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1
Title Page
Title:
Pyrroloquinoline quinone ameliorates oxidative stress and lipid peroxidation in the brain of streptozotocin-induced diabetic mice
Authors:
Corresponding author:
Narendra Kumar*, Anand Kar
Narendra Kumar School of Life Sciences, Devi Ahilya University, Takshashila Campus, Khandwa Road, Indore, Madhya Pradesh, India. E.mail: [email protected] Phone +91 9425481242, Fax +91 731 2360026
Running title:
Effects of PQQ in diabetic mice brain
Acknowledgement:
Council of Scientific and Industrial Research (CSIR), New Delhi,
Key words:
Antioxidants; Diabetes mellitus; PQQ; TBA-RS
Page 2 of 39
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Abstract: Diabetes, characterized by hyperglycemia leads to several complication through generation of reactive oxygen species and initiates tissue damages. Pyrroloquinoline quinone (PQQ) is believed to be a strong antioxidant as it protects cells from oxidative damage. In this study, we elucidated the hitherto unknown potential of PQQ to ameliorate the brain damage caused by diabetes mellitus and associated hyperglycemia-induced oxidative damage. Administration of single dose of streptozotocin (STZ), i.e.150 mg/kg body weight significantly enhanced the brain tissue lipid peroxidation and hydroperoxidation with a decrease in the levels of antioxidants. It also increased the serum glucose, cholesterol and triglycerides levels. However, when STZ treated animals received PQQ (20 mg/kg body weight/d, for 15 days), it significantly decreased the levels of serum glucose, lipid peroxidation products and increased the activities of antioxidants in the diabetic mice brain. These findings suggest that PQQ has the potential to ameliorate STZ-induced oxidative damage in brain, as well as the STZ-induced diabetes. Key Words: Antioxidants; Diabetes Mellitus; PQQ; TBA-RS
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Introduction Diabetes mellitus (DM) is one of the most common health problems in the world. DM most commonly results from defective insulin secretion or resistance to insulin action or both (Gavin et al. 1997). DM is thought to increase oxidative stress in several organs including brain, which in turn is believed to be the result of the increased production of reactive oxygen species (ROS) and decreased antioxidant defense system (Wiernsperger 2003; Ahmed 2005; Annuziata et al. 2005). DM-induced hyperglycemia is believed to generate free radicals which act as factors for pathogenesis and complications of the disorder. DM-induced hyperglycemia increases the risk of central nervous system (CNS) problems and it is strongly associated with degenerative and functional disorders of the CNS (Northam et al. 2009). Gradual damage to CNS is a common observation in chronic patients of DM (Mastrocola et al. 2005; Cardoso et al. 2010; Qi et al. 2012). Further, an association between DM and several brain abnormalities is well known (Northam et al. 2009; Languren et al. 2013). A previous report suggests that oxidative stress is the final common pathway through which risk factors of several diseases including diabetes, exert their deleterious effects (Rains and Jain 2011). In DM, insulin deficiency or resistance and related adverse conditions in the liver and peripheral tissues ultimately cause hyperglycemia, which generate reactive oxygen species (ROS) and results in the development of oxidative stress in the body and consequently leads to neuronal death (Biessels et al. 2002; Biessels and Gispen 2005; Russell et al. 2008; Tomlinson and Gardiner 2008). Furthermore, hyperglycemia reduces the levels of superoxide dismutase (SOD), a key antioxidative enzyme (Stanely et al. 2011). It also increases lipid peroxidation (LPO) and free radicals such as nitric oxide (NO) (Pitocco et al. 2010). In fact, DM-induced
Page 4 of 39
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pathological changes in the CNS very often leads to vascular complications causing cognitive and affective deficits (Magarinos and McEwen 2000; Biessels and Gispen 2005; Jung et al. 2010). Oxidative stress-characterized by increased production of free radicals and impaired antioxidant defenses act as key factors in the development and progression of diabetes and its complications (Ceriello 2003). At high concentrations, ROS can lead to damage to the major components of the cellular structure, including nucleic acids, proteins, amino acids, and lipids (Valko et al. 2007). Such oxidative modification in the diabetic condition affects several cell functions, metabolism, and gene expression; which in turn can develop other pathological conditions (Young and Woodside 2001). An ubiquitous molecule, pyrroloquinoline quinone (PQQ) is reported to be beneficial for growth and stress tolerance (Guo et al. 2009). Recently, it has been demonstrated as a strong antioxidant and as a cofactor of many enzymes (Rajpurohit et al. 2008; Mishra et al. 2012). PQQ has also