Metab Brain Dis DOI 10.1007/s11011-015-9652-6
RESEARCH ARTICLE
The neuroprotective effect of berberine in mercury-induced neurotoxicity in rats Ahmed E. Abdel Moneim
Received: 23 October 2014 / Accepted: 13 January 2015 # Springer Science+Business Media New York 2015
Abstract The central nervous system is one of the most vulnerable organs affected by mercury toxicity. Both acute and chronic exposure to mercury is also known to cause a variety of neurological or psychiatric disorders. Here, the neuroprotective effect of berberine (BN; 100 mg/kg bwt) on mercuric chloride (HgCl2; 0.4 mg/kg bwt) induced neurotoxicity and oxidative stress was examined in rats. Adult male albino Wistar rats were injected with HgCl2 for 7 days. HgCl2 treatment induced oxidative stress by increasing lipid peroxidation (LPO) and nitrite/nitrate (nitric oxide; NO) production along with a concomitant decrease in glutathione (GSH) and various antioxidant enzymes, namely superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. Pretreatment of rats with BN inhibited LPO and NO production, whereas it increased GSH content. Activities of antioxidant enzymes were also restored concomitantly when compared to the control rats after BN administration. Berberine also caused decrease in TNF-α level and caspase-3 activity which was higher with HgCl2. Furthermore, treatment with BN inhibited apoptosis, as indicated by the reduction of Bax/Bcl-2 ratio in brain tissue. These data indicated that BN augments antioxidant defense with anti-inflammatory and anti-apoptotic activities against HgCl2-induced neurotoxicity and provides evidence that it has a therapeutic potential as neuroprotective agent.
Keywords Berberine . Mercuric chloride . Neurotoxicity . Neuroprotection . Rats
A. E. Abdel Moneim (*) Zoology and Entomology Department, Faculty of Science, Helwan University, Cairo, Egypt e-mail: [email protected]
Introduction Mercury (Hg) naturally occurs in several physical and chemical forms, all of which can produce toxic effects even in low doses. It is a highly reactive and toxic transition element (Sharma et al. 2014). Organic mercury compounds, such as methylmercury (MeHg), have been extensively studied because they are able to reach high levels in the central nervous system (CNS), leading to neurotoxicity (Farina et al. 2011). In contrast to MeHg, much less is known about the toxic effect of other forms of Hg like inorganic mercury, HgCl2 (Xu et al. 2012). Notwithstanding that mercury neurotoxicity has been well reported in both humans and mammalian models. Inorganic mercury [Hg(II)] cannot normally penetrate the blood brain barrier (BBB). However, it still has neurotoxicity in the brain, and it is trapped inside the brain without the ability to way back (Sharma et al. 2014). The key process of Hg(II) to penetrate BBB is bacterial methylation. Evidence suggests that Hg(II) is a substrate for mercuric reductase (MerA), a novel Hg resistance gene in intestinal microbiota, and will be rapidly reduced to elemental Hg(0) which is very lipid soluble and can diffuse through the gut epithelium membrane back into circulation (Bridges and Zalups 2010). The mechanism by which Hg inducing neurological damage is still unclear, its ability to react with and deplete sulfhydryl groups as well as to disrupt cell cycle progression and/or induce apoptosis in several tissues is well recognized (Sutton and Tchounwou 2006). Moreover, Hg-induced neurotoxicity is known to be mediated by reactive oxygen species (ROS) in different models (Mieiro et al. 2011) by alternating Na+/K+ ATPase activity and mitochondrial function. Disruption of the glutathione system by Hg leading to the depletion of glutathione content, and the inhibition of glutathione reductase (GR) and glutathione peroxidase (GPx) activities. This is frequently
Metab Brain Dis
suggested to be an expression of Hg neurotoxicity (Mieiro et al. 2011; Roos et al. 2009). Brain has a high rate of oxidative metabolism, consuming ~20 % of the cardiac output. At the same time, the brain compared to lung, liver and other organs, contains relatively low levels of enzymatic and non-enzymatic antioxidants and high amounts of unsaturated lipids, rendering it more vulnerable to oxidative stress compared to other tissues (Abdel Moneim 2012). Increasing evidences suggested that, excessive production of free radicals in brain and the imbalance between oxidative species and antioxidant defenses are related to the pathogenesis of neurodegenerative diseases. Berberine (BN) is one of protoberberine isoquinoline alkaloid extracted from the roots and barks of many plants of the Berberis species (Zhang et al. 2011). BN has long been used as a traditional remedy as it has many medicinal properties that have attracted the attention of researchers over the last decade. These properties include antibacterial, antiviral and antifungal activities (Siow et al. 2011). Moreover, it possesses anti-inflammatory, antioxidant, hepato- and reno-protective activities (Othman et al. 2014; Singh and Mahajan 2013). Due to its high blood brain barrier permeability (Wang et al. 2005), the beneficial neuroprotective effects of BN in Alzheimer’s disease and diabetic neuropathy have been demonstrated in animal studies (Hsu et al. 2013; Ji and Shen 2012). In brain, BN could down-regulate the caspase-3 and nuclear factor-κB (NF-κB) to suppress the proapoptosis signal and can also stimulate the expression of Bcl-2 (Chai et al. 2014; Simoes Pires et al. 2014). Therefore, the aim of the present study was to evaluate the neurotoxic effect of mercuric chloride in rats based on oxidative stress, inflammation and apoptosis induction as well as to verify the possible mitigating effect of berberine against acute Hg neurotoxicity.
Materials and methods Chemicals
rats per group) and housed in wire bottomed cages in a room under standard conditions of illumination with a 12-h light– dark cycle at 25±1 °C. They were provided with water and a balanced diet ad libitum. The experiment has followed the European Community Directive (86/609/EEC) and national rules on animal care that was carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals 8th edition. Experimental protocol Rats were randomized and divided into four groups (seven rats each); Group 1 served as control and received distilled water (300 μl/rat) by oral administration. Group 2 (Hg group) received oral administration of 300 μl of 0.4 mg/kg bwt HgCl2 according to Glaser et al. (2010) for 7 days. Group 3 (BN group) received 300 μl BN by gavage (orally) once daily for 7 days at a dose of 100 mg/kg (Bhutada et al. 2011), and the animals of group 4 received 300 μl BN by gavage once daily for 7 days at a dose of 100 mg/kg bwt. An hour after the treatment with BN, group 4 was orally administrated 300 μl of HgCl2 (0.4 mg/kg bwt) for 7 days and the rats of this group were classified as the BN-Hg group. After 24 h of last administration, the animals were euthanized under mild ether anesthesia. The animals of all groups were sacrificed by fast decapitation; blood samples were collected, and set aside to stand for half an hour and then centrifuged at 3000 rpm for 15 min at 4 °C to separate serum which was stored at −20 °C for the different biochemical measurements. Brains of rats were carefully removed, and washed twice in ice-cold 50 mM Tris–HCl, pH 7.4. After then, each brain was weighed and homogenized immediately to give 10 % (w/v) homogenate in ice-cold medium that contained 50 mM Tris– HCl, pH 7.4. The homogenates were centrifuged at 3000 rpm for 10 min at 4 °C. The supernatants were used for the various biochemical determinations. The total protein content of the homogenized brain was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
Berberine chloride hydrate (CAS Number: 141433-60-5), Mercury(II) chloride (CAS Number: 7487-94-7), nitro blue tetrazolium, N-(1–naphthyl) ethylenediamine and Tris–HCl were purchased from Sigma (St. Louis, MO, USA). Thiobarbituric acid and trichloroacetic acid were purchased from Merck. All other chemicals and reagents used in this study were of analytical grade. Double-distilled water was used as the solvent.
Oxidative stress markers
Experimental animals
Enzymatic antioxidant status
Twenty eight Wistar adult albino rats weighing 150–180 g were obtained from The Holding Company for Biological Products and Vaccines (VACSERA). After an acclimatization period of 1 week, the animals were divided into four groups (7
Homogenates of brain were used for the determination of superoxide dismutase activity (SOD) according to Nishikimi et al. (1972), catalase activity (CAT) as described by Aebi (1984), glutathione peroxidase activity (GPx) according to
Serum and homogenates of brain were used to determine lipid peroxidation (LPO) by reaction of thiobarbituric acid (Ohkawa et al. 1979). Similarly, nitrite/nitrate (nitric oxide; NO) and glutathione (GSH) were assayed by the methods of Green et al. (1982) and Ellman (1959), respectively.
Metab Brain Dis Fig. 1 Effects of berberine on lipid peroxidation formation in brain (a) and serum (b) of rats treated with mercuric chloride for 7 days, data expressed as malondialdehyde formed. Values are means±SEM. a Significant change at p
RESEARCH ARTICLE
The neuroprotective effect of berberine in mercury-induced neurotoxicity in rats Ahmed E. Abdel Moneim
Received: 23 October 2014 / Accepted: 13 January 2015 # Springer Science+Business Media New York 2015
Abstract The central nervous system is one of the most vulnerable organs affected by mercury toxicity. Both acute and chronic exposure to mercury is also known to cause a variety of neurological or psychiatric disorders. Here, the neuroprotective effect of berberine (BN; 100 mg/kg bwt) on mercuric chloride (HgCl2; 0.4 mg/kg bwt) induced neurotoxicity and oxidative stress was examined in rats. Adult male albino Wistar rats were injected with HgCl2 for 7 days. HgCl2 treatment induced oxidative stress by increasing lipid peroxidation (LPO) and nitrite/nitrate (nitric oxide; NO) production along with a concomitant decrease in glutathione (GSH) and various antioxidant enzymes, namely superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. Pretreatment of rats with BN inhibited LPO and NO production, whereas it increased GSH content. Activities of antioxidant enzymes were also restored concomitantly when compared to the control rats after BN administration. Berberine also caused decrease in TNF-α level and caspase-3 activity which was higher with HgCl2. Furthermore, treatment with BN inhibited apoptosis, as indicated by the reduction of Bax/Bcl-2 ratio in brain tissue. These data indicated that BN augments antioxidant defense with anti-inflammatory and anti-apoptotic activities against HgCl2-induced neurotoxicity and provides evidence that it has a therapeutic potential as neuroprotective agent.
Keywords Berberine . Mercuric chloride . Neurotoxicity . Neuroprotection . Rats
A. E. Abdel Moneim (*) Zoology and Entomology Department, Faculty of Science, Helwan University, Cairo, Egypt e-mail: [email protected]
Introduction Mercury (Hg) naturally occurs in several physical and chemical forms, all of which can produce toxic effects even in low doses. It is a highly reactive and toxic transition element (Sharma et al. 2014). Organic mercury compounds, such as methylmercury (MeHg), have been extensively studied because they are able to reach high levels in the central nervous system (CNS), leading to neurotoxicity (Farina et al. 2011). In contrast to MeHg, much less is known about the toxic effect of other forms of Hg like inorganic mercury, HgCl2 (Xu et al. 2012). Notwithstanding that mercury neurotoxicity has been well reported in both humans and mammalian models. Inorganic mercury [Hg(II)] cannot normally penetrate the blood brain barrier (BBB). However, it still has neurotoxicity in the brain, and it is trapped inside the brain without the ability to way back (Sharma et al. 2014). The key process of Hg(II) to penetrate BBB is bacterial methylation. Evidence suggests that Hg(II) is a substrate for mercuric reductase (MerA), a novel Hg resistance gene in intestinal microbiota, and will be rapidly reduced to elemental Hg(0) which is very lipid soluble and can diffuse through the gut epithelium membrane back into circulation (Bridges and Zalups 2010). The mechanism by which Hg inducing neurological damage is still unclear, its ability to react with and deplete sulfhydryl groups as well as to disrupt cell cycle progression and/or induce apoptosis in several tissues is well recognized (Sutton and Tchounwou 2006). Moreover, Hg-induced neurotoxicity is known to be mediated by reactive oxygen species (ROS) in different models (Mieiro et al. 2011) by alternating Na+/K+ ATPase activity and mitochondrial function. Disruption of the glutathione system by Hg leading to the depletion of glutathione content, and the inhibition of glutathione reductase (GR) and glutathione peroxidase (GPx) activities. This is frequently
Metab Brain Dis
suggested to be an expression of Hg neurotoxicity (Mieiro et al. 2011; Roos et al. 2009). Brain has a high rate of oxidative metabolism, consuming ~20 % of the cardiac output. At the same time, the brain compared to lung, liver and other organs, contains relatively low levels of enzymatic and non-enzymatic antioxidants and high amounts of unsaturated lipids, rendering it more vulnerable to oxidative stress compared to other tissues (Abdel Moneim 2012). Increasing evidences suggested that, excessive production of free radicals in brain and the imbalance between oxidative species and antioxidant defenses are related to the pathogenesis of neurodegenerative diseases. Berberine (BN) is one of protoberberine isoquinoline alkaloid extracted from the roots and barks of many plants of the Berberis species (Zhang et al. 2011). BN has long been used as a traditional remedy as it has many medicinal properties that have attracted the attention of researchers over the last decade. These properties include antibacterial, antiviral and antifungal activities (Siow et al. 2011). Moreover, it possesses anti-inflammatory, antioxidant, hepato- and reno-protective activities (Othman et al. 2014; Singh and Mahajan 2013). Due to its high blood brain barrier permeability (Wang et al. 2005), the beneficial neuroprotective effects of BN in Alzheimer’s disease and diabetic neuropathy have been demonstrated in animal studies (Hsu et al. 2013; Ji and Shen 2012). In brain, BN could down-regulate the caspase-3 and nuclear factor-κB (NF-κB) to suppress the proapoptosis signal and can also stimulate the expression of Bcl-2 (Chai et al. 2014; Simoes Pires et al. 2014). Therefore, the aim of the present study was to evaluate the neurotoxic effect of mercuric chloride in rats based on oxidative stress, inflammation and apoptosis induction as well as to verify the possible mitigating effect of berberine against acute Hg neurotoxicity.
Materials and methods Chemicals
rats per group) and housed in wire bottomed cages in a room under standard conditions of illumination with a 12-h light– dark cycle at 25±1 °C. They were provided with water and a balanced diet ad libitum. The experiment has followed the European Community Directive (86/609/EEC) and national rules on animal care that was carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals 8th edition. Experimental protocol Rats were randomized and divided into four groups (seven rats each); Group 1 served as control and received distilled water (300 μl/rat) by oral administration. Group 2 (Hg group) received oral administration of 300 μl of 0.4 mg/kg bwt HgCl2 according to Glaser et al. (2010) for 7 days. Group 3 (BN group) received 300 μl BN by gavage (orally) once daily for 7 days at a dose of 100 mg/kg (Bhutada et al. 2011), and the animals of group 4 received 300 μl BN by gavage once daily for 7 days at a dose of 100 mg/kg bwt. An hour after the treatment with BN, group 4 was orally administrated 300 μl of HgCl2 (0.4 mg/kg bwt) for 7 days and the rats of this group were classified as the BN-Hg group. After 24 h of last administration, the animals were euthanized under mild ether anesthesia. The animals of all groups were sacrificed by fast decapitation; blood samples were collected, and set aside to stand for half an hour and then centrifuged at 3000 rpm for 15 min at 4 °C to separate serum which was stored at −20 °C for the different biochemical measurements. Brains of rats were carefully removed, and washed twice in ice-cold 50 mM Tris–HCl, pH 7.4. After then, each brain was weighed and homogenized immediately to give 10 % (w/v) homogenate in ice-cold medium that contained 50 mM Tris– HCl, pH 7.4. The homogenates were centrifuged at 3000 rpm for 10 min at 4 °C. The supernatants were used for the various biochemical determinations. The total protein content of the homogenized brain was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
Berberine chloride hydrate (CAS Number: 141433-60-5), Mercury(II) chloride (CAS Number: 7487-94-7), nitro blue tetrazolium, N-(1–naphthyl) ethylenediamine and Tris–HCl were purchased from Sigma (St. Louis, MO, USA). Thiobarbituric acid and trichloroacetic acid were purchased from Merck. All other chemicals and reagents used in this study were of analytical grade. Double-distilled water was used as the solvent.
Oxidative stress markers
Experimental animals
Enzymatic antioxidant status
Twenty eight Wistar adult albino rats weighing 150–180 g were obtained from The Holding Company for Biological Products and Vaccines (VACSERA). After an acclimatization period of 1 week, the animals were divided into four groups (7
Homogenates of brain were used for the determination of superoxide dismutase activity (SOD) according to Nishikimi et al. (1972), catalase activity (CAT) as described by Aebi (1984), glutathione peroxidase activity (GPx) according to
Serum and homogenates of brain were used to determine lipid peroxidation (LPO) by reaction of thiobarbituric acid (Ohkawa et al. 1979). Similarly, nitrite/nitrate (nitric oxide; NO) and glutathione (GSH) were assayed by the methods of Green et al. (1982) and Ellman (1959), respectively.
Metab Brain Dis Fig. 1 Effects of berberine on lipid peroxidation formation in brain (a) and serum (b) of rats treated with mercuric chloride for 7 days, data expressed as malondialdehyde formed. Values are means±SEM. a Significant change at p
