Toxicology and Applied Pharmacology 273 (2013) 68–76
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Salvianolic acid A preconditioning confers protection against concanavalin A-induced liver injury through SIRT1-mediated repression of p66shc in mice Xiaomei Xu a, Yan Hu a, Xiaohan Zhai a, Musen Lin a, Zhao Chen b, Xiaofeng Tian b, Feng Zhang b, Dongyan Gao a, Xiaochi Ma a, Li Lv a,⁎, Jihong Yao a,⁎ a b
Department of Pharmacology, Dalian Medical University, Dalian 116044, China Department of General Surgery, Second Affiliated Hospital of Dalian Medical University, Dalian 116023, China
a r t i c l e
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Article history: Received 28 March 2013 Revised 15 July 2013 Accepted 16 August 2013 Available online 28 August 2013 Keywords: Concanavalin A Hepatitis Salvianolic acid A SIRT1
a b s t r a c t Salvianolic acid A (SalA) is a phenolic carboxylic acid derivative extracted from Salvia miltiorrhiza. It has many biological and pharmaceutical activities. The purpose of this study was to investigate the effect of SalA on concanavalin A (ConA)-induced acute hepatic injury in Kunming mice and to explore the role of SIRT1 in such an effect. The results showed that in vivo pretreatment with SalA significantly reduced ConA-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and decreased levels of the hepatotoxic cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Moreover, the SalA pretreatment ameliorated the increases in NF-κB and in cleaved caspase-3 caused by ConA exposure. Whereas, the pretreatment completely reversed expression of the B-cell lymphoma-extra large (Bcl-xL). More importantly, the SalA pretreatment significantly increased the expression of SIRT1, a NAD+-dependent deacetylase, which was known to attenuate acute hypoxia damage and metabolic liver diseases. In our study, the increase in SIRT1 was closely associated with down-regulation of the p66 isoform (p66shc) of growth factor adapter Shc at both protein and mRNA levels. In HepG2 cell culture, SalA pretreatment increased SIRT1 expression in a time and dose-dependent manner and such an increase was abrogated by siRNA knockdown of SIRT1. Additionally, inhibition of SIRT1 significantly reversed the decreased expression of p66shc, and attenuated SalA-induced p66shc down-regulation. Collectively, the present study indicated that SalA may be a potent activator of SIRT and that SalA can alleviate ConA-induced hepatitis through SIRT1-mediated repression of the p66shc pathway. © 2013 Elsevier Inc. All rights reserved.
Introduction Autoimmune hepatitis (AIH) is a type of chronic inflammatory liver disease. There is currently no specific drugs for treatment of the illness, as its pathogenic mechanisms have not been clearly elucidated (Wang et al., 2012b). In studying AIH, concanavalin A (ConA)-induced hepatitis is a well established experimental mouse model where ConA, a kind of lectin, is purified from Canavalia ensiformis. The ConA-induced hepatitis model is widely used to investigate the pathophysiology of immunemediated liver injury and the various aspects of T cell-mediated hepatic diseases e.g., autoimmune or viral hepatitis. In this experimental hepatitis model, T cells, especially CD4+ T helper cells and natural killer T (NKT) cells are activated in abundance, resulting in production of
Abbreviations: SalA, salvianolic acid A; ConA, concanavalin A; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TNF-α, tumor necrosis factor-α; IFN-γ, interferon gamma; SIRT1, sirtuin1; Bcl-xL, B-cell lymphoma-extra large; NF-κB, nuclear factor κB; AIH, autoimmune hepatitis; p66shc, the 66 kDa isoform of the growth factor adapter Shc. ⁎ Corresponding authors. E-mail addresses:
[email protected] (L. Lv),
[email protected] (J. Yao). 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.08.021
inflammatory cytokine, adhesion molecule and chemokine such as tumor necrosis factor-α (TNF-α), interferon gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-4, IL-6, and intercellular adhesion molecule-1 (ICAM-1), among which TNF-α and IFN-γ are the main contributor cytokines. These factors, in turn, recruit and activate more immune cells, leading to severe hepatic damage. The necrosis and apoptosis of hepatocytes and the neutrophilic infiltration are a characteristic feature of ConA-induced hepatitis (Amin et al., 2007; Shi et al., 2012; Sternak et al., 2010). It is not known whether ConA-induced hepatitis is related to sirtuin1 (SIRT1), a NAD-dependent class III histone deacetylase (HDAC), which is indicated to be an important regulator of immunologic balance in recent studies (Kong et al., 2012). Among the seven sirtuin genes in mammals, SIRT1 is a homolog of SIR2 in yeast Saccharomyces cerevisiae. SIRT1 is mostly found in the nucleus, where it plays a vital role in transcriptional repression via histone deacetylation. It is also well-known that SIRT1 regulates cell apoptosis, senescence, metabolism, and proliferation, and thereby influences multiple biological situations including longevity, obesity, age-related diseases and cancers (Alcendor et al., 2004; Hong et al., 2012; Kelly, 2010a,b). Recent studies showed that SIRT1-
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null mice developed an autoimmune-like disease, accompanied by accumulation of immune complexes in the liver and kidney (Sequeira et al., 2008). Additionally, it has been proven that failure of T-cell anergy may be involved in the development and progression of some autoimmune diseases in mammals, such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis. Concurrently, SIRT1 mRNA and protein expression levels were found increased in activated T cells, especially in anergic T cells, while loss of SIRT1 function results in an abnormal increase in T cell activation and a breakdown of CD4 + T cell tolerance. These findings suggest that SIRT1 plays an essential role in attenuating T cell activation and maintaining T cell tolerance (Gao et al., 2012; Kong et al., 2011; Zhang et al., 2009). A number of studies have revealed an important role of SIRT1 in metabolic liver diseases. For example, a recent study demonstrated that the up-regulation