Original Paper Pharmacology 2014;94:123–130 DOI: 10.1159/000363062
Received: January 27, 2014 Accepted after revision: April 22, 2014 Published online: September 17, 2014
Effects of L-Carnitine on High Glucose-Induced Oxidative Stress in Retinal Ganglion Cells Yu Cao a Xin Li a Ping Shi a Le-xin Wang b Zhong-guo Sui a a
The Affiliated Hospital of Qingdao University, Qingdao, China; b School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, N.S.W., Australia
Abstract Background: Oxidative stress plays a role in diabetic retinopathy. L-Carnitine is an endogenous mitochondrial membrane compound. Objective: To elucidate the protective effects of L-carnitine on high glucose-induced oxidative stress in retinal ganglion cells (RGCs). Methods: Hoechst 33258 staining was used to estimate cell loss. Mitochondrial function was predicted by mitochondrial membrane potential (ΔΨm) measurement. The expression of apoptosis-related protein was measured by Western blotting. Assays for reactive oxygen species (ROS) accumulation, lipid peroxidation, total antioxidative capacity (T-AOC) and antioxidant defense enzymes were completed to explain the antioxidative capacity of L-carnitine. Results: L-Carnitine (12 h) inhibited high glucose-mediated cell loss and restored mitochondrial function including a reversion of ΔΨm loss and cytochrome c release. Cell apoptosis triggered by high glucose was also inhibited by L-carnitine, characterized by the downregulation of caspase-9, caspase-3 and Bax/Bcl-2. Furthermore, Lcarnitine inhibited high glucose-induced ROS production and lipid peroxidation and promoted endogenous antioxidant defense components including superoxide dismutase,
© 2014 S. Karger AG, Basel 0031–7012/14/0944–0123$39.50/0 E-Mail [email protected] www.karger.com/pha
glutathione peroxidase, catalase and T-AOC in a concentration-dependent manner. Conclusions: L-Carnitine may protect RGCs from high glucose-induced injury through the inhibition of oxidative damage, mitochondrial dysfunction and, ultimately, cell apoptosis. © 2014 S. Karger AG, Basel
Introduction
Diabetes mellitus affects approximately 285 million individuals worldwide [1, 2]. Approximately 21% of patients with diabetes mellitus have retinopathy, a leading cause of blindness, at the first time of diagnosis [3]. The degeneration of retinal ganglion cells (RGCs) is a primary pathology in diabetic retinopathy [4, 5]. It has been suggested that oxidative stress is involved in the pathogenesis of RGC degeneration and apoptosis. Several biochemical changes have been proposed to be essential events that commit a cell to undergo apoptosis, which can be mediated by reactive oxygen species (ROS), caspase activation, loss of mitochondrial membrane potential (ΔΨm) and cytochrome c redistribution [6].
Prof. Zhong-guo Sui The Affiliated Hospital of Qingdao University Qingdao 266003 (China) E-Mail buxie75 @ 163.com Prof. Le-xin Wang School of Biomedical Sciences, Charles Sturt University Wagga Wagga, NSW 2678 (Australia) E-Mail lwang @ csu.edu.au
Downloaded by: University of Tokyo 157.82.153.40 - 5/20/2015 3:05:49 PM
Key Words Oxidative stress · L-Carnitine · Glucose · Diabetic retinopathy · Apoptosis
Carnitine is synthesized in vivo from L-lysine and Lmethionine, mostly in the liver and kidneys [7]. The main functions of L-carnitine [3-hydroxy-4-(trimethylazaniumyl)butanoate] include transferring the long-chain fatty acids through the mitochondrial barrier, the protection of membrane structures and the reduction of lactate production [8]. Lipid metabolism disorder in ischemia and anoxia can lead to accumulation of fatty acyl-CoA, which in turn may lead to membrane structure damage and cell death [9, 10]. In vitro studies have shown that L-carnitine as a free radical scavenger is an effective antioxidant [11, 12]. Whether L-carnitine can effectively protect against damage induced by hyperglycemia in RGCs is unknown. In the present study, the effect of L-carnitine on high glucose-induced apoptosis in RGCs was investigated. Materials and Methods Cell Culture, Treatment, Purification