|Year : 2013 | Volume
| Issue : 3 | Page : 289-293
Diosgenin prevents hepatic oxidative stress, lipid peroxidation and molecular alterations in chronic renal failure rats
Jeganathan Manivannan1, Pandiyan Arunagiri1, Jeganathan Sivasubramanian2, Elumalai Balamurugan1
1 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar, Tamil Nadu, India
2 Department of Physics, Faculty of Science, Annamalai University, Annamalai Nagar, Tamil Nadu, India
|Date of Submission||29-Jan-2013|
|Date of Acceptance||13-Feb-2013|
|Date of Web Publication||10-Jul-2013|
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: Chronic renal failure (CRF) is one of the major contributors of cardiovascular pathological events and CRF associated uremic condition in rats elevates liver oxidative stress. The aim of the present study was to evaluate the preventive potential of diosgenin on antioxidant system and molecular protection potential in liver of CRF rats. Materials and Methods: CRF in rats was induced by feeding rats with diet containing 0.75% adenine for 5 weeks. Diosgenin was given orally at a dose of 40 mg/kg body weight (bw) of animal each and every day. The activities of antioxidant enzymes and lipid peroxidation level were determined by biochemical assays. Fourier transform infrared spectroscopy (FTIR) was employed to illustrate the molecular protection potential of diosgenin. Results: This study has shown adenine containing diet induced CRF in rats elevates liver oxidative stress by suppressing the activity of enzymatic antioxidant system, increased lipid peroxidation, and macromolecular structural alterations. In this study, treatment of diosgenin 40 mg/kg bw of rat significantly restores the enzymatic antioxidant system and reduces the lipid peroxidation level. Moreover, based on the FTIR study, we confirmed that diosgenin administration significantly protected the macromolecular changes including, the protein structural damage that occurred in liver. Conclusion: This study has proved the hepato protective action of diosgenin through antioxidant and molecular protection effect in CRF condition.
Keywords: Antioxidant, chronic renal failure, diosgenin, fourier transform infrared spectroscopy
|How to cite this article:|
Manivannan J, Arunagiri P, Sivasubramanian J, Balamurugan E. Diosgenin prevents hepatic oxidative stress, lipid peroxidation and molecular alterations in chronic renal failure rats. Int J Nutr Pharmacol Neurol Dis 2013;3:289-93
|How to cite this URL:|
Manivannan J, Arunagiri P, Sivasubramanian J, Balamurugan E. Diosgenin prevents hepatic oxidative stress, lipid peroxidation and molecular alterations in chronic renal failure rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2020 Aug 15];3:289-93. Available from: http://www.ijnpnd.com/text.asp?2013/3/3/289/114870
| Introduction|| |
Cardiovascular (CV) complications are the leading cause of death in patients with chronic kidney disease (CKD).  The elevated parathyroid hormone and disordered mineral metabolism (hyperphosphatemia) associated with secondary hyperparathyroidism complicate the clinical course of most patients with CKD and when advanced, are associated with markedly increased morbidity and mortality. 
Recent findings demonstrated that acute renal failure after kidney injury or bilateral nephrectomy triggers oxidative stress and causes damage to the liver and there was a gender difference with regard to the severity of hepatic oxidative stress and inflammatory response in acute uremia.  Chronic renal failure (CRF) patients have an increased plasma level of urea, which can be a source of cyanate, which can cause protein carbamoylation thereby changing biological activity of proteins. Therefore, in renal failure patients, cyanate can disturb metabolism and functioning of the liver. 
A recent evidence have shown that the most important frontline defense system composed of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) actively prevents the oxidative stress generated in liver during renal failure related pathological conditions with the help of an antioxidant molecule.  When reactive oxygen species (ROS) are produced at high-levels, mitochondrial derived ROS also causes deleterious effects by massive and irreversible oxidation of their principal targets (lipids, proteins, and DNA). 
Diosgenin is a naturally occurring steroidal saponin present in a variety of plants including, fenugreek (Trigonella foenum graecum) and roots of wild yam (Dioscorea villosa).  Molecular structure of diosgenin was shown in [Figure 1]. Diosgenin has been used in traditional medicine as an anti-hypercholesterolemia, anti-hypertriacylglycerolemia, anti-diabetes, and anti-hyperglycemia agent. ,,, Recent study on effect of diosgenin on vascular smooth muscle cells under Tumor necrosis factor (TNF-α) induced condition reports that, diosgenin abrogated TNF-α induced production of intracellular ROS and phosphorylation of mitogen activated protein kinases (MAPK).  Another finding suggested that diosgenin could have a beneficial role against aortic remodeling induced by oxidative stress in diabetic state, which was evidenced from the propensity of diosgenin to modulate the antioxidant defense and to decrease the lipid peroxidation in aorta.  Further, a study on membrane stabilizing effect of diosgenin, in experimentally induced myocardial infarction shown that anti lipid peroxidative potential of diosgenin may be due to its anti-oxidative activity. 