of SIRT1 protects against carbon-tetrachloride (CCl4)-induced acute liver injury (Xie et al., 2013). Yet, the role of SIRT1 in ConA-induced liver injury has not been investigated. The growth factor adapter p66shc is one of three isoforms of ShcA adaptors p46shc, p52shc and p66shc. In contrast to smaller ShcA isoforms, which are constitutively and ubiquitously expressed, pro-apoptotic factor p66shc only exists in the cytosol and mitochondria (Migliaccio et al., 2006). Recent reports have shown that p66shc plays a crucial role in regulating cellular responses to oxidative stress, apoptosis and aging. The p66shc knockout (KO) mice are protected against aging, diabetes, vascular, cardiac, and ischemia reperfusion impairment (Cosentino et al., 2008). The studies have also revealed that enhanced p66shc expression contributes to the progression of alcoholinduced liver damage or nonalcoholic steatohepatitis in a likelihood via a mechanism, which involves the reduced expression of antioxidant defenses and exaggerated apoptosis (Koch et al., 2008; Tomita et al., 2012). Interestingly, SIRT1 overexpression was found to decrease highglucose-induced p66shc expression in human umbilical vein endothelial cells, suggesting that SIRT1 may be a regulator of p66shc (Chen et al., 2012; Zhou et al., 2011). Therefore, it is possible that SIRT1 suppresses p66shc expression in acute hepatitis induced by ConA, thereby affording protection against the disease. The present study was to evaluate whether SIRT1 expression is altered in ConA-induced acute liver injury and whether salvianolic acid A (SalA) has a protective effect on such an acute liver injury. SalA is a phenolic carboxylic acid derivative, a biologically active compound extracted from Salvia miltiorrhiza (Fig. 1). SalA possesses a variety of biological activities, such as protecting effects against impaired vascular responsiveness in streptozotocin (STZ)-induced diabetic rats (Wang et al., 2009b), the antiplatelet and antithrombotic activities in an arterio-venous shunt model (Fan et al., 2010), and the cardioprotective effects on animal models of heart hypoxia/reoxygenation injury (Zhang et al., 2010). Interestingly, in a recent study, treatment with SalA conferred potent protective effects against (CCl4)-induced acute liver damage in rats and such effects may mainly be attributed to its antioxidative
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and anti-apoptotic properties (Wu et al., 2007). Currently, it has been reported that SalB (Lee et al., 2011) and several other polyphenols, including resveratrol (Yu et al., 2009), quercetin (Hong et al., 2012), and icariine (Wang et al., 2009a), were able to up-regulate SIRT1 activity. Besides, SalA was reported to protect against diabetic neuropathy in rats, and these beneficial effects are associated with the up-regulation of SIRT3 (Yu et al., 2012). The above observations on biological activities of SalA impetus us to investigate whether SalA can attenuate ConA-induced severe liver damage. In the present study, we examined SIRT1 expression in ConAinduced acute liver hepatitis, determined the up-regulation of SIRT1 expression by SalA in the same experimental disease model, and further elucidated the correlation between SIRT1 and p66shc during ConAinduced liver injury. We also assessed the expression of the apoptosisrelated factors including the cleaved caspase-3, the B-cell lymphomaextra large (Bcl-xL), and the pro-inflammatory mediator NF-κB. The regulation of these factors explains the hepatoprotective effects of SalA in ConA-induced injury. Materials and methods Experimental animals and reagents. Male Kunming mice, with weight ranging from 18 to 22 g, were obtained from the Experimental Animal Center of Dalian Medical University (Dalian, China). The mice were kept with standard laboratory conditions with free access to food and water. The animals were set to adapt to the new environment for one week before experimental procedures, which were performed in accordance with the local institutional guidelines. ConA was purchased from Sigma-Aldrich (St. Louis, MO, USA). The SalA with more than 98% of purity was obtained from Shanghai Winherb Medical Technology Co. (Shanghai, China). Both ConA and SalA were dissolved in pathogenfree saline. Animal treatment and experimental design. Mice were administrated with ConA at a dose of 18 mg/kg by tail vein injection to induce acute hepatic injury 12 h after the injection. SalA, at dose of 15 and/or 25 mg/kg, was intraperitoneally injected to the animals 30 min before ConA was used. All these dosages were determined by preliminary experiments and the control mice were treated with the equal volume of saline. The experimental mice were randomly divided into five groups. The first group was the vehicle control, in which mice were administrated with saline instead of ConA and/or SalA. In the second group, mice were given SalA at a dose of 25 mg/kg without ConA treatment. In the third group, mice were treated with ConA without SalA treatment. In the fourth and fifth groups, mice were pretreated with SalA at a dose of 15 mg/kg and 25 mg/kg respectively for 30 min. Both the fourth and fifth groups were then challenged with ConA for 12 h after SalA treatment. All animals were euthanized and, the liver, blood and other tissue samples were harvested for further analysis. Cell culture. The human hepatoma cell line HepG2 was grown in minimum essential medium (MEM), containing 10% (v/v) fetal bovine serum (FBS) in a humidified incubator with 5% CO2 at 37 °C. Both MEM and FBS are Invitrogen products from Life Biotechnologies (Carlsbad, CA, USA). The experiment protocols were as described in the legends of the corresponding figures.
Fig. 1. Chemical structure of salvianolic acid A.
Measurement of serum levels of ALT and AST. The blood samples were harvested by drawing the blood from the eyeball. Serum samples were then isolated from the blood by centrifugation at 3000 rpm for 15 min, after blood coagulation for 2 h on ice. The activities of ALT and AST in serum were measured using commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), according to the manufacturer's instructions.
