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Salvianolic acid B attenuates apoptosis and inflammation via SIRT1 activation in experimental stroke rats
Brain Research Bulletin 115 (2015) 30–36
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Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Salvianolic acid B attenuates apoptosis and inflammation via SIRT1 activation in experimental stroke rats Hongdi Lv a , Ling Wang b , Jinchang Shen c,∗ , Shaojun Hao d , Aimin Ming e , Xidong Wang d , Feng Su a , Zhengchen Zhang d a
Department of Cardiology, No. 371 Central Hospital of PLA, Xinxiang, Henan 453000, China Department of Nursing, No. 371 Central Hospital of PLA, Xinxiang, Henan 453000, China c Department of Interventional Radiology, No. 371 Central Hospital of PLA, Xinxiang, Henan 453000, China d Department of Drugs and Equipment, No. 371 Central Hospital of PLA, Xinxiang, Henan 453000, China e Department of Urology, No. 371 Central Hospital of PLA, Xinxiang, Henan 453000, China b
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Article history: Received 15 April 2015 Received in revised form 7 May 2015 Accepted 8 May 2015 Available online 14 May 2015 Keywords: Salvianolic acid B Ischemic stroke SIRT1 Apoptosis Inflammation
a b s t r a c t Silent information regulator 1 (SIRT1), a histone deacetylase, has been suggested to be effective in ischemic brain diseases. Salvianolic acid B (SalB) is a polyphenolic and one of the active components of Salvia miltiorrhiza Bunge. Previous studies suggested that SalB is protective against ischemic stroke. However, the role of SIRT1 in the protective effect of SalB against cerebral ischemia has not been explored. In this study, the rat brain was subjected to middle cerebral artery occlusion (MCAO). Before this surgery, rats were intraperitoneally administrated SalB with or without EX527, a specific SIRT1 inhibitor. The infarct volume, neurological score and brain water content were assessed. In addition, levels of TNF␣ and IL-1 in the brain tissues were detected by commercial ELISA kits. And the expression levels of SIRT, Ac-FOXO1, Bcl-2 and Bax were detected by Western blot. The results suggested that SalB exerted a cerebral-protective effect, as shown by reduced infarct volume, lowered brain edema and increased neurological scores. SalB also exerted anti-inflammatory effects as indicated by the decreased TNF-␣ and IL-1 levels in the brain tissue. Moreover, SalB upregulated the expression of SIRT1 and Bcl-2 and downregulated the expression of Ac-FOXO1 and Bax. These effects of SalB were abolished by EX527 treatment. In summary, our results demonstrate that SalB treatment attenuates brain injury induced by ischemic stoke via reducing apoptosis and inflammation through the activation of SIRT1 signaling. © 2015 Published by Elsevier Inc.
1. Introduction Ischemic stroke remains a leading cause of death and is associated with a high incidence of mortality and morbidity worldwide (Donnan et al., 2008; Hankey, 2012). Ischemic stroke results from the occlusion of a cerebral vessel and the middle cerebral artery (MCA) is often involved. When blood supply is damaged, a series of pathophysiologic events occur, such as glutamate excitotoxicity, calcium overload, oxidative stress, inflammation, apoptosis and mitochondrial dysfunction (Mattiasson et al., 2003; Reiter et al., 2007; Sahota and Savitz, 2011; Thompson et al., 2015). Although thrombolytic agents have been used for clinical treatment of ischemic stroke, their application is limited due to the narrow therapeutic window and other safety concerns (Mattiasson et al.,
∗ Corresponding author. Tel.: +86 373 3541018; fax: +86 373 3541018. E-mail address: [email protected] (J. Shen). http://dx.doi.org/10.1016/j.brainresbull.2015.05.002 0361-9230/© 2015 Published by Elsevier Inc.
2003; Yepes et al., 2009). Therefore, there is an urgent need for the development of novel therapeutic agents for treatment of ischemic stroke. Danshen, the dried root of Salvia miltiorrhiza Bunge, is a Chinese medicinal herb. It has been widely used for treatment of hepatitis, hemorrhage, menstrual abnormalities, as well as ischemic brain and heart diseases (Ji et al., 2000; Wang et al., 2013). It is one of the main components of Xinnaoning, a drug used for cardiovascular and cerebral vascular diseases. Salvianolic acid B (SalB, Fig. 1), the most abundant and bioactive compound of Danshen, has been suggested to exert various pharmacological effects, such as antioxidation, anti-inflammation and anti-tumor (Li et al., 2014; Tang et al., 2014; Wang et al., 2013). Previous studies have suggested that SalB has protective effects in ischemic stroke. However, the underlying mechanisms have not been fully elucidated. Silent information regulator 1 (SIRT1) is a histone deacetylase, and its activity is dependent on nicotinamide adenine dinucleotide (NAD+). SIRT1 expression decreases with age and is known
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2.3. Rat model of middle cerebral artery occlusion A middle cerebral artery occlusion (MCAO) model was established as previously described (Longa et al., 1989). Briefly, rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (350 mg/kg). The right common carotid artery was then exposed and isolated. The MCA was occluded by inserting a monofilament nylon suture with a heat-rounded tip into the internal carotid artery, which was advanced further until it closed the origin of the MCA. Thirty minutes after induction of ischemia, reperfusion was initiated by withdrawal of the monofilament. Sham-operated control rats received the same surgical procedure without MCA occlusion. Rectal temperature was maintained at 37 ◦ C using a heating blanket and heating lamp during the surgery. 2.4. Experimental protocol
Fig. 1. Structure of salvianolic acid B.
to plays a key role in the longevity effects induced by calorie restriction (Kilic et al., 2015; Ramis et al., 2015). SIRT1 deacetylates and activates forkhead box O (FOXO), which synthesizes antioxidants, such as manganese superoxide dismutase and catalase, promoting cellular resistance against oxidative stress (Brunet et al., 2004). In addition, SIRT1 has been suggested to be critical in cerebral protection. Ischemia preconditioning (Thompson et al., 2013) and hyperbaric oxygen preconditioning (Herskovits and Guarente, 2014) reportedly decrease cerebral ischemia-reperfusion injury via SIRT1 activation. Intriguingly, a previous study suggested that SalB protects against ischemic stroke (Tian et al., 2009; Zhu et al., 2013). In addition, SalB is suggested to protect against acute ethanol-induced liver injury through SIRT1-mediated deacetylation of p53 (Li et al., 2014). However, the role of SIRT1 in the protective effect of SalB against ischemic stroke remains unclear.