been shown to play a role as an inducer of cellular redox signaling pathway and is involved in regulating various physiological processes (Wojciechowski et al. 2010; Mishra et al. 2012). Both in vivo and in vitro studies have shown that PQQ can protect against several types of oxidative damages including neurotoxicity (Hamagishi et al. 1990; Smidt et al. 1991; Zhang and Rosenberg 2002; Nunome et al. 2008; Zhang et al. 2009; Kumar and Kar 2013). However, its role in diabetes-induced oxidative damages in brain tissue remains uninvestigated. Streptozotocin (STZ) is widely used in experimental diabetes, because it selectively destroys pancreatic β cells through the generation of ROS (Szkudelski 2001; Coskun et al. 2005; Hayashi et al. 2006; Lenzen 2008). As the current therapeutic strategies for DM are limited
Page 5 of 39
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5
(Srinivasan and Ramarao 2007) and chemically induced DM is very often associated with ROS production, in this investigation ameliorating effects of PQQ has been evaluated in STZ-induced diabetic mice, assuming that a free radical scavenger and antioxidant properties may offer protection from the STZ-induced diabetes and associated pathological problems. Hyperglycemia causes overproduction of NO which reacts with superoxide resulting in the generation of highly reactive peroxynitrite which induces massive tissue damage including neurotoxicity (Haluzik and Nedvidkova 2000). In fact, oxidative stress and related pathways have major contributory role in the development of diabetic complications (Fiorentino et al. 2013). On the other hand, several compounds with antioxidant properties have been found to protect DM-induced oxidative damages (Armagan et al. 2006; Kuhad and Chopra 2008; Aybek et al. 2008; Stanely et al. 2011). In recent years, PQQ has also been reported to exhibit beneficial effects on neurotoxicity (Zhang et al. 2009; 2013). However, as described above, the effects of PQQ on diabetes-induced neural dysfunction had not been investigated so far. Therefore, the primary aim of this study was to test whether administration of PQQ could reduce oxidative stress and improve antioxidant levels in the brain of STZ-induced diabetic mice.
Materials and Methods Animals Swiss albino mice weighing 30 ± 2 g were obtained from institutional animal house. They were housed in polypropylene cages in a standard photoperiod (14 h light:10 h dark) and temperature (27 ± 1°C) controlled room with the provision of laboratory feed (Gold Mohur Feed, Hindustan Lever Limited, Mumbai, India) and water ad libitum. Animals were maintained in
Page 6 of 39
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6
accordance with the guidelines of committee for the purpose of control and supervision of experiments on animals (CPCSEA), Ministry of Social justice and Empowerment, Govt. of India (Reg.No.-779). Chemicals PQQ was purchased from Quality of Life Lab, USA, and STZ was obtained from SigmaAldrich chemicals (St. Louis, MO, USA). Ellman’s reagent, m-phosphoric acid, thio-barbituric acid (TBA), sodium dodecyl sulphate, tri carboxylic acid (TCA) and hydrogen peroxide (H2O2) were obtained from E. Merck Ltd., Mumbai, India. Kits for the determination of lipids, glucose, urea, and creatinine were procured from Transasia Bio-Medicals ltd., Solan, India. All other chemicals were of reagent grade and were obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Induction of diabetes mellitus to experimental mice DM was induced in mice (fasted for 24 hours) by a single intraperitoneal (i.p) injection of STZ at 150 mg/kg dissolved in citrate buffer (0.1 M citrate, pH 4.5) (Li et al. 2013). After 72 h of STZ administration, the tail vein blood was collected to determine fasting blood glucose level. Only mice with fasting blood glucose over 220 mg/dl were considered in the experiments. Experimental design Thirty-five healthy male mice were divided into five groups of seven each. Group I animals received single intraperitoneal injection of citrate buffer (0.1ml, 0.1 M citrate, pH 4.5) and served as control; whereas the group II, III, IV and V received STZ. After 72 h of STZ administration and ensuring hyperglycemia, animals of group III, IV and V were treated
Page 7 of 39
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intraperitoneal with three different doses of PQQ (5, 10 and 20 mg/kg/day, respectively for 15 days). These doses were selected from the previous studies (Zhu et al. 2006; Takada et al. 2012). A supplementary experiment was also done to find out the adverse effects of 20 mg/kg of PQQ, if any, in the normoglycemic aimals, considering all the above indices. Fourteen healthy male mice were divided into two groups of seven each. Group one received single (i.p.) injection of citrate buffer (0.1ml, 0.1 M citrate, pH 4.5) and served as control; whereas group II received single i.p injection of citrate buffer along with 20 mg/ kg PQQ (i.p.) injection for 15 days.
