecific permeability transition pore in the mitochondrial membrane.43 Intracellular accumulation of calcium during hypoxia has been reported by Vecchiet et al39 earlier. In the present study, an increase in intracellular calcium accumulation and a decrease in the ΔψM were observed in CoCl2-treated cells, which were prevented in the neferine-pretreated group. From this result it was clear that neferine treatment protects muscle cells from CoCl2-induced mitochondrial membrane damage and calcium accumulation.
The present study reveals that neferine pretreatment ameliorated CoCl2-induced ROS generation, restored cellular antioxidant pool, attenuated mitochondrial transmembrane potential loss, and maintained intracellular calcium levels in muscle cells. These findings support the hypothesis that cytoprotection mediated by neferine is due, in part, to the inhibition of the oxidative stress-mediated cell death.
Conflicts of interest
All authors have no conflicts of interest to declare.
Acknowledgments
Rathinasamy Baskaran greatly acknowledges Defence Institute of Physiology and Allied Sciences, New Delhi, India, for granting financial support to the project. Dr P.V.L Rao, Director, and Dr K. Kathirvelu, Joint Director, DRDO-BU Center for Life Sciences, Coimbatore, are also acknowledged for their support.
References
- 1.Radák Z., Asano K., Inoue M., Kizaki T., Oh-Ishi S., Suzuki K. Superoxide dismutase derivative reduces oxidative damage in skeletal muscle of rats during exhaustive exercise. J Appl Physiol. 1995;79:129–135. doi: 10.1152/jappl.1995.79.1.129. [DOI] [PubMed] [Google Scholar]
- 2.Ward M.P., Milledge J.S., West J.B. Arnold; London: 2000. High altitude medicine and physiology. [Google Scholar]
- 3.Irani K., Goldschmidt-Clermont P.J. Ras, superoxide and signal transduction. Biochem Pharmacol. 1998;55:1339–1346. doi: 10.1016/s0006-2952(97)00616-3. [DOI] [PubMed] [Google Scholar]
- 4.Pahlmark K., Folbergrová J., Smith M.L., Siesjö B.K. Effects of dimethyl thiourea on selective neuronal vulnerability in forebrain ischemia in rats. Stroke. 1993;24:731–736. doi: 10.1161/01.str.24.5.731. [DOI] [PubMed] [Google Scholar]
- 5.Patt A., Harken A.H., Burton L.K., Rodell T.C., Piermattei D., Schorr W.J. Xanthine oxidase-derived hydrogen peroxide contributes to ischemia reperfusion-induced edema in gerbil brains. J Clin Invest. 1988;81:1556–1562. doi: 10.1172/JCI113488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Atamaniuk J., Vidotto C., Tschan H., Bachl N., Stuhlmeier K.M., Muller M.M. Increased concentrations of cell-free plasma DNA after exhaustive exercise. Clin Chem. 2004;50:1668–1670. doi: 10.1373/clinchem.2004.034553. [DOI] [PubMed] [Google Scholar]
- 7.Medved I., Brown M.J., Bjorksten A.R., Leppik J.A., Sostaric S., McKenna M.J. N-acetylcysteine infusion alters blood redox status but not time to fatigue during intense exercise in humans. J Appl Physiol. 2003;94:1572–1582. doi: 10.1152/japplphysiol.00884.2002. [DOI] [PubMed] [Google Scholar]
- 8.Bakonyi T., Radák Z. High altitude and free radicals. J Sport Sci Med. 2004;3:64–69. [PMC free article] [PubMed] [Google Scholar]
- 9.Liu X., Chen Y., Wu Y., Ha T., Li C. The cardioprotection induced by lipopolysaccharide involves phosphoinositide 3-kinase/Akt and high mobility group box 1 pathways. J Biomed Res. 2010;24:324–331. doi: 10.1016/S1674-8301(10)60045-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Chopra R.N., Nayar S.L., Chopra I.C. Council of Scientific Industrial Research; New Delhi, India: 1956. Glossary of Indian medicinal plants. [Google Scholar]
