|Year : 2011 | Volume
| Issue : 6 | Page : 671-675
Hepatoprotection through regulation of voltage dependent anion channel expression by Amomum subulatum Roxb seeds extract
Mihir Y Parmar1, Purvi A Shah1, Jing Gao2, Tejal R Gandhi1
1 Anand Pharmacy College, Opp. Town Hall, Anand, Gujarat, India
2 School of Pharmacy, Jiangsu University, Zhenjiang, P. R., China
|Date of Submission||25-Nov-2010|
|Date of Decision||01-Jul-2011|
|Date of Acceptance||02-Sep-2011|
|Date of Web Publication||14-Nov-2011|
Tejal R Gandhi
Anand Pharmacy College, Opp. Town Hall, Anand, Gujarat
Source of Support: None, Conflict of Interest: None
Background and Purpose : Voltage dependent anion channel (VDAC) plays an important role in triggering the opening of the mitochondrial permeability transition pore (PTP) that leads to mitochondrial damage and induce apoptic or necrotic cell death. In the present study, the methanolic extract of Amomum subulatum Roxb. seeds (MEAS) was used to examine its effect on VDAC. Aminotransferase activity, mitochondrial membrane potential, calcium-induced liver MPT, and VDAC expression were used to evaluate the hepato protective effect of MEAS.
Results : Pretreatment of mice with MEAS (100 and 300 mg/kg) significantly blocked the CCl 4 -induced increase in AST and ALT activities. Pretreatment with MEAS showed significant preservation of mitochondrial membrane potential as compared to CCl 4 control demonstrating the mitochondrial protection. In addition, pretreatment with MEAS at various concentrations exerted a dose-dependent effect against sensitivity to mitochondrial swelling induced by calcium. In addition, MEAS (300 mg/kg) significantly increased the transcription and translation of VDAC.
Conclusion : Our data suggest that MEAS significantly prevents the damage to liver mitochondria through regulation of VDAC expression.
Keywords: CCl 4 , Amomum subulatum Roxb seeds, liver mitochondria, mitochondrial permeability transition, voltage dependent anion channel
|How to cite this article:|
Parmar MY, Shah PA, Gao J, Gandhi TR. Hepatoprotection through regulation of voltage dependent anion channel expression by Amomum subulatum Roxb seeds extract. Indian J Pharmacol 2011;43:671-5
|How to cite this URL:|
Parmar MY, Shah PA, Gao J, Gandhi TR. Hepatoprotection through regulation of voltage dependent anion channel expression by Amomum subulatum Roxb seeds extract. Indian J Pharmacol [serial online] 2011 [cited 2021 Jul 31];43:671-5. Available from: https://www.ijp-online.com/text.asp?2011/43/6/671/89824
| » Introduction|| |
It is well accepted that cell death is the most crucial step in the development of all kinds of liver injury. ,, Mitochondria play a key role in controlling cell death not only by providing ATP by oxidative phosphorylation, but also by modulating intracellular Ca +2 homeostasis and induction of cell death. , Mitochondrial permeability transition (MPT) is the opening of a permeation pathway of the inner mitochondrial membrane allowing the diffusion of solutes of molecular mass up to ~1500 Kilo Dalton caused by an opening of specific nonselective proteinaceous pores in the inner mitochondrial membrane. , The realization that opening of the mitochondrial permeability transition pore (PTP) is critical to the release of both pro and anti-apoptotic factors which result in the attenuation of mitochondrial membrane potential and the mitochondrial swelling has stimulated research on components of the PTP complex.
The mitochondrial PTP composed of a voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT) etc., has been recognized as a major complex in MPT. , The PTP opening is also accompanied by the release of cytochrome-c (Cyt-c), a strong activator of caspase-9, apoptotic protease activating factor-1,  and subsequently, caspase-3 which results in apoptosis.  VDAC is a key protein that regulates basic mitochondrial functions such as energy transduction, substance metabolism, and intracellular Ca +2 homeostasis as well as it plays a very important role in cell death control, especially apoptosis initiation.
Amomum subulatum Roxb (commonly known as: greater cardamom, Family: Zingiberaceae) is a perennial herb which grows widely in moist tropical countries.  Recently, in our study, methanolic extracts of Amomum subulatum seed was demonstrated to possess hepato protective activity.  However, the mechanism underlying the hepato protective activity has not been investigated. In this paper, we addressed the possible action of Amomum subulatum Roxb seeds extract on mitochondrial PTP by regulation of VDAC, the most important proteins on the outer membrane of mitochondria, to search for the possible mitochondrial mechanism underlying its hepato protective activity.
| » Methods and Methods|| |
Plant Material and Extraction
Seeds of Amomum subulatum were purchased from a commercial supplier, identified, and authenticated by Dr. A. S. Reddy, Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India, where a voucher specimen (No. MP-1 : 28/7/2007) was kept for the future reference.