Blood sampling and preparation of tissue homogenate At the end of experimentation, mice were sacrificed under mild ether anesthesia and blood from each animal was collected from the heart. Blood samples were centrifuged at 3000g for 5 min; serum was separated and stored at −20◦C until determination of different biochemical parameters including glucose, cholesterol and triglyceride. Brain tissue homogenates were prepared in ice cold phosphate buffered saline (PBS, 0.1M, pH 7.4) using a homogenizer. The homogenate was centrifuged at 15,000g for 30 min at 4ºC and the supernatant was used for the determination of lipid peroxidation, superoxide dismutase, catalase and glutathione peroxidase activities as well as glutathione content and lipid hydroperoxide.
Thiobarbituric acid reactive substances (TBARS) assay Lipid peroxidation (LPO) level in the tissues was measured by the method of Ohkawa et al. (1979) which is based on the thiobarbituric acid reaction with malondialdehyde (MDA), a product formed due to the peroxidation of membrane lipids. The amount of MDA was measured
Page 8 of 39
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by taking the absorbance at 532 nm (extinction coefficient, E = 1.56x105), using a Shimadzu UV-170 spectrophotometer. LPO was finally expressed as nM MDA formed /h /mg protein.
Lipid Hydroperoxide (LOOH) assay The LOOH activity was calculated according to the method of Griffiths et al. (2000). LOOH present in the sample reacts with ferrous ions and oxidizes it to ferric form under acidic condition. The ferric ion indicator dye xylenol orange when binds with ferric ions produce a blue color complex and the absorbance was measured at 560 nm.
Superoxide dismutase (SOD) assay Activity of SOD was determined following the pyrogallol auto-oxidation inhibition assay method of Marklund and Marklund (1974). The rate of auto-oxidation is calculated from the increase in absorbance at 420 nm. The enzyme activity was expressed as units/mg protein and one unit is defined as the enzyme activity that inhibits auto-oxidation of pyrogallol by 50%.
Catalase (CAT) assay Determination of catalase activity was based on the principle of decomposition of H2O2 which is measured spectrophotometrically from the changes in absorbance at 240 nm (Aebi 1983). The values were expressed as µM of H2O2 decomposed/min/mg protein.
Glutathione (GSH) assay For the assay of tissue GSH content the protocol of Ellman (1959) was followed in which the –SH group of GSH reacts with Ellman's reagent to produce a yellow-colored 2-nitro-5-
Page 9 of 39
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mercaptobenzoic acid and the absorbance was measured at 412 nm. The GSH content is expressed as µM GSH/mg protein.
Glutathion peroxidase (GPx) assay The GPx activity was calculated according to the method of Mohandas et al. (1984) with some modifications. The mixture consisted of 0.4 ml phosphate (PO4) buffer (0.4 M, pH 7.0), 0.2 ml EDTA (0.8 mM), 0.10 ml sodium azide (10 mM), 0.1 ml GSH (4 mM), 0.1 ml H2O2 (30 mM), and 0.1 ml TCA (10% w/v) with a total volume of 1.0 ml. Disappearance of GSH at 420 nm was recorded at room temperature.
Protein, glucose and total cholesterol determinations Protein concentrations were determined by Lowry method (1951), and the fasting serum glucose concentration was measured by glucose oxidase /peroxidase method based on the protocol of Trinder (1969), where 4-amino antipyrine and phenol react with glucose to produce a pink colored quinoneimine dye. The intensity of the color developed is proportional to glucose concentration in the sample. The quantitation of serum total cholesterol was done by spectrophotometry (Allain et al. 1974).