- 11.Chen Y., Fan G., Wu H., Wu Y., Mitchell A. Separation, identification and rapid determination of liensine, isoliensinine and neferine from embryo of the seed of Nelumbo nucifera Gaertn. by liquid chromatography coupled to diode array detector and tandem mass spectrometry. J Pharm Biomed Anal. 2007;43:99–104. doi: 10.1016/j.jpba.2006.06.016. [DOI] [PubMed] [Google Scholar]
- 12.Qian J.Q. Cardiovascular pharmacological effects of bisbenzyl isoquinoline alkaloid derivatives. Acta Pharmacol Sin. 2002;23:1086–1092. [PubMed] [Google Scholar]
- 13.Wang J., Kan Q., Li J., Zhang X., Qi Y. Effect of neferine on liver ischemia–reperfusion injury in rats. Transplant Proc. 2011;43:2536–2539. doi: 10.1016/j.transproceed.2011.04.013. [DOI] [PubMed] [Google Scholar]
- 14.Zhao L., Wang X., Chang Q., Xu J., Huang Y., Guo Q. Neferine, a bisbenzylisoquinoline alkaloid attenuates bleomycin-induced pulmonary fibrosis. Eur J Pharmacol. 2010;627:304–312. doi: 10.1016/j.ejphar.2009.11.007. [DOI] [PubMed] [Google Scholar]
- 15.Lalitha G., Poornima P., Archanah A., Padma V.V. Protective effect of neferine against isoproterenol-induced cardiac toxicity. Cardiovasc Toxicol. 2013;13:168–179. doi: 10.1007/s12012-012-9196-5. [DOI] [PubMed] [Google Scholar]
- 16.Poornima P., Quency R.S., Padma V.V. Neferine induces reactive oxygen species mediated intrinsic pathway of apoptosis in HepG2 cells. Food Chem. 2013;136:659–667. doi: 10.1016/j.foodchem.2012.07.112. [DOI] [PubMed] [Google Scholar]
- 17.Poornima P., Weng C.F., Padma V.V. Neferine from Nelumbo nucifera induces autophagy through the inhibition of PI3K/Akt/mTOR pathway and ROS hyper generation in A549 cells. Food Chem. 2013;141:3598–3605. doi: 10.1016/j.foodchem.2013.05.138. [DOI] [PubMed] [Google Scholar]
- 18.Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
- 19.Ulmer D.D., Vallee B.L., Wacker W.E. Metalloenzymes and myocardial infarction. II. Malic and lactic dehydrogenase activities and zinc concentrations in serum. N Engl J Med. 1956;255:450–456. doi: 10.1056/NEJM195609062551001. [DOI] [PubMed] [Google Scholar]
- 20.Stuehr D.J., Marletta M.A. Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma. J Immunol. 1987;139:518–525. [PubMed] [Google Scholar]
- 21.Ohkawa H., Ohishi N., Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95:351–358. doi: 10.1016/0003-2697(79)90738-3. [DOI] [PubMed] [Google Scholar]
- 22.Hissin P.J., Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 1976;74:214–226. doi: 10.1016/0003-2697(76)90326-2. [DOI] [PubMed] [Google Scholar]
- 23.Marklund S., Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47:469–474. doi: 10.1111/j.1432-1033.1974.tb03714.x. [DOI] [PubMed] [Google Scholar]
- 24.Aebi H. Catalase. In: Methods of enzymatic analysis. Bergmeyer H., editor. Academic Press; New York: 1974. [Google Scholar]
- 25.Habig W.H., Pabst M.J., Jakoby W.B. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249:7130–7139. [PubMed] [Google Scholar]
- 26.Rotruck J.T., Pope A.L., Ganther H.E., Swanson A.B., Hafeman D.G., Hoekstra W.G. Selenium: biochemical role as a component of glutathione peroxidase. Science. 1973;179:588–590. doi: 10.1126/science.179.4073.588. [DOI] [PubMed] [Google Scholar]
- 27.Levine R.L., Garland D., Oliver C.N., Amici A., Climent I., Lenz A.G. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990;186:464–478. doi: 10.1016/0076-6879(90)86141-h. [DOI] [PubMed] [Google Scholar]
- 28.Pialoux V., Mounier R., Rock E., Mazur A., Schmitt L., Richalet J.P. Effects of acute hypoxic exposure on prooxidant/antioxidant balance in elite endurance athletes. Int J Sports Med. 2009;30:87–93. doi: 10.1055/s-0028-1103284. [DOI] [PubMed] [Google Scholar]
- 29.Kayser B. Nutrition and high altitude exposure. Int J Sports Med. 1992;13(Suppl 1):S129–S132. doi: 10.1055/s-2007-1024616. [DOI] [PubMed] [Google Scholar]
