Original Article
495
Authors
W. Zhang, L. Cheng, Y. Hou, M. Si, Y.-P. Zhao, L. Nie
Affiliation
Department of Orthopedics, Qilu Hospital, Shandong University, Jinan, Shandong, China
Key words ▶ cytokines ● ▶ reactive oxygen species ● ▶ antioxidant ● ▶ neuroprotection ●
Abstract
received 04.08.2014 accepted 21.08.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1389950 Published online: September 22, 2014 Drug Res 2015; 65: 495–499 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence L. Nie Department of Orthopedics Qilu Hospital Shandong University No. 107 Wenhua West Road Jinan Shandong 250012 China Tel.: + 86/635/8505 608 Fax: + 86/531/82169 114 [email protected]
▼
Background: Spinal cord injury causes posttraumatic degeneration through series of biochemical events. This study aims to evaluate the possible protective mechanism of Plumbagin against Spinal cord injury induced oxidative stress and inflammation. Plumbagin is a potent antioxidant and shows anti-carcinogenic, antiinflammatory and analgesic activities. However, its exact molecular mechanism of action has yet to be explored. Methods: We tested the effects of Plumbagin on spinal cord injury induced ROS generation and lipid peroxidation content in wistar rats. Additionally, the expression of 2 important transcription factors NF-κB and Nrf-2 was investigated.
Introduction
▼
Spinal cord injury induced post-traumatic degeneration is mediated through multi-factorial secondary injury. Secondary pathogenesis includes dysregulation of ion balance, oxidative damage, excitotoxicity and inflammation [1]. One of the early effects of spinal injury includes deregulation of calcium homeostasis and release of proinflammatory mediators prostraglandins and leukotrienes through cycloxygenase and lipoxygenase pathways [2]. In addition, dysregulation of redox homeostasis and damage to the macromolecules plays as an important mediator in the pathophysiology of secondary spinal cord injury. Oxidative stress and inflammation exuberates adversity of spinal cord injury through interrelated mechanisms [3]. Pharmacologic intervention at manageable events of oxidative stress and inflammatory responses using antioxidants could effectively ameliorate adverse and damaging effects of spinal cord injury. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is an analogue of vitamin K3, is isolated
Results: Plumbagin treatment significantly ameliorated oxidative stress through inhibition of ROS and lipid peroxidation with a concomitant increase in antioxidant status. Western blot analysis revealed enhanced nuclear levels of Nrf-2, while NF-κB expression was suppressed during Plumbagin administration. Enzyme linked immunosorbent assay for pro-inflammatory cytokines (TNF-α, IL-1β) showed a significant downregulation followed by Plumbagin treatment in spinal cord injury rats. Conclusion: Taken together, the data suggests potential and novel role of Plumbagin in cytoprotection by modulating NF-κB and Nrf-2 levels against spinal cord injury.
from the root of Plumbago zeylanica L. Plumbagin shows various protective activities and it is used traditionally in Chinese medicine. Plumbagin mediates anti-proliferative, chemopreventive, anti-metastatic activities and anti-inflammatory effects [4–9]. The chemical structure of Plumbagin is derived from naphthoquinone and suggested to be involved in its anti-cancer activity. The present study was designed to investigate the possible protective role of Plumbagin against oxidative stress and inflammatory responses in spinal cord injury in wistar rats.
Materials and Methods
▼
Animals and treatment schedule
Male Sprague-Dawley rats weighing 180–210 g were used for the present experimental study. Food and water were made available ad libitum. Animals were maintained in the windowless animal quarter with automatic temperature (22 ± 1 °C) and light (12 h light/dark cycle). The humidity ranged from 60–70 %. Animals received
Zhang W et al. Plumbagin Protects Spinal Cord Injury … Drug Res 2015; 65: 495–499
Downloaded by: University of Liverpool. Copyrighted material.
Plumbagin Protects Against Spinal Cord Injuryinduced Oxidative Stress and Inflammation in Wistar Rats through Nrf-2 Upregulation
496 Original Article
Induction of SCI: The spinal cord injury was induced as described by Ersahin et al., [10] and Allen, [11]. After the complete treatment schedule spinal tissue was homogenized using ice-cold Tris-HCl buffer (50 mM, pH 7.4) and used for biochemical assays. Isolation of nuclear and whole cell protein from tissue samples are described under western blot section. Protein estimation was performed according to the method of Lowry et al. [12].