SalB was dissolved in normal saline and injected intraperitoneally at a dose of 25 mg/kg administrated twice (immediately after induction of ischemia and at the beginning of reperfusion). As reported previously (Yang et al., 2013), the SIRT1 inhibitor EX527 was first dissolved in dimethyl sulphoxide (DMSO) and diluted to the final concentration with normal saline (the final DMSO concentration <2%). EX527 was intraperitoneally injected at the dose of 5 mg/kg or the same volume of vehicle was performed every 2 days for four times before MCAO. All the rats were randomly assigned to the following groups (n = 10 in each group): Sham, MCAO, MCAO + SalB, MCAO + SalB + EX527 and MCAO + EX527. 2.5. Evaluation of neurological deficit Neurological deficit scores were evaluated by an observer blinded to the experimental groups at 24 h after MCAO as reported previously (Longa et al., 1989). The scores are as follows: 0, no motor deficits (normal); 1, forelimb weakness and torso turning to the ipsilateral side when held by tail (mild); 2, circling to the contralateral side but normal posture at rest (moderate); 3, unable to bear weight on the affected side at rest (severe); 4, no spontaneous locomotor activity or barrel rolling (critical).
2. Materials and methods 2.6. Evaluation of infarct volume 2.1. Reagents SalB (purity >98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). 2,3,5-Triphenyltetrazolium chloride (TTC) was purchased from Sigma–Aldrich (St. Louis, MO, USA). EX527 was purchased from Tocris Bioscience (Bristol, UK). Antibodies against acetylateFOXO1 (Ac-FOXO1), Bcl2, Bax, and -actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against SIRT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit anti-goat, goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from the Zhongshan Company (Beijing, China).
Infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) at 24 h after reperfusion. Animals were euthanized and the brains were quickly removed. Then the brain was sliced into five coronal sections (3 mm thick each) and stained with 2% solution of TTC at 37 ◦ C for 20 min, followed by fixation in 4% paraformaldehyde. TTC-stained sections were photographed and the digital images were analyzed using image analysis software (Image-Pro Plus 5.1). The lesion volume was calculated by multiplying the area by the thickness of slices. The percentage hemisphere lesion volume (%HLV) was calculated by the following formula (Tatlisumak, 1998): %HLV = ([total infarct volume − (the volume of intact ipsilateral hemisphere − the volume of intact contralateral hemisphere)])/contralateral hemisphere volume × 100%.
2.2. Animals 2.7. Brain water content Male Sprague-Dawley rats (230–280 g), purchased from the Laboratory Animal Center of Xinxiang Medical University, were kept under a controlled environment (12/12 h light/dark cycle, 60 ± 5% humidity, 22 ± 3 ◦ C). And they have free access to water and food. All procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), revised 1996.
As described previously (Yang et al., 2015), after the wet weight of the brain tissues was quantified, the red and white parts of these brains were desiccated at 105 ◦ C for 48 h until the weight was constant. The total weight of the dried TTC-stained brains was obtained by measuring the desiccated red and white parts of these brains together, and the water content of each brain was calculated as follows: (wet weight − dried weight)/wet weight × 100%.
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Fig. 2. Effects of salvianolic acid B on infarct volume, neurological score, and brain water content in rats subjected to MCAO. (A) Infarct volume. (B) Neurological score. (C) Brain water content. The results are expressed as the mean ± S.E.M., n = 10, * P < 0.05 vs. the MCAO group.
2.8. Detection of inflammatory cytokines
2.9. Western blot
The inflammatory cytokines in brain tissue were measured using commercially ELISA kits for TNF-␣ and IL-1 (Beyotime, China). All spectrophotometric readings were performed with a microplate reader (Multiskan MK3, Thermo, USA). All procedures were performed according to the instructions.
The ischemic penumbra of cerebral cortex was collected. The brain tissue was then lysed in ice-cold lysis buffer (50 mM Tris–HCl, 1 mM EDTA, 1 mM EGTA, 0.5 mMNa3 VO4 , 0.1% 2-mercaptoethanol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium -glyceropyrophosphate, 0.1 mM phenylmethanesulfonyl
Fig. 3. Effects of salvianolic acid B on SIRT1, Ac-FOXO1, Bcl-2 and Bax expression in rats subjected to MCAO. The representative images of SIRT1, Ac-FOXO1, Bcl-2, and Bax are shown. The results are expressed as the mean ± S.E.M., n = 10, * P < 0.05 vs. the MCAO group.