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Assessment of liver histopathology. The isolated left lateral liver lobes were fixed in 4% formaldehyde. After the tissues were being embedded in paraffin, the tissue sections were obtained and stained with hematoxylin and eosin (H&E). The specimens were examined under light microscopy. Determination of serum cytokine levels by ELISA. IFN-γ and TNF-α levels were measured by enzyme-linked immunosorbent assay (ELISA), using commercial kits from Boster Biological Technology Co. Ltd (Wuhan, China). All the procedures were carried out according to the protocols provided by the manufactures. RNA isolation and RT-PCR analysis. The total RNAs were extracted from the frozen liver samples using RNAiso Plus Reagents from TaKaRa (TaKaRa, Dalian, China) following the manufacturer's instructions. The RNAs were dissolved in diethyl pyrocarbonate-treated deionized water, and RNA concentrations were determined using an ND UV1102 Nanodrop Spectrophotometer (Shanghai, China). Reverse transcription was performed using a TaKaRa RNA PCR Kit with AMV ver. 3.0. The PCR primers and their sequences were as follows: SIRT1, forward (F) 5′-GCAACAGCATCTTGCCTGAT-3′ and reverse (R) GTGCTACTGG TCTCACTT-3′; p66shc, F 5′-ACTACCCTGTGTTCCTTCTTTC-3′ and R 5′-TCGGTGGATTCCTGAGATACTGT-3′; β-actin, F 5′-AGAGGGAAATCG TGCGTGAC-3′ and R 5′-CAATAGTGATGACCTGGCCGT-3′. The two-step RT-PCR was carried out as described in the TaKaRa RNA PCR protocol. The conditions for reverse transcription were: 42 °C for 30 min, 99 °C for 5 min, and then 5 °C for 5 min. The PCR was performed at the condition as follows: 30 s at 94 °C, for denaturation; 30 s at 55 °C for annealing, and 90 s at 72 °C for extension. The PCR products were separated by electrophoresis using 1.5% agarose gels. The DNA bands were visualized using the BioSpectrum-410 multispectral imaging system with Chemi HR camera 410 (Upland, CA, USA). Western blotting analysis. Nuclear and cytosolic proteins were extracted from liver tissues or HepG2 cells with protein extraction kit (KeyGen Biotech, Nanjing, China), according to the manufacturer's instructions. Protein concentrations were determined using BCA protein assay kit (Beyotime Biotech, Shanghai, China). Nuclear proteins were used for determining SIRT1, NF-κB, and Bcl-xL proteins. Cytosolic proteins were used for examining p66shc and cleaved caspase-3 protein. For Western blotting, the same amount of sample proteins, 50 μg per lane, was separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane (Millipore, Bedford, USA). The membrane was blocked with 5% fat-free milk in TTBS buffer, then incubated at 4 °C overnight, with a proper primary antibody including antiSIRT1 (Abcam Ltd., Cambridge, UK), p66shc (Abcam Ltd., Cambridge, UK), NF-κB (Proteintech Group Inc., Wuhan, China), Bcl-xL, and cleaved caspase-3 (Bioworld Technology, Inc.). The membranes were further washed with TTBS buffer for three times, incubated with HRPconjugated secondary antibodies at 37 °C for 2 h. After adequately washing with TTBS, the membranes were finally developed with chemiluminescence-plus reagents (Beyotime Institute of Biotechnology, Hangzhou, China). The images of protein blots were captured using BioSpectrum-410 multispectral imaging system with a Chemi HR camera 410. The density of interested protein bands was determined using Gel-Pro Analyzer Version 4.0 software from Media Cybernetics Inc. (Rockville, MD, USA). Western blotting using antiβ-actin antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA) served as a loading control. siRNA transfection. SiRNAs, both gene-specific and nonspecific control, were obtained from Shanghai GenePharma Co. (Shanghai, China). The SIRT1 siRNA sequences are sense 5′-CCCUGUAAAGCUUUCAGAA (dTdT)-3′ and antisense 5′-UUCUGAAAGCUUUACAGGG(dTdT)-3′. In siRNA transfection experiments, HepG2 cells were seeded at 2 × 105 cells/ml in 6-well plates and the cells were transfected at a
final concentration of 0.1 μM of siRNA using LipofectamineTM 2000 reagent from Invitrogen, according to the manufacturer's instructions. The transfected cells were incubated at 37 °C in serum free MEM and FBS was added to 10% 6 h after transfection. After gowning for additional 48 h, the cells were treated with 20 μM SalA in serum-free MEM for another 6 h and then the proteins were extracted for Western blotting. TUNEL assay. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed with an In Situ Cell Death Detection Kit, TMR red (Roche, NJ, USA), according to the manufacturer's instructions. Liver samples were treated as described in the Assessment of liver histopathology section. Briefly, the liver tissue sections were pretreated with 0.1% triton X-100 for 8 min, washed with PBS buffer, and then stained by TUNEL reaction mix for 1 h at 37 °C. The FITC-labeled TUNEL-positive cells were imaged under a Leica DMI 4000 B fluorescent microscope (Leica, Germany), using 488 nm excitation and 530 nm emission. Dual immunofluorescence labeling. HepG2 cells were grown on sixwell chamber slides, fixed in 4% paraformaldehyde in PBS for 20 min. After rinsing with PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 2% BSA in PBS for 30 min. The specimen slides were incubated with primary anti-SIRT1 or antip66shc antibody at 4 °C overnight. The specimens were then washed for three times with PBS, incubated with a proper FITC-conjugated and Cy3-tagged secondary antibodies (Santa Cruz Biotechnology) at 37 °C for 1 h. After additional washes, the specimens were counterstained for nuclei with DAPI and the immunofluorescent images were captured by 80i Nikon microscope (Tokyo, Japan). Statistical analysis. All the statistical analysis was carried out by using SPSS19.0 software (Chicago, USA). Student's t-test was performed between two groups and one-way analysis of variance with Student–Newman–Kuels multiple comparison was used for experiments involving more than two groups. p b 0.05 was considered statistically significant and the mean ± SEM values were used to express the measurements. Results SalA preconditioning attenuates ConA-induced hepatitis To explore whether SalA has a protective effect on ConA-induced hepatitis, the experimental hepatitis model on Kunming mice was established. Twelve hours after intravenous injection of ConA, severe hepatic injury was elicited in the animals. Compared to saline or SalA control groups, serum ALT and AST levels clearly increased in response to ConA treatment (Figs. 2A and B). However, SalA treatment significantly inhibited ALT and AST activities in a dose-dependent manner (p b 0.01), but did not affect ALT and AST levels in control mice, suggesting a protective effect of SalA on ConA-induced hepatitis. The protective effect of SalA was further confirmed by morphological analysis (Fig. 2C). ConA administration led to a massive infiltration of T lymphocytes in the liver, particularly in portal area, and large areas of necrosis within the liver lobules. In addition, large numbers of apoptotic hepatocytes were found and such a liver histopathologic injury correlated with elevated serum levels of liver enzymes. In contrast, SalA pretreatment dramatically reduced liver damage caused by ConA challenge, attenuating hepatocyte apoptosis and T lymphocyte infiltration, and ameliorating liver necrosis. These results indicated that SalA is effective in protecting against ConA-induced liver injury. SalA preconditioning inhibits the release of pro-inflammatory cytokines in ConA-treated mice It has been suggested that TNF-α and IFN-γ play important roles in the progression of ConA-induced hepatitis (Wang et al., 2012a).