- 30.Radák Z., Asano K., Lee K.C., Ohno H., Nakamura A., Nakamoto H. High altitude training increases reactive carbonyl derivatives but not lipid peroxidation in skeletal muscle of rats. Free Radic Biol Med. 1997;22:1109–1114. doi: 10.1016/s0891-5849(96)00350-4. [DOI] [PubMed] [Google Scholar]
- 31.Frank A., Rauen U., de Groot H. Protection by glycine against hypoxic injury of rat hepatocytes: inhibition of ion fluxes through nonspecific leaks. J Hepatol. 2000;32:58–66. doi: 10.1016/s0168-8278(00)80190-7. [DOI] [PubMed] [Google Scholar]
- 32.Zou W., Yan M., Xu W., Huo H., Sun L., Zheng Z. Cobalt chloride induces PC12 cells apoptosis through reactive oxygen species and accompanied by AP-1 activation. J Neurosci Res. 2001;64:646–653. doi: 10.1002/jnr.1118. [DOI] [PubMed] [Google Scholar]
- 33.Satoh T., Enokido Y., Aoshima H., Uchiyama Y., Hatanaka H. Changes in mitochondrial membrane potential during oxidative stress-induced apoptosis in PC12 cells. J Neurosci Res. 1997;50:413–420. doi: 10.1002/(SICI)1097-4547(19971101)50:3<413::AID-JNR7>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
- 34.Hou R.C., Huang H.M., Tzen J.T., Jeng K.C. Protective effects of sesamin and sesamolin on hypoxic neuronal and PC12 cells. J Neurosci Res. 2003;74:123–133. doi: 10.1002/jnr.10749. [DOI] [PubMed] [Google Scholar]
- 35.Ramyaa P., Krishnaswamy R., Padma V.V. Quercetin modulates OTA-induced oxidative stress and redox signalling in HepG2 cells—up regulation of Nrf2 expression and down regulation of NF-κB and COX-2. Biochim Biophys Acta. 2014;1840:681–692. doi: 10.1016/j.bbagen.2013.10.024. [DOI] [PubMed] [Google Scholar]
- 36.Dalle-Donne I., Rossi R., Colombo R., Giustarini D., Milzani A. Biomarkers of oxidative damage in human disease. Clin Chem. 2006;52:601–623. doi: 10.1373/clinchem.2005.061408. [DOI] [PubMed] [Google Scholar]
- 37.Sevanian A., Hochstein P. Mechanisms and consequences of lipid peroxidation in biological systems. Annu Rev Nutr. 1985;5:365–390. doi: 10.1146/annurev.nu.05.070185.002053. [DOI] [PubMed] [Google Scholar]
- 38.Fulle S., Mecocci P., Fanó G., Vecchiet I., Vecchini A., Racciotti D. Specific oxidative alterations in vastuslateralis muscle of patients with the diagnosis of chronic fatigue syndrome. Free Radic Biol Med. 2000;29:1252–1259. doi: 10.1016/s0891-5849(00)00419-6. [DOI] [PubMed] [Google Scholar]
- 39.Vecchiet J., Cipollone F., Falasca K., Mezzetti A., Pizzigallo E., Bucciarelli T. Relationship between musculoskeletal symptoms and blood markers of oxidative stress in patients with chronic fatigue syndrome. Neurosci Lett. 2003;335:151–154. doi: 10.1016/s0304-3940(02)01058-3. [DOI] [PubMed] [Google Scholar]
- 40.Vina J. CRC; Boca Raton, FL: 1990. Glutathione: metabolism and physiological functions. [Google Scholar]
- 41.Joanny P., Steinberg J., Robach P., Richalet J.P., Gortan C., Gardette B. Operation Everest III (Comex’97): the effect of simulated sever hypobaric hypoxia on lipid peroxidation and antioxidant defence systems in human blood at rest and after maximal exercise. Resuscitation. 2001;49:307–314. doi: 10.1016/s0300-9572(00)00373-7. [DOI] [PubMed] [Google Scholar]
- 42.Saggu S., Kumar R. Modulatory effect of seabuckthorn leaf extract on oxidative stress parameters in rats during exposure to cold, hypoxia and restraint (C-H-R) stress and post stress recovery. J Pharm Pharmacol. 2007;59:1739–1745. doi: 10.1211/jpp.59.12.0018. [DOI] [PubMed] [Google Scholar]
- 43.Griffiths E.J. Mitochondria—potential role in cell life and death. Cardiovasc Res. 2000;46:24–27. doi: 10.1016/s0008-6363(00)00020-1. [DOI] [PubMed] [Google Scholar]