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fluoride and protease inhibitor mixture) for 10 min. The lysates were centrifuged for 15 min at 12,000 × g, and the resulting supernatant was collected and boiled. Protein concentrations of the extracts were measured by BCA assay. The whole protein 50 g was resolved on 8–12% SDS polyacrylamide gel, then transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBST (150 mM NaCl, 50 mM Tris [pH 7.5], 0.1% Tween-20) and then incubated with antibodies against SIRT1 (1:500), Ac-FOXO1, Bcl2, Bax and -actin (1:1000) at 4 ◦ C overnight, followed by washes with TBST. The membranes were then probed with the appropriate secondary antibodies (1:5000) at room temperature for 2 h and washed with TBST. The protein bands were detected using a BioRad imaging system (BioRad, Hercules, CA, USA) and quantified using the Quantity One software package (West Berkeley, CA, USA). The value for the MCAO group was defined as 100%. 2.10. Statistical analysis Data were reported as mean ± standard error of the mean (S.E.M.). One-way analysis of variance (ANOVA) followed by the Dunnett’s test was performed to compare the differences (SPSS 13.0, Chicago, IL, USA). A value of P < 0.05 was considered to be statistically significant. 3. Results 3.1. SalB reduced brain injury after MCAO To investigate the potential protective effects of SalB against MCAO-induced brain injury, we examined the effect of SalB on brain infarct volume, neurological score and cerebral edema. Compared with the MCAO group, SalB significantly reduced infarct volume, neurological score and brain water content in MCAO + SalB group (Fig. 2) (P < 0.05). 3.2. Effect of SalB on the expressions of SIRT1, Ac-FOXO1, Bcl-2 and Bax To investigate the key proteins involved in the protective role of SalB, we analyzed the expression of SIRT1, Ac-FOXO1, Bcl-2 and Bax. As shown in Fig. 3, SalB treatment significantly increased SIRT1 and Bcl-2 expression in MCAO + SalB compared with their expression in MCAO group (P < 0.05). SalB also decreased the Ac-FOXO1, and Bax expression in MCAO + SalB group compared with their expression in MCAO group (Fig. 3) (P < 0.05). 3.3. Effect of SalB on the production of inflammatory cytokines in brain tissue To elucidate the effect of SalB on inflammation induce by MCAO, we evaluated the production of TNF-␣ and IL-1 in the brain tissue. SalB dramatically decreased the production of TNF-␣ and IL-1 in the brain tissue in MCAO + SalB group compared with those in the MCAO group (Fig. 4) (P < 0.05). 3.4. EX527 abolished the protective effect of SalB We used EX527, a specific SIRT1 inhibitor, to investigate whether SIRT1 is a key molecule in the protective effect of SalB and to elucidate the underlying mechanisms. Compared with the MCAO group, SalB significantly reduced infarct volume, neurological score and brain water content in MCAO + SalB group. However, EX527 administration abolished the protective effect of SalB; it increased the infarct volume, neurological score and brain edema in
Fig. 4. Effects of salvianolic acid B and EX527 on TNF-␣ and IL-1 in the brain tissue. (A) The production of TNF-␣ in the brain tissue. (B) The production of IL-1 in the brain tissue. The results are expressed as the mean ± S.E.M., n = 10, ** P < 0.05 vs. the MCAO group, ## P < 0.05 vs. the MCAO + SalB group, $$ P < 0.05 vs. the MCAO + SalB + EX527 group.
MCAO + SalB + EX527 group compared with the MCAO + SalB group (Fig. 5) (P < 0.05). Western blot results suggested that SalB treatment significantly increased SIRT1 and Bcl-2 expression. SalB also decreased the expression of Ac-FOXO1 and Bax. However, EX527 significantly decreased SIRT1 and Bcl-2 expression and increased Ac-FOXO1 and Bax expression in the MCAO + SalB + EX527 group compared with that in the MCAO + SalB group (Fig. 6) (P < 0.05). 4. Discussion The major findings in the present study are: (1) SalB protects the brain from ischemia. (2) SalB reduces inflammation in the ischemic brain. (3) SalB decreases apoptosis in the ischemic brain. (4) The activation of SIRT1 is involved in the neuroprotective effect of SalB. S. miltiorrhiza has been widely used for treatment of various diseases in China (Zhou et al., 2005). Many studies have demonstrated that SalB exerts various pharmacological activities, such as antiapoptosis, anti-inflammation, anti-diabetes, promotion of cellular proliferation, differentiation and bone formation and preservation of normal cell functions (He and Shen, 2014; Huang et al., 2015; Shi et al., 2007; Wang et al., 2010; Xu et al., 2015; Zeng et al., 2010).
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Fig. 5. Effects of salvianolic acid B and EX527 on infarct volume, neurological score and brain water content in rats subjected to MCAO. (A) Infarct volume. (B) Neurological score. (C) Brain water content. The results are expressed as the mean ± S.E.M., n = 10, ** P < 0.05 vs. the MCAO group, ## P < 0.05 vs. the MCAO + SalB group, $$ P < 0.05 vs. the MCAO + SalB + EX527 group.
SalB has been shown to be beneficial in the central nervous system. Zhu et al. suggest that SMND-309, a novel derivative of SalB, protects rat brains ischemia and reperfusion injury by targeting the JAK2/STAT3 pathway (Zhu et al., 2013). SalB confers neuroprotection via anti-inflammatory and anti-oxidative effects in Alzheimer’s disease (Lee et al., 2013). And SalB attenuates cognitive dysfunctions in Alzheimer’s disease (Kim et al., 2011). In addition, SalB has been suggested to protect against acute ethanol-induced liver injury via SIRT1 activation (Li et al., 2014). These findings suggest that SalB regulates inflammation and apoptosis in many diseases and SIRT1 may be involved in this activity of SalB. SIRT1, a member of the class III group of histone deacetylases, has been suggested to be protective in cerebral ischemia (Yang et al., 2013, 2015). SIRT1 is thought to play an important role in neuroprotection against brain ischemia by deacetylation and subsequent inhibition of p53 and nuclear factor B-induced inflammatory and apoptotic pathways (Hernandez-Jimenez et al., 2013). It also mediates hyperbaric oxygen preconditioning-induced ischemic tolerance in the rat brain (Herskovits and Guarente, 2014). Consistent with these findings, we found that SalB augmented SIRT1 expression and inhibited apoptosis and inflammation. Our results suggest that SIRT1 activation by SalB plays a critical role in protection against cerebral ischemic injury. In this study, we first investigated the effect of SalB on infarct volume, neurological score and brain water content. Our results suggest that SalB significantly reduced infarct volume, neurological score and brain water content. Additionally, SalB dramatically up-regulated SIRT1 expression, leading to down-regulation of AcFOXO1, which is downstream of SIRT1. Moreover, to investigate the role of SalB in neuronal apoptosis, the expression of Bax and
Bcl-2 was analyzed. The results demonstrate that SalB significantly increased Bcl-2 and decreased Bax expression, indicating that SalB inhibits apoptosis in ischemic stroke. To further investigate the role of SIRT1 in the protective effect of SalB, we used EX527, a specific SIRT1, inhibitor to elucidate the underlying mechanisms. Our results suggest that EX527 abolishes the protective effect of SalB. EX527 significantly increased infarct volume, neurological score and brain water content. In addition, EX527 abolished the antiapoptotic effect of SalB, as evidenced by the increased Bax and decreased Bcl-2 expression. Therefore, these results suggest that SIRT1 activation by SalB contributes to the attenuation of brain injury induced by ischemic stroke. Ischemic stroke is associated with the production and release of pro-inflammatory cytokines such as TNF-␣ and IL-1 (Palencia et al., 2015), and this triggers additional inflammatory processes such as activation of cyclooxygenase-2 (COX-2), NOS, and NF-B (Mohammadi et al., 2012; Ridder and Schwaninger, 2009). The inflammation in turn may aggravate infarction, brain edema and neuronal death. The present results suggest that SalB alleviates inflammation as evidenced by the decrease of TNF-␣ and IL-1 levels in brain tissue. However, this effect was abolished by EX527, indicating that the anti-inflammatory effect of SalB is associated with SIRT1 signaling. In summary, the present study suggested for the first time that SalB has a protective effect against ischemic stroke via the activation of SIRT1. SalB activates SIRT1 signaling, accompanied by reduced expression of Ac-FOXO1, leading to increased Bcl-2 and decreased Bax expression. Taken together, these findings suggest that SalB may be an important therapeutic strategy for attenuating brain injury associated with ischemic stroke.