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Fig. 2. SalA pretreatment attenuates ConA-induced liver injury. Mice were treated with SalA for 30 min before ConA was administrated. The serum and livers of control and experimental mice were collected 12 h after ConA injection. (A) Serum ALT levels. (B) Serum AST levels. (C) Histopathology of liver indicated by H&E staining of experimental groups, a. control; b. control + SalA (25 mg/kg); c. ConA (18 mg/kg); d. ConA + SalA (15 mg/kg) and e. ConA + SalA (25 mg/kg). H&E stained sections were photographed at 40× magnification. Data represent the mean ± SEM (n = 8). *p b 0.01 vs. control group, #p b 0.01 vs. ConA group.
Hence, the serum levels of these pro-inflammatory cytokines were analyzed. As shown in Figs. 3A and B, TNF-α and IFN-γ levels greatly increased in response to ConA exposure. Conversely SalA pretreatment inhibited such an increase in a dose-dependent manner (p b 0.01). Thus, prevention of ConA-induced hepatitis by SalA is associated with reduction of TNF-α and IFN-γ. SalA-mediated protection against ConA-induced hepatitis involves SIRT1 up-regulation In the model of ConA-induced hepatitis, a large number of T cells are activated, resulting in the enhancement of immunological reactions. Moreover, recent studies have indicated that SIRT1 plays an important role in immune regulation through attenuating T cell activation and maintaining T cell tolerance. Hence, we investigated whether SIRT1 expression was altered during ConA-induced acute liver injury. As shown in Figs. 4A and B, after administration of ConA, hepatic SIRT1 mRNA and protein levels were up regulated in comparison to that in control group. Additionally, SalA pretreatment enhanced mRNA and protein expression of SIRT1 in a dose-dependent manner in comparison to that in ConA group. Thus, it appeared that SIRT1 activation was associated with SalA-mediated protection against ConA-induced hepatitis.
We next used Human hepatoma G2 (HepG2) cells to further confirm that the SalA treatment causes SIRT1 up regulation in vitro. HepG2 cell line is a well differentiated transformed cell line and it represents a well-characterized, reliable in vitro model. HepG2 cell line has been widely used to study biochemical and nutritional variations in antioxidant defense systems (Alia et al., 2005). To this end, HepG2 cells were treated with SalA at various doses for different time periods, from 0 to 3, 6, 12, 24 or 48 h. The results showed that SalA enhanced SIRT1 expression in a time-dependent manner, with a peak at 6 h (Fig. 5A). SalA also increased SIRT1 expression in a dose-dependent manner at 6 h period (Fig. 5B). The effect of SalA in activating SIRT1 was reversed upon siRNA knockdown of SIRT1 in HepG2 cells (Fig. 5C). Together, these data demonstrated that ConA administration enhanced SIRT1 expression, and SalA treatment led to SIRT1 up-regulation, indicating that SalA is a potential activator of SIRT1. SalA induced SIRT1 plays an anti-apoptotic role in ConA-induced hepatitis by inhibiting p66Shc expression It is well known that ConA administration causes hepatocyte apoptosis. We therefore performed a TUNEL assay to measure the extent of apoptosis. As shown in Fig. 6A, a massive hepatocyte apoptosis was observed
Fig. 3. SalA pretreatment inhibits cytokine release in ConA-treated mice. The animals were treated with SalA for 30 min before ConA was administrated. The serum samples were collected 12 h after ConA injection. The tumor necrosis factor (TNF-α) (A) and interferon-γ (IFN-γ) (B) were measured. Data were expressed as mean ± SEM (n = 8). *p b 0.01 vs. control group, #p b 0.01 vs. ConA group.
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Fig. 4. Effects of SalA on SIRT1 protein and mRNA expressions in ConA-induced hepatitis. The mice were treated with SalA for 30 min before ConA was administrated. The liver samples were collected 12 h after ConA treatment and the protein and mRNA expressions of SIRT1 were measured. (A) Western blot analysis for protein levels of hepatic SIRT1. (B) RT-PCR analysis for mRNA levels of hepatic SIRT1. Values are presented as means ± SEM (n = 3). *p b 0.01 vs. control group, #p b 0.01 vs. ConA group.
in the liver of the mice treated with ConA. Whereas, in SalA pretreatment group, ConA-induced apoptosis was dramatically attenuated. The pro-apoptotic factor p66shc plays a crucial role in regulating cellular responses to apoptosis, oxidative stress and aging. For example, it was shown that SIRT1 overexpression suppressed high-glucoseinduced p66shc expression in human umbilical vein endothelial cells (Zhou et al., 2011). We further explored the regulation of p66shc expression by SIRT1 in ConA-induced hepatitis. As shown in Figs. 6B and C, p66shc mRNA and protein levels in the liver were significantly increased in the ConA treatment group compared to the control group. Whereas, SalA pretreatment effectively repressed p66shc mRNA and protein expressions in a dose-dependent manner (Fig. 6C). These results indicated that the protective effects of SalA against ConA-induced liver injury may be related to SIRT1-mediated inhibition of p66shc expression. In order to investigate the above hypothesis further, we examined the effect of SalA on p66shc expression following siRNA knockdown of SIRT1 in HepG2 cells. We found that knockdown of SIRT1 increased p66shc expression compared to control siRNA. This was in accordance with data from a previous study, which showed that SIRT1 repressed high-glucose-induced p66Shc expression through epigenetic chromatin modification (Zhou et al., 2011). Interestingly, SalA-mediated downregulation of p66shc in HepG2 cells was attenuated by inhibition of SIRT1 expression (Fig. 7A). In our immunofluorescence experiments,
GFP-tagged SIRT1 and RFP-p66shc clearly co-localized in nucleus of the cells treated with control siRNA with or without SalA pretreatment. Whereas, when SIRT1 was knocked down, SIRT1 fluorescence clearly decreased, which was closely associated with an increase in p66shc fluorescence in comparison to control siRNA groups. As expected, SalAinduced up-regulation of SIRT1 and p66shc down-regulation were all attenuated in SIRT1 knockdown cells that were pretreated with SalA in comparison to that in control siRNA cells pretreated with SalA (Fig. 7B). These findings revealed that SalA may enhance SIRT1-mediated repression of p66Shc expression, and have an anti-apoptotic role in ConAinduced hepatitis.