Statistical analysis Data are expressed as mean ± SEM. For the statistical evaluation, analysis of variance and Student ‘t’ test were used using Graph Pad prism 5 (trial version) and Microsoft excel 2003. P value of 0.05 or less is considered to be significant.
Page 10 of 39
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Results Results of the present investigation clearly indicated the therapeutic potential of test drug, PQQ in the regulation of hyperglycemia and related oxidative problems in the brain tissues of mice as evidenced by improvements in serum glucose and other diabetes related indices. STZ administration increased the brain tissue LPO as well as LOOH level significantly (p < 0.001 and p < 0.01 respectively), with a decrease in SOD activity (p < 0.01). It also decreased CAT, GPx and GSH (p < 0.001, p < 0.001 and p < 0.05 respectively). Following the administration of PQQ all the aforesaid adverse effects of STZ were reversed. However, out of the three doses (5, 10 and 20 mg/kg/d) of PQQ, tested 20 mg/kg was found to be the most effective dose, as it not only significantly (p < 0.001) decreased the LPO in tissues (Fig. 1), the percentage decrease was found to be very high (67%). Of course other two doses were also found to be significantly effective. With respect to LOOH level, 20 mg/kg of PQQ decreased the LOOH level maximally with a decrease of 63% (Fig. 2). With respect to SOD activity, again the highest dose, i.e. 20 mg/kg was most effective (p < 0.001) (Fig. 3), as it restored the SOD activity to control levels. Other two doses were also found to be effective. Catalase enzyme activity also significantly increased (p < 0.01) in brain tissue by the administration of 20 mg/kg of PQQ (Fig. 4), as it normalized the CAT activity to control levels. Similarly, GPx activity was significantly increased in brain tissue (p < 0.001) at a dose of 20 mg/kg with a percent increase of 216% (Fig. 5). STZ significantly depleted GSH levels in brain tissue (p < 0.05). Although 10 and 20 mg/kg PQQ increased GSH content significantly (p < 0.05 & p
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1
Title Page
Title:
Pyrroloquinoline quinone ameliorates oxidative stress and lipid peroxidation in the brain of streptozotocin-induced diabetic mice
Authors:
Corresponding author:
Narendra Kumar*, Anand Kar
Narendra Kumar School of Life Sciences, Devi Ahilya University, Takshashila Campus, Khandwa Road, Indore, Madhya Pradesh, India. E.mail: [email protected] Phone +91 9425481242, Fax +91 731 2360026
Running title:
Effects of PQQ in diabetic mice brain
Acknowledgement:
Council of Scientific and Industrial Research (CSIR), New Delhi,
Key words:
Antioxidants; Diabetes mellitus; PQQ; TBA-RS
Page 2 of 39
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2
Abstract: Diabetes, characterized by hyperglycemia leads to several complication through generation of reactive oxygen species and initiates tissue damages. Pyrroloquinoline quinone (PQQ) is believed to be a strong antioxidant as it protects cells from oxidative damage. In this study, we elucidated the hitherto unknown potential of PQQ to ameliorate the brain damage caused by diabetes mellitus and associated hyperglycemia-induced oxidative damage. Administration of single dose of streptozotocin (STZ), i.e.150 mg/kg body weight significantly enhanced the brain tissue lipid peroxidation and hydroperoxidation with a decrease in the levels of antioxidants. It also increased the serum glucose, cholesterol and triglycerides levels. However, when STZ treated animals received PQQ (20 mg/kg body weight/d, for 15 days), it significantly decreased the levels of serum glucose, lipid peroxidation products and increased the activities of antioxidants in the diabetic mice brain. These findings suggest that PQQ has the potential to ameliorate STZ-induced oxidative damage in brain, as well as the STZ-induced diabetes. Key Words: Antioxidants; Diabetes Mellitus; PQQ; TBA-RS
Page 3 of 39
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3
Introduction Diabetes mellitus (DM) is one of the most common health problems in the world. DM most commonly results from defective insulin secretion or resistance to insulin action or both (Gavin et al. 1997). DM is thought to increase oxidative stress in several organs including brain, which in turn is believed to be the result of the increased production of reactive oxygen species (ROS) and decreased antioxidant defense system (Wiernsperger 2003; Ahmed 2005; Annuziata et al. 2005). DM-induced hyperglycemia is believed to generate free radicals which act as factors for pathogenesis and complications of the disorder. DM-induced hyperglycemia increases the risk of central nervous system (CNS) problems and it is strongly associated with degenerative and functional disorders of the CNS (Northam et al. 2009). Gradual damage to CNS is a common observation in chronic patients of DM (Mastrocola et al. 2005; Cardoso et al. 2010; Qi et al. 2012). Further, an association between DM and several brain abnormalities is well known (Northam et al. 2009; Languren et al. 2013). A previous report suggests that oxidative stress is the final common pathway through which risk factors of several diseases including diabetes, exert their deleterious effects (Rains and Jain 2011). In DM, insulin deficiency or resistance and related adverse conditions in the liver and peripheral tissues ultimately cause hyperglycemia, which generate reactive oxygen species (ROS) and results in the development of oxidative stress in the body and consequently leads to neuronal death (Biessels et al. 2002; Biessels and Gispen 2005; Russell et al. 2008; Tomlinson and Gardiner 2008). Furthermore, hyperglycemia reduces the levels of superoxide dismutase (SOD), a key antioxidative enzyme (Stanely et al. 2011). It also increases lipid peroxidation (LPO) and free radicals such as nitric oxide (NO) (Pitocco et al. 2010). In fact, DM-induced
Page 4 of 39
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4
pathological changes in the CNS very often leads to vascular complications causing cognitive and affective deficits (Magarinos and McEwen 2000; Biessels and Gispen 2005; Jung et al. 2010). Oxidative stress-characterized by increased production of free radicals and impaired antioxidant defenses act as key factors in the development and progression of diabetes and its complications (Ceriello 2003). At high concentrations, ROS can lead to damage to the major components of the cellular structure, including nucleic acids, proteins, amino acids, and lipids (Valko et al. 2007). Such oxidative modification in the diabetic condition affects several cell functions, metabolism, and gene expression; which in turn can develop other pathological conditions (Young and Woodside 2001). An ubiquitous molecule, pyrroloquinoline quinone (PQQ) is reported to be beneficial for growth and stress tolerance (Guo et al. 2009). Recently, it has been demonstrated as a strong antioxidant and as a cofactor of many enzymes (Rajpurohit et al. 2008; Mishra et al. 2012). PQQ has also been shown to play a role as an inducer of cellular redox signaling pathway and is involved in regulating various physiological processes (Wojciechowski et al. 2010; Mishra et al. 2012). Both in vivo and in vitro studies have shown that PQQ can protect against several types of oxidative damages including neurotoxicity (Hamagishi et al. 1990; Smidt et al. 1991; Zhang and Rosenberg 2002; Nunome et al. 2008; Zhang et al. 2009; Kumar and Kar 2013). However, its role in diabetes-induced oxidative damages in brain tissue remains uninvestigated. Streptozotocin (STZ) is widely used in experimental diabetes, because it selectively destroys pancreatic β cells through the generation of ROS (Szkudelski 2001; Coskun et al. 2005; Hayashi et al. 2006; Lenzen 2008). As the current therapeutic strategies for DM are limited
Page 5 of 39
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5
(Srinivasan and Ramarao 2007) and chemically induced DM is very often associated with ROS production, in this investigation ameliorating effects of PQQ has been evaluated in STZ-induced diabetic mice, assuming that a free radical scavenger and antioxidant properties may offer protection from the STZ-induced diabetes and associated pathological problems. Hyperglycemia causes overproduction of NO which reacts with superoxide resulting in the generation of highly reactive peroxynitrite which induces massive tissue damage including neurotoxicity (Haluzik and Nedvidkova 2000). In fact, oxidative stress and related pathways have major contributory role in the development of diabetic complications (Fiorentino et al. 2013). On the other hand, several compounds with antioxidant properties have been found to protect DM-induced oxidative damages (Armagan et al. 2006; Kuhad and Chopra 2008; Aybek et al. 2008; Stanely et al. 2011). In recent years, PQQ has also been reported to exhibit beneficial effects on neurotoxicity (Zhang et al. 2009; 2013). However, as described above, the effects of PQQ on diabetes-induced neural dysfunction had not been investigated so far. Therefore, the primary aim of this study was to test whether administration of PQQ could reduce oxidative stress and improve antioxidant levels in the brain of STZ-induced diabetic mice.
Materials and Methods Animals Swiss albino mice weighing 30 ± 2 g were obtained from institutional animal house. They were housed in polypropylene cages in a standard photoperiod (14 h light:10 h dark) and temperature (27 ± 1°C) controlled room with the provision of laboratory feed (Gold Mohur Feed, Hindustan Lever Limited, Mumbai, India) and water ad libitum. Animals were maintained in
Page 6 of 39
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6
accordance with the guidelines of committee for the purpose of control and supervision of experiments on animals (CPCSEA), Ministry of Social justice and Empowerment, Govt. of India (Reg.No.-779). Chemicals PQQ was purchased from Quality of Life Lab, USA, and STZ was obtained from SigmaAldrich chemicals (St. Louis, MO, USA). Ellman’s reagent, m-phosphoric acid, thio-barbituric acid (TBA), sodium dodecyl sulphate, tri carboxylic acid (TCA) and hydrogen peroxide (H2O2) were obtained from E. Merck Ltd., Mumbai, India. Kits for the determination of lipids, glucose, urea, and creatinine were procured from Transasia Bio-Medicals ltd., Solan, India. All other chemicals were of reagent grade and were obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Induction of diabetes mellitus to experimental mice DM was induced in mice (fasted for 24 hours) by a single intraperitoneal (i.p) injection of STZ at 150 mg/kg dissolved in citrate buffer (0.1 M citrate, pH 4.5) (Li et al. 2013). After 72 h of STZ administration, the tail vein blood was collected to determine fasting blood glucose level. Only mice with fasting blood glucose over 220 mg/dl were considered in the experiments. Experimental design Thirty-five healthy male mice were divided into five groups of seven each. Group I animals received single intraperitoneal injection of citrate buffer (0.1ml, 0.1 M citrate, pH 4.5) and served as control; whereas the group II, III, IV and V received STZ. After 72 h of STZ administration and ensuring hyperglycemia, animals of group III, IV and V were treated
Page 7 of 39
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7
intraperitoneal with three different doses of PQQ (5, 10 and 20 mg/kg/day, respectively for 15 days). These doses were selected from the previous studies (Zhu et al. 2006; Takada et al. 2012). A supplementary experiment was also done to find out the adverse effects of 20 mg/kg of PQQ, if any, in the normoglycemic aimals, considering all the above indices. Fourteen healthy male mice were divided into two groups of seven each. Group one received single (i.p.) injection of citrate buffer (0.1ml, 0.1 M citrate, pH 4.5) and served as control; whereas group II received single i.p injection of citrate buffer along with 20 mg/ kg PQQ (i.p.) injection for 15 days.
Blood sampling and preparation of tissue homogenate At the end of experimentation, mice were sacrificed under mild ether anesthesia and blood from each animal was collected from the heart. Blood samples were centrifuged at 3000g for 5 min; serum was separated and stored at −20◦C until determination of different biochemical parameters including glucose, cholesterol and triglyceride. Brain tissue homogenates were prepared in ice cold phosphate buffered saline (PBS, 0.1M, pH 7.4) using a homogenizer. The homogenate was centrifuged at 15,000g for 30 min at 4ºC and the supernatant was used for the determination of lipid peroxidation, superoxide dismutase, catalase and glutathione peroxidase activities as well as glutathione content and lipid hydroperoxide.
Thiobarbituric acid reactive substances (TBARS) assay Lipid peroxidation (LPO) level in the tissues was measured by the method of Ohkawa et al. (1979) which is based on the thiobarbituric acid reaction with malondialdehyde (MDA), a product formed due to the peroxidation of membrane lipids. The amount of MDA was measured
Page 8 of 39
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8
by taking the absorbance at 532 nm (extinction coefficient, E = 1.56x105), using a Shimadzu UV-170 spectrophotometer. LPO was finally expressed as nM MDA formed /h /mg protein.