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Fig. 6. The effects of salvianolic acid B and EX527 on Ac-FOXO1, Bcl-2 and Bax expression in rats subjected to MCAO. The representative images of SIRT1, Ac-FOXO1, Bcl-2, and Bax are shown. The results are expressed as the mean ± S.E.M., n = 10, ** P < 0.05 vs. the MCAO group, ## P < 0.05 vs. the MCAO + SalB group, $$ P < 0.05 vs. the MCAO + SalB + EX527 group.
Conflict of interest There is no conflict of interest. References Brunet, A., Sweeney, L.B., Sturgill, J.F., Chua, K.F., Greer, P.L., Lin, Y., Tran, H., Ross, S.E., Mostoslavsky, R., Cohen, H.Y., Hu, L.S., Cheng, H.L., Jedrychowski, M.P., Gygi, S.P., Sinclair, D.A., Alt, F.W., Greenberg, M.E., 2004. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303, 2011–2015. Donnan, G.A., Fisher, M., Macleod, M., Davis, S.M., 2008. Stroke. Lancet 371, 1612–1623. Hankey, G.J., 2012. Anticoagulant therapy for patients with ischaemic stroke. Nat. Rev. Neurol. 8, 319–328. He, X., Shen, Q., 2014. Salvianolic acid B promotes bone formation by increasing activity of alkaline phosphatase in a rat tibia fracture model: a pilot study. BMC Complement. Altern. Med. 14, 493. Hernandez-Jimenez, M., Hurtado, O., Cuartero, M.I., Ballesteros, I., Moraga, A., Pradillo, J.M., McBurney, M.W., Lizasoain, I., Moro, M.A., 2013. Silent information regulator 1 protects the brain against cerebral ischemic damage. Stroke J. Cereb. Circ. 44, 2333–2337. Herskovits, A.Z., Guarente, L., 2014. SIRT1 in neurodevelopment and brain senescence. Neuron 81, 471–483. Huang, M., Wang, P., Xu, S., Xu, W., Xu, W., Chu, K., Lu, J., 2015. Biological activities of salvianolic acid B from Salvia miltiorrhiza on type 2 diabetes induced by high-fat diet and streptozotocin. Pharm. Biol., 1–8. Ji, X.Y., Tan, B.K., Zhu, Y.Z., 2000. Salvia miltiorrhiza and ischemic diseases. Acta Pharmacol. Sin. 21, 1089–1094.
Kilic, U., Gok, O., Erenberk, U., Dundaroz, M.R., Torun, E., Kucukardali, Y., Elibol-Can, B., Uysal, O., Dundar, T., 2015. A remarkable age-related increase in SIRT1 protein expression against oxidative stress in elderly: SIRT1 gene variants and longevity in human. PLOS ONE 10, e0117954. Kim, D.H., Park, S.J., Kim, J.M., Jeon, S.J., Kim, D.H., Cho, Y.W., Son, K.H., Lee, H.J., Moon, J.H., Cheong, J.H., Ko, K.H., Ryu, J.H., 2011. Cognitive dysfunctions induced by a cholinergic blockade and Abeta 25-35 peptide are attenuated by salvianolic acid B. Neuropharmacology 61, 1432–1440. Lee, Y.W., Kim, D.H., Jeon, S.J., Park, S.J., Kim, J.M., Jung, J.M., Lee, H.E., Bae, S.G., Oh, H.K., Son, K.H., Ryu, J.H., 2013. Neuroprotective effects of salvianolic acid B on an Abeta25-35 peptide-induced mouse model of Alzheimer’s disease. Eur. J. Pharmacol. 704, 70–77. Li, M., Lu, Y., Hu, Y., Zhai, X., Xu, W., Jing, H., Tian, X., Lin, Y., Gao, D., Yao, J., 2014. Salvianolic acid B protects against acute ethanol-induced liver injury through SIRT1-mediated deacetylation of p53 in rats. Toxicol. Lett. 228, 67–74. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke J. Cereb. Circ. 20, 84–91. Mattiasson, G., Shamloo, M., Gido, G., Mathi, K., Tomasevic, G., Yi, S., Warden, C.H., Castilho, R.F., Melcher, T., Gonzalez-Zulueta, M., Nikolich, K., Wieloch, T., 2003. Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma. Nat. Med. 9, 1062–1068. Mohammadi, M.T., Shid-Moosavi, S.M., Dehghani, G.A., 2012. Contribution of nitric oxide synthase (NOS) in blood-brain barrier disruption during acute focal cerebral ischemia in normal rat. Pathophysiol.: Off. J. Int. Soc. Pathophysiol./ISP 19, 13–20. Palencia, G., Medrano, J.A., Ortiz-Plata, A., Farfan, D.J., Sotelo, J., Sanchez, A., TrejoSolis, C., 2015. Anti-apoptotic, anti-oxidant, and anti-inflammatory effects of thalidomide on cerebral ischemia/reperfusion injury in rats. J. Neurol. Sci. 351, 78–87.