Effects of SalA on cleaved caspase-3, Bcl-xL, and NF-κB expressions We subsequently investigated the effect of SalA on expression of apoptosis-related factors, such as cleaved caspase-3, Bcl-xL, and proinflammatory mediator NF-κB. As shown in Fig. 8, ConA administration enhanced the expressions of cleaved caspase-3 and NF-κB, but down regulated Bcl-xL expression, as compared to the control. Moreover, pretreatment with SalA prior to ConA exposure resulted in increased Bcl-xL accumulation, but conversely attenuated expressions of cleaved caspase3 and NF-κB in a dose-dependent manner. These results suggested that
Fig. 5. Dose and time-dependent effects of SalA on SIRT1 induction in HepG2 cells. (A) HepG2 cell cultures were treated with SalA from 0 up to 48 h (0, 3, 6, 12, 24, and 48 h). The SIRT1 protein was determined by Western blotting using specific antibodies. *p b 0.01 vs. control group at corresponding time points. (B) HepG2 cells were pretreated with various doses of SalA, from 0.2, 2 to 20 μM for 6 h and the figure showed a dose-dependent response of SIRT1 to SalA treatment. *p b 0.01 vs. control group. (C) HepG2 cells were transfected with a SIRT1 siRNA or control siRNA and transfected cells were exposed to 20 μM SalA for 6 h as described in the Materials and methods section. Protein level of SIRT1 was evaluated by Western blotting. *p b 0.01 vs. si-control group, #p b 0.01 vs. si-control + SalA group.
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Fig. 6. The induction of SIRT1 by SalA decreases ConA-induced up-regulation of p66shc and exerts an anti-apoptotic role. (A) SalA attenuates ConA-induced hepatocyte apoptosis in vivo. Liver tissues were examined by TUNEL assay and were imaged by fluorescent microscopy. The content of TUNEL-positive cells was equal to the number of green points in the photograph. Results were obtained from 10 to 12 frames/group, from three or four animals/group and presented as means ± SEM. The groups were a. control; b. control + SalA (25 mg/kg); c. ConA; d. ConA + SalA (15 mg/kg); and e. ConA + SalA (25 mg/kg). (B) ConA-induced up-regulation of p66shc is decreased by SalA. The mice were treated with SalA for 30 min before ConA administrated. The proteins in liver samples were analyzed for p66shc. (C) mRNA expression of p66shc from the liver samples as described for (B). Values are means ± SEM (n = 3). *p b 0.01 vs. control group, #p b 0.01 vs. ConA group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the effect of SalA on ConA-induced acute liver injury may be related to repression of hepatocyte apoptosis and inflammation. Discussion Salvianolic acids, including SalA, SalB and a rosmarinic acid, are the major active constituents of water-soluble compounds extracted from S. miltiorrhiza. It is well known that SalA is a potent free radical scavenger, and acts as the most effective polyphenolic antioxidant among the salvianolic acids (Wang et al., 2011). It was recently shown that SalA prevents ischemia/reperfusion-induced myocardial damage by reducing apoptosis and necrosis both in vivo and in vitro (Fan et al., 2012; Pan et al., 2011). At cellular level, SalA regulates NF-κB-dependent inflammatory pathways via inhibition of IKKβ (Oh et al., 2011). These properties indicate that in addition to being a reactive oxygen species (ROS) scavenger, SalA also possesses an ability to modulate the expression of the genes that regulate apoptosis and inflammation. Besides, it has been reported that SalA may be a potential immunomodulator in cardiovascular disease, as it shares the core structure of rosmarinic acid, which inhibits T cell receptor (TCR)-induced T cell activation in the Lck SH2 domain of the Src family protein of tyrosine kinases (Ho and Hong, 2011; Won et al., 2003). Nevertheless, the mechanisms under protective effects of SalA on ConA-induced hepatitis remain unexplored. In this study, we observed that SalA pretreatment protected mice from ConA-induced hepatic injury, as determined by several measurements: (a) the attenuated serum ALT and AST levels, and the ameliorated histopathological injury; (b) the decreased serum cytokine IFN-γ and TNF-α levels; (c) the up-regulation of SIRT1 expression; (d) the down-regulation of p66shc; and (e) the decreased expression of cleaved caspase-3 and the increased Bcl-xL expression, as well as the decreased NF-κB expression. Taken together, these findings suggest that SalA pretreatment is beneficial for acute liver injury induced by ConA. We were interested in determining the mechanism by which SalA exerts its protective effects against ConA-induced hepatitis. It has been
reported that SIRT1 expression is highly induced in activated or anergic T cells (Zhang et al., 2009). When mice were immunized with myelin oligodendrocyte glycoprotein 35–55 peptide to induce experimental autoimmune encephalomyelitis (EAE), sirt1−/− mice were more sensitive to EAE as compared to sirt1+/− mice, suggesting that SIRT1 negatively regulates T cell activation, and is possibly required for T cell tolerance. Likewise, in our study, ConA administration led to an increase in SIRT1 expression. The phenomena, SIRT1 up regulation, manifested itself in the model of ConA-induced hepatitis where T cells were activated in abundance. In other words, increased SIRT1 expression is an adaptive response to ConA-induced hepatitis. More importantly, SalA pretreatment enhanced SIRT1 expression, indicating that SalA may be a potential activator of SIRT1, and may protect mice from ConA-induced T cell-mediated hepatic injury through SIRT1 up regulation. We entertained such a possibility via siRNA knockdown of SIRT1 expression in HepG2 cells. As expected, the results showed that SIRT1 knockdown inhibited the effect of SalA in the up-regulation of SIRT1, confirming that SalA is a potential activator of SIRT1. Thus, SIRT1 activation by SalA contributes to the prevention of ConA-induced hepatitis. Since hepatocyte apoptosis plays an important role in ConA-induced hepatitis, we further explored the anti-apoptotic pathway associated with SIRT1. The p66shc adaptor protein regulates ROS levels, apoptosis induction, and the lifespan of mammals (Galimov, 2010). Indeed, p66Shc−/− fibroblasts have increased resistance to treatment with oxidants, which correlate with a reduction in apoptotic responses to these stimuli. Conversely, p66Shc overexpression results in enhanced stress-induced apoptosis (Migliaccio et al., 1999). In the same vein, p66shc knockdown cells exhibited lower cellular ROS levels and less apoptosis compared to control cells when exposed to hypoxia/reoxygenation (H/R) injury (Haga et al., 2008). In our study, p66shc expression significantly increased in the ConA treatment group, while pretreatment with SalA led to repression of p66shc expression in a dose-dependent manner. Collectively, these results confirmed that p66shc plays an important role in apoptosis induced by various agents (Galimov, 2010).