Oxidative stress parameters
Reactive oxygen species generation: The method was followed as described by Hashimoto et al. [13] using 2', 7' – dichlorofluorescein diacetate DCF-DA. ROS levels were measured spectrofluorimetrically at excitation (485 nm) and emission (528 nm) wavelength. Lipid peroxidation: Tissue samples were assayed for products of lipid peroxidation as described by Ohkawa et al. [14]. Absorbance was measured at 532 nm and results expressed as nM TBARS/mg of protein.
Antioxidant status
Glutathione (GSH) content: The GSH content was determined as described by Beutler, [15] with some modifications. The absorbance was measured at 412 nm in a spectrophotometer. GSH content was calculated from the standard curve and the results were expressed as nmoles of GSH/mg of protein. NAD(P)H:Quinone oxidoreductase (NQO1) activity: Endo genous NQO1 enzymatic activity was measured using mena dione as a substrate [16]. The absorbance was measured at 570 nm spectrophometrically. The results are expressed as change in U/mg protein. Catalase (CAT) activity: The catalase (CAT) activity was determined according to the method of Aebi et al. [17]. The measurements were performed spectrophotometrically at 240 nm. Total (Cu–Zn and Mn) superoxide dismutase activity: SOD activity was determined according to the method of Sun et al. [18]. The principle involves inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine/xanthine oxidase system as a superoxide generator. Glutathione peroxdiase (GSH-Px) activity: Glutathione peroxidase (GSH-Px) activity was determined as described by Paglia and Valentine, [19]. The reaction mixture contained reduced nicoti- namide adenine dinucleotide phosphate, reduced glutathione, sodium azide and glutathione reductase. The reaction Zhang W et al. Plumbagin Protects Spinal Cord Injury … Drug Res 2015; 65: 495–499
was initiated by addition of hydrogen peroxide and the change in absorbance at 340 nm.
Glutathione-S-Transferase (GST) activity: The GST activity was determined as described by Habig et al. [20]. The assay involves reaction between 1-chloro-2, 4-dinitro benzene (CDNB) and reduced glutathione which ultimately results in formation of dinitrophenylthioether. The product formed was measured spectrophometrically at 340 nm.
Western blot analysis
Cytosolic and nuclear extract Preparation: For protein expression studies cytosolic and nuclear extracts were isolated as described by Kwon et al., [21] and Kawamori et al. [22] respectively. The extracts were aliquoted and stored at − 80 °C until further analysis. Sample proteins from cytosolic (50 µg) and nuclear extracts (50 µg) were loaded onto 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins separated were transferred to a nitrocellulose membrane (Bio-Rad, Munich, Germany) and blocked with 3 % non-fat milk in Trisbuffered saline (pH 7.4) containing 0.1 % Tween (TBST) for 1 h. The membranes were washed thrice in TBST and incubated at RT with appropriate primary antibody (anti- NF-κB p65 and antiNrf-2, Santa Cruz Biotechnology) for 1 h. The blots were washed with TBST thrice and incubated overnight at 4 ℃ with secondary antibody, horseradish peroxidase-linked anti-rabbit IgG (Santa Cruz Biotechnology). Followed by which the blots were enhanced with chemiluminescence reagent (ECL plus Western-blotting detection system, GE Healthcare). Loading control was maintained fusing β-actin for cytosolic extract and GAPDH for nuclear extracts. The intensity of the bands was calculated with image J software [23].
ELISA: TNF- α and IL-1β levels
The levels of TNF-α (KMC3011) and IL-1β (KMC0011) were detected using an ELISA kit (Invitrogen, Carlsbad, CA, USA). The concentration of serum interleukins was expressed as pg/ml.
Statistical analysis
The data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.