36
H. Lv et al. / Brain Research Bulletin 115 (2015) 30–36
Ramis, M.R., Esteban, S., Miralles, A., Tan, D.X., Reiter, R.J., 2015. Caloric restriction, resveratrol and melatonin: role of SIRT1 and implications for aging and relateddiseases. Mech. Ageing Dev. 146–148C, 28–41. Reiter, R.J., Tan, D.X., Manchester, L.C., Tamura, H., 2007. Melatonin defeats neurallyderived free radicals and reduces the associated neuromorphological and neurobehavioral damage. J. Physiol. Pharmacol.: Off. J. Pol. Physiol. Soc. 58 (Suppl. 6), 5–22. Ridder, D.A., Schwaninger, M., 2009. NF-kappaB signaling in cerebral ischemia. Neuroscience 158, 995–1006. Sahota, P., Savitz, S.I., 2011. Investigational therapies for ischemic stroke: neuroprotection and neurorecovery. Neurotherap.: J. Am. Soc. Exp. Neurotherap. 8, 434–451. Shi, C.S., Huang, H.C., Wu, H.L., Kuo, C.H., Chang, B.I., Shiao, M.S., Shi, G.Y., 2007. Salvianolic acid B modulates hemostasis properties of human umbilical vein endothelial cells. Thromb. Res. 119, 769–775. Tang, Y., Jacobi, A., Vater, C., Zou, X., Stiehler, M., 2014. Salvianolic acid B protects human endothelial progenitor cells against oxidative stress-mediated dysfunction by modulating Akt/mTOR/4EBP1, p38 MAPK/ATF2, and ERK1/2 signaling pathways. Biochem. Pharmacol. 90, 34–49. Tatlisumak, T., Carano, R.A., Takano, K., Opgenorth, T.J., Sotak, C.H., Fisher, M., 1998. A novel endothelin antagonist, A-127722, attenuates ischemic lesion size in rats with temporary middle cerebral artery occlusion: a diffusion and perfusion MRI study. Stroke J. Cereb. Circ. 29, 850–857 (discussion 857–858). Thompson, J.W., Dave, K.R., Saul, I., Narayanan, S.V., Perez-Pinzon, M.A., 2013. Epsilon PKC increases brain mitochondrial SIRT1 protein levels via heat shock protein 90 following ischemic preconditioning in rats. PLOS ONE 8, e75753. Thompson, J.W., Narayanan, S.V., Koronowski, K.B., Morris-Blanco, K., Dave, K.R., Perez-Pinzon, M.A., 2015. Signaling pathways leading to ischemic mitochondrial neuroprotection. J. Bioenerg. Biomembr. 47, 101–110.
Tian, J., Fu, F., Li, G., Wang, Y., Gao, Y., Liu, Z., Zhang, S., 2009. SMND-309, a novel derivate of salvianolic acid B, ameliorates cerebral infarction in rats: characterization and role. Brain Res. 1263, 114–121. Wang, S.X., Hu, L.M., Gao, X.M., Guo, H., Fan, G.W., 2010. Anti-inflammatory activity of salvianolic acid B in microglia contributes to its neuroprotective effect. Neurochem. Res. 35, 1029–1037. Wang, Z.S., Luo, P., Dai, S.H., Liu, Z.B., Zheng, X.R., Chen, T., 2013. Salvianolic acid B induces apoptosis in human glioma U87 cells through p38-mediated ROS generation. Cell. Mol. Neurobiol. 33, 921–928. Xu, S., Zhong, A., Bu, X., Ma, H., Li, W., Xu, X., Zhang, J., 2015. Salvianolic acid B inhibits platelets-mediated inflammatory response in vascular endothelial cells. Thromb. Res. 135, 137–145. Yang, Y., Duan, W., Li, Y., Yan, J., Yi, W., Liang, Z., Wang, N., Yi, D., Jin, Z., 2013. New role of silent information regulator 1 in cerebral ischemia. Neurobiol. Aging 34, 2879–2888. Yang, Y., Jiang, S., Dong, Y., Fan, C., Zhao, L., Yang, X., Li, J., Di, S., Yue, L., Liang, G., Reiter, R.J., Qu, Y., 2015. Melatonin prevents cell death and mitochondrial dysfunction via a SIRT1-dependent mechanism during ischemic-stroke in mice. J. Pineal Res. 58, 61–70. Yepes, M., Roussel, B.D., Ali, C., Vivien, D., 2009. Tissue-type plasminogen activator in the ischemic brain: more than a thrombolytic. Trends Neurosci. 32, 48–55. Zeng, G., Tang, T., Wu, H.J., You, W.H., Luo, J.K., Lin, Y., Liang, Q.H., Li, X.Q., Huang, X., Yang, Q.D., 2010. Salvianolic acid B protects SH-SY5Y neuroblastoma cells from 1-methyl-4-phenylpyridinium-induced apoptosis. Biol. Pharm. Bull. 33, 1337–1342. Zhou, L., Zuo, Z., Chow, M.S., 2005. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J. Clin. Pharmacol. 45, 1345–1359. Zhu, H., Zou, L., Tian, J., Du, G., Gao, Y., 2013. SMND-309, a novel derivative of salvianolic acid B, protects rat brains ischemia and reperfusion injury by targeting the JAK2/STAT3 pathway. Eur. J. Pharmacol. 714, 23–31.
