IN-VITRO NEUROPROTECTIVE ACTIVITY OF SILIBININ AGAINST MPTP (1-METHYL-4-PHENYL-1, 2, 3, 6-TETRAHYDROPYRIDINE) INDUCED NEUROTOXICITY MODELHTML Full Text
IN-VITRO NEUROPROTECTIVE ACTIVITY OF SILIBININ AGAINST MPTP (1-METHYL-4-PHENYL-1, 2, 3, 6-TETRAHYDROPYRIDINE) INDUCED NEUROTOXICITY MODEL
G. Devala Rao 2, S. Praveen Begum * 1 and Hanumanthu Penchalaiah 1
University College of Pharmaceutical Sciences 1, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur - 522510, Andhra Pradesh, India.
K. V. S. R. Siddhartha College of Pharmaceutical Sciences 2, Vijayawada - 520008, Andhra Pradesh, India.
ABSTRACT: In the present study, we examined the effect of Silibinin on the accumulation of oxidative stress induced by MPTP by in-vitro. After decapitation, healthy rat brain was removed rapidly from the skull and rinsed with cold artificial cerebrospinal fluid (ACSF) which has been equilibrated with 95% O2 / 5% CO2 gas mixture. Group I brain was incubated in CSF serve as normal, Group II lenses brain was incubated in CSF and DMSO (10%) serve as disease, Group III brain was incubated with MPTP (1 ng/mL) Group IV brain was incubated in MPTP with bromocriptine (10 µg/mL), Group V brain was incubated in MPTP with Silibinin (10 µg/mL) Group VI brain was incubated in MPTP with Silibinin (20 µg/mL). After 1 h of incubation brain slices were homogenized in PBS buffer, pH 7.4 and estimated for protein content, LPO* and GSH as per the procedures. MPTP incubated brain LPO* levels were increased, and GSH activity decreased compared to normal group. Silibinin treated group brain LPO* levels were significantly decreased and GSH activity was increased. The research results were concluded that the Silibinin exhibited significant neuroprotective effect against MPP + free radicals due to their antioxidant activity.
Silibinin, Cerebrospinal fluid, MPTP, Brain slices, Lipid peroxidation (LPO), Glutathione (GSH)
INTRODUCTION: Glials, a specialized type of non-neuronal cell, regulate the neuronal microenvironment and provide support to the nervous system 1, 2. Amongst the glial cells, astrocytes are abundantly present with a close connection to neurons in the brain and spinal cord 3 regulating various physiological and pathological conditions. Astrocyte metabolism is a key feature on which the neurons are functionally dependent, including its role in energy metabolism and synthesis of neurotransmitters by maintaining the amino acid homeostasis 4.
Astrocytes play a dynamic role in the brain 5 and are associated with apoptosis, ischemia and various neurodegenerative disorders 6, 7. The brain’s vulnerability towards oxidative stress is highly dependent on astrocytes, and thus astrogliosis may critically impair the survival of neurons 8, 9. Astrocyte activation releases neuroinflammatory molecules like the proinflammatory cytokines tumor necrosis factor α (TNFα); interleukin (IL-1β and IL-6) 10.
These neuroinflammatory cytokines modulate the astroglia dependent apoptosis resulting in malignant glioma development. Reactive astrocytes are a key feature for formation of the ‘glial scar’ expressing the glial fibrillary acidic protein and ultimate consequence of neuronal death 11. Silibinin, a flavonolignan obtained from the fruit and seed extracts of ‘milk thistle’, (Silybum marianum, Asteraceae) is one of the most biologically active component among the three isomers (Silibinin, silychristin and silidianin) which collectively form ‘Silymarin.’ It is well known for its excellent hepatoprotective effect and has been reported to act as cardioprotective, having anti-cancer activity, immunomodulatory effect and an excellent antioxidant, inhibiting lipid peroxidation and scavenging free radicals 12, 13, 14, 15. Clinical studies assessing the effect of Silibinin and Silymarin on the human liver cirrhosis have provided promising results in normalizing the hepatic markers.
Moreover, the ongoing clinical trials with a positive outcome include prostate cancer treatment and death cap (Amanita phalloides) poisoning 17. It has been shown to decrease the microglia and astrocyte activation in the brain of Parkinson’s and Alzheimer’s patients 18. Moreover, Silibinin has shown a protective mechanism on heavy metal-induced- neurotoxicity 19, oxaliplatin-induced oxidative stress 20 and diabetes-associated neuronal injury. In the present study was evaluated the potential neuroprotective potential of Silymarin on MPTP induced neurotoxicity using in-vitro model.
MATERIAL AND METHODS:
Experimental Procedure for Neuroprotective Effect of Silibinin by in In-vitro:
Materials: MPTP (Sigma Aldrich) Silibinin (Yarrow Chem Pvt.Ltd) sucrose (Loba Chem Pvt. Ltd.,) Bromocriptine (Para Chem Pvt. Ltd.,).
Methods: Preparation of artificial cerebrospinal fluid (ACSF), pH 7.4 contains sodium chloride (122 mM), potassium chloride (3.1 mM), calcium chloride (1.3 mM), magnesium sulphate (1.2 mM), glucose (10 mM), and glycylglycine (30 mM). Sodium chloride, 122 mM: 1.425 g of NaCl was weighed potassium chloride (3.1 mM): 46.22 mg of KCl was weighed calcium chloride (1.3 mM): 28.85 mg of CaCl2 was weighed magnesium sulfate (1.2 mM): 59.136 of Mg sulfate was weighed glucose (10 mM): 0.36 g of glucose was weighed glycylglycine (30 mM): 0.792 g of glycylglycine was weighed. All the above chemicals (in their specified amounts) were dissolved in 200 ml of distilled water. The solution of salts can be prepared and kept in refrigerator and glucose with glycylglycine can be added later on the day of the experiment.
Methodology: After decapitation, the brain was removed rapidly from the skull and rinsed with cold artificial cerebrospinal fluid (ACSF) which has been equilibrated with 95% O2 / 5% CO2 gas mixture.
Treatment and Evaluation of Selected Bio-markers: In-vitro studies were performed using sagittal slices of male mouse brain of 1 mm thickness. The slices were incubated at 37 °C in ACSF of pH 7.4, in an oxygen-rich atmosphere as described by Mcllwain with or without MPTP (1 nM) for 1 h 21. In one group of experiments, slices were pretreated with Silibinin (10 and 20 µg/mL) for 0.5 h before incubation with MPTP (1 nM) for 1 h). The brain slices were homogenized in PBS buffer, pH 7.4 and estimated for protein content, LPO* and GSH as per the procedures.
Statistical Analysis: All data are expressed as means ± SEM. Statistical differences among the experimental groups were tested by using a one-way analysis of variance (ANOVA) and Dunnet test was employed for multiple comparisons. P-values less than 0.05 were accepted as significant.
Effects of Silibinin on Protein Content, GSH, and LPO on Brain Slices: The effects of Silibinin on biochemical parameters in the sagittal brain slices are tabulated as follows.
TABLE 1: EFFECT OF SILIBININ ON SELECTED BIOMARKERS TESTED ON SAGITTAL BRAIN SLICES
[nmoles TBARS/mg protein]
[µmoles/ mg protein]
|Normal control||15.43 ± 2.3||3.65 ± 0.9||0.036 ± 0.007|
|DMSO control||14.12 ± 1.2||3.21 ± 1.3||0.032 ± 0.004|
|MPTP (1 ng/ml)||4.56 ± 1.3***||13.4 ± 1.7***||0.0098 ± 0.0006**|
|Bromocriptine (10 µg/mL)||8.43 ± 1.8||9.25 ± 1.1||0.027 ± 0.008|
|Silibinin (10 µg/mL)||6.45 ± 1.3||78 ± 1.2**||0.018 ± 0.006|
|Silibinin (20 µg/mL)||8.26 ± 1.3**||7.32 ± 1.4||0.021 ± 0.007**|
All values are expressed in Mean ± SEM. Statistical analysis determined by ANOVA followed by Dunnet’s method of comparison. b denotes treated groups were compared against MPTP group. while the a denotes, MPTP control group was compared against the DMSO control.
There was a considerable decrease in the protein content (4.56 ± 1.2 μg/mg tissue, P<0.001***) and GSH content (0.0098 ± 0.0006) μmoles/mg protein, P<0.001***) while increase in the lipid peroxidation products was observed (123.4 ± 1.7 nmoles TBARS/mg protein, P<0.001***) in the MPTP group. Silibinin at 20 µg/mL showed considerable neuroprotective properties in term of restored GSH levels of 0.021 ± 0.007 μmoles/mg protein (P<0.01**) and decreased LPO levels of 7.32 ± 1.4 nmoles TBARS/mg protein (P<0.01**) and improved protein content 8.26 ± 1.3*. Thus, Silibinin showed better results than bromocriptine in terms of GSH and LPO, while it improved Table 1 and Fig 1, 2.
DISCUSSION AND CONCLUSION: In human and nonhuman primates MPTP produces clinical, biochemical, and neuropathologic changes analogous to those observed in idiopathic Parkinson's disease. The neurotoxic effects of MPTP are thought to be initiated by MPP+, which is a metabolite formed by the monoamine oxidase (MAO) B-mediated oxidation of MPTP 22. MPP+ is selectively taken up by high-affinity dopamine and noradrenaline uptake systems and is subsequently accumulated within mitochondria of dopaminergic neurons. There it disrupts oxidative phosphorylation by inhibiting complex I of the mitochondrial electron transport chain 23.
The interruption of oxidative phosphorylation results in decreased levels of ATP 24, which may lead to partial neuronal depolarization and secondary activation of voltage-dependent NMDA receptors, resulting in excitotoxic neuronal cell death 25. Although excitotoxic neuronal damage has been linked to Ca" influxes, the subsequent crucial steps that lead to cell death remain unknown. Recent evidence has implicated both oxygen free radicals and nitric oxide (NO'). The entry of calcium through NMDA receptor channels into cells stimulates nitric oxide synthase (NOS) activity by binding to calmodulin, a cofactor for NOS. Studies in dissociated cell cultures showed that NOS inhibitors effectively blocked NMDA-induced cell death 26. Furthermore, NO' may react with superoxide (OZ) to generate peroxynitrite 27, which may promote nitration of tyrosine 26 and produce hydroxyl radicals 28.
In the present study, results revealed that Silibinin exhibited significant neuroprotection against MPP+ free radicals due to the neutralization of LPO* free radicals and enhanced GSH activity. Although the mechanism by which Silibinin regulates MPTP induced oxidative stress remains to be determined, there are several possible explanations. Firstly, as a polyphenolic flavonoid, Silibinin has strong free radical-scavenging activity 29. Silibinin reacts with a damaging free radical and forms a flavonoid radical, which has greater stability and then breaks the free radical chain reaction 30.
It is possible that Silibinin prevents oxidative damage directly by scavenging free radicals. The results from the present study confirm, for the first time, that Silibinin could alleviate the neurotoxicity induced by MPTP in-vitro method. The effect of Silibinin may be attributed to the prevention of oxidative damage, measured in terms of the amount of peroxidized lipid and the level of GSH. Therefore, Silibinin is a potential candidate for a further preclinical study aimed at the treatment of neurotoxicity.
ACKNOWLEDGEMENT: Authors thankful to University College of Pharmaceutical Sciences, Acharya Nagarjuna University, for providing the necessary lab facility to carry out research.
CONFLICT OF INTEREST: The authors state no conflict of interest.
- Jessen KR and Mirsky R: Glial cells in the enteric nervous system contain glial fibrillary acidic protein. Nature 1980; 286(5774): 736-37.
- Glia SN: The other brain cells. Discover Magazine 2011.
- Tiwari V, Guan Y, and Raja SN: Modulating the delicate glial-neuronal interactions in neuropathic pain: promises and potential caveats. Neuroscience and Biobehavioral Reviews 2014; 45: 19-27.
- Schousboe A, Sickmann HM, Bak LK, Schousboe I, Jajo FS, Faek SA and Waagepetersen HS: Neuron-glia interactions in glutamatergic neurotransmission: Roles of oxidative and glycolytic adenosine triphosphate as an energy source. Journal of Neuroscience Research 2011; 89(12): 1926-34.
- Liebner S, Czupalla CJ and Wolburg H: Current concepts of blood-brain barrier development. International Journal of Developmental Biology 2011; 55(4-5): 467-76.
- Birgner C, Nordenankar K, Lundblad M, Mendez JA, Smith C, le Grevès M, Galter D, Olson L, Fredriksson A, Trudeau LE and Kullander K: VGLUT2 in dopamine neurons is required for psychostimulant-induced behavioral activation. Proceedings of the National Academy of Sciences 2010; 107(1): 389-94.
- Barreto G, White ER and Ouyang Y: Astrocytes: targets for neuroprotection in stroke. Central Nervous System Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Central Nervous System Agents) 2011; 11(2): 164-73.
- Masmoudi‐Kouki O, Douiri S, Hamdi Y, Kaddour H, Bahdoudi S, Vaudry D, Basille M, Leprince J, Fournier A, Vaudry H and Tonon MC: Pituitary adenylate cyclise ‐activating polypeptide protects astroglial cells against oxidative stress‐induced apoptosis. Journal of Neuro-chemistry 2011; 117(3): 403-11.
- Hu LF, Lu M, Tiong CX, Dawe GS, Hu G and Bian JS: Neuroprotective effects of hydrogen sulfide on Parkinson’s disease rat models. Aging Cell 2010; 9(2): 135-46.
- Sun W, Depping R and Jelkmann W: Interleukin-1β promotes hypoxia-induced apoptosis of glioblastoma cells by inhibiting hypoxia-inducible factor-1 mediated adreno-medullin production. Cell Death & Disease 2014; 5(1): 1020.
- Salewski R and Emrani H: Neural Stem/Progenitor Cells for Spinal Cord Regeneration 2013.
- Bansal N, Gill R and Gupta GD: Silymarin: a flavonolignan with antidepressant activity. International Journal of Pharmaceutical Innovations 2013; 93-8.
- Jin G, Bai D, Yin S, Yang Z, Zou D, Zhang Z, Li X, Sun Y and Zhu Q: Silibinin rescues learning and memory deficits by attenuating microglia activation and preventing neuroinflammatory reactions in SAMP8 mice. Neuroscience Letters 2016; 629: 256-61
- Lee Y, Chun HJ and Lee KM: Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Res 2015; 1627: 233-42.
- Cheng H and Tsai MJ: Use of silymarin and silybin in the treatment of the neural injury. Google Patents 2013.
- Gordon A, Hobbs DA, Bowden DS, Bailey MJ, Mitchell J, Francis AJ and Roberts SK: Effects of Silybum marianum on serum hepatitis C virus RNA, alanine aminotransferase levels and well‐being in patients with chronic hepatitis C. Journal of Gastroenterology and Hepatology 2006; 21(1): 275-80.
- Wang MJ, Lin WW and Chen HL: Silymarin protects dopaminergic neurons against lipopolysaccharide‐induced neurotoxicity by inhibiting microglia activation. European Journal of Neuroscience 2002; 16(11): 2103-12.
- Khazim K, Gorin Y, Cavaglieri RC, Abboud HE and Fanti P: The antioxidant silybin prevents high glucose-induced oxidative stress and podocyte injury in-vitro and in-vivo. American Journal of Physiology-Renal Physiology 2013; 305(5): F691-00.
- Mannelli DCL, Zanardelli M and Failli P: Oxaliplatin-induced neuropathy: Oxidative stress as a pathological mechanism. Protective effect of Silibinin. The Journal of Pain: Official Journal of the American Pain Society 2012; 13(3): 276-84.
- Marrazzo G, Bosco P and La Delia F: Neuroprotective effect of Silibinin in diabetic mice. Neuroscience Letters 2011; 504(3): 252-6
- McIlwain H: Preparing neural tissue for metabolic studies in isolation. In Practical Neurochemistry; Churchill Livingstone: London, 1975: 105-32.
- Tipton KF and Singer TP: Advances in our understanding of the mechanisms of the neurotoxicity of MPTP and related compounds. Jou of Neuroch 1993; 61(4): 1191-06.
- Gluck MR, Krueger MJ, Ramsay RR, Sablin SO, Singer TP and Nicklas WJ: Characterization of the inhibitory mechanism of 1-methyl-4-phenylpyridinium and 4-phenyl pyridine analogs in inner membrane preparations. Journal of Biological Chemistry 1994; 269(5): 3167-74.
- Chan P, DeLanney LE, Irwin I, Langston JW and Di Monte D: Rapid ATP loss caused by 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine in mouse brain. Journal of Neurochemistry 1991; 57(1): 348-51.
- Beal MF: Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses?. Annals of Neurology: Official Journal of the American Neurological Association and the Child Neurology Society 1992; 31(2): 119-30.
- Dawson VL, Dawson TM, London ED, Bredt DS and Snyder SH: Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proceedings of the National Academy of Sciences 1991; 88(14): 6368-71.
- Beckman JS, Ischiropoulos H, Zhu L, van der Woerd M, Smith C, Chen J, Harrison J, Martin JC and Tsai M: Kinetics of superoxide dismutase-and iron-catalyzed nitration of phenolics by peroxynitrite. Archives of Biochemistry and Biophysics 1992; 298(2): 438-45.
- van der Vliet A, O'Neill CA, Halliwell B, Cross CE and Kaur H: Aromatic hydroxylation and nitration of phenyl alanine and tyrosine by peroxynitrite. Febs Letters 1994; 339(1-2): 89-92.
- Trouillas P, Marsal P, Svobodová A, Vostalova J, Gažák R, Hrbacˇ J, Sedmera P, Křen V, Lazzaroni R, Duroux JL and Walterova D: Mechanism of the antioxidant action of silybin and 2, 3-dehydrosilybin flavonolignans: a joint experimental and theoretical study. The Journal of Physical Chemistry A 2008; 112(5): 1054-63.
- Weber KC, Honório KM, Bruni AT and da Silva AB: The use of classification methods for modeling the antioxidant activity of flavonoid compounds. Journal of Molecular Modeling 2006; 12(6): 915-20.
How to cite this article:
Rao GD, Begum SP and Penchalaiah H: In-vitro neuroprotective activity of Silibinin against MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydro pyridine) induced neurotoxicity model. Int J Pharm Sci & Res 2019; 10(5): 2301-05. doi: 10.13040/IJPSR.0975-8232.10(5).2301-05.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
G. D. Rao, S. P. Begum * and H. Penchalaiah
University College of Pharmaceutical Sciences, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India.
21 August 2018
03 November 2018
09 November 2018
01 May 2019