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Fig. 7. SIRT1 negatively regulates p66shc expression in HepG2 cells. (A) HepG2 cells were cultured, transfected with control or SIRT1 siRNA and exposed to 20 μM SalA for 6 h as described in the Materials and methods section. The p66shc protein expression was detected by Western blotting. β-actin was used as a loading control. *p b 0.01 vs. si-control group, #p b 0.01 vs. si-control + SalA group. (B) The expressions of SIRT1 and p66shc were detected by immunofluorescence. SIRT1 was labeled with an antibody conjugated to FITC (green), and p66shc was labeled with an antibody conjugated to Cy3 (red). DAPI was used to counterstain nucleus. Scale bar equals to 100 μm. The images are representative of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
In order to study the potential mechanisms underlying p66shcmediated cell apoptosis in ConA-induced hepatitis, we also evaluated cleaved caspase-3 and Bcl-xL expressions. Cleaved caspase-3 is a pro-apoptotic protein that acts as an early indicator of apoptosis in
p66shc-associated injury both in vivo and in vitro (Ben et al., 2012; Clark et al., 2010). While Bcl-xL, an isoform of the Bcl-2 family, can inhibit apoptosis induced by p66shc knockdown in hepatocyte H/R-induced cellular apoptosis (Haga et al., 2008). Our findings
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Fig. 8. Effects of SalA on hepatoprotective molecules in the liver. Mice were treated with SalA for 30 min before ConA was administrated and the liver samples were collected 12 h after ConA treatment. Cleaved caspase-3, Bcl-xL and NF-κB protein expressions were measured by Western blotting using specific antibodies. β-actin was used as a loading control. Values are means ± SEM (n = 3). *p b 0.01 vs. control group, #p b 0.01 vs. ConA group.
showed that the expression of cleaved caspase-3 significantly increased in ConA treatment group, while the pretreatment with SalA markedly reduced its expression. However, Bcl-xL expression was completely opposed to the expression of cleaved caspase-3 as anticipated. Thus, it can be concluded that p66shc plays a vital role in hepatocyte apoptosis induced by ConA. It has been well documented that SIRT1 can affect the promoter of its target gene through deacetylation of histone H3 lysine 9 (Shankaranarayana et al., 2003). In accord, it was recently shown that SIRT1 overexpression inhibited high-glucose-induced p66Shc expression at both the mRNA and protein levels through modification of histone H3, which was accompanied by reduced oxidative stress and improved endothelial function (Chen et al., 2012; Zhou et al., 2011). Consistent with these observations, we found that pretreatment with SalA augmented SIRT1 expression and reversed the up-regulation of p66shc expression caused by ConA. These results suggest that SIRT1 may suppress p66Shc expression during ConA-induced acute liver injury. Thus, the SalA-up-regulated SIRT1 plays a crucial hepatoprotective role in ConA-induced hepatitis. To further explain the repression of p66Shc expression by SIRT1 during pretreatment with SalA, our siRNA experiments with HepG2 cells showed that SIRT1 siRNA significantly reversed the decreased expression of p66shc in HepG2 cells compared to the control siRNA group, indicating that SIRT1 is essential for repression of p66Shc expression. Coherently, the SIRT1 overexpression by SalA pretreatment decreased p66shc expression in cells transfected with control siRNA, whereas transfection of SIRT1 siRNA attenuated the effect of SalA on p66shc expression (Fig. 7A). These findings were confirmed by immunofluorescence analysis (Fig. 7B). Together, these data suggest that p66shc is a target of SIRT1, and activation of SIRT1 by SalA confers protection against ConA-induced hepatitis, which may be related to repression of p66shc expression. It is known that ConA-induced hepatic injury is associated with the release of pro-inflammatory cytokines such as TNF-α and IFN-γ (Oh et al., 2011). Production of these and other inflammatory mediators mainly depends upon activation of NF-κB, a ubiquitous transcription factor that regulates a number of genes involved in inflammation. Our examination on NF-κB expression after pretreatment with SalA in ConA-induced hepatitis further suggests that the protective effect of SalA is also related to inhibition of NF-κB-mediated inflammation. In summary, the present study for the first time revealed that SalA has a protective effect against ConA-induced hepatitis, which is a wellcharacterized model of T cell-mediated hepatitis. The protective effects of SalA are associated with the up-regulation of SIRT1 expression and accompanied by down-regulation of p66shc, resulting in a profound reduction in cleaved caspase-3 and NF-κB levels, and up-regulation of
Bcl-xL. These results indicate that SalA confers protection against ConA-induced hepatitis, at least in part through SIRT1-mediated repression of p66shc expression. Further studies are needed to determine the precise mechanism through which SalA modulates SIRT1 expression. Given that SalA is beneficial in prevention of ConA-induced hepatitis, which mimics many clinical diseases, such as fulminant hepatitis, viral hepatitis and autoimmune hepatitis, it would be interesting to explore whether SalA can be used for treatment of experimental hepatitis in other proper animal models. Conflict of interest The authors declared no conflict of interest. Acknowledgments This work was supported by grants from the Chinese National Natural Science Foundation (No. 81171850) and the Key Laboratory Foundation of Liaoning Province, China (No. LS2010052). References Alcendor, R.R., Kirshenbaum, L.A., Imai, S., Vatner, S.F., Sadoshima, J., 2004. Silent information regulator 2alpha, a longevity factor and class III histone deacetylase, is an essential endogenous apoptosis inhibitor in cardiac myocytes. Circ. Res. 95, 971–980. Alia, M., Ramos, S., Mateos, R., Bravo, L., Goya, L., 2005. Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2). J. Biochem. Mol. Toxicol. 19, 119–128. Amin, A.R., Paul, R.K., Thakur, V.S., Agarwal, M.L., 2007. A novel role for p73 in the regulation of Akt-Foxo1a-Bim signaling and apoptosis induced by the plant lectin, concanavalin A. Cancer Res. 67, 5617–5621. Ben, D.F., Yu, X.Y., Ji, G.Y., Zheng, D.Y., Lv, K.Y., Ma, B., Xia, Z.F., 2012. TLR4 mediates lung injury and inflammation in intestinal ischemia–reperfusion. J. Surg. Res. 174, 326–333. Chen, H., Wan, Y., Zhou, S., Lu, Y., Zhang, Z., Zhang, R., Chen, F., Hao, D., Zhao, X., Guo, Z., Liu, D., Liang, C., 2012. Endothelium-specific SIRT1 overexpression inhibits hyperglycemiainduced upregulation of vascular cell senescence. Sci. China Life Sci. 55, 467–473. Clark, J.S., Faisal, A., Baliga, R., Nagamine, Y., Arany, I., 2010. Cisplatin induces apoptosis through the ERK-p66shc pathway in renal proximal tubule cells. Cancer Lett. 297, 165–170. Cosentino, F., Francia, P., Camici, G.G., Pelicci, P.G., Luscher, T.F., Volpe, M., 2008. Final common molecular pathways of aging and cardiovascular disease: role of the p66Shc protein. Arterioscler. Thromb. Vasc. Biol. 28, 622–628. Fan, H.Y., Fu, F.H., Yang, M.Y., Xu, H., Zhang, A.H., Liu, K., 2010. Antiplatelet and antithrombotic activities of salvianolic acid A. Thromb. Res. 126, e17–e22. Fan, H., Yang, L., Fu, F., Xu, H., Meng, Q., Zhu, H., Teng, L., Yang, M., Zhang, L., Zhang, Z., Liu, K., 2012. Cardioprotective effects of salvianolic acid a on myocardial ischemia– reperfusion injury in vivo and in vitro. Evid.-Based Complement. Altern. Med. 2012, 508938. Galimov, E.R., 2010. The role of p66shc in oxidative stress and apoptosis. Acta Naturae 2, 44–51. Gao, B., Kong, Q., Kemp, K., Zhao, Y.S., Fang, D., 2012. Analysis of sirtuin 1 expression reveals a molecular explanation of IL-2-mediated reversal of T-cell tolerance. Proc. Natl. Acad. Sci. U. S. A. 109, 899–904.
76
X. Xu et al. / Toxicology and Applied Pharmacology 273 (2013) 68–76
Haga, S., Terui, K., Fukai, M., Oikawa, Y., Irani, K., Furukawa, H., Todo, S., Ozaki, M., 2008. Preventing hypoxia/reoxygenation damage to hepatocytes by p66(shc) ablation: up-regulation of anti-oxidant and anti-apoptotic proteins. J. Hepatol. 48, 422–432. Ho, J.H., Hong, C.Y., 2011. Salvianolic acids: small compounds with multiple mechanisms for cardiovascular protection. J. Biomed. Sci. 18, 30. Hong, K.S., Park, J.I., Kim, M.J., Kim, H.B., Lee, J.W., Dao, T.T., Oh, W.K., Kang, C.D., Kim, S.H., 2012. Involvement of SIRT1 in hypoxic down-regulation of c-Myc and beta-catenin and hypoxic preconditioning effect of polyphenols. Toxicol. Appl. Pharmacol. 259, 210–218. Kelly, G.S., 2010a. A review of the sirtuin system, its clinical implications, and the potential role of dietary activators like resveratrol: part 2. Altern. Med. Rev. 15, 313–328. Kelly, G., 2010b. A review of the sirtuin system, its clinical implications, and the potential role of dietary activators like resveratrol: part 1. Altern. Med. Rev. 15, 245–263. Koch, O.R., Fusco, S., Ranieri, S.C., Maulucci, G., Palozza, P., Larocca, L.M., Cravero, A.A., Farre', S.M., De Spirito, M., Galeotti, T., Pani, G., 2008. Role of the life span determinant P66(shcA) in ethanol-induced liver damage. Lab. Invest. 88, 750–760. Kong, S., Kim, S.J., Sandal, B., Lee, S.M., Gao, B., Zhang, D.D., Fang, D., 2011. The type III histone deacetylase Sirt1 protein suppresses p300-mediated histone H3 lysine 56 acetylation at Bclaf1 promoter to inhibit T cell activation. J. Biol. Chem. 286, 16967–16975. Kong, S., McBurney, M.W., Fang, D., 2012. Sirtuin 1 in immune regulation and autoimmunity. Immunol. Cell Biol. 90, 6–13. Lee, B.W., Chun, S.W., Kim, S.H., Lee, Y., Kang, E.S., Cha, B.S., Lee, H.C., 2011. Lithospermic acid B protects beta-cells from cytokine-induced apoptosis by alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1. Toxicol. Appl. Pharmacol. 252, 47–54. Migliaccio, E., Giorgio, M., Mele, S., Pelicci, G., Reboldi, P., Pandolfi, P.P., Lanfrancone, L., Pelicci, P.G., 1999. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 402, 309–313. Migliaccio, E., Giorgio, M., Pelicci, P.G., 2006. Apoptosis and aging: role of p66Shc redox protein. Antioxid. Redox Signal. 8, 600–608. Oh, K.S., Oh, B.K., Mun, J., Seo, H.W., Lee, B.H., 2011. Salvianolic acid A suppress lipopolysaccharide-induced NF-kappaB signaling pathway by targeting IKKbeta. Int. Immunopharmacol. 11, 1901–1906. Pan, H., Li, D., Fang, F., Chen, D., Qi, L., Zhang, R., Xu, T., Sun, H., 2011. Salvianolic acid A demonstrates cardioprotective effects in rat hearts and cardiomyocytes after ischemia/ reperfusion injury. J. Cardiovasc. Pharmacol. 58, 535–542. Sequeira, J., Boily, G., Bazinet, S., Saliba, S., He, X., Jardine, K., Kennedy, C., Staines, W., Rousseaux, C., Mueller, R., McBurney, M.W., 2008. sirt1-null mice develop an autoimmune-like condition. Exp. Cell Res. 314, 3069–3074. Shankaranarayana, G.D., Motamedi, M.R., Moazed, D., Grewal, S.I., 2003. Sir2 regulates histone H3 lysine 9 methylation and heterochromatin assembly in fission yeast. Curr. Biol. 13, 1240–1246. Shi, G., Zhang, Z., Zhang, R., Zhang, X., Lu, Y., Yang, J., Zhang, D., Zhang, Z., Li, X., Ning, G., 2012. Protective effect of andrographolide against concanavalin A-induced liver injury. Naunyn Schmiedebergs Arch. Pharmacol. 385, 69–79.