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Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Salvianolic acid B attenuates apoptosis and inflammation via SIRT1 activation in experimental stroke rats Hongdi Lv a , Ling Wang b , Jinchang Shen c,∗ , Shaojun Hao d , Aimin Ming e , Xidong Wang d , Feng Su a , Zhengchen Zhang d a
Department of Cardiology, No. 371 Central Hospital of PLA, Xinxiang, Henan 453000, China Department of Nursing, No. 371 Central Hospital of PLA, Xinxiang, Henan 453000, China c Department of Interventional Radiology, No. 371 Central Hospital of PLA, Xinxiang, Henan 453000, China d Department of Drugs and Equipment, No. 371 Central Hospital of PLA, Xinxiang, Henan 453000, China e Department of Urology, No. 371 Central Hospital of PLA, Xinxiang, Henan 453000, China b
a r t i c l e
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Article history: Received 15 April 2015 Received in revised form 7 May 2015 Accepted 8 May 2015 Available online 14 May 2015 Keywords: Salvianolic acid B Ischemic stroke SIRT1 Apoptosis Inflammation
a b s t r a c t Silent information regulator 1 (SIRT1), a histone deacetylase, has been suggested to be effective in ischemic brain diseases. Salvianolic acid B (SalB) is a polyphenolic and one of the active components of Salvia miltiorrhiza Bunge. Previous studies suggested that SalB is protective against ischemic stroke. However, the role of SIRT1 in the protective effect of SalB against cerebral ischemia has not been explored. In this study, the rat brain was subjected to middle cerebral artery occlusion (MCAO). Before this surgery, rats were intraperitoneally administrated SalB with or without EX527, a specific SIRT1 inhibitor. The infarct volume, neurological score and brain water content were assessed. In addition, levels of TNF␣ and IL-1 in the brain tissues were detected by commercial ELISA kits. And the expression levels of SIRT, Ac-FOXO1, Bcl-2 and Bax were detected by Western blot. The results suggested that SalB exerted a cerebral-protective effect, as shown by reduced infarct volume, lowered brain edema and increased neurological scores. SalB also exerted anti-inflammatory effects as indicated by the decreased TNF-␣ and IL-1 levels in the brain tissue. Moreover, SalB upregulated the expression of SIRT1 and Bcl-2 and downregulated the expression of Ac-FOXO1 and Bax. These effects of SalB were abolished by EX527 treatment. In summary, our results demonstrate that SalB treatment attenuates brain injury induced by ischemic stoke via reducing apoptosis and inflammation through the activation of SIRT1 signaling. © 2015 Published by Elsevier Inc.
1. Introduction Ischemic stroke remains a leading cause of death and is associated with a high incidence of mortality and morbidity worldwide (Donnan et al., 2008; Hankey, 2012). Ischemic stroke results from the occlusion of a cerebral vessel and the middle cerebral artery (MCA) is often involved. When blood supply is damaged, a series of pathophysiologic events occur, such as glutamate excitotoxicity, calcium overload, oxidative stress, inflammation, apoptosis and mitochondrial dysfunction (Mattiasson et al., 2003; Reiter et al., 2007; Sahota and Savitz, 2011; Thompson et al., 2015). Although thrombolytic agents have been used for clinical treatment of ischemic stroke, their application is limited due to the narrow therapeutic window and other safety concerns (Mattiasson et al.,
∗ Corresponding author. Tel.: +86 373 3541018; fax: +86 373 3541018. E-mail address: [email protected] (J. Shen). http://dx.doi.org/10.1016/j.brainresbull.2015.05.002 0361-9230/© 2015 Published by Elsevier Inc.
2003; Yepes et al., 2009). Therefore, there is an urgent need for the development of novel therapeutic agents for treatment of ischemic stroke. Danshen, the dried root of Salvia miltiorrhiza Bunge, is a Chinese medicinal herb. It has been widely used for treatment of hepatitis, hemorrhage, menstrual abnormalities, as well as ischemic brain and heart diseases (Ji et al., 2000; Wang et al., 2013). It is one of the main components of Xinnaoning, a drug used for cardiovascular and cerebral vascular diseases. Salvianolic acid B (SalB, Fig. 1), the most abundant and bioactive compound of Danshen, has been suggested to exert various pharmacological effects, such as antioxidation, anti-inflammation and anti-tumor (Li et al., 2014; Tang et al., 2014; Wang et al., 2013). Previous studies have suggested that SalB has protective effects in ischemic stroke. However, the underlying mechanisms have not been fully elucidated. Silent information regulator 1 (SIRT1) is a histone deacetylase, and its activity is dependent on nicotinamide adenine dinucleotide (NAD+). SIRT1 expression decreases with age and is known
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2.3. Rat model of middle cerebral artery occlusion A middle cerebral artery occlusion (MCAO) model was established as previously described (Longa et al., 1989). Briefly, rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (350 mg/kg). The right common carotid artery was then exposed and isolated. The MCA was occluded by inserting a monofilament nylon suture with a heat-rounded tip into the internal carotid artery, which was advanced further until it closed the origin of the MCA. Thirty minutes after induction of ischemia, reperfusion was initiated by withdrawal of the monofilament. Sham-operated control rats received the same surgical procedure without MCA occlusion. Rectal temperature was maintained at 37 ◦ C using a heating blanket and heating lamp during the surgery. 2.4. Experimental protocol
Fig. 1. Structure of salvianolic acid B.
to plays a key role in the longevity effects induced by calorie restriction (Kilic et al., 2015; Ramis et al., 2015). SIRT1 deacetylates and activates forkhead box O (FOXO), which synthesizes antioxidants, such as manganese superoxide dismutase and catalase, promoting cellular resistance against oxidative stress (Brunet et al., 2004). In addition, SIRT1 has been suggested to be critical in cerebral protection. Ischemia preconditioning (Thompson et al., 2013) and hyperbaric oxygen preconditioning (Herskovits and Guarente, 2014) reportedly decrease cerebral ischemia-reperfusion injury via SIRT1 activation. Intriguingly, a previous study suggested that SalB protects against ischemic stroke (Tian et al., 2009; Zhu et al., 2013). In addition, SalB is suggested to protect against acute ethanol-induced liver injury through SIRT1-mediated deacetylation of p53 (Li et al., 2014). However, the role of SIRT1 in the protective effect of SalB against ischemic stroke remains unclear.