Lipid Hydroperoxide (LOOH) assay The LOOH activity was calculated according to the method of Griffiths et al. (2000). LOOH present in the sample reacts with ferrous ions and oxidizes it to ferric form under acidic condition. The ferric ion indicator dye xylenol orange when binds with ferric ions produce a blue color complex and the absorbance was measured at 560 nm.
Superoxide dismutase (SOD) assay Activity of SOD was determined following the pyrogallol auto-oxidation inhibition assay method of Marklund and Marklund (1974). The rate of auto-oxidation is calculated from the increase in absorbance at 420 nm. The enzyme activity was expressed as units/mg protein and one unit is defined as the enzyme activity that inhibits auto-oxidation of pyrogallol by 50%.
Catalase (CAT) assay Determination of catalase activity was based on the principle of decomposition of H2O2 which is measured spectrophotometrically from the changes in absorbance at 240 nm (Aebi 1983). The values were expressed as µM of H2O2 decomposed/min/mg protein.
Glutathione (GSH) assay For the assay of tissue GSH content the protocol of Ellman (1959) was followed in which the –SH group of GSH reacts with Ellman's reagent to produce a yellow-colored 2-nitro-5-
Page 9 of 39
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mercaptobenzoic acid and the absorbance was measured at 412 nm. The GSH content is expressed as µM GSH/mg protein.
Glutathion peroxidase (GPx) assay The GPx activity was calculated according to the method of Mohandas et al. (1984) with some modifications. The mixture consisted of 0.4 ml phosphate (PO4) buffer (0.4 M, pH 7.0), 0.2 ml EDTA (0.8 mM), 0.10 ml sodium azide (10 mM), 0.1 ml GSH (4 mM), 0.1 ml H2O2 (30 mM), and 0.1 ml TCA (10% w/v) with a total volume of 1.0 ml. Disappearance of GSH at 420 nm was recorded at room temperature.
Protein, glucose and total cholesterol determinations Protein concentrations were determined by Lowry method (1951), and the fasting serum glucose concentration was measured by glucose oxidase /peroxidase method based on the protocol of Trinder (1969), where 4-amino antipyrine and phenol react with glucose to produce a pink colored quinoneimine dye. The intensity of the color developed is proportional to glucose concentration in the sample. The quantitation of serum total cholesterol was done by spectrophotometry (Allain et al. 1974).
Statistical analysis Data are expressed as mean ± SEM. For the statistical evaluation, analysis of variance and Student ‘t’ test were used using Graph Pad prism 5 (trial version) and Microsoft excel 2003. P value of 0.05 or less is considered to be significant.
Page 10 of 39
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Results Results of the present investigation clearly indicated the therapeutic potential of test drug, PQQ in the regulation of hyperglycemia and related oxidative problems in the brain tissues of mice as evidenced by improvements in serum glucose and other diabetes related indices. STZ administration increased the brain tissue LPO as well as LOOH level significantly (p < 0.001 and p < 0.01 respectively), with a decrease in SOD activity (p < 0.01). It also decreased CAT, GPx and GSH (p < 0.001, p < 0.001 and p < 0.05 respectively). Following the administration of PQQ all the aforesaid adverse effects of STZ were reversed. However, out of the three doses (5, 10 and 20 mg/kg/d) of PQQ, tested 20 mg/kg was found to be the most effective dose, as it not only significantly (p < 0.001) decreased the LPO in tissues (Fig. 1), the percentage decrease was found to be very high (67%). Of course other two doses were also found to be significantly effective. With respect to LOOH level, 20 mg/kg of PQQ decreased the LOOH level maximally with a decrease of 63% (Fig. 2). With respect to SOD activity, again the highest dose, i.e. 20 mg/kg was most effective (p < 0.001) (Fig. 3), as it restored the SOD activity to control levels. Other two doses were also found to be effective. Catalase enzyme activity also significantly increased (p < 0.01) in brain tissue by the administration of 20 mg/kg of PQQ (Fig. 4), as it normalized the CAT activity to control levels. Similarly, GPx activity was significantly increased in brain tissue (p < 0.001) at a dose of 20 mg/kg with a percent increase of 216% (Fig. 5). STZ significantly depleted GSH levels in brain tissue (p < 0.05). Although 10 and 20 mg/kg PQQ increased GSH content significantly (p < 0.05 & p