Sternak, M., Khomich, T.I., Jakubowski, A., Szafarz, M., Szczepanski, W., Bialas, M., Stojak, M., Szymura-Oleksiak, J., Chlopicki, S., 2010. Nicotinamide N-methyltransferase (NNMT) and 1-methylnicotinamide (MNA) in experimental hepatitis induced by concanavalin A in the mouse. Pharmacol. Rep. 62, 483–493. Tomita, K., Teratani, T., Suzuki, T., Oshikawa, T., Yokoyama, H., Shimamura, K., Nishiyama, K., Mataki, N., Irie, R., Minamino, T., Okada, Y., Kurihara, C., Ebinuma, H., Saito, H., Shimizu, I., Yoshida, Y., Hokari, R., Sugiyama, K., Hatsuse, K., Yamamoto, J., Kanai, T., Miura, S., Hibi, T., 2012. p53/p66Shc-mediated signaling contributes to the progression of non-alcoholic steatohepatitis in humans and mice. J. Hepatol. 57, 837–843. Wang, L., Zhang, L., Chen, Z.B., Wu, J.Y., Zhang, X., Xu, Y., 2009a. Icariin enhances neuronal survival after oxygen and glucose deprivation by increasing SIRT1. Eur. J. Pharmacol. 609, 40–44. Wang, S.B., Yang, X.Y., Tian, S., Yang, H.G., Du, G.H., 2009b. Effect of salvianolic acid A on vascular reactivity of streptozotocin-induced diabetic rats. Life Sci. 85, 499–504. Wang, T., Shan, S.Y., Han, B., Zhang, L.M., Fu, F.H., 2011. Salvianolic acid A exerts antiamnesic effect on diazepam-induced anterograde amnesia in mice. Neurochem. Res. 36, 103–108. Wang, C., Nie, H., Li, K., Zhang, Y.X., Yang, F., Li, C.B., Wang, C.F., Gong, Q., 2012a. Curcumin inhibits HMGB1 releasing and attenuates concanavalin A-induced hepatitis in mice. Eur. J. Pharmacol. 697, 152–157. Wang, H.X., Liu, M., Weng, S.Y., Li, J.J., Xie, C., He, H.L., Guan, W., Yuan, Y.S., Gao, J., 2012b. Immune mechanisms of concanavalin A model of autoimmune hepatitis. World J. Gastroenterol. 18, 119–125. Won, J., Hur, Y.G., Hur, E.M., Park, S.H., Kang, M.A., Choi, Y., Park, C., Lee, K.H., Yun, Y., 2003. Rosmarinic acid inhibits TCR-induced T cell activation and proliferation in an Lckdependent manner. Eur. J. Immunol. 33, 870–879. Wu, Z.M., Wen, T., Tan, Y.F., Liu, Y., Ren, F., Wu, H., 2007. Effects of salvianolic acid a on oxidative stress and liver injury induced by carbon tetrachloride in rats. Basic Clin. Pharmacol. Toxicol. 100, 115–120. Xie, J., Wan, J., Jiang, R., Lu, H., Peng, X., Zhang, L., 2013. Upregulation of Sirt1 in carbontetrachloride-induced acute liver injury. Drug Chem. Toxicol. 36, 277–283. Yu, W., Fu, Y.C., Zhou, X.H., Chen, C.J., Wang, X., Lin, R.B., Wang, W., 2009. Effects of resveratrol on H(2)O(2)-induced apoptosis and expression of SIRTs in H9c2 cells. J. Cell. Biochem. 107, 741–747. Yu, X., Zhang, L., Yang, X., Huang, H., Huang, Z., Shi, L., Zhang, H., Du, G., 2012. Salvianolic acid A protects the peripheral nerve function in diabetic rats through regulation of the AMPK–PGC1alpha–Sirt3 axis. Molecules 17, 11216–11228. Zhang, J., Lee, S.M., Shannon, S., Gao, B., Chen, W., Chen, A., Divekar, R., McBurney, M.W., Braley-Mullen, H., Zaghouani, H., Fang, D., 2009. The type III histone deacetylase Sirt1 is essential for maintenance of T cell tolerance in mice. J. Clin. Invest. 119, 3048–3058. Zhang, Y.Q., Tang, Y., Wu, A.L., Zhu, H.B., 2010. Salvianolic acid A displays cardioprotective effects in in vitro models of heart hypoxia/reoxygenation injury. J. Asian Nat. Prod. Res. 12, 899–915. Zhou, S., Chen, H.Z., Wan, Y.Z., Zhang, Q.J., Wei, Y.S., Huang, S., Liu, J.J., Lu, Y.B., Zhang, Z.Q., Yang, R.F., Zhang, R., Cai, H., Liu, D.P., Liang, C.C., 2011. Repression of p66Shc expression by SIRT1 contributes to the prevention of hyperglycemia-induced endothelial dysfunction. Circ. Res. 109, 639–648.