Results
▼
Plumbagin exerts neuroprotection by reducing spinal cord injury induced ROS and Lipid peroxide levels
We investigated whether Plumbagin would ameliorate spinal cord injury-induced ROS levels and associated damage of lipid peroxidation. The results show that spinal cord injury caused a significant increase in ROS and Lipid peroxide content when compared to sham operated animals. Contrarily, intraperitoneal administration of Plumbagin after SCI-induction significantly lowered oxidative stress when compared to SCI-induced rats ▶ Fig. 1, 2). ( ●
Plumbagin attenuates oxidative stress by enhancing antioxidant status
Phase II antioxidant enzymes are battery of critical proteins regulated by Nrf-2/ARE pathway and show protective effects in conditions of oxidative stress [24]. In order to determine the effect of Plumbagin on redox imbalance caused during spinal cord
Downloaded by: University of Liverpool. Copyrighted material.
human care, according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the NIH. The experimental procedures for the present study were approved by Department of Orthopedics, Qilu Hospital, Shandong University, China. The animals were acclimatized to their environment for 10 days. After the process of acclimatization, the rats were randomly divided into 3 groups. Group (I) Sham (n = 6) – underwent sham surgery. Group (II) SCI group – underwent SCI induction and injected with ip saline. Group (III) – SCI-induction and Plumbagin- SCI (day 1) and intraperitoneal injection of Plumbagin (20 mg/kg) was given after 1 h of SCI on day 1 which was followed for 5 consecutive days.
Original Article
497
pared to sham operated rats. However, a significant increase in antioxidant pool was observed in rats treated with Plumbagin ▶ Table 1). followed by spinal cord injury ( ●
Plumbagin modulates nuclear Nrf-2 and NF-κB expressions
Plumbagin reduces spinal cord injury induced proinflammatory cytokines
Spinal cord-injury induced inflammation could up regulate cytokine levels. As Plumbagin showed to suppress NF-κB expression, we further tested if cytokine production in SCI rat model could be ameliorated by Plumbagin treatment. The results showed a significant upregulation of pro-inflammatory cytokines (TNF-α, IL-1β) when compared to sham operated rats. In addition, Plumbagin treatment significantly reduced TNF-α, IL-1β levels when compared to rats with spinal cord injury. This shows that Plumbagin is a potent suppressor of SCI-induced ▶ Fig. 4). inflammation ( ●
Discussion
▼ Fig. 2 Plumbagin inhibits lipid peroxidation levels in SCI rats: The results are expressed as nanomoles of TBA reactants formed/mg of protein. **p
495
Authors
W. Zhang, L. Cheng, Y. Hou, M. Si, Y.-P. Zhao, L. Nie
Affiliation
Department of Orthopedics, Qilu Hospital, Shandong University, Jinan, Shandong, China
Key words ▶ cytokines ● ▶ reactive oxygen species ● ▶ antioxidant ● ▶ neuroprotection ●
Abstract
received 04.08.2014 accepted 21.08.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1389950 Published online: September 22, 2014 Drug Res 2015; 65: 495–499 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence L. Nie Department of Orthopedics Qilu Hospital Shandong University No. 107 Wenhua West Road Jinan Shandong 250012 China Tel.: + 86/635/8505 608 Fax: + 86/531/82169 114 [email protected]
▼
Background: Spinal cord injury causes posttraumatic degeneration through series of biochemical events. This study aims to evaluate the possible protective mechanism of Plumbagin against Spinal cord injury induced oxidative stress and inflammation. Plumbagin is a potent antioxidant and shows anti-carcinogenic, antiinflammatory and analgesic activities. However, its exact molecular mechanism of action has yet to be explored. Methods: We tested the effects of Plumbagin on spinal cord injury induced ROS generation and lipid peroxidation content in wistar rats. Additionally, the expression of 2 important transcription factors NF-κB and Nrf-2 was investigated.
Introduction
▼
Spinal cord injury induced post-traumatic degeneration is mediated through multi-factorial secondary injury. Secondary pathogenesis includes dysregulation of ion balance, oxidative damage, excitotoxicity and inflammation [1]. One of the early effects of spinal injury includes deregulation of calcium homeostasis and release of proinflammatory mediators prostraglandins and leukotrienes through cycloxygenase and lipoxygenase pathways [2]. In addition, dysregulation of redox homeostasis and damage to the macromolecules plays as an important mediator in the pathophysiology of secondary spinal cord injury. Oxidative stress and inflammation exuberates adversity of spinal cord injury through interrelated mechanisms [3]. Pharmacologic intervention at manageable events of oxidative stress and inflammatory responses using antioxidants could effectively ameliorate adverse and damaging effects of spinal cord injury. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is an analogue of vitamin K3, is isolated
Results: Plumbagin treatment significantly ameliorated oxidative stress through inhibition of ROS and lipid peroxidation with a concomitant increase in antioxidant status. Western blot analysis revealed enhanced nuclear levels of Nrf-2, while NF-κB expression was suppressed during Plumbagin administration. Enzyme linked immunosorbent assay for pro-inflammatory cytokines (TNF-α, IL-1β) showed a significant downregulation followed by Plumbagin treatment in spinal cord injury rats. Conclusion: Taken together, the data suggests potential and novel role of Plumbagin in cytoprotection by modulating NF-κB and Nrf-2 levels against spinal cord injury.
from the root of Plumbago zeylanica L. Plumbagin shows various protective activities and it is used traditionally in Chinese medicine. Plumbagin mediates anti-proliferative, chemopreventive, anti-metastatic activities and anti-inflammatory effects [4–9]. The chemical structure of Plumbagin is derived from naphthoquinone and suggested to be involved in its anti-cancer activity. The present study was designed to investigate the possible protective role of Plumbagin against oxidative stress and inflammatory responses in spinal cord injury in wistar rats.
Materials and Methods
▼
Animals and treatment schedule
Male Sprague-Dawley rats weighing 180–210 g were used for the present experimental study. Food and water were made available ad libitum. Animals were maintained in the windowless animal quarter with automatic temperature (22 ± 1 °C) and light (12 h light/dark cycle). The humidity ranged from 60–70 %. Animals received
Zhang W et al. Plumbagin Protects Spinal Cord Injury … Drug Res 2015; 65: 495–499
Downloaded by: University of Liverpool. Copyrighted material.
Plumbagin Protects Against Spinal Cord Injuryinduced Oxidative Stress and Inflammation in Wistar Rats through Nrf-2 Upregulation
496 Original Article
Induction of SCI: The spinal cord injury was induced as described by Ersahin et al., [10] and Allen, [11]. After the complete treatment schedule spinal tissue was homogenized using ice-cold Tris-HCl buffer (50 mM, pH 7.4) and used for biochemical assays. Isolation of nuclear and whole cell protein from tissue samples are described under western blot section. Protein estimation was performed according to the method of Lowry et al. [12].
Oxidative stress parameters
Reactive oxygen species generation: The method was followed as described by Hashimoto et al. [13] using 2', 7' – dichlorofluorescein diacetate DCF-DA. ROS levels were measured spectrofluorimetrically at excitation (485 nm) and emission (528 nm) wavelength. Lipid peroxidation: Tissue samples were assayed for products of lipid peroxidation as described by Ohkawa et al. [14]. Absorbance was measured at 532 nm and results expressed as nM TBARS/mg of protein.
Antioxidant status
Glutathione (GSH) content: The GSH content was determined as described by Beutler, [15] with some modifications. The absorbance was measured at 412 nm in a spectrophotometer. GSH content was calculated from the standard curve and the results were expressed as nmoles of GSH/mg of protein. NAD(P)H:Quinone oxidoreductase (NQO1) activity: Endo genous NQO1 enzymatic activity was measured using mena dione as a substrate [16]. The absorbance was measured at 570 nm spectrophometrically. The results are expressed as change in U/mg protein. Catalase (CAT) activity: The catalase (CAT) activity was determined according to the method of Aebi et al. [17]. The measurements were performed spectrophotometrically at 240 nm. Total (Cu–Zn and Mn) superoxide dismutase activity: SOD activity was determined according to the method of Sun et al. [18]. The principle involves inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine/xanthine oxidase system as a superoxide generator. Glutathione peroxdiase (GSH-Px) activity: Glutathione peroxidase (GSH-Px) activity was determined as described by Paglia and Valentine, [19]. The reaction mixture contained reduced nicoti- namide adenine dinucleotide phosphate, reduced glutathione, sodium azide and glutathione reductase. The reaction Zhang W et al. Plumbagin Protects Spinal Cord Injury … Drug Res 2015; 65: 495–499
was initiated by addition of hydrogen peroxide and the change in absorbance at 340 nm.
Glutathione-S-Transferase (GST) activity: The GST activity was determined as described by Habig et al. [20]. The assay involves reaction between 1-chloro-2, 4-dinitro benzene (CDNB) and reduced glutathione which ultimately results in formation of dinitrophenylthioether. The product formed was measured spectrophometrically at 340 nm.
Western blot analysis
Cytosolic and nuclear extract Preparation: For protein expression studies cytosolic and nuclear extracts were isolated as described by Kwon et al., [21] and Kawamori et al. [22] respectively. The extracts were aliquoted and stored at − 80 °C until further analysis. Sample proteins from cytosolic (50 µg) and nuclear extracts (50 µg) were loaded onto 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins separated were transferred to a nitrocellulose membrane (Bio-Rad, Munich, Germany) and blocked with 3 % non-fat milk in Trisbuffered saline (pH 7.4) containing 0.1 % Tween (TBST) for 1 h. The membranes were washed thrice in TBST and incubated at RT with appropriate primary antibody (anti- NF-κB p65 and antiNrf-2, Santa Cruz Biotechnology) for 1 h. The blots were washed with TBST thrice and incubated overnight at 4 ℃ with secondary antibody, horseradish peroxidase-linked anti-rabbit IgG (Santa Cruz Biotechnology). Followed by which the blots were enhanced with chemiluminescence reagent (ECL plus Western-blotting detection system, GE Healthcare). Loading control was maintained fusing β-actin for cytosolic extract and GAPDH for nuclear extracts. The intensity of the bands was calculated with image J software [23].
ELISA: TNF- α and IL-1β levels
The levels of TNF-α (KMC3011) and IL-1β (KMC0011) were detected using an ELISA kit (Invitrogen, Carlsbad, CA, USA). The concentration of serum interleukins was expressed as pg/ml.
Statistical analysis
The data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.
Results
▼
Plumbagin exerts neuroprotection by reducing spinal cord injury induced ROS and Lipid peroxide levels
We investigated whether Plumbagin would ameliorate spinal cord injury-induced ROS levels and associated damage of lipid peroxidation. The results show that spinal cord injury caused a significant increase in ROS and Lipid peroxide content when compared to sham operated animals. Contrarily, intraperitoneal administration of Plumbagin after SCI-induction significantly lowered oxidative stress when compared to SCI-induced rats ▶ Fig. 1, 2). ( ●
Plumbagin attenuates oxidative stress by enhancing antioxidant status
Phase II antioxidant enzymes are battery of critical proteins regulated by Nrf-2/ARE pathway and show protective effects in conditions of oxidative stress [24]. In order to determine the effect of Plumbagin on redox imbalance caused during spinal cord
Downloaded by: University of Liverpool. Copyrighted material.
human care, according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the NIH. The experimental procedures for the present study were approved by Department of Orthopedics, Qilu Hospital, Shandong University, China. The animals were acclimatized to their environment for 10 days. After the process of acclimatization, the rats were randomly divided into 3 groups. Group (I) Sham (n = 6) – underwent sham surgery. Group (II) SCI group – underwent SCI induction and injected with ip saline. Group (III) – SCI-induction and Plumbagin- SCI (day 1) and intraperitoneal injection of Plumbagin (20 mg/kg) was given after 1 h of SCI on day 1 which was followed for 5 consecutive days.
Original Article
497
pared to sham operated rats. However, a significant increase in antioxidant pool was observed in rats treated with Plumbagin ▶ Table 1). followed by spinal cord injury ( ●
Plumbagin modulates nuclear Nrf-2 and NF-κB expressions
Plumbagin reduces spinal cord injury induced proinflammatory cytokines
Spinal cord-injury induced inflammation could up regulate cytokine levels. As Plumbagin showed to suppress NF-κB expression, we further tested if cytokine production in SCI rat model could be ameliorated by Plumbagin treatment. The results showed a significant upregulation of pro-inflammatory cytokines (TNF-α, IL-1β) when compared to sham operated rats. In addition, Plumbagin treatment significantly reduced TNF-α, IL-1β levels when compared to rats with spinal cord injury. This shows that Plumbagin is a potent suppressor of SCI-induced ▶ Fig. 4). inflammation ( ●
Discussion
▼ Fig. 2 Plumbagin inhibits lipid peroxidation levels in SCI rats: The results are expressed as nanomoles of TBA reactants formed/mg of protein. **p