SalB was dissolved in normal saline and injected intraperitoneally at a dose of 25 mg/kg administrated twice (immediately after induction of ischemia and at the beginning of reperfusion). As reported previously (Yang et al., 2013), the SIRT1 inhibitor EX527 was first dissolved in dimethyl sulphoxide (DMSO) and diluted to the final concentration with normal saline (the final DMSO concentration <2%). EX527 was intraperitoneally injected at the dose of 5 mg/kg or the same volume of vehicle was performed every 2 days for four times before MCAO. All the rats were randomly assigned to the following groups (n = 10 in each group): Sham, MCAO, MCAO + SalB, MCAO + SalB + EX527 and MCAO + EX527. 2.5. Evaluation of neurological deficit Neurological deficit scores were evaluated by an observer blinded to the experimental groups at 24 h after MCAO as reported previously (Longa et al., 1989). The scores are as follows: 0, no motor deficits (normal); 1, forelimb weakness and torso turning to the ipsilateral side when held by tail (mild); 2, circling to the contralateral side but normal posture at rest (moderate); 3, unable to bear weight on the affected side at rest (severe); 4, no spontaneous locomotor activity or barrel rolling (critical).
2. Materials and methods 2.6. Evaluation of infarct volume 2.1. Reagents SalB (purity >98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). 2,3,5-Triphenyltetrazolium chloride (TTC) was purchased from Sigma–Aldrich (St. Louis, MO, USA). EX527 was purchased from Tocris Bioscience (Bristol, UK). Antibodies against acetylateFOXO1 (Ac-FOXO1), Bcl2, Bax, and -actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against SIRT1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit anti-goat, goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from the Zhongshan Company (Beijing, China).
Infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) at 24 h after reperfusion. Animals were euthanized and the brains were quickly removed. Then the brain was sliced into five coronal sections (3 mm thick each) and stained with 2% solution of TTC at 37 ◦ C for 20 min, followed by fixation in 4% paraformaldehyde. TTC-stained sections were photographed and the digital images were analyzed using image analysis software (Image-Pro Plus 5.1). The lesion volume was calculated by multiplying the area by the thickness of slices. The percentage hemisphere lesion volume (%HLV) was calculated by the following formula (Tatlisumak, 1998): %HLV = ([total infarct volume − (the volume of intact ipsilateral hemisphere − the volume of intact contralateral hemisphere)])/contralateral hemisphere volume × 100%.
2.2. Animals 2.7. Brain water content Male Sprague-Dawley rats (230–280 g), purchased from the Laboratory Animal Center of Xinxiang Medical University, were kept under a controlled environment (12/12 h light/dark cycle, 60 ± 5% humidity, 22 ± 3 ◦ C). And they have free access to water and food. All procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), revised 1996.
As described previously (Yang et al., 2015), after the wet weight of the brain tissues was quantified, the red and white parts of these brains were desiccated at 105 ◦ C for 48 h until the weight was constant. The total weight of the dried TTC-stained brains was obtained by measuring the desiccated red and white parts of these brains together, and the water content of each brain was calculated as follows: (wet weight − dried weight)/wet weight × 100%.
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Fig. 2. Effects of salvianolic acid B on infarct volume, neurological score, and brain water content in rats subjected to MCAO. (A) Infarct volume. (B) Neurological score. (C) Brain water content. The results are expressed as the mean ± S.E.M., n = 10, * P < 0.05 vs. the MCAO group.
2.8. Detection of inflammatory cytokines
2.9. Western blot
The inflammatory cytokines in brain tissue were measured using commercially ELISA kits for TNF-␣ and IL-1 (Beyotime, China). All spectrophotometric readings were performed with a microplate reader (Multiskan MK3, Thermo, USA). All procedures were performed according to the instructions.
The ischemic penumbra of cerebral cortex was collected. The brain tissue was then lysed in ice-cold lysis buffer (50 mM Tris–HCl, 1 mM EDTA, 1 mM EGTA, 0.5 mMNa3 VO4 , 0.1% 2-mercaptoethanol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium -glyceropyrophosphate, 0.1 mM phenylmethanesulfonyl
Fig. 3. Effects of salvianolic acid B on SIRT1, Ac-FOXO1, Bcl-2 and Bax expression in rats subjected to MCAO. The representative images of SIRT1, Ac-FOXO1, Bcl-2, and Bax are shown. The results are expressed as the mean ± S.E.M., n = 10, * P < 0.05 vs. the MCAO group.
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fluoride and protease inhibitor mixture) for 10 min. The lysates were centrifuged for 15 min at 12,000 × g, and the resulting supernatant was collected and boiled. Protein concentrations of the extracts were measured by BCA assay. The whole protein 50 g was resolved on 8–12% SDS polyacrylamide gel, then transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBST (150 mM NaCl, 50 mM Tris [pH 7.5], 0.1% Tween-20) and then incubated with antibodies against SIRT1 (1:500), Ac-FOXO1, Bcl2, Bax and -actin (1:1000) at 4 ◦ C overnight, followed by washes with TBST. The membranes were then probed with the appropriate secondary antibodies (1:5000) at room temperature for 2 h and washed with TBST. The protein bands were detected using a BioRad imaging system (BioRad, Hercules, CA, USA) and quantified using the Quantity One software package (West Berkeley, CA, USA). The value for the MCAO group was defined as 100%. 2.10. Statistical analysis Data were reported as mean ± standard error of the mean (S.E.M.). One-way analysis of variance (ANOVA) followed by the Dunnett’s test was performed to compare the differences (SPSS 13.0, Chicago, IL, USA). A value of P < 0.05 was considered to be statistically significant. 3. Results 3.1. SalB reduced brain injury after MCAO To investigate the potential protective effects of SalB against MCAO-induced brain injury, we examined the effect of SalB on brain infarct volume, neurological score and cerebral edema. Compared with the MCAO group, SalB significantly reduced infarct volume, neurological score and brain water content in MCAO + SalB group (Fig. 2) (P < 0.05). 3.2. Effect of SalB on the expressions of SIRT1, Ac-FOXO1, Bcl-2 and Bax To investigate the key proteins involved in the protective role of SalB, we analyzed the expression of SIRT1, Ac-FOXO1, Bcl-2 and Bax. As shown in Fig. 3, SalB treatment significantly increased SIRT1 and Bcl-2 expression in MCAO + SalB compared with their expression in MCAO group (P < 0.05). SalB also decreased the Ac-FOXO1, and Bax expression in MCAO + SalB group compared with their expression in MCAO group (Fig. 3) (P < 0.05). 3.3. Effect of SalB on the production of inflammatory cytokines in brain tissue To elucidate the effect of SalB on inflammation induce by MCAO, we evaluated the production of TNF-␣ and IL-1 in the brain tissue. SalB dramatically decreased the production of TNF-␣ and IL-1 in the brain tissue in MCAO + SalB group compared with those in the MCAO group (Fig. 4) (P < 0.05). 3.4. EX527 abolished the protective effect of SalB We used EX527, a specific SIRT1 inhibitor, to investigate whether SIRT1 is a key molecule in the protective effect of SalB and to elucidate the underlying mechanisms. Compared with the MCAO group, SalB significantly reduced infarct volume, neurological score and brain water content in MCAO + SalB group. However, EX527 administration abolished the protective effect of SalB; it increased the infarct volume, neurological score and brain edema in
Fig. 4. Effects of salvianolic acid B and EX527 on TNF-␣ and IL-1 in the brain tissue. (A) The production of TNF-␣ in the brain tissue. (B) The production of IL-1 in the brain tissue. The results are expressed as the mean ± S.E.M., n = 10, ** P < 0.05 vs. the MCAO group, ## P < 0.05 vs. the MCAO + SalB group, $$ P < 0.05 vs. the MCAO + SalB + EX527 group.