and Identification For cell culture, 25-mm2 culture flasks and plates were incubated with 100 μg/ml polylysine at 37 ° C overnight and washed 3 times with PBS. Briefly, retinas from 1- to 3-day-old Wistar rats were incubated at 37 ° C for 10–15 min in 0.125% trypsinase solution [13, 14]. To yield a suspension of single cells, the tissue was triturated sequentially through a narrow-bore Pasteur pipette in DMEM/F12 solution containing 10% bovine serum albumin. After centrifugation at 1,000 r.p.m. for 5 min, the cells were rewashed in another DMEM/F12 solution, and the cell density was adjusted to 1 × 106/ml [13, 14]. The retinal suspension was incubated in the flasks at 37 ° C in a 5% CO2 incubator for 24 h. Then 5-bromo-2′-deoxyuridine (BrdU) was added to prevent the growth of nonneuronal cells. Cell morphology was observed under an inverted phase contrast microscope every day for 5 days. The coverslips were washed in PBS 3 times and fixed for an additional 4 h in 4% paraformaldehyde. The sections were blocked with 5% (w/v) bovine serum albumin and 0.1% Triton X-100 for 30 min at 37 ° C and washed in PBS 3 times. The slips were incubated with both polyclonal antibodies specific for Thy1.1 (1:100; Abcam) and Map2 (1:200; Abcam) for 12–24 h at 4 ° C. After having been washed in PBS 3 times, the secondary antibodies (TRITC Goat Anti-Mouse and FITC Goat AntiRabbit; Beijing Zhong Shan Golden Bridge Bio-Tech, Beijing, China) were added in a dark room and incubated for 1 h at 37 ° C. The coverslips were washed in PBS 3 times and sealed with buffer glycerol. The images were captured with a Leica fluorescence microscope. The purity of the nerve cells of the retina was determined by dividing the number of double-positive cells by the total number of cells in the corresponding field. The purity was calculated from 5 fields of vision in each experiment.
High-Glucose Stimulation High-glucose conditions are defined as 30 mmol/l glucose. This glucose level is commonly used to induce glucose-related dysfunction while maintaining cell viability. The cultured cells were randomized as follows: 100 μmol/l L-carnitine (LC), 30 mmol/l glucose (Glu), 30 mmol/l glucose + 50 μmol/l L-carnitine [Glu +
124
ΔΨm Measurement The mitochondrial membrane was monitored using the fluorescent dye Rh123, a cell-permeable cationic dye. It was added to cell cultures to obtain a final concentration of 10 μmol/l for 30 min at 37 ° C. The cells were collected by pipetting, washed twice with PBS and then analyzed by flow cytometry and CellQuest software within 1 h. The laser was adjusted to emit at 480 nm, and a 530-nm long-pass filter was used [15].
Hoechst 33258 Staining Cells were seeded in 6-well plates at a density of 5 × 105/ml. Each group was cultured for 24 h. When 80% confluent, cells were treated and cultured for 48 h. They were fixed with 4% paraformaldehyde for 30 min at room temperature and then washed and stained with 0.5 ml Hoechst 33258 at 37 ° C for 30 min. They were observed under a fluorescence microscope equipped with a UV filter. The images were recorded on a computer with a digital camera attached to the microscope. The Hoechst reagent was taken up by the nuclei of the cells, and apoptotic cells exhibited a bright blue fluorescence. Three fields were randomly selected for cell counting. The apoptosis rate was the number of apoptotic cells to the total number of cells in the field.
Pharmacology 2014;94:123–130 DOI: 10.1159/000363062
Preparation of Total Cell Lysates as well as Cytosolic and Mitochondrial Fractions Cells were harvested and centrifuged at 800 g at 4 ° C for 10 min; the cell suspension was then placed into a glass homogenizer and homogenized with 30 strokes using a tight pestle on ice. Homogenates were centrifuged at 800 g at 4 ° C for 10 min to collect the supernatant. The resulting supernatants were further centrifuged at 10,000 g at 4 ° C for 20 min to obtain the cytosol (supernatant) and mitochondrial (deposition) fraction. Protein concentrations were determined using the BCA Protein Assay Kit.
Western Blotting This analysis was performed on 40 mg of protein from each cell lysate. Proteins were electroblotted onto a PVDF membrane after having been fractionated by SDS-PAGE. The membranes were incubated overnight at 4 ° C to probe possible cross-contamination in cytosolic and mitochondrial fractions. The primary antibodies were cytochrome c (1: 600), anti-Bax (1: 400), anti-Bcl-2 (1: 400) and anti-β-actin (1: 700). The secondary antibody (PeroxidaseConjugated AffiniPure Goat Anti-Rabbit IgG; 1:5,000) was diluted in blocking solution and incubated with the membranes, followed by blocking with 5% nonfat dried milk. Excess antibody was washed off with 20 mmol/l TBST [20 mmol/l Tris, 150 mmol/l NaCl (pH 7.5) and 0.1% Tween 20] before incubation in ECL Advance. The bands were scanned and densitometrically analyzed using an automatic image analysis system (Alpha Innotech Corporation, San Leandro, Calif., USA). These quantitative analyses were normalized to β-actin (after stripping).
Assays for ROS Accumulation, Lipid Peroxidation, Total Antioxidative Capacity and Antioxidant Defense Enzymes RGCs were initially plated in triplicate at a density of 5 × 105 cells/well in 6-well plates for 24 h. The cells were then preincubated
Cao /Li /Shi /Wang /Sui
Downloaded by: University of Tokyo 157.82.153.40 - 5/20/2015 3:05:49 PM
LC(L)], 30 mmol/l glucose + 100 μmol/l L-carnitine [Glu + LC(M)], 30 mmol/l glucose + 200 μmol/l L-carnitine [Glu + LC(H)] and 30 mmol/l glucose + 5 mmol/l N-acetyl-L-cysteine [Glu + NAC].
a
b
Map2
Merge
c
d
with or without L-carnitine following incubation with high glucose. To monitor the intracellular accumulation of ROS, the treated cells were incubated with 5 μmol/l DCFDA for 30 min at 37 ° C. The cells were subsequently washed twice with D-Hanks solution. The DCF fluorescence intensity of 100 μl cell suspension was quantified with a fluorometer (GENios, USA) using 485-nm excitation and 535-nm emission filters. The results are given as percentages relative to the oxidative stress of the control cells (set at 100%). All experiments were performed in triplicate. Malonyldialdehyde (MDA), a terminal product of lipid peroxidation, was measured to estimate the content of lipid peroxidation in RGCs. The MDA concentration in cell homogenates was determined with commercial kits purchased from Jiancheng Bioengineering (Nanjing, China) using the thiobarbituric acid method. The assay was based on the conjugation ability of MDA with thiobarbituric acid to form a red product which has maximum absorbance at 532 nm [15]. The RGCs were washed 3 times with ice-cold D-Hanks solution and lysed in the extraction buffer [50 mmol/l Tris-HCl (pH 7.4), 1 mmol/l ethylene glycol-bis(2-aminoethylether)tetraacetic acid, 150 mmol/l NaCl, 1% (v/v) Triton X-100, 1 mmol/l phenylmethylsulfonyl, 10 μg/ml aprotinin, 10 mmol/l EDTA, 1 mmol/l NaF and 1 mmol/l Na3VO4] on ice for 30 min. The cells were scraped from the plates, and the lysates were subjected to 20,000 g centrifugation at 4 ° C for 10 min. The amount of proteins in the cleared lysates was quantified with a bicinchoninic acid assay (Beyotime Biotechnology, Jiangsu, China). After determining the amount of total proteins in the supernatants, the total antioxidative capacity (T-AOC) was detected, and the endogenous antioxidant defense components such as glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) were measured with reagent kits (Jiancheng Bioengineering). Data were defined as the amount of protein that inhibits the rate of nitroblue tetrazolium reduction by 50%. Values were calculated using absorption (520 nm for T-AOC, 412 nm for GPx, 405 nm for CAT and 550 nm for SOD) and are expressed as units per milligram protein. The activities of T-AOC,
Effects of L-Carnitine on High GlucoseInduced Oxidative Stress in RGCs
Color version available online
Thy1.1
GPx, CAT and SOD are expressed as units per milligram protein. The data were calculated and are represented as percentages of control cells. All experiments were performed in triplicate. Statistical Analysis Data are expressed as means ± SD. Statistical comparisons were made using one-way ANOVA. The Student-Newman-Keuls method was used as a post hoc test. p values
Received: January 27, 2014 Accepted after revision: April 22, 2014 Published online: September 17, 2014
Effects of L-Carnitine on High Glucose-Induced Oxidative Stress in Retinal Ganglion Cells Yu Cao a Xin Li a Ping Shi a Le-xin Wang b Zhong-guo Sui a a
The Affiliated Hospital of Qingdao University, Qingdao, China; b School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, N.S.W., Australia
Abstract Background: Oxidative stress plays a role in diabetic retinopathy. L-Carnitine is an endogenous mitochondrial membrane compound. Objective: To elucidate the protective effects of L-carnitine on high glucose-induced oxidative stress in retinal ganglion cells (RGCs). Methods: Hoechst 33258 staining was used to estimate cell loss. Mitochondrial function was predicted by mitochondrial membrane potential (ΔΨm) measurement. The expression of apoptosis-related protein was measured by Western blotting. Assays for reactive oxygen species (ROS) accumulation, lipid peroxidation, total antioxidative capacity (T-AOC) and antioxidant defense enzymes were completed to explain the antioxidative capacity of L-carnitine. Results: L-Carnitine (12 h) inhibited high glucose-mediated cell loss and restored mitochondrial function including a reversion of ΔΨm loss and cytochrome c release. Cell apoptosis triggered by high glucose was also inhibited by L-carnitine, characterized by the downregulation of caspase-9, caspase-3 and Bax/Bcl-2. Furthermore, Lcarnitine inhibited high glucose-induced ROS production and lipid peroxidation and promoted endogenous antioxidant defense components including superoxide dismutase,
© 2014 S. Karger AG, Basel 0031–7012/14/0944–0123$39.50/0 E-Mail [email protected] www.karger.com/pha
glutathione peroxidase, catalase and T-AOC in a concentration-dependent manner. Conclusions: L-Carnitine may protect RGCs from high glucose-induced injury through the inhibition of oxidative damage, mitochondrial dysfunction and, ultimately, cell apoptosis. © 2014 S. Karger AG, Basel
Introduction
Diabetes mellitus affects approximately 285 million individuals worldwide [1, 2]. Approximately 21% of patients with diabetes mellitus have retinopathy, a leading cause of blindness, at the first time of diagnosis [3]. The degeneration of retinal ganglion cells (RGCs) is a primary pathology in diabetic retinopathy [4, 5]. It has been suggested that oxidative stress is involved in the pathogenesis of RGC degeneration and apoptosis. Several biochemical changes have been proposed to be essential events that commit a cell to undergo apoptosis, which can be mediated by reactive oxygen species (ROS), caspase activation, loss of mitochondrial membrane potential (ΔΨm) and cytochrome c redistribution [6].
Prof. Zhong-guo Sui The Affiliated Hospital of Qingdao University Qingdao 266003 (China) E-Mail buxie75 @ 163.com Prof. Le-xin Wang School of Biomedical Sciences, Charles Sturt University Wagga Wagga, NSW 2678 (Australia) E-Mail lwang @ csu.edu.au
Downloaded by: University of Tokyo 157.82.153.40 - 5/20/2015 3:05:49 PM
Key Words Oxidative stress · L-Carnitine · Glucose · Diabetic retinopathy · Apoptosis
Carnitine is synthesized in vivo from L-lysine and Lmethionine, mostly in the liver and kidneys [7]. The main functions of L-carnitine [3-hydroxy-4-(trimethylazaniumyl)butanoate] include transferring the long-chain fatty acids through the mitochondrial barrier, the protection of membrane structures and the reduction of lactate production [8]. Lipid metabolism disorder in ischemia and anoxia can lead to accumulation of fatty acyl-CoA, which in turn may lead to membrane structure damage and cell death [9, 10]. In vitro studies have shown that L-carnitine as a free radical scavenger is an effective antioxidant [11, 12]. Whether L-carnitine can effectively protect against damage induced by hyperglycemia in RGCs is unknown. In the present study, the effect of L-carnitine on high glucose-induced apoptosis in RGCs was investigated. Materials and Methods Cell Culture, Treatment, Purification and Identification For cell culture, 25-mm2 culture flasks and plates were incubated with 100 μg/ml polylysine at 37 ° C overnight and washed 3 times with PBS. Briefly, retinas from 1- to 3-day-old Wistar rats were incubated at 37 ° C for 10–15 min in 0.125% trypsinase solution [13, 14]. To yield a suspension of single cells, the tissue was triturated sequentially through a narrow-bore Pasteur pipette in DMEM/F12 solution containing 10% bovine serum albumin. After centrifugation at 1,000 r.p.m. for 5 min, the cells were rewashed in another DMEM/F12 solution, and the cell density was adjusted to 1 × 106/ml [13, 14]. The retinal suspension was incubated in the flasks at 37 ° C in a 5% CO2 incubator for 24 h. Then 5-bromo-2′-deoxyuridine (BrdU) was added to prevent the growth of nonneuronal cells. Cell morphology was observed under an inverted phase contrast microscope every day for 5 days. The coverslips were washed in PBS 3 times and fixed for an additional 4 h in 4% paraformaldehyde. The sections were blocked with 5% (w/v) bovine serum albumin and 0.1% Triton X-100 for 30 min at 37 ° C and washed in PBS 3 times. The slips were incubated with both polyclonal antibodies specific for Thy1.1 (1:100; Abcam) and Map2 (1:200; Abcam) for 12–24 h at 4 ° C. After having been washed in PBS 3 times, the secondary antibodies (TRITC Goat Anti-Mouse and FITC Goat AntiRabbit; Beijing Zhong Shan Golden Bridge Bio-Tech, Beijing, China) were added in a dark room and incubated for 1 h at 37 ° C. The coverslips were washed in PBS 3 times and sealed with buffer glycerol. The images were captured with a Leica fluorescence microscope. The purity of the nerve cells of the retina was determined by dividing the number of double-positive cells by the total number of cells in the corresponding field. The purity was calculated from 5 fields of vision in each experiment.
High-Glucose Stimulation High-glucose conditions are defined as 30 mmol/l glucose. This glucose level is commonly used to induce glucose-related dysfunction while maintaining cell viability. The cultured cells were randomized as follows: 100 μmol/l L-carnitine (LC), 30 mmol/l glucose (Glu), 30 mmol/l glucose + 50 μmol/l L-carnitine [Glu +
124
ΔΨm Measurement The mitochondrial membrane was monitored using the fluorescent dye Rh123, a cell-permeable cationic dye. It was added to cell cultures to obtain a final concentration of 10 μmol/l for 30 min at 37 ° C. The cells were collected by pipetting, washed twice with PBS and then analyzed by flow cytometry and CellQuest software within 1 h. The laser was adjusted to emit at 480 nm, and a 530-nm long-pass filter was used [15].
Hoechst 33258 Staining Cells were seeded in 6-well plates at a density of 5 × 105/ml. Each group was cultured for 24 h. When 80% confluent, cells were treated and cultured for 48 h. They were fixed with 4% paraformaldehyde for 30 min at room temperature and then washed and stained with 0.5 ml Hoechst 33258 at 37 ° C for 30 min. They were observed under a fluorescence microscope equipped with a UV filter. The images were recorded on a computer with a digital camera attached to the microscope. The Hoechst reagent was taken up by the nuclei of the cells, and apoptotic cells exhibited a bright blue fluorescence. Three fields were randomly selected for cell counting. The apoptosis rate was the number of apoptotic cells to the total number of cells in the field.
Pharmacology 2014;94:123–130 DOI: 10.1159/000363062
Preparation of Total Cell Lysates as well as Cytosolic and Mitochondrial Fractions Cells were harvested and centrifuged at 800 g at 4 ° C for 10 min; the cell suspension was then placed into a glass homogenizer and homogenized with 30 strokes using a tight pestle on ice. Homogenates were centrifuged at 800 g at 4 ° C for 10 min to collect the supernatant. The resulting supernatants were further centrifuged at 10,000 g at 4 ° C for 20 min to obtain the cytosol (supernatant) and mitochondrial (deposition) fraction. Protein concentrations were determined using the BCA Protein Assay Kit.
Western Blotting This analysis was performed on 40 mg of protein from each cell lysate. Proteins were electroblotted onto a PVDF membrane after having been fractionated by SDS-PAGE. The membranes were incubated overnight at 4 ° C to probe possible cross-contamination in cytosolic and mitochondrial fractions. The primary antibodies were cytochrome c (1: 600), anti-Bax (1: 400), anti-Bcl-2 (1: 400) and anti-β-actin (1: 700). The secondary antibody (PeroxidaseConjugated AffiniPure Goat Anti-Rabbit IgG; 1:5,000) was diluted in blocking solution and incubated with the membranes, followed by blocking with 5% nonfat dried milk. Excess antibody was washed off with 20 mmol/l TBST [20 mmol/l Tris, 150 mmol/l NaCl (pH 7.5) and 0.1% Tween 20] before incubation in ECL Advance. The bands were scanned and densitometrically analyzed using an automatic image analysis system (Alpha Innotech Corporation, San Leandro, Calif., USA). These quantitative analyses were normalized to β-actin (after stripping).
Assays for ROS Accumulation, Lipid Peroxidation, Total Antioxidative Capacity and Antioxidant Defense Enzymes RGCs were initially plated in triplicate at a density of 5 × 105 cells/well in 6-well plates for 24 h. The cells were then preincubated
Cao /Li /Shi /Wang /Sui
Downloaded by: University of Tokyo 157.82.153.40 - 5/20/2015 3:05:49 PM
LC(L)], 30 mmol/l glucose + 100 μmol/l L-carnitine [Glu + LC(M)], 30 mmol/l glucose + 200 μmol/l L-carnitine [Glu + LC(H)] and 30 mmol/l glucose + 5 mmol/l N-acetyl-L-cysteine [Glu + NAC].
a
b
Map2
Merge
c
d
with or without L-carnitine following incubation with high glucose. To monitor the intracellular accumulation of ROS, the treated cells were incubated with 5 μmol/l DCFDA for 30 min at 37 ° C. The cells were subsequently washed twice with D-Hanks solution. The DCF fluorescence intensity of 100 μl cell suspension was quantified with a fluorometer (GENios, USA) using 485-nm excitation and 535-nm emission filters. The results are given as percentages relative to the oxidative stress of the control cells (set at 100%). All experiments were performed in triplicate. Malonyldialdehyde (MDA), a terminal product of lipid peroxidation, was measured to estimate the content of lipid peroxidation in RGCs. The MDA concentration in cell homogenates was determined with commercial kits purchased from Jiancheng Bioengineering (Nanjing, China) using the thiobarbituric acid method. The assay was based on the conjugation ability of MDA with thiobarbituric acid to form a red product which has maximum absorbance at 532 nm [15]. The RGCs were washed 3 times with ice-cold D-Hanks solution and lysed in the extraction buffer [50 mmol/l Tris-HCl (pH 7.4), 1 mmol/l ethylene glycol-bis(2-aminoethylether)tetraacetic acid, 150 mmol/l NaCl, 1% (v/v) Triton X-100, 1 mmol/l phenylmethylsulfonyl, 10 μg/ml aprotinin, 10 mmol/l EDTA, 1 mmol/l NaF and 1 mmol/l Na3VO4] on ice for 30 min. The cells were scraped from the plates, and the lysates were subjected to 20,000 g centrifugation at 4 ° C for 10 min. The amount of proteins in the cleared lysates was quantified with a bicinchoninic acid assay (Beyotime Biotechnology, Jiangsu, China). After determining the amount of total proteins in the supernatants, the total antioxidative capacity (T-AOC) was detected, and the endogenous antioxidant defense components such as glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) were measured with reagent kits (Jiancheng Bioengineering). Data were defined as the amount of protein that inhibits the rate of nitroblue tetrazolium reduction by 50%. Values were calculated using absorption (520 nm for T-AOC, 412 nm for GPx, 405 nm for CAT and 550 nm for SOD) and are expressed as units per milligram protein. The activities of T-AOC,
Effects of L-Carnitine on High GlucoseInduced Oxidative Stress in RGCs
Color version available online
Thy1.1
GPx, CAT and SOD are expressed as units per milligram protein. The data were calculated and are represented as percentages of control cells. All experiments were performed in triplicate. Statistical Analysis Data are expressed as means ± SD. Statistical comparisons were made using one-way ANOVA. The Student-Newman-Keuls method was used as a post hoc test. p values