The seeds were dried at room temperature and mechanically powdered to obtain a coarse powder. The powdered seeds of A. subulatum after defatting with petroleum ether (60-80°C) were extracted with methanol/water (70/30, v/v) and filtered. The filtrate was evaporated under vacuum using a rotary evaporator to obtain a dark brown powder (9.5% w/w yield). The methanolic extract of A. subulatum (MEAS) was dried and stored in a cool place.
CCl 4, JC-1 (5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'- tetraethyl-benzimidazolyl carbocyanine iodide), succinate, rotenone, cyclosporine A (CsA), anti-VDAC antibody were purchased from Sigma (St Louis, MO, USA) and tripure reagent from Roche Diagnostics Corporation (Indianapolis, USA). AMV reverse transcriptase, RNase inhibitor, dNTP, Oligo(dT)15, and Taq polymerase were procured from Promega (Madison, USA). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) kits were procured from Nanjing sunshine biotech Ltd. (Nanjing, East china). All other chemicals were of high purity and bought from the commercial sources.
Male ICR mice (Experiment Animal Center of School of Pharmacy, Zhenjiang, P. R. China, Protocol certificate No. SCXK [Jiangsu] 2007-0001), each weighing 18-22 g, were used. All animals were fed a standard diet ad libitum and housed at a temperature of 20-25°C under a 12-h light/dark cycle throughout the experiment. The study protocol complied with the guidelines of Jiangsu University, China.
Carbon Tetrachloride (CCl 4 )-Induced Hepatotoxicity in Mice 
ICR mice (18-22 g) randomly selected were allocated into four groups, each containing six animals. Group I (normal control) received olive oil (10 ml/kg, i. p.) and group II (CCl 4 control) received 0.15% CCl 4 in olive oil (10 ml/kg, i. p.) for five consecutive days. Group III (test 1) and group IV (test 2) animals were given MEAS at a dose of 100 and 300 mg/kg, p. o., respectively, for five days. Additionally, after 30 min of drug treatment, groups III and IV also received 0.15% CCl 4 in olive oil (10 ml/kg, i.p.) for five days.
After 18 hours of the last dose, blood was collected and mice were euthanized. The blood was allowed to clot at room temperature and serum was separated by centrifugation at 3,000 g for 20 min. Meanwhile, the whole liver was excised, liver lobes intended for mRNA and protein analyses were frozen immediately and stored in liquid nitrogen before extraction.
Aminotransferase Activity Determination
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, markers for hepatotoxicity, were determined using an automatic analyzer (Hitachi 7600-020, Japan).
Isolation of Liver Mitochondria
Mitochondria were prepared from mouse liver.  Livers were excised and homogenized in isolation buffer containing 225 mM D-mannitol, 75 mM sucrose, 0.05 mM EDTA, and 10 mM Tris-HCl (pH 7.4) at 4°C. The homogenates were centrifuged at the speed of 600 g for 5 min and supernatants were separated and centrifuged at 8,800 g for 10 min. The pellet was washed twice with the same buffer. Protein concentration was determined using Coomassie brilliant blue.
Measurement of Mitochondrial Membrane Potential
The mitochondrial membrane potential (Ψm) was evaluated following the uptake of the fluorescent dye JC-1 (5, 5', 6, 6'-tetrachloro-1 1', 3, 3'-tetraethyl-benzimidazolyl carbocyanine iodide).  Isolated liver mitochondria were suspended in the assay buffer (0.5 mg protein/ml) containing 225 mM mannitol, 70 mM sucrose, and 5 mM HEPES (N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid), p H 7.2. The mitochondrial membrane potential was assessed spectrophotometrically (Hitachi 850) at 25°C with excitation at 505 nm and detection at 534 nm after addition of 0.3 μM JC-1. The distribution of JC-1 between mitochondria and medium follows the Nernst equation.  The membrane potential was calculated as follows:
Measurement of Mitochondrial Swelling
Mitochondrial swelling was assessed by measuring the absorbance of their suspension at 540 nm. Liver mitochondria were prepared in 3 ml of the assay buffer (0.5 mg protein/ml) containing 125 mM sucrose, 50 mM KCl, 2 mM KH 2 PO 4 , 10 mM HEPES, and 5 mM succinate. Ca +2 (50 μM) was added to the assay buffer to initiate the mitochondrial swelling at 30°C. CsA (5 μM) was used as a positive reference  and various concentrations of MEAS (1, 10, 50, 80 and 100 μM) were added to mitochondrial solution 3 min before incubation with 50 μM of Ca +2. The extent of mitochondrial swelling was assayed by measuring the decrease in absorbance (A) 0-10 min at every 30 sec after the addition of 50 μM of Ca +2 and the inhibitory rate of mitochondrial swelling was calculated as follows. 
Where, ΔA= A 0 min − A 10 min
Evaluation of VDAC mRNA Level by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Assay
Total RNA was extracted from liver using Tripure reagent. Reverse transcription was started by incubating 2 μg of total RNA at 42°C for 60 min in a 20 μl reaction mixture containing 20 U RNase inhibitor, 0.25 mM each of dNTP, 0.5 μg Oligo(dT)15, and 15 U AMV reverse transcriptase. The reaction was terminated by incubation at 95°C for 5 min. Polymerase chain reaction (PCR) amplification was performed for 30 cycles, including 4 μl cDNA by adding 5 mM MgCl 2 , 2.5 U Taq polymerase, 0.25 mM each of dNTP and 5'-and 3'-sequence-specific oligonucleotide primers for VDAC and β-actin in 1ΧTaq polymerase reaction buffer, respectively. Each PCR cycle was comprised of 94°C, 50 sec; 60°C, 1 min; 72°C, 1 min; and finally 72°C, 8 min. The internal standard was set with β-actin. The amplified fragments were detected by agarose gel electrophoresis and visualized by ethidium bromide (EB) staining.
The oligonucleotide primers used were as follows:
For VDAC, sense 5'- GGC TAC GGC TTT GGC TTA AT -3' and anti-sense 5'- CCC TCT TGT ACC CTG TCT TGA -3', yielding a deduced amplification product of 321 base pairs (bps).
For β-actin, sense 5'- TGC TAT CCC TGT ACG CCT CT -3' and anti-sense 5'- GGA GGA GCA ATG ATC TTG A -3' yielding a deduced amplification product of 601 bps.
Western Blot Analysis for VDAC
Liver samples were homogenized in ice-cold lysis buffer. Homogenates were centrifuged at 12,000 g for 10 min and the supernatants were collected and the protein concentration was determined using Coomassie brilliant blue dye. The samples (40 μg/lane) were dissolved in the sample buffer and separated by 12% sodiumdodecylsulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel and electrophoretically transferred onto a polyvinylidene-difluoride (PVDF) membrane. The membrane was incubated with VDAC primary antibody (1:4,000) and β-actin antibody (1:80,000). The membrane was then exposed to the enhanced chemiluminescence (ECL) solution.
Differences among all groups were analyzed by one-way analysis of variance, (ANOVA) followed by Dunnett's multiple comparison test. Differences were considered to be statistically significant when P < 0.05.
| » Results|| |
Effect of MEAS on ALT and AST Levels
Administration of CCl 4 caused a marked elevation (P < 0.05) of serum ALT (727.23 ± 3.45) and AST (902.10 ± 5.30) levels in the CCl 4 control mice in comparison with the normal control [Figure 1]. Pretreatment of mice with MEAS at doses of 100 and 300 mg/kg significantly inhibited the elevation in ALT (331.15 ± 3.67; 264.84 ± 2.89) and AST (322.10 ± 4.68; 275.70 ± 2.42) levels.
Effect of MEAS on Mitochondrial Membrane Potential Dissipation
The mitochondrial membrane potential of normal (control) mice was -188.0 ± 2.5 mV. CCl 4 ingestion in mice significantly (P < 0.05) changed the mitochondrial membrane potential value to -156.8 ± 3.0 mV [Figure 2]. Pretreatment with MEAS (100 and 300 mg/kg) showed significant (P < 0.05) preservation of mitochondrial membrane potential as compared to CCl 4 control.
|Figure 2: Effects of MEAS on prevention of mitochondrial membrane potential dissipation induced by CCl4|
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Inhibitory Effects of MEAS on Ca +2 -Induced Mitochondrial Swelling
Addition of 50 μM Ca +2 induced a remarkable mitochondrial swelling as shown in [Figure 3]. Pretreatment with increasing concentrations of MEAS exerted a dose-dependent effect against Ca +2 -induced mitochondrial swelling. At 7 min, the inhibitory effect of 1, 10, 50, 80, and 100 μM of MEAS was maximum and reached up to 5.60, 24.88, 34.72, 58.06, and 52.90%, respectively. Similarly, maximum inhibitory mitochondrial swelling effect of CsA positive reference was found to be 90.64% at 7 min.
Effect of MEAS on Mitochondrial VDAC Expression in CCl 4 -Treated Mouse Liver
Down-regulation of liver VDAC mRNA level induced by CCl 4
The effect of MEAS on VDAC mRNA transcription was examined by reverse transcriptase (RT-PCR). As shown in [Figure 4]a, higher level of VDAC mRNA expression was detected in mice treated with CCl 4 compared to normal control group. While treatment with 300 mg/kg MEAS significantly blocked the CCl 4 -induced VDAC mRNA elevation.
|Figure 4: Effects of MEAS on mitochondrial VDAC expression in CCl4- treated mouse livers|
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Down-regulation of liver VDAC protein level induced by CCl 4
Down-regulation of VDAC protein expression mediated by MEAS was demonstrated by Western blot analysis [Figure 4]b. Normal control animals showed a significant signal for VDAC, and mice receiving CCl 4 showed a significantly stronger signal for VDAC. In contrast, in mice pretreated with MEAS at 300 mg/kg dose, a lower level of VDAC protein signal similar to that of normal mice was evident 18 h following CCl 4 treatment compared to mice treated with CCl 4 alone.
| » Discussion|| |
Liver damage induced by CCl 4 is a well-characterized paradigm for acute hepatic failure and is often used to screen drugs for hepato protective activities.  CCl 4 -induced acute liver injury may be initiated by the *CCl 3 radical, which is formed by a metabolic enzyme (cytochrome P450) and could induce peroxidation of the unsaturated fatty acids of cell membrane and lead to membrane injury and leakage of sensitive markers of hepatocellular injury such as serum ALT and AST.  This study revealed a significant increase in ALT and AST levels following exposure to CCl 4 indicating considerable hepatocellular injury which could be inhibited by the oral administration of MEAS at doses of 100 and 300 mg/kg demonstrating its hepato protective effect. At the same time, the effect of MEAS on AST also suggests its possible roles on mitochondria because 80% of AST was released from mitochondria.
Another sensitive marker of mitochondrial injury is the dissipation of the mitochondrial membrane potential. In the present study, the protective effect of MEAS on liver mitochondrial membrane potential in CCl 4 -intoxicated mice was evaluated. CCl 4 ingestion in mice damaged liver mitochondria as demonstrated by the dissipation of mitochondrial membrane potential which is in accordance with previous findings of Gao et al., and Tang et al., , Pretreatment with MEAS (100 and 300 mg/kg) could significantly prevent the dissipation of the mitochondrial membrane potential confirming the protective effect of MEAS against mitochondrial damage. Hepatocellular Ca +2 overload can activate the mitochondrial Ca +2 uni-porter in the mitochondrial inner membrane and induce a mitochondrial Ca +2 influx. However, an excessive intra-mitochondrial Ca +2 can lead to the opening of mitochondrial PTP and finally damage it and induce apoptotic or necrotic cell death. Thus, Ca +2 -induced liver MPT has become a widely used model for evaluating the effect of drugs or other substances on mitochondria. Our data has revealed that MEAS could act on mitochondria PTP directly against Ca +2 -induced mitochondrial swelling which suggests that MEAS may protect mitochondria. Indeed, it has been believed that inhibition of mitochondrial PTP opening might constitute a relevant pharmacological approach to protect cells from death and the search for novel PTP inhibitors should be an important strategy for the treatment of liver diseases. ,
VDAC play an important role in triggering the opening of the PTP. There is accumulating evidence that there are changes in the levels of expression of the mitochondrial VDAC, one of the most important proteins on the outer membrane with regard to the process of apoptosis. ,,, VDAC levels increased significantly after CCl 4 administration and pretreatment of MEAS (300 mg/kg) could inhibit the elevation of both transcriptional and translational levels of VDAC in acute liver injury process. This suggests a protective effect of MEAS on liver mitochondria in mice might be related to a down-regulation of the expression of mitochondrial VDAC which was up-regulated by CCl 4 .
In conclusion, the above data indicate that MEAS has hepato protective activity and the mechanisms underlying its protective effects may be related to mitochondrial protection and especially the regulation of VDAC expression.
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