Steroidal saponin diosgenin from Dioscorea bulbifera protects cardiac cells from hypoxia-reoxygenation injury through modulation of pro-survival and pro-death molecules
Karuppiah Shanmugasundarapandian Jayachandran1, A Hannah Rachel Vasanthi2, Narasimman Gurusamy3
1 Department of Biotechnology, Centre for Advanced Research in Indian System of Medicine, SASTRA University, Thanjavur, Tamil Nadu, India
2 Department of Biotechnology, Pondicherry University, Puducherry, India
3 Department of Pharmacology, College of Pharmacy, King Khalid University, Abha, Kingdom of Saudi Arabia
|Date of Web Publication||10-Feb-2016|
A Hannah Rachel Vasanthi
Department of Biotechnology, Pondicherry University, Puducherry - 605 014
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Diosgenin, a steroidal saponin from plants, exhibits many biological potentials. Herein, the cardioprotective role of diosgenin is studied. Materials and Methods: The effect of diosgenin, isolated from Dioscorea bulbifera, was studied on hypoxia-reoxygenation (HR) in H9c2 cardiomyoblast cells. The amount of diosgenin in the plant extract was analyzed by high-performance thin layer chromatography using a solvent system comprising of chloroform:methanol:acetic acid:formic acid (13:4.5:1.5:1). Cardioprotection was checked by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Further, the release of lactate dehydrogenase, an enzyme released during cell death was checked. The proteins responsible for cell death (Bax) and cell survival (Bcl-2, hemeoxygenase-1 and Akt) were analyzed using Western blot to check the cardioprotective role of diosgenin. Conclusion:Supplementation of diosgenin mitigates HR injury, thereby exhibiting cardioprotective potential.
Keywords: Cardioprotection, diosgenin, H9c2, hypoxia, reoxygenation
|How to cite this article:|
Jayachandran KS, Vasanthi A H, Gurusamy N. Steroidal saponin diosgenin from Dioscorea bulbifera protects cardiac cells from hypoxia-reoxygenation injury through modulation of pro-survival and pro-death molecules. Phcog Mag 2016;12, Suppl S1:14-20
|How to cite this URL:|
Jayachandran KS, Vasanthi A H, Gurusamy N. Steroidal saponin diosgenin from Dioscorea bulbifera protects cardiac cells from hypoxia-reoxygenation injury through modulation of pro-survival and pro-death molecules. Phcog Mag [serial online] 2016 [cited 2021 May 12];12, Suppl S1:14-20. Available from: http://www.phcog.com/text.asp?2016/12/45/14/176114
- The cardioprotective effect of Diosgenin was evidenced from the improved cell survival after hypoxia-.reoxygenation injury demonstrated through MTT cell survival assay.
- The release of lactate dehydrogenase, an enzyme released during cell death was decreased by Diosgenin.
- Diosgenin upregulated the pro-survival molecules like B-cell lymphoma 2 (Bcl-2), heme oxygenase-1 and the phosphorylation of ATK (at serine 473); and at the same time pro-.death molecules like Bax was downregulated.
- Thus, Diosgenin as a plant based steroidal saponin is confirmed to mitigate ischemic reperfusion injury.
| Introduction|| |
Inefficient vascular supply and the resultant reduction in tissue oxygen tension often lead to neovascularization in order to satisfy the needs of the tissue. The reliance of many cells on aerobic respiration as a mandatory energy source requires a variety of responses to oxygen lack or “hypoxia.” Both hypoxia (lack of oxygen relative to metabolic needs) and reoxygenation (reintroduction of oxygen to hypoxic tissue) are important in human pathophysiology because they occur in a wide variety of important clinical conditions. Prominent examples of tissue hypoxia that predispose to injury during reoxygenation include circulatory shock, myocardial ischemia, stroke, and transplantation of organs.,,, At the cellular level, hypoxia activates numerous major signaling pathways, resulting in changes in gene expression, which influence the cellular ability to survive or die. Severe hypoxia, occurring at partial pressure of oxygen below 20 mm Hg, impairs cellular energy production and ion homeostasis, leading to cell injury and cell death. In contrast, a lower degree of hypoxia, defined as between 50 and 100 mm Hg, may activate mechanisms that could produce cellular phenotype more resistant to acute severe oxidative stress., This phenomenon is one of the most important components of different forms of ischemic heart diseases which include myocardial infarction also. As diseases due to ischemia (e.g. myocardial infarction and stroke) are exceedingly common causes of morbidity and mortality which involves production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), understanding the role of ROS and RNS in reoxygenation injury has the potential to lead to therapies that could improve public health. Cellular models of hypoxia-reoxygenation (HR) have provided useful tools for the study of reactive species mediated mechanisms of cellular dysfunction in ischemia-reperfusion injury. H9c2 cells are derived from embryonic rat heart and are generally accepted to be a good model for cardiomyoblast cells. These cells have been successfully implemented to study mechanisms of cellular and cardiac protection.,
Dioscorea bulbifera commonly known as air potato belonging to a class of yam contains a large number of polyphenols including organic acids, some of which may be potential antioxidants, furthermore, D. bulbifera yam also contains the steroidal saponin diosgenin, the principal material for the synthesis of corticosteroids, estrogen, and contraceptives. Diosgenin is among the ten most important sources of steroids and the most often prescribed medicine of plant origin. Among the steroidal saponins, diosgenyl saponins are the most abundant and exert a large variety of biological functions. Diosgenin is the primary furostanol saponin [Figure 1] found in several plants, including Dioscorea species (yams), fenugreek, and Costus speciosus. This compound has been shown to be useful for the maintenance of healthy blood cholesterol levels, it has been reported that diosgenin retarded the progression of osteoporosis  and possessed anti-inflammatory effects in rats. Also, diosgenin glycosides altered myocardial mechanical activities, probably, through the modulation of extracellular calcium (Ca 2+) flux across the plasma membrane. Another study demonstrated the vasodilator effect of diosgenin in rat superior mesenteric artery. Diosgenin has been found to ameliorate myocardial infarction by its lipoperoxidative activity. Our earlier study on the hydroalcoholic extract of D. bulbifera on myocardial ischemic reperfusion injury  further kindled us to study the quantity of diosgenin in the various fractions of D. bulbifera and study its role in HR injury in in vitro condition.
In the present study, we quantified the amount of diosgenin present in D. bulbifera by high-performance thin layer chromatography (HPTLC) profile. Also, we report that in H9c2 rat myocardium-derived cardiomyoblast, apoptosis is inhibited in conditions of simulated ischemia, represented by HR and are prevented by diosgenin, from D. bulbifera. The protective effect of diosgenin correlates with a marked down-regulation of pro-apoptotic protein Bax and death signaling kinases (phospho-c-Jun N-terminal kinases and phospho-Src), with an up-regulation of cell survival molecules (p-Akt, and hemeoxygenase-1 [HO-1]) and anti-apoptotic protein Bcl-2 in H9c2 cardiomyoblast cells.
| Materials and Methods|| |
Collection of plant material and processing
D. bulbifera Linn. tuber was purchased from Ayurmed Biotech Pvt. Ltd., Mumbai, and it was authenticated at the Centre for Advanced Research in Indian System of Medicine (CARISM) by qualified botanists. The tubers were cut into pieces of 5–8 mm thickness, and it was air-dried. The air-dried tubers were coarsely pulverized.
Extraction and fractionation of Dioscorea bulbifera Linn. tuber
The extraction and fractionation procedures were carried out as described by Gao et al., with slight modifications. About 2 kg of the coarsely powdered tuber was extracted with 5 L of 85% ethanol by cold maceration process for 72 h with occasional shaking. The extracts were drained and this process was repeated thrice. The obtained extract was pooled and concentrated in vacuo to obtain the crude extract (CE). The total ethanolic CE was suspended in water, and it was subjected to partitioning using chloroform and ethyl acetate to yield chloroform, ethyl acetate and water-soluble fraction. The individual fractions were concentrated in vacuo, and they were stored in a desiccator to free from moisture for further analysis.
High-performance thin layer chromatography fingerprinting analysis of alcoholic extract of Dioscorea bulbifera
The fingerprinting analysis of the hydroalcoholic extract and the different fractions were carried out at the CARISM, SASTRA University, Thanjavur using HPTLC, CAMAG, Switzerland.
Preparation of sample
Two hundred milligram extract of D. bulbifera was dissolved in 10 ml of 99.9% ethanol, and it was filtered through Whatmann filter paper 1 and the filtrate was used for analysis. Similarly, the different solvent fractions were dissolved in their respective solvents, and the filtrate was used for the analysis.
Sample: Hydroalcoholic extract, choloroform fraction, ethyl acetate fraction,
Solvent system: Chloroform:methanol:acetic acid:formic acid (13:4.5:1.5:1)
Application: Linomat V
Saturation time: 15 min
Volume for application: 10 and 20 µl
Plate: Silica gel GF 254
Plate size: 10 cm × 10 cm
Application position: 15 mm
Solvent front: 80 mm.
Quantification of diosgenin using high-performance thin layer chromatography
Purified diosgenin isolated from Dioscorea species was purchased from Sigma Aldrich (St. Louis, MO, USA) and was used as the standard. Diosgenin content in the alcoholic extract and fractions were estimated using a modified procedure of Suthar and Mulani. Silica gel G 60 F254 precoated plates were used with chloroform: Acetone (80:20) as solvent system. CE and the different fractions were spotted on precoated TLC plates. The ascending mode was used for the development of thin layer chromatography. TLC plates were developed up to 8 cm. After development, the plate was derivatized dipping in 10% methanolic sulfuric acid reagent (10 ml concentrated sulfuric acid added in 90 ml of methanol with cooling and the reagent must be freshly prepared), heated at 110°C for 2–3 min and brought to room temperature. Brownish spot with Rf >>0.54–0.57 was visible and scanned under visible light at 535 nm.
Standard curve of diosgenin was also run simultaneously at concentration of 500–1500 ng. The results are expressed as percentage of diosgenin present in CE, chloroform soluble fraction, ethyl acetate soluble fraction and water-soluble fraction.
Hypoxia-reoxygenation injury in H9c2 cells
Rat myocardium-derived H9c2 cardiac myoblast cell line was purchased from the American Type Culture Collection (ATCC; Manasses, VA, USA). Cells were grown on Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Grand Island, NY, USA) containing 4 mM L-glutamine, 4.5 g/L glucose, and 10% fetal bovine serum (Invitrogen) and incubated at 37°C in a humidified chamber with 5% CO2. To prevent the loss of differentiation potential, cells were not allowed to become confluent.
Induction of hypoxia
H9c2 culture plates were subjected to either 30 min. of hypoxia or 30 min of hypoxia followed by 1 h of reoxygenation (H/R) or hypoxic adaptation, where the cells were subjected to three cyclic episodes of 5 min hypoxia and 5 min reoxygenation, followed by H/R. Hypoxia was given by placing the cultured plates into an air-tight hypoxic chamber placed in a humidified 37°C CO2 incubator, and passing the mixture of 95% nitrogen and 5% CO2. Reoxygenation was given by placing back the cultured plates in a humidified 37°C CO2 incubator saturated with air.
Cell survival assay
At the end of the experimentation, cells were washed with phosphate buffered saline. The viability of cells was studied using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) as described by the manufacturer. Since diosgenin was dissolved in ethanol, a separate group treated with ethanol was also subjected to HR.
Cell death assay
Cell death analysis was performed with the spent culture medium as substrate with lactate dehydrogenase (LDH) cytotoxicity assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) as mentioned by the manufacturer.
Western blot analysis
The respective cytosolic or nuclear proteins were separated by SDS-PAGE and transferred to nitrocellulose filters. Filters were blocked in 5% nonfat dry milk and probed with primary antibody overnight. Primary antibodies such as HO-1, Akt, phospho-Akt (Ser 473), glyceraldehyde-6-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA; whereas Bax, Bcl-2, Src and phospho-Src (Tyr416), were obtained from Cell Signaling Technology, Beverly, MA, USA. All primary antibodies were used at a dilution of 1:1000. Protein bands were identified with horseradish peroxidase conjugated secondary antibody (1:2000 dilutions) and Western blotting luminol reagent (Santa Cruz Biotechnology). GAPDH was used as loading control for cytosolic and nuclear fraction. The resulting blots were digitized, subjected to densitometric scanning using a QuantiOne software (Bio-Rad), and normalized against loading control.
Results are expressed as the mean ± standard error of the mean (SEM), and Bonferroni's correction was also performed. The results were considered to be significant if P < 0.05. Western blotting was performed with QuantiOne software with H9c2 cell lysate for the expression of protein markers mean ± SEM *P = 0.05 versus normal, ethanol (EtOH) + HR and diosgenin + HR;#P = 0.05 versus HR.
| Results|| |
Quantification of diosgenin
[Figure 2]a shows the fingerprinting of the various fractions of D. bulbifera Linn. which shows the presence of diosgenin in the different fractions, whereas [Figure 2]b chromatogram profile of the various fractions and the CE showing the peak area of diosgenin in the different fractions. The percentage of diosgenin quantified from crude and different fractions is given in [Table 1]. The ethyl acetate fraction possesses the maximum quantity of diosgenin of 0.66% whereas the crude ethanolic extract used for the study contains 0.069%.
|Figure 2: (a) High-performance thin layer chromatography profile of the various fractions of Dioscorea bulbifera Linn. for the estimation of diosgenin (b) High-performance thin layer chromatography of various fractions of Dioscorea bulbifera Linn. showing diosgenin peak area|
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|Table 1: Percentage of diosgenin in crude and different fractions of Dioscorea bulbifera Linn.|
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Diosgenin prevents H/R-induced cell death and increases cell viability
HR injury in H9c2 cardiac myoblast cells increased cell death as assessed by quantitative MTT assay [Figure 3]. The cytoprotective effect of the saponin diosgenin was evidenced from the improved cell survival [Figure 3]. We also measured the release of LDH in the culture media to determine the extent of the leakage of the cytosolic components during oxidative insult. Hypoxia and H/R resulted in a significant increase in LDH level compared with the normal cells [Figure 4].
|Figure 3: Diosgenin induces cell survival. H9c2 cells were cultured in (1 × 104 in a 96-well plate). H9c2 cells were treated with 30 μM of diosgenin followed by hypoxia and reoxygenation. (3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide cell survival assay was performed with (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide Cell Proliferation Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). Results are expressed as mean ± standard error of the mean *P = 0.05 versus normal, ethanol + hypoxia-reoxygenation and Dios + hypoxia-reoxygenation; #P = 0.05 versus hypoxia-reoxygenation|
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|Figure 4: Diosgenin induces cell survival. H9c2 cells were cultured in (1 × 104 in a 96-well plate). H9c2 cells were treated with 30 μM of diosgenin, followed by hypoxia and reoxygenation. The release of lactate dehydrogenase as a marker of cell death was performed with lactate dehydrogenase cytotoxicity assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). Results are expressed as mean ± standard error of the mean *P = 0.05 versus Normal, ethanol + hypoxia-reoxygenation and Dios + hypoxia-reoxygenation; #P = 0.05 versus hypoxia-reoxygenation|
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Anti-apoptotic effect of diosgenin phosphorylates Akt
To verify whether Akt is involved, the Akt phosphorylation pattern was examined in normal, H/R control, EtoH + H/R as well as in diosgenin treated groups. It was explored that diosgenin-treated cells show an upstream effector's pattern of phosphorylated Akt during HR [Figure 5].
|Figure 5: Diosgenin induces cell survival signals and attenuates death protein levels. H9c2 cells were cultured in (3 × 105 in a 10 cm dish). H9c2 cells were treated with 30 μM of diosgenin, followed by hypoxia and reoxygenation. Western blotting was performed with QuantiOne software with H9c2 cell lysate for the expression of protein markers mean ± standard error of the mean *P = 0.05 versus Normal, ethanol + hypoxia-reoxygenation and Dios + hypoxia-reoxygenation; #P = 0.05 versus hypoxia-reoxygenation|
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Effect of Akt overexpression on Bcl-2/Bax
Bax and Bcl-2 play an important role in the regulation of apoptosis; so in order to ascertain whether the anti-apoptotic effects conferred by hypoxia were due to altered expression of Bcl-2 family proteins, the Bcl-2/Bax ratio was studied. Akt blocked the expression of mitochondrial Bcl-2 family protein, Bax and its overexpression inhibited H/R-induced loss of Bcl-2 protein on treating with diosgenin [Figure 5].
Effect of diosgenin on mitogen-activated protein kinase family protein, c-Jun N-terminal kinases
The role of c-Jun N-terminal kinases (JNK), a mitogen-activated protein kinase (MAPK) family protein in HR condition, has been controversial with studies, suggesting both a defensive and detrimental facet to JNK activation. Because of the extreme clinical importance of this family of proteins, their regulation by oxidative stress has been under intense study for many years. In order to reveal its role in H/R, we examined JNK and p-JNK in normal, Control hypoxia, EtoH + H/R and diosgenin + H/R groups. It was observed that there was a down-regulation of JNK in diosgenin + H/R group [Figure 5].
Effect of diosgenin on Src kinases
Src kinases are key upstream mediators of MAPK signaling pathways and have been shown to have important roles in cell proliferation, migration and survival. It was observed that the Src kinase activity was augmented in the control H/R group which was seen attenuated in the diosgenin + H/R group [Figure 5].
Effect of diosgenin on hemeoxygenase-1
HO-1, a stress-inducible protein and Phase II enzyme, is an important cytoprotective agent in various models of HR injury and was found to be up regulated in diosgenin treated group during HR [Figure 5].
| Discussion|| |
Diosgenin, a steroidal saponin isolated from D. bulbifera, matches a standard obtained from Sigma Aldrich (purity 93%) [Figure 1]. Sapogenins are aglycones of glycosidic saponins; the latter are so called because they have the property of forming soapy lather in water. The steroidal sapogenins have 27 carbons and are internal ketals of 16, 26-dihydroxy-22-keto-steroids. Diosgenin, chemically named as (25R)-spirost-5-en-3 β-ol, is prominent among the sapogenins used in steroid industry. Diosgenin has a parent steroid (cyclopentano perhydro phenanthrene [CPPP]) ring structure and it consists of two components namely the hydrophilic sugar moiety and a hydrophobic steroid (CPPP) unit. The structural conformation has revealed that the carbons at 5 and 25 are responsible for Diosgenin's biological activity. Based on its chemical nature to bind to various target proteins, it was identified that diosgenin mediates pro-survival and pro-death protein molecules thereby alleviating HR injury hypoxia followed by re-oxygenation (H/R) is associated with both apoptotic and necrotic cell death, whereas hypoxia contributes to only a minimal amount of cell death. The mechanisms by which the ischemic insult induces apoptosis are not well understood. Furthermore, only little knowledge exists concerning pharmacological agents that target the hypoxia-induced apoptotic pathways in the heart and thereby exerting a cardioprotective effect. Therapeutic reperfusion is currently performed without any measure to protect the myocardium from apoptosis during the treatment of myocardial infarction. In the present study, we show that HR induces apoptosis in H9c2 myoblast cells that is accompanied by Bax up-regulation, down-regulation of Bcl-2 and cell survival molecules (p-Akt and HO-1), activation of death signaling kinases (p-JNK and p-Src). Our study demonstrates that reduced apoptosis and death signaling kinases through Akt-mediated signaling molecules may be a possible mechanism in diosgenin treatment during HR.
The Akt signaling pathway plays a central role in mediating protection in HR conditions. There is abundant evidence that activated Akt prevents cardiomyocyte apoptosis induced by multiple pathological insults including ischemia/reperfusion, pressure overload, hypoxia, hypoglycemia, or cardiotoxic drugs.,,,,, Pharmacological activation of Akt has been demonstrated to provide protection against various stressors by providing mitochondrial integrity and function. Akt activation has also been shown to decrease apoptosis, as well as to reduce infarct size and to improve cardiac function after H/R., These findings have described mechanisms by which the Akt pathway promotes cell proliferation and inhibits cell death. Activated Akt phosphorylates various substrates, activating anti-apoptotic factors and inactivating pro-apoptotic factors. A significant expression of phosphorylated Akt was observed in the diosgenin treated cells during HR. Comparison between diosgenin + H/R and EtOH + H/R revealed that diosgenin displayed significant protection compared with EtOH.
The anti-apoptotic role of Bcl-2 is well documented in the myocardium. It was established that genetic modification of the myocardium with the anti-apoptotic human Bcl-2 gene conferred myocardial protection against HR-induced apoptosis., Furthermore, several studies have reported a reduction of apoptosis by H/R via altering the expression of Bcl-2 and/or Bax protein, thus increasing the ratio of Bcl-2/Bax protein prevents the progression of apoptosis in myocardium after HR., Akt has also been reported to phosphorylate and inhibit Bax. Recently we have shown that Dioscorea bulibifera extract ameliorates rat ischemia/reperfusion injury with down regulated expression of Bax activity resulting in considerable reduction in the Bax/Bcl-2 ratio. In the present study, it was shown that apoptosis occurred in H9c2 cardiomyoblast cells subjected to 30 min of hypoxia followed by 60 min of reoxygenation. Treatment with cells pretreated with 30 µM of diosgenin and subjected to hypoxia (30 min) and reoxygenation (1 h) stimulates phosphorylation of Akt, a kinase directly downstream of PI-3 kinase. Phosphorylated Akt activates Bcl-2, an intracellular anti-apoptotic membrane protein. Up-regulation of Bcl-2 has an inverse relationship with Bax, which shows a significant down-regulation. The MAPK pathways are also known to play both pro and anti-apoptotic roles in cardiomyocyte cell death. There are three principal MAPK terminal effector kinases: The extracellular signal-regulated protein kinases, p38, and the JNK. p38, MAPK and JNK are stress-activated protein kinases that are known to regulate cell viability and are phosphorylated by MAPK kinases MKK3/6 and MKK4/7, respectively, in response to several cellular stresses including ischemia (p38), reperfusion (JNK), as well as UV radiation and cytokines. Bax-dependent activation of the mitochondrial pathway of apoptosis is mediated by early JNK activation. It is possible that the Akt pathway down regulates the JNK pathway, as reported in some studies., JNK is strongly induced by oxidative stress in cultured cardiac myocytes and in intact hearts , and may be a critical determinant of ischemic injury in the myocardium as well as the brain.,,, Our results obtained were in accordance with the previous literature that exhibited pronounced activation of JNK after HR which seemed to be well prevented by diosgenin treatment during the same.
Among the large number of tyrosine kinases, in the present study, the Src family of tyrosine kinases was focused as a candidate responsible for triggering or mediating ischemic preconditioning. c-Src has been implicated in mechanisms of cell survival and death, which was regulated by complex signal transduction processes. Several studies show the increased activity of c-Src in case of cellular damage. The present study probed that distinct activation of c-Src after HR was well prevented by diosgenin compared to that of the control as well as EtOH + H/R treated cells.
The Phase II enzyme HO-1 is an inducible isoform of the first and rate-limiting enzyme of the degradation of heme into iron, carbon mono oxide, and biliverdin, the latter being subsequently converted into bilirubin. Several positive biological effects exerted by this enzyme have gained attention such as anti-inflammatory, anti-apoptotic, angiogenic, and cytoprotective functions and are attributable to carbon monoxide and/or bilirubin. Thus, the physiological induction of HO-1 may be an adaptive and beneficial response to several possibly noxious stimuli, including heme itself, suggesting a potentially autoprotective and autodefensive role in several pathophysiological states including acute coronary syndromes and stroke 9. The results of our study showed an up-regulation of HO-1 in diosgenin treated H9c2 cardiomyoblast cells.
| Conclusion|| |
Altogether, the results of the present study substantiate that diosgenin, a steroidal saponin present in D. bulbifera, modulates HR injury in H9c2 cells via Akt-mediated signaling pathway. Cardioprotection by diosgenin was further demonstrated through MTT cell viability assay and LDH release assay thereby contributing to the protective role of D. bulbifera in experimental HR. At this instance, it is interesting to identify that dioscin, which is the glycoside form of diosgenin, promotes autophagy in lung tumors. Likewise, cardioprotection by adaptation to ischemia augments autophagy. Hence, further studies are worth studying to study the role of diosgenin in D. bulbifera as a potential functional food ingredient.
The authors acknowledge (Late) Dr. Dipak K. Das, for providing the facilities for executing this work in the Cardiovascular Research Centre at UConn Health Centre, Farmington, CT. HRV acknowledges Department of Science and Technology, Govt. of India for the BOYSCAST Fellowship (2007-2008) to work at UConn Health Centre.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992;359:843-5.
Martindale JL, Holbrook NJ. Cellular response to oxidative stress: Signaling for suicide and survival. J Cell Physiol 2002;192:1-15.
Wenger RH. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J 2002;16:1151-62.
Levinson RM, Shure D, Moser KM. Reperfusion pulmonary edema after pulmonary artery thromboendarterectomy. Am Rev Respir Dis 1986;134:1241-5.
Brady AH, Brady NR, Gottileb RA. Title missing? Cardiovasc Drugs Ther 2006;20:445-62.
McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159-63.
Robin ED, Theodore J. Are there ischemic lung diseases? Arch Intern Med 1982;142:1791-3.
Watkins MT, Haudenschild CC, al-Badawi H, Velazquez FR, Larson DM. Immediate responses of endothelial cells to hypoxia and reoxygenation: An in vitro
model of cellular dysfunction. Am J Physiol 1995;268 (2 Pt 2):H749-58.
Ekhterae D, Lin Z, Lundberg MS, Crow MT, Brosius FC rd, Núñez G. ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells. Circ Res 1999;85:e70-7.
Ranki HJ, Budas GR, Crawford RM, Davies AM, Jovanovic A. 17Beta-estradiol regulates expression of K (ATP) channels in heart-derived H9c2 cells. J Am Coll Cardiol 2002;40:367-74.
Kamboj VP. Title missing? Herb Med Curr Sci 2000;78:35-9.
Fowler MW, Mantell SH, Smith H. Plant Biotechnology. Cambridge: Cambridge University Press; 1984. p. 3-37.
Francis G, Kerem Z, Makkar HP, Becker K. The biological action of saponins in animal systems: A review. Br J Nutr 2002;88:587-605.
Komesaroff PA, Black CV, Cable V, Sudhir K. Effects of wild yam extract on menopausal symptoms, lipids and sex hormones in healthy menopausal women. Climacteric 2001;4:144-50.
Higdon K, Scott A, Tucci M, Benghuzzi H, Tsao A, Puckett A, et al.
The use of estrogen, DHEA, and diosgenin in a sustained delivery setting as a novel treatment approach for osteoporosis in the ovariectomized adult rat model. Biomed Sci Instrum 2001;37:281-6.
Au AL, Kwok CC, Lee AT, Kwan YW, Lee MM, Zhang RZ, et al.
Activation of iberiotoxin-sensitive, Ca2+-activated K+ channels of porcine isolated left anterior descending coronary artery by diosgenin. Eur J Pharmacol 2004;502:123-33.
Lísias GD, Azevedo CN, Karyne GP, Maria BF. Mechanisms involved in the vasodilator effect induced by diosgenin in rat superior mesenteric artery.
Eur J Pharmacol 2007;574:172-8.
Jayachandran KS, Vasanthi HR, Rajamanickam GV. Antilipoperoxidative and membrane stabilizing effect of diosgenin, in experimentally induced myocardial infarction. Mol Cell Biochem 2009;327:203-10.
Vasanthi HR, Mukherjee S, Ray D, Pandian Jayachandran KS, Lekli I, Das DK. Protective role of air potato (Dioscorea bulbifera
) of yam family in myocardial ischemic reperfusion injury. Food Funct 2010;1:278-83.
Gao H, Kuroyanagi M, Wu L, Kawahara N, Yasuno T, Nakamura Y. Antitumor-promoting constituents from Dioscorea bulbifera
L. in JB6 mouse epidermal cells. Biol Pharm Bull 2002;25:1241-3.
Suthar A, Mulani R. A high performance thin layer chromatography method for quantitative estimation of diosgenin in Solanum nigrum
Linn. Pharmacogn Mag 2008;4:112-5.
Gurusamy N, Lekli I, Gorbunov NV, Gherghiceanu M, Popescu LM, Das DK. Cardioprotection by adaptation to ischaemia augments autophagy in association with BAG-1 protein. J Cell Mol Med 2009;13:373-87.
Jayadev R, Rao VC. Diosgenin, a steroid saponin constituent of yams and fenugreek emerging evidence for applications in medicine. Bioact Compd Phytomed 2012;2011:126-42.
Kang PM, Haunstetter A, Aoki H, Usheva A, Izumo S. Morphological and molecular characterization of adult cardiomyocyte apoptosis during hypoxia and reoxygenation. Circ Res 2000;87:118-25.
Uchiyama T, Engelman RM, Maulik N, Das DK. Role of Akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning. Circulation 2004;109:3042-9.
Fujio Y, Nguyen T, Wencker D, Kitsis RN, Walsh K. Akt promotes survival of cardiomyocytes in vitro
and protects against ischemia-reperfusion injury in mouse heart. Circulation 2000;101:660-7.
Miao W, Luo Z, Kitsis RN, Walsh K. Intracoronary, adenovirus-mediated Akt gene transfer in heart limits infarct size following ischemia-reperfusion injury in vivo
. J Mol Cell Cardiol 2000;32:2397-402.
Tsujita Y, Muraski J, Shiraishi I, Kato T, Kajstura J, Anversa P, et al.
Nuclear targeting of Akt antagonizes aspects of cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A 2006;103:11946-51.
Matsui T, Li L, del MonteF, Fukui Y, Franke TF, Hajjar RJ, et al.
Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro
. Circulation 1999;100:2373-9.
Ricci C, Jong CJ, Schaffer SW. Proapoptotic and antiapoptotic effects of hyperglycemia: Role of insulin signaling. Can J Physiol Pharmacol 2008;86:166-72.
Negoro S, Oh H, Tone E, Kunisada K, Fujio Y, Walsh K, et al.
Glycoprotein 130 regulates cardiac myocyte survival in doxorubicin-induced apoptosis through phosphatidylinositol 3-kinase/Akt phosphorylation and Bcl-xL/caspase-3 interaction. Circulation 2001;103:555-61.
Matsui T, Tao J, del Monte F, Lee KH, Li L, Picard M, et al.
Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo
. Circulation 2001;104:330-5.
Cantley LC, Neel BG. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci U S A 1999;96:4240-5.
Brocheriou V, Hagège AA, Oubenaïssa A, Lambert M, Mallet VO, Duriez M, et al.
Cardiac functional improvement by a human Bcl-2 transgene in a mouse model of ischemia/reperfusion injury. J Gene Med 2000;2:326-33.
Chen Z, Chua CC, Ho YS, Hamdy RC, Chua BH. Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice. Am J Physiol Heart Circ Physiol 2001;280:H2313-20.
Maulik N, Engelman RM, Rousou JA, Flack JE 3rd
, Deaton D, Das DK. Ischemic preconditioning reduces apoptosis by upregulating anti-death gene Bcl-2. Circulation 1999;100 19 Suppl: II369-75.
Nakamura M, Wang NP, Zhao ZQ, Wilcox JN, Thourani V, Guyton RA, et al.
Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc Res 2000;45:661-70.
Shaw J, Kirshenbaum LA. Prime time for JNK-mediated Akt reactivation in hypoxia-reoxygenation. Circ Res 2006;98:7-9.
Autret A, Martin-Latil S, Mousson L, Wirotius A, Petit F, Arnoult D, et al.
Poliovirus induces Bax-dependent cell death mediated by c-Jun NH2-terminal kinase. J Virol 2007;81:7504-16.
Aikin R, Maysinger D, Rosenberg L. Cross-talk between phosphatidylinositol 3-kinase/AKT and c-jun NH2-terminal kinase mediates survival of isolated human islets. Endocrinology 2004;145:4522-31.
Kim AH, Khursigara G, Sun X, Franke TF, Chao MV. Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol Cell Biol 2001;21:893-901.
Laderoute KR, Webster KA. Hypoxia/reoxygenation stimulates Jun kinase activity through redox signaling in cardiac myocytes. Circ Res 1997;80:336-44.
Kubasiak LA, Hernandez OM, Bishopric NH, Webster KA. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3. Proc Natl Acad Sci U S A 2002;99:12825-30.
Das DK. Redox regulation of cardiomyocyte survival and death. Antioxid Redox Signal 2001;3:23-37.
Cook SA, Sugden PH, Clerk A. Activation of c-Jun N-terminal kinases and p38-mitogen-activated protein kinases in human heart failure secondary to ischaemic heart disease. J Mol Cell Cardiol 1999;31:1429-34.
Herdegen T, Claret FX, Kallunki T, Martin-Villalba A, Winter C, Hunter T, et al.
Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury. J Neurosci 1998;18:5124-35.
Sadoshima J, Montagne O, Wang Q, Yang G, Warden J, Liu J, et al.
The MEKK1-JNK pathway plays a protective role in pressure overload but does not mediate cardiac hypertrophy. J Clin Invest 2002;110:271-9.
Hattori R, Otani H, Uchiyama T, Imamura H, Cui J, Maulik N, et al.
Src tyrosine kinase is the trigger but not the mediator of ischemic preconditioning. Am J Physiol Heart Circ Physiol 2001;281:H1066-74.
Vasanthi HR, Mukherjee S, Lekli I, Ray D, Veeraraghavan G, Das DK. Potential role of Borreria hispida in ameliorating cardiovascular risk factors. J Cardiovasc Pharmacol 2009;53:499-506.
Idriss NK, Blann AD, Lip GY. Hemoxygenase-1 in cardiovascular disease. J Am Coll Cardiol 2008;52:971-8.
Hsieh MJ, Tsai TL, Hsieh YS, Wang CJ, Chiou HL. Dioscin-induced autophagy mitigates cell apoptosis through modulation of PI3K/Akt and ERK and JNK signaling pathways in human lung cancer cell lines. Arch Toxicol 2013;87:1927-37.
| Authors|| |
Dr. A. Hannah Rachel Vasanthi, Associate Professor Department of Biotechnology Pondicherry University Puducherry 605 014
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]