From this point of view, this present study intend to evaluate the preventive potential of diosgenin against CRF induced hepatic oxidative stress by means of fourier transform infrared spectroscopy (FTIR). A previous study reports that protective role of borneol, a natural terpene on liver metabolism in a nitric oxide deficient model of hypertension through interpretation of FTIR spectral information and results demonstrate that FTIR can successfully indicate the molecular changes that occur in liver during oxidative stress.  In this study, activity of enzymatic antioxidants, level of lipid peroxidation products, and macromolecular structure related alterations in liver of CRF rats treated with diosgenin were assessed.
| Materials and Methods|| |
Animals and chemicals
Male albino Wistar rats, 7-10 weeks old (180-220 g) were procured from the Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University. The experimental study was approved by the Ethical Committee of Rajah Muthiah Medical College and Hospital, Annamalai Nagar, Tamil Nadu, India. Diosgenin was purchased from Sigma Aldrich (USA) and all other chemicals in this study used were of analytical grade.
Experimental groups and diosgenin treatment
CRF in Wistar rats was induced by fed the animals with diet containing 0.75% adenine for 5 weeks. Each of the following groups were consists of six animals. Diosgenin 40 mg/kg was administered orally every day by dissolved in oil as vehicle throughout the experiment.
Group I: Control (animals fed with rat chow).
Group II: Control + diosgenin 40 mg/kg body weight (bw) of animals.
Group III: Control animals fed with 0.75% adenine-CRF animals.
Group IV: CRF + diosgenin 40 mg/kg bw of animals.
Liver tissue homogenization
Liver tissues were sliced into pieces and homogenized in appropriate buffer in cold condition (pH 7.0) to give 20% homogenate (w/v). The homogenate was centrifuged at 560 × g for 10 min at 4°C in refrigerated centrifuge. The supernatant was separated and used for various biochemical estimations.
Antioxidant enzyme activity and lipid peroxidation products in liver
Liver antioxidant enzymes assay and lipid peroxidation assays were done for all the groups . SOD, CAT, GPx, were assayed with the methods of Kakkar et al.,  Sinha,  Rotruck et al.  respectively. Total protein was assayed by the method of Lowry et al.  The level of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides were estimated by the methods of Niehaus and Samuelson,  Jiang et al. 
FTIR analysis was carried out as described previously.  A small and equal amount of liver samples were homogenized and freeze dried. Samples were stored under −80°C until used. For FTIR analysis, the samples were mixed with Potassium Bromide (KBr) at ratio of 1:100. The mixture was then subjected to a pressure of 1,100 kg/cm 2 to produce KBr pellets for use in FTIR spectrometer. Pellets of the same thickness were prepared by taking the same amount of sample and applying the same pressure. FTIR spectra of the region 4,000-400 cm−1 were recorded at the temperature of 25 ± 1°C on a Nicolet-Avatar-360 FTIR spectrometer.
Values are given as mean ± SD for six rats in each group. Data were analyzed by one-way analysis of variance followed by Duncan's multiple range test using SPSS version 11.5 (SPSS, Chicago, IL). The limit of statistical significance was set at P < 0.05.
| Results|| |
Effect of diosgenin on enzymatic antioxidants and lipid peroxidation products
The activities of SOD, CAT, and GPx in liver of all the groups are presented in [Table 1]. The activities of these enzymatic antioxidants significantly decreased in CRF rats. Treatment with 40 mg/kg diosgenin significantly ( P < 0.05) restored the activity of these enzymatic antioxidants in liver.
[Table 1] also portrays the levels of TBARS, lipid hydro peroxides. Levels of lipid peroxidation products in liver were significantly elevated in CRF rats, whereas treatment of diosgenin significantly reduced the lipid peroxidation products.
|Table 1: Effect of diosgenin on liver enzymatic antioxidants and lipid peroxidation|
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The representative FTIR spectra of the control liver have shown important macromolecular bonding frequency regions [Figure 2]. The spectral wave numbers were assigned from previous studies. , Frequency shifts of macromolecules were illustrated in [Table 2]. The bands in 3,393 cm−1 region arise from N-H and O-H stretching modes of proteins, and intermolecular hydrogen bonding. In the experiment, Amide A appeared at 3,393 cm−1 in control and the band frequency was significantly shift in renal failure rats, whereas the diosgenin treatment protects the changes significantly and there was no significant change between control and diosgenin alone treated group. CH 2 symmetric stretching of lipids/fatty acids appears at 2853 cm−1 in control and in renal failure it was shifted increase in wave number significantly. However, the diosgenin treated group was significantly brought back the shifted peak to control level. The most important molecular bonds of proteins, amide linkages were significantly altered in renal failure group, in which the Amide I band was appeared in 1650 cm−1 in control. In the wave number of Amide II linkage, the control group appeared at 1542 cm − 1 and the significant change in frequency shift was observed in CRF rat liver. However, this change was significantly protected by diosgenin in Amide I but not in Amide II region.
|Figure 2: Fourier transform infrared spectroscopy of control liver with important macromolecular bond frequency regions|
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| Discussion|| |
The high-rate of CV complications observed in hemodialysis patients is only partly explained by traditional risk-factors, such as ageing, gender, hypertension, diabetes, smoking, dyslipidemia, and obesity. Indeed, non-traditional risk-factors including, malnutrition, inflammation, and oxidative stress have emerged. , The oxidative stress generated during renal failure after kidney injury or bilateral nephrectomy activates oxidative stress and causes damage to the liver.  In this study, the enzymatic antioxidant system was focused in liver during the CRF condition.
Free radical scavenging enzymes such as SOD, CAT, and GPx are the first line of cellular defense against oxidative injury. SOD reduces superoxide anion to form H 2 O 2 and oxygen. CAT removes H 2 O 2 by breaking it down directly to oxygen.  The observed declined activities of SOD, CAT in renal failure rat liver may be due to the involvement of free radicals and oxidative stress, which is consistent with a previous study.  A decrease in activity of these enzymes leads to accumulation of superoxide anion and H 2 O 2 which in turn can form the toxic hydroxyl radical. Reduced activities of GPx might be due to reduced substrate because of increased utilization for protecting proteins from oxidative damage. Our results show that diosgenin prevented the decrease in the activity of all the above enzymic antioxidants in liver tissue of renal failure rats.
Lipid peroxidation, arising from the reaction of free radicals with lipids, has been linked with altered membrane structure and enzyme inactivation. Its end products measured as TBARS, lipid hydroperoxides were seen increased in liver tissue clearly indicating increased oxidative stress in renal failure. Diosgenin inhibits lipid peroxidation, which may be due to its radical scavenging and anti lipidperoxidative property. 
The FTIR spectroscopy monitors the vibration modes of functional groups present in proteins, lipids, polysaccharides, and nucleic acids in liver tissue. Shifts in peak positions indicate the molecular changes associated with macromolecules in a condition. In this analysis, the N-H stretching of proteins was changed significantly during renal failure, this indicates that the intra molecular hydrogen bonding in proteins of liver tissue may be disturbed.  This effect might be due to the oxidative stress generated during uremic condition as previously reported.  This effect was protected by diosgenin treated group indicates its antioxidant potential against oxidative stress generated during renal failure.
The wave number of fatty acid species changed significantly in renal failure liver, this indicates the acyl chain modification of membrane lipids. The frequency of the CH 2 bands of acyl chains depends on the degree of conformational disorder and level of flexibility. The position of these bands provides information about the lipid acyl chain flexibility (order/disorder state of lipids).  The shift of the peak position of these bands to higher values indicates that increase lipid order and acyl chain flexibility. A previous study indicates that this kind of changes may associate with oxidative stress generated in liver.  The above shift was significantly protected by diosgenin which indicates its protective effect on lipid species through its antioxidant and anti-lipid peroxidative potential in liver as already reported in aorta.  This effect further supports anti-lipid peroxidation potential of diosgenin on liver in uremia related oxidative stress and lipid peroxidation in an indirect way, which is consistent with lipid peroxidation parameter results.
Amide bands are the most important indicators of oxidative stress. The shift in Amide I and Amide II in renal failure group liver compared with control indicates the excess production of free radicals and production of free radical associated protein damage. This structural change might also occur due to elevated urea induced cyanate related toxicity as described previously.  However, diosgenin treatment significantly protects the Amide I and Amide II associated changes. According to findings of a previous study, all the constituent amino acid side-chains in proteins are susceptible to free radicals, but some are more vulnerable than others. Thus, exposure of proteins to free radical generating systems may induce secondary structural changes. Secondary structure is stabilized by hydrogen bonding of the peptide backbone and interference with the functional groups of the peptide bonds may cause structural modifications. Further the shift in Amide I and II regions corresponds to the alpha-helix protein conformational change.  The above effect may directly indicate the antioxidant potential of diosgenin, which actively scavenge free radicals and activate the defense system through which it protects the macromolecular damage and liver dysfunction.
Previous studies have shown that, natural products have promising strategy to protect tissue from oxidative stress and lipid peroxidation under various pathological conditions. ,, Consistent with the previous studies, , this work also proven that natural molecules enhances the activity of enzymatic antioxidants thereby protects the tissue against oxidative stress.
| Conclusion|| |
Over all study proves that the antioxidant potential of diosgenin prevents oxidative stress, lipid peroxidation mediated hepatic damage and macromolecular structural alterations in CRF rats. Thus, in future, diosgenin can be used as a hepato protective agent in renal failure patients.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]