MCAO + SalB + EX527 group compared with the MCAO + SalB group (Fig. 5) (P < 0.05). Western blot results suggested that SalB treatment significantly increased SIRT1 and Bcl-2 expression. SalB also decreased the expression of Ac-FOXO1 and Bax. However, EX527 significantly decreased SIRT1 and Bcl-2 expression and increased Ac-FOXO1 and Bax expression in the MCAO + SalB + EX527 group compared with that in the MCAO + SalB group (Fig. 6) (P < 0.05). 4. Discussion The major findings in the present study are: (1) SalB protects the brain from ischemia. (2) SalB reduces inflammation in the ischemic brain. (3) SalB decreases apoptosis in the ischemic brain. (4) The activation of SIRT1 is involved in the neuroprotective effect of SalB. S. miltiorrhiza has been widely used for treatment of various diseases in China (Zhou et al., 2005). Many studies have demonstrated that SalB exerts various pharmacological activities, such as antiapoptosis, anti-inflammation, anti-diabetes, promotion of cellular proliferation, differentiation and bone formation and preservation of normal cell functions (He and Shen, 2014; Huang et al., 2015; Shi et al., 2007; Wang et al., 2010; Xu et al., 2015; Zeng et al., 2010).
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Fig. 5. Effects of salvianolic acid B and EX527 on infarct volume, neurological score and brain water content in rats subjected to MCAO. (A) Infarct volume. (B) Neurological score. (C) Brain water content. The results are expressed as the mean ± S.E.M., n = 10, ** P < 0.05 vs. the MCAO group, ## P < 0.05 vs. the MCAO + SalB group, $$ P < 0.05 vs. the MCAO + SalB + EX527 group.
SalB has been shown to be beneficial in the central nervous system. Zhu et al. suggest that SMND-309, a novel derivative of SalB, protects rat brains ischemia and reperfusion injury by targeting the JAK2/STAT3 pathway (Zhu et al., 2013). SalB confers neuroprotection via anti-inflammatory and anti-oxidative effects in Alzheimer’s disease (Lee et al., 2013). And SalB attenuates cognitive dysfunctions in Alzheimer’s disease (Kim et al., 2011). In addition, SalB has been suggested to protect against acute ethanol-induced liver injury via SIRT1 activation (Li et al., 2014). These findings suggest that SalB regulates inflammation and apoptosis in many diseases and SIRT1 may be involved in this activity of SalB. SIRT1, a member of the class III group of histone deacetylases, has been suggested to be protective in cerebral ischemia (Yang et al., 2013, 2015). SIRT1 is thought to play an important role in neuroprotection against brain ischemia by deacetylation and subsequent inhibition of p53 and nuclear factor B-induced inflammatory and apoptotic pathways (Hernandez-Jimenez et al., 2013). It also mediates hyperbaric oxygen preconditioning-induced ischemic tolerance in the rat brain (Herskovits and Guarente, 2014). Consistent with these findings, we found that SalB augmented SIRT1 expression and inhibited apoptosis and inflammation. Our results suggest that SIRT1 activation by SalB plays a critical role in protection against cerebral ischemic injury. In this study, we first investigated the effect of SalB on infarct volume, neurological score and brain water content. Our results suggest that SalB significantly reduced infarct volume, neurological score and brain water content. Additionally, SalB dramatically up-regulated SIRT1 expression, leading to down-regulation of AcFOXO1, which is downstream of SIRT1. Moreover, to investigate the role of SalB in neuronal apoptosis, the expression of Bax and
Bcl-2 was analyzed. The results demonstrate that SalB significantly increased Bcl-2 and decreased Bax expression, indicating that SalB inhibits apoptosis in ischemic stroke. To further investigate the role of SIRT1 in the protective effect of SalB, we used EX527, a specific SIRT1, inhibitor to elucidate the underlying mechanisms. Our results suggest that EX527 abolishes the protective effect of SalB. EX527 significantly increased infarct volume, neurological score and brain water content. In addition, EX527 abolished the antiapoptotic effect of SalB, as evidenced by the increased Bax and decreased Bcl-2 expression. Therefore, these results suggest that SIRT1 activation by SalB contributes to the attenuation of brain injury induced by ischemic stroke. Ischemic stroke is associated with the production and release of pro-inflammatory cytokines such as TNF-␣ and IL-1 (Palencia et al., 2015), and this triggers additional inflammatory processes such as activation of cyclooxygenase-2 (COX-2), NOS, and NF-B (Mohammadi et al., 2012; Ridder and Schwaninger, 2009). The inflammation in turn may aggravate infarction, brain edema and neuronal death. The present results suggest that SalