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Protective effects of the aqueous extract of Scutellaria baicalensis against acrolein-induced oxidative stress in cultured human umbilical vein endothelial cells

Protective effects of the aqueous extract of Scutellaria baicalensis against acrolein-induced... PHB Context: Scutellaria baicalensis Georgi (Labiatae) (SbG), one of the fifty fundamental herbs of Chinese herbology, has been reported to have anti-asthmatic, antifungal, antioxidative, and anti-inflammatory activities. Objective: This study was designed to determine the protective effects of the extract of SbG against the acrolein- induced oxidative stress in cultured human umbilical vein endothelial cells (HUVEC). Materials and methods: The MTT reduction assay was employed to determine cell viability. The total cellular glutathione (GSH) level was detected using a colorimetric GSH assay kit. Cellular GSH production was conducted by detecting the mRNA expression levels of γ-glutamylcysteine ligase catalytic subunit and modifier subunit. Results: Concentration-dependent cytotoxic effects of acrolein were observed while SbG could effectively protect the acrolein-induced oxidative damage. The protective mechanism was investigated, showing that the increased GSH content in the SbG-incubated HUVE cells was associated with the protective effects of SbG-treated cells. Further RT-PCR data confirmed the elevated mRNA expressions of GSH synthesis enzymes. Discussion and conclusion: The current study strongly indicated that SbG could be a potential antioxidant against oxidative stress in treating cardiovascular diseases. Keywords: Scutellaria baicalensis Georgi, cytotoxicity, glutathione, endothelial cell. Introduction the neuroprotective ee ff cts on ROS-induced cytotoxicity in neuronal HT-22 cells, neuroblastoma SH-SY5Y cells and Acrolein is a highly reactive unsaturated aldehydic PC12 cells (Choi et al., 2002; Gao et al., 2001; Shang et al., product of lipid peroxidation that could implicate vari- 2006; Wang et al., 2009). SbG contains numerous a fl vone ous degenerative pathogenesis, oxidative DNA damage derivatives and the active components were studied (Ding and mutagenesis and is widely used as a positive con- et al., 2009; Jia et al., 2007; Yu et al., 2007). It is worthwhile trol of reactive oxygen species (ROS) experiments (Jia to investigate whether the SbG also had protective effects et al., 2007; LoPachin et al., 2009; Luo & Shi, 2005; Pan on endothelial cells, which is of significant clinical mean- et al., 2009; Roy et al., 2009; Wang et al., 2009; Zitting & ing in treating endothelial cell related diseases. Heinonen, 1980). However, protection against acrolein toxicity in endothelial cells by using the aqueous extract of Scutellaria baicalensis Georgi (Labiatae) (SbG) has Materials and methods not been investigated, though SbG has been reported to have anti-asthmatic, anti-fungal, antioxidative, and anti- Materials inflammatory activities (Huang et al., 2006; Koda et al., Human umbilical vein endothelial (HUVE) cells were 1972; Wong & Tsang, 2009). Past studies of SbG showed purchased from BD Biosciences (San Jose, CA). The MTT Address for Correspondence: Yue-liang Shen, Department of Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058, P.R. China. Tel./Fax: +86 571 88208250; E-mail: [email protected] (Received 01 March 2010; revised 00 00 0000; accepted 14 June 2010) 256 Scutellaria protection against oxidative stress 257 kit was purchased from Invitrogen (Calscard, CA). Glu- GST activity measurement tathione (GSH) and glutathione S-transferase (GST) kits Cellular GST activity was measured by using a GST were from BioVision (Mountain View, CA) and all the fluorometric assay kit (BioVision). Briefly, appropriate chemicals were purchased from Sigma (St. Louis, MO). samples were prepared in a total of 100 µL volume with GST sample buffer from the kit, including the negative Cell culture and preparation of aqueous extract of SbG control of 100 µL of GST sample buffer alone and a posi- The human umbilical vein endothelial cells were grown tive control of 2 µL of GST and 98 µL of sample buffer. All to confluence in CS-C medium (Sigma) supplemented the samples were made in three dilutions and loaded with 5% fetal bovine serum (FBS) and 1% penicillin/ into a 96-well plate in triplicates for each experiment. Af- streptomycin at 37 °C in a humidified atmosphere of 5% ter adding GSH and substrate mix, the plate was read at CO . The culture media was changed every week. SbG Ex/Em 380/460 nm at 30 min time point to compare the was purchased from Hangzhou Chinese Pharmaceuti- fluorescence level of treated versus control samples. cal (Zhejiang, China) and extracted as reported (Wong & Tsang, 2009; Yoon et al., 2009). Briefly, the extractions RT-PCR of GCLC and GCLM were started with 50 g of material in 1000 mL of distilled Molecular level of cellular GSH production was con- water, boiled for 2 h, filtered through 0.22 µm syringe fil- ducted by detecting the mRNA expression levels of ter, and then lyophilized with a yield of 28.86% (w/w). γ-glutamylcysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM), since the synthesis of GSH re- quires two ATP-dependent enzymes, γ-glutamylcysteine Acrolein exposure and SbG supplementation ligase (GCL) and GSH synthase. GCL is the rate-limiting When HUVE cells were confluent in the 96-well plate enzyme in the overall GSH synthesis process which and 8-well cell culture chamber (Fisher scientific, USA), is composed of a catalytic (GCLC) and modulatory acrolein was dissolved in 1× PBS and loaded onto the (GCLM) subunit. GCLC has the catalytic activity while cells for 24 h. The protective effects of SbG were studied GCLM could enhance the catalytic activity. Total RNA with the acute toxicity experiment by pretreating the cells was extracted using TRIzol reagent (Life Technolo- with SbG for 24 h. gies, USA) following the manufacturer’s protocol. RNA was quantitated and 5 μg of the total RNA isolated was Cell viability assay reverse transcribed to cDNA at 37°C for 1 h in a 30 μL MTT reduction assay was employed to determine cell reaction mix containing 200 U of MMLV Reverse Tran- viability. Briefly, the cells were plated into the 96-well scriptase (Promega) as per the manufacturer’s pro- plate. For the SbG protection groups, the appropri- tocol. All the PCR amplifications were done in a 50 μL ate wells in triplicates were loaded with SbG for 24 h. reaction mix containing 2.5 U Taq DNA Polymerase Then the cells were incubated with acrolein in DMEM (Promega, USA) in 1× reaction buffer, 1.5 mM MgCl , supplemented with 0.5% FBS at 37 °C in a humidi- 150 μM dNTP mix, 5 to 25 pmol of forward and reverse fied atmosphere of 5% CO for 24 h, after which time primers (Genosys, Sigma) and 3 μL of template cDNA. the media were discarded, followed by addition to The primers used in quantitative real-time PCR were each well of 0.1 mL of fresh medium containing MTT GCLC (forward, 5′-GGCGATGAGGTGGAATAC-3′; (0.2 mg/mL). The plate was incubated for another 4 h at reverse, 5′-AAAGGGTAGGATGGTTTGG-3′), GCLM 37 °C. After solubilizing the formazon crystals by add- (forward, 5′-ATCAAACTCTTCATCATCAAC-3′; re- ing DMSO, optical densities were detected at 570 nm verse, 5′-GATTAACTCCATCTTCAATAGG-3′) and using a microplate spectrophotometer (Bio-Tek Instru- glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ments, USA). forward, 5′-ACAGTCAGCCGCATCTTC-3′; reverse, 5′- GCCCAATACGACCAAATCC-3′). Real-time reactions Glutathione level measurement were conducted on an Applied Biosystems 5700 using The total cellular GSH level was detected by using a colo- a hot start, followed by 40 cycles at 94°C for 15 s, 56°C rimetric GSH assay kit (BioVision). This kit makes use of a for 30 s, 72°C for 30 s, and a final extension step at 72°C kinetic enzymatic recycling assay based on the oxidation for 3 min. Analysis of the relative gene expression data of GSH by 5, 5′-dithiobis-2-nitrobenzoic acid (DTNB) and was performed using the △C method. For each primer glutathione reductase to measure the total GSH content pair, a plot of the log cDNA dilution versus △C was in cells. Addition of NADPH2 initiates the progressive re- generated to validate the quantitative PCR results. The duction of DTNB by GSH, causing a color increase that is mean quantities of GCLC and GCLM transcripts were monitored at 405 nm. The rate of color change, typically normalized based on the mean quantity of the control monitored over a 5 min period, is proportional to the total gene (GAPDH). GSH concentration. This assay is specific for GSH; other thiols do not cause interference in the assay. Cellular Statistical analysis GSH content was calculated by using the standard GSH All data are expressed as mean ± SEM from three inde- from the kit and expressed as nanomoles of GSH per mg pendent experiments. Data were analyzed with SPSS. of cellular protein. Significant differences were examined using analysis © 2011 Informa Healthcare USA, Inc. 258 Xing-Wei Zhang et al. of variance (ANOVA) and Student’s t-test was used to viability when treated with acrolein at 20 µM; Interest- determine the difference between the two groups and ingly, SbG at 100 µg/mL had better protection than SbG the paired t-test for within-group differences. A value of at 50 µg/mL when acrolein concentrations were 40 and P <0.05 was considered statistically significant. 60 µM, respectively (P <0.01 and P <0.05, respectively, Figure 2). Results Eec ff ts of SbG on cellular GSH level in HUVE cells Eec ff ts of SbG on cell viability When HUVE cells were incubated with SbG at 50 and Figure 1 shows the cell viability of the HUVE cells when 100 µg/ mL for 24 h, we found that the cellular GSH contents they were treated with a series of SbG for 24 h. No sig- were signic fi antly increased. However, no statistically nificant cytotoxicity was observed when SbG was be- signic fi ant GSH increase was observed between the two low 100 µg/mL, but the cell viability was significantly concentrations of SbG treatment (Figure 3). decreased when the SbG was over 200 µg/mL, and no dose-response relationship was observed. In the follow- Eec ff ts of SbG on GST activity ing cotreatment of SbG, 50 and 100µg/mL were employed GST assay was conducted according to the kit instruc- for the subsequent experiments. tion (BioVision).The assay utilizes monochlorobimane (MCB), a dye that reacts with GSH. The free form Eec ff ts of acrolein on cell viability of MCB is almost non-fluorescent, whereas the dye HUVE cell viability in the presence of acrolein is shown fluoresces blue (Ex/Em = 380/461 nm) when reacting in Figure 1. Acrolein below 10 µM for 24 h had no effect with GSH. GST catalyzes the MCB-GSH reactions and on the cell viability. However, significant decreased cell the fluorescence levels are proportional to the amounts viability was observed when the concentration was over of GST presence in the reaction. The sample- and re- 20 µM and there was a concentration-dependent man- agent-loaded assay plate was incubated for 2 h at 37°C ner in the cytotoxicity when the cells were incubated with with gentle rotation. Thirty minutes after loading the acrolein for 24 h (Figure 1). substrate mix, the plate was read in Applied Biosystem CytoFluor plate Reader at Ex/Em = 380/460 nm. Our Protective effects of SbG on acrolein-induced results showed that the cellular GST activities were not cytotoxicity in HUVE cells significantly changed when comparing the GST activity In order to elucidate the protective role of SbG against of SbG pretreated groups (50 and 100 µg/mL for 24 h) acrolein-induced cytotoxicity, HUVE cells were cultured with the control group (Figure 3). in the 96-well plate at the density of 5 × 10 for each well. SbG at different concentrations were loaded into the ap- Eec ff ts of acrolein on GSH content in HUVE cells propriate wells and incubated for 24 h followed by an- The effects of acrolein on GSH reduction is shown in other 24 h incubation with acrolein at the serial concen- Figure 4. HUVE cells were treated with different concen- trations. The MTT cell viability results showed that SbG trations of acrolein for 24 h followed by GSH assay. The at 50 and 100 µg/mL could significantly improve the cell results showed that acrolein could significantly decrease the cellular GSH contents. p < 0.05 * * ** p < 0.05 ** ** ** acrolein (µM) 0 20 20 20 40 40 40 60 60 60 SbG (µg/mL) 0 050100 050100 050100 0 25 50 100 200 400 05 10 20 40 60 Figure 2. Protective effects of SbG pretreatment at 50 and 100 μg/ SbG (µg/mL) acrolein (µM) ml showing the protection against acrolein-induced cytotoxicity in the HUVE cells. Cells were plated in the 96-well plate and SbG at different concentrations were loaded into the appropriate Figure 1. Effects of SbG on HUVE cell viability and cytotoxic wells and incubated for 24 h. The media was discarded and the effect of acrolein on HUVE cells. The cells were treated with SbG cells were continuously cultured in the presence or absence of or acrolein for 24 h at appropriate concentrations. MTT was acrolein for another 24 h. MTT was conducted for cell viability. conducted for the cell viability assay. Data were presented as Data were expressed as mean±SEM of the results of three mean±SEM of results in three independent experiments. Each independent experiments. *P < 0.05, **P < 0.01 vs acrolein alone experiment was performed in triplicate. *P< 0.05, **P <0.001 groups. versus control (black bar). Pharmaceutical Biology Cell Viability (% of normal) Cell Viability (% of normal) Scutellaria protection against oxidative stress 259 of 100 µg/mL SbG, in which GCLC had 2.8-fold elevation. Protective effects of SbG on cellular GSH contents Interestingly, pretreatment of SbG followed by another Because GSH plays an important role in the cellular an- 24 h incubation of 40 µM acrolein significantly reduced tioxidant defense (Pocernich et al., 2001), we thus inves- the GCLC mRNA level (P <0.05), while no statistical dif- tigated the cytoprotective effects of the SbG pretreated ference was found for the GCLM mRNA levels after the on acrolein-induced cytotoxicity in HUVE cells. Briefly, 24 h acrolein treatment (P >0.05). HUVE cells were treated with 50 and 100 µg/mL for 24 h followed by another 24 h of different concentrations of acrolein treatment. GSH assay was performed. As shown Discussion in Figure 4, incubation of the cells with SbG for 24 h could Protection against oxidative stress in cardiovascular significantly inhibit the GSH decrease. disorders by using phytotherapy and western medi- cines has been extensively studied (Banerjee et al., RT-PCR for GCLC and GCLM 2003; Chen et al., 2009; Costa et al., 2007; Csiszar et al., Since GSH synthesis requires two ATP-dependent 2008; He et al., 2008a; 2008b; Kobayashi et al., 2002; enzymes of GCL and GSH synthase while GCL is the Lakomkin et al., 2005; Liao et al., 2009; Nemoto et al., rate-limiting enzyme including GCLC and GCLM (Inoue 2007; Saada et al., 2009; Vaage et al., 1997; Venardos et al., 2003; Lu, 2009; Sekhar et al., 2003), we used a sensi- et al., 2007; Yang et al., 1995). HUVE cells have been tive real-time PCR assay to detect their mRNA changes. extensively employed to study cardiovascular diseases As shown in Figure 5, the mRNA levels for both GCLC and (Breymann et al., 2006; Chen et al., 2009; Erdbrugger GCLM were significantly increased after 24 h incubation et al., 1989; Morikawa et al., 2002). The HUVEC model we used had duplicatable results when they were exposed GSH to the cytotoxic acrolein, which is a major byproduct of 120 GST oxidative stress and lipid peroxidation. GSH and GST have been suggested to play a crucial role in the de- toxification of acrolein ( Jaeschke et al., 1987; Mitchell & Petersen, 1989; Monteil et al., 1999). Although acro- lein toxicity has been recently investigated in endothe- lial cells, herbal extract protection and their molecular 20 mechanism have not been investigated (Misonou et al., 2005; Park & Taniguchi, 2008; Patel & Block, 1993; Wu et al., 2006). The current study was aimed to elucidate SbG (µg/mL) 050 100 050 100 the protective effects of SbG and explore the possible mechanism. Our data showed that SbG-incubated Figure 3. Effects of SbG on cellular GSH levels and GST activity. HUVE cells had higher levels of cellular GSH and the HUVE cells were incubated with 50 and 100µg/ml SBG for 24 h. elevated mRNA expressions of GCLC and GCLM, which GSH contents were evaluated (*P < 0.05 vs the control). However, no significant GST activity changes were observed in the SbG treated were the catalytic and modulatory subunits of the GSH when compared with the control group. Data were presented as synthesis process. Surprisingly, SbG did not affect GST mean ±SEM of the results of three independent experiments. activity when the cells were incubated with SbG for 24 h. The protective effect of SbG was significant when GCLC GCLM ** 40 # * # 20 ** ** 0 0 acrolein (µM) 0 20 20 40 40 60 60 SbG − + + − + + acrolein SbG (µg/mL) 0 0 100 0 100 0 100 − − + − − + Figure 4. Protective effects of SbG on HUVE cells. The cells were Figure 5. After 24 h pretreatment of 100 µg/mL SbG, mRNA treated with acrolein for 24 h at different concentrations. In the levels of both GCLC and GCLM were elevated. Acrolein (40 µM) another set of the experiment, the cells were pretreated with treatment inhibited the SbG induced GCLC mRNA up-regulation 100 µg/mL SbG. Data were presented as mean±SEM of three levels. Data were mean±SEM of three independent experiments. independent experiments. *P <0.05, ** P < 0.01 versus the control, *P <0.05, ** P < 0.01 versus the control; # P < 0.05, versus the SBG # P < 0.05, versus the acrolein alone group. alone group. © 2011 Informa Healthcare USA, Inc. Cell GSH level (nmole/mg protein) Cellular GSH (nmole/mg protein) and GST activity (normalized to control) mRNA levels (normalized to GAPDH) 260 Xing-Wei Zhang et al. ligase (GCLM) is a molecular target for amelioration of cisplatin the cells were pretreated with SbG for 24 h, followed by resistance in lung cancer. Int J Oncol, 23, 1333–1339. exposing the cells to the cytotoxic acrolein incubation Jaeschke H, Kleinwaechter C, Wendel A. (1987). The role of acrolein in for another 24 h. The protection also showed even at the allyl alcohol-induced lipid peroxidation and liver cell damage in 50 µg/mL concentration. The present data suggest that mice. Biochem Pharmacol, 36, 51–57. SbG could be a potential antioxidant against oxidative Jia L, Liu Z, Sun L, Miller SS, Ames BN, Cotman CW, Liu J. (2007). Acrolein, a toxicant in cigarette smoke, causes oxidative damage stress in cardiovascular diseases. and mitochondrial dysfunction in RPE cells: Protection by (R)- alpha-lipoic acid. 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Protective effects of the aqueous extract of Scutellaria baicalensis against acrolein-induced oxidative stress in cultured human umbilical vein endothelial cells

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Taylor & Francis
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© 2011 Informa Healthcare USA, Inc.
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1744-5116
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10.3109/13880209.2010.501803
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Abstract

PHB Context: Scutellaria baicalensis Georgi (Labiatae) (SbG), one of the fifty fundamental herbs of Chinese herbology, has been reported to have anti-asthmatic, antifungal, antioxidative, and anti-inflammatory activities. Objective: This study was designed to determine the protective effects of the extract of SbG against the acrolein- induced oxidative stress in cultured human umbilical vein endothelial cells (HUVEC). Materials and methods: The MTT reduction assay was employed to determine cell viability. The total cellular glutathione (GSH) level was detected using a colorimetric GSH assay kit. Cellular GSH production was conducted by detecting the mRNA expression levels of γ-glutamylcysteine ligase catalytic subunit and modifier subunit. Results: Concentration-dependent cytotoxic effects of acrolein were observed while SbG could effectively protect the acrolein-induced oxidative damage. The protective mechanism was investigated, showing that the increased GSH content in the SbG-incubated HUVE cells was associated with the protective effects of SbG-treated cells. Further RT-PCR data confirmed the elevated mRNA expressions of GSH synthesis enzymes. Discussion and conclusion: The current study strongly indicated that SbG could be a potential antioxidant against oxidative stress in treating cardiovascular diseases. Keywords: Scutellaria baicalensis Georgi, cytotoxicity, glutathione, endothelial cell. Introduction the neuroprotective ee ff cts on ROS-induced cytotoxicity in neuronal HT-22 cells, neuroblastoma SH-SY5Y cells and Acrolein is a highly reactive unsaturated aldehydic PC12 cells (Choi et al., 2002; Gao et al., 2001; Shang et al., product of lipid peroxidation that could implicate vari- 2006; Wang et al., 2009). SbG contains numerous a fl vone ous degenerative pathogenesis, oxidative DNA damage derivatives and the active components were studied (Ding and mutagenesis and is widely used as a positive con- et al., 2009; Jia et al., 2007; Yu et al., 2007). It is worthwhile trol of reactive oxygen species (ROS) experiments (Jia to investigate whether the SbG also had protective effects et al., 2007; LoPachin et al., 2009; Luo & Shi, 2005; Pan on endothelial cells, which is of significant clinical mean- et al., 2009; Roy et al., 2009; Wang et al., 2009; Zitting & ing in treating endothelial cell related diseases. Heinonen, 1980). However, protection against acrolein toxicity in endothelial cells by using the aqueous extract of Scutellaria baicalensis Georgi (Labiatae) (SbG) has Materials and methods not been investigated, though SbG has been reported to have anti-asthmatic, anti-fungal, antioxidative, and anti- Materials inflammatory activities (Huang et al., 2006; Koda et al., Human umbilical vein endothelial (HUVE) cells were 1972; Wong & Tsang, 2009). Past studies of SbG showed purchased from BD Biosciences (San Jose, CA). The MTT Address for Correspondence: Yue-liang Shen, Department of Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058, P.R. China. Tel./Fax: +86 571 88208250; E-mail: [email protected] (Received 01 March 2010; revised 00 00 0000; accepted 14 June 2010) 256 Scutellaria protection against oxidative stress 257 kit was purchased from Invitrogen (Calscard, CA). Glu- GST activity measurement tathione (GSH) and glutathione S-transferase (GST) kits Cellular GST activity was measured by using a GST were from BioVision (Mountain View, CA) and all the fluorometric assay kit (BioVision). Briefly, appropriate chemicals were purchased from Sigma (St. Louis, MO). samples were prepared in a total of 100 µL volume with GST sample buffer from the kit, including the negative Cell culture and preparation of aqueous extract of SbG control of 100 µL of GST sample buffer alone and a posi- The human umbilical vein endothelial cells were grown tive control of 2 µL of GST and 98 µL of sample buffer. All to confluence in CS-C medium (Sigma) supplemented the samples were made in three dilutions and loaded with 5% fetal bovine serum (FBS) and 1% penicillin/ into a 96-well plate in triplicates for each experiment. Af- streptomycin at 37 °C in a humidified atmosphere of 5% ter adding GSH and substrate mix, the plate was read at CO . The culture media was changed every week. SbG Ex/Em 380/460 nm at 30 min time point to compare the was purchased from Hangzhou Chinese Pharmaceuti- fluorescence level of treated versus control samples. cal (Zhejiang, China) and extracted as reported (Wong & Tsang, 2009; Yoon et al., 2009). Briefly, the extractions RT-PCR of GCLC and GCLM were started with 50 g of material in 1000 mL of distilled Molecular level of cellular GSH production was con- water, boiled for 2 h, filtered through 0.22 µm syringe fil- ducted by detecting the mRNA expression levels of ter, and then lyophilized with a yield of 28.86% (w/w). γ-glutamylcysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM), since the synthesis of GSH re- quires two ATP-dependent enzymes, γ-glutamylcysteine Acrolein exposure and SbG supplementation ligase (GCL) and GSH synthase. GCL is the rate-limiting When HUVE cells were confluent in the 96-well plate enzyme in the overall GSH synthesis process which and 8-well cell culture chamber (Fisher scientific, USA), is composed of a catalytic (GCLC) and modulatory acrolein was dissolved in 1× PBS and loaded onto the (GCLM) subunit. GCLC has the catalytic activity while cells for 24 h. The protective effects of SbG were studied GCLM could enhance the catalytic activity. Total RNA with the acute toxicity experiment by pretreating the cells was extracted using TRIzol reagent (Life Technolo- with SbG for 24 h. gies, USA) following the manufacturer’s protocol. RNA was quantitated and 5 μg of the total RNA isolated was Cell viability assay reverse transcribed to cDNA at 37°C for 1 h in a 30 μL MTT reduction assay was employed to determine cell reaction mix containing 200 U of MMLV Reverse Tran- viability. Briefly, the cells were plated into the 96-well scriptase (Promega) as per the manufacturer’s pro- plate. For the SbG protection groups, the appropri- tocol. All the PCR amplifications were done in a 50 μL ate wells in triplicates were loaded with SbG for 24 h. reaction mix containing 2.5 U Taq DNA Polymerase Then the cells were incubated with acrolein in DMEM (Promega, USA) in 1× reaction buffer, 1.5 mM MgCl , supplemented with 0.5% FBS at 37 °C in a humidi- 150 μM dNTP mix, 5 to 25 pmol of forward and reverse fied atmosphere of 5% CO for 24 h, after which time primers (Genosys, Sigma) and 3 μL of template cDNA. the media were discarded, followed by addition to The primers used in quantitative real-time PCR were each well of 0.1 mL of fresh medium containing MTT GCLC (forward, 5′-GGCGATGAGGTGGAATAC-3′; (0.2 mg/mL). The plate was incubated for another 4 h at reverse, 5′-AAAGGGTAGGATGGTTTGG-3′), GCLM 37 °C. After solubilizing the formazon crystals by add- (forward, 5′-ATCAAACTCTTCATCATCAAC-3′; re- ing DMSO, optical densities were detected at 570 nm verse, 5′-GATTAACTCCATCTTCAATAGG-3′) and using a microplate spectrophotometer (Bio-Tek Instru- glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ments, USA). forward, 5′-ACAGTCAGCCGCATCTTC-3′; reverse, 5′- GCCCAATACGACCAAATCC-3′). Real-time reactions Glutathione level measurement were conducted on an Applied Biosystems 5700 using The total cellular GSH level was detected by using a colo- a hot start, followed by 40 cycles at 94°C for 15 s, 56°C rimetric GSH assay kit (BioVision). This kit makes use of a for 30 s, 72°C for 30 s, and a final extension step at 72°C kinetic enzymatic recycling assay based on the oxidation for 3 min. Analysis of the relative gene expression data of GSH by 5, 5′-dithiobis-2-nitrobenzoic acid (DTNB) and was performed using the △C method. For each primer glutathione reductase to measure the total GSH content pair, a plot of the log cDNA dilution versus △C was in cells. Addition of NADPH2 initiates the progressive re- generated to validate the quantitative PCR results. The duction of DTNB by GSH, causing a color increase that is mean quantities of GCLC and GCLM transcripts were monitored at 405 nm. The rate of color change, typically normalized based on the mean quantity of the control monitored over a 5 min period, is proportional to the total gene (GAPDH). GSH concentration. This assay is specific for GSH; other thiols do not cause interference in the assay. Cellular Statistical analysis GSH content was calculated by using the standard GSH All data are expressed as mean ± SEM from three inde- from the kit and expressed as nanomoles of GSH per mg pendent experiments. Data were analyzed with SPSS. of cellular protein. Significant differences were examined using analysis © 2011 Informa Healthcare USA, Inc. 258 Xing-Wei Zhang et al. of variance (ANOVA) and Student’s t-test was used to viability when treated with acrolein at 20 µM; Interest- determine the difference between the two groups and ingly, SbG at 100 µg/mL had better protection than SbG the paired t-test for within-group differences. A value of at 50 µg/mL when acrolein concentrations were 40 and P <0.05 was considered statistically significant. 60 µM, respectively (P <0.01 and P <0.05, respectively, Figure 2). Results Eec ff ts of SbG on cellular GSH level in HUVE cells Eec ff ts of SbG on cell viability When HUVE cells were incubated with SbG at 50 and Figure 1 shows the cell viability of the HUVE cells when 100 µg/ mL for 24 h, we found that the cellular GSH contents they were treated with a series of SbG for 24 h. No sig- were signic fi antly increased. However, no statistically nificant cytotoxicity was observed when SbG was be- signic fi ant GSH increase was observed between the two low 100 µg/mL, but the cell viability was significantly concentrations of SbG treatment (Figure 3). decreased when the SbG was over 200 µg/mL, and no dose-response relationship was observed. In the follow- Eec ff ts of SbG on GST activity ing cotreatment of SbG, 50 and 100µg/mL were employed GST assay was conducted according to the kit instruc- for the subsequent experiments. tion (BioVision).The assay utilizes monochlorobimane (MCB), a dye that reacts with GSH. The free form Eec ff ts of acrolein on cell viability of MCB is almost non-fluorescent, whereas the dye HUVE cell viability in the presence of acrolein is shown fluoresces blue (Ex/Em = 380/461 nm) when reacting in Figure 1. Acrolein below 10 µM for 24 h had no effect with GSH. GST catalyzes the MCB-GSH reactions and on the cell viability. However, significant decreased cell the fluorescence levels are proportional to the amounts viability was observed when the concentration was over of GST presence in the reaction. The sample- and re- 20 µM and there was a concentration-dependent man- agent-loaded assay plate was incubated for 2 h at 37°C ner in the cytotoxicity when the cells were incubated with with gentle rotation. Thirty minutes after loading the acrolein for 24 h (Figure 1). substrate mix, the plate was read in Applied Biosystem CytoFluor plate Reader at Ex/Em = 380/460 nm. Our Protective effects of SbG on acrolein-induced results showed that the cellular GST activities were not cytotoxicity in HUVE cells significantly changed when comparing the GST activity In order to elucidate the protective role of SbG against of SbG pretreated groups (50 and 100 µg/mL for 24 h) acrolein-induced cytotoxicity, HUVE cells were cultured with the control group (Figure 3). in the 96-well plate at the density of 5 × 10 for each well. SbG at different concentrations were loaded into the ap- Eec ff ts of acrolein on GSH content in HUVE cells propriate wells and incubated for 24 h followed by an- The effects of acrolein on GSH reduction is shown in other 24 h incubation with acrolein at the serial concen- Figure 4. HUVE cells were treated with different concen- trations. The MTT cell viability results showed that SbG trations of acrolein for 24 h followed by GSH assay. The at 50 and 100 µg/mL could significantly improve the cell results showed that acrolein could significantly decrease the cellular GSH contents. p < 0.05 * * ** p < 0.05 ** ** ** acrolein (µM) 0 20 20 20 40 40 40 60 60 60 SbG (µg/mL) 0 050100 050100 050100 0 25 50 100 200 400 05 10 20 40 60 Figure 2. Protective effects of SbG pretreatment at 50 and 100 μg/ SbG (µg/mL) acrolein (µM) ml showing the protection against acrolein-induced cytotoxicity in the HUVE cells. Cells were plated in the 96-well plate and SbG at different concentrations were loaded into the appropriate Figure 1. Effects of SbG on HUVE cell viability and cytotoxic wells and incubated for 24 h. The media was discarded and the effect of acrolein on HUVE cells. The cells were treated with SbG cells were continuously cultured in the presence or absence of or acrolein for 24 h at appropriate concentrations. MTT was acrolein for another 24 h. MTT was conducted for cell viability. conducted for the cell viability assay. Data were presented as Data were expressed as mean±SEM of the results of three mean±SEM of results in three independent experiments. Each independent experiments. *P < 0.05, **P < 0.01 vs acrolein alone experiment was performed in triplicate. *P< 0.05, **P <0.001 groups. versus control (black bar). Pharmaceutical Biology Cell Viability (% of normal) Cell Viability (% of normal) Scutellaria protection against oxidative stress 259 of 100 µg/mL SbG, in which GCLC had 2.8-fold elevation. Protective effects of SbG on cellular GSH contents Interestingly, pretreatment of SbG followed by another Because GSH plays an important role in the cellular an- 24 h incubation of 40 µM acrolein significantly reduced tioxidant defense (Pocernich et al., 2001), we thus inves- the GCLC mRNA level (P <0.05), while no statistical dif- tigated the cytoprotective effects of the SbG pretreated ference was found for the GCLM mRNA levels after the on acrolein-induced cytotoxicity in HUVE cells. Briefly, 24 h acrolein treatment (P >0.05). HUVE cells were treated with 50 and 100 µg/mL for 24 h followed by another 24 h of different concentrations of acrolein treatment. GSH assay was performed. As shown Discussion in Figure 4, incubation of the cells with SbG for 24 h could Protection against oxidative stress in cardiovascular significantly inhibit the GSH decrease. disorders by using phytotherapy and western medi- cines has been extensively studied (Banerjee et al., RT-PCR for GCLC and GCLM 2003; Chen et al., 2009; Costa et al., 2007; Csiszar et al., Since GSH synthesis requires two ATP-dependent 2008; He et al., 2008a; 2008b; Kobayashi et al., 2002; enzymes of GCL and GSH synthase while GCL is the Lakomkin et al., 2005; Liao et al., 2009; Nemoto et al., rate-limiting enzyme including GCLC and GCLM (Inoue 2007; Saada et al., 2009; Vaage et al., 1997; Venardos et al., 2003; Lu, 2009; Sekhar et al., 2003), we used a sensi- et al., 2007; Yang et al., 1995). HUVE cells have been tive real-time PCR assay to detect their mRNA changes. extensively employed to study cardiovascular diseases As shown in Figure 5, the mRNA levels for both GCLC and (Breymann et al., 2006; Chen et al., 2009; Erdbrugger GCLM were significantly increased after 24 h incubation et al., 1989; Morikawa et al., 2002). The HUVEC model we used had duplicatable results when they were exposed GSH to the cytotoxic acrolein, which is a major byproduct of 120 GST oxidative stress and lipid peroxidation. GSH and GST have been suggested to play a crucial role in the de- toxification of acrolein ( Jaeschke et al., 1987; Mitchell & Petersen, 1989; Monteil et al., 1999). Although acro- lein toxicity has been recently investigated in endothe- lial cells, herbal extract protection and their molecular 20 mechanism have not been investigated (Misonou et al., 2005; Park & Taniguchi, 2008; Patel & Block, 1993; Wu et al., 2006). The current study was aimed to elucidate SbG (µg/mL) 050 100 050 100 the protective effects of SbG and explore the possible mechanism. Our data showed that SbG-incubated Figure 3. Effects of SbG on cellular GSH levels and GST activity. HUVE cells had higher levels of cellular GSH and the HUVE cells were incubated with 50 and 100µg/ml SBG for 24 h. elevated mRNA expressions of GCLC and GCLM, which GSH contents were evaluated (*P < 0.05 vs the control). However, no significant GST activity changes were observed in the SbG treated were the catalytic and modulatory subunits of the GSH when compared with the control group. Data were presented as synthesis process. Surprisingly, SbG did not affect GST mean ±SEM of the results of three independent experiments. activity when the cells were incubated with SbG for 24 h. The protective effect of SbG was significant when GCLC GCLM ** 40 # * # 20 ** ** 0 0 acrolein (µM) 0 20 20 40 40 60 60 SbG − + + − + + acrolein SbG (µg/mL) 0 0 100 0 100 0 100 − − + − − + Figure 4. Protective effects of SbG on HUVE cells. The cells were Figure 5. After 24 h pretreatment of 100 µg/mL SbG, mRNA treated with acrolein for 24 h at different concentrations. In the levels of both GCLC and GCLM were elevated. Acrolein (40 µM) another set of the experiment, the cells were pretreated with treatment inhibited the SbG induced GCLC mRNA up-regulation 100 µg/mL SbG. Data were presented as mean±SEM of three levels. Data were mean±SEM of three independent experiments. independent experiments. *P <0.05, ** P < 0.01 versus the control, *P <0.05, ** P < 0.01 versus the control; # P < 0.05, versus the SBG # P < 0.05, versus the acrolein alone group. alone group. © 2011 Informa Healthcare USA, Inc. 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Journal

Pharmaceutical Biology – Taylor & Francis

Published: Mar 1, 2011

Keywords: Scutellaria baicalensis Georgi; cytotoxicity; glutathione; endothelial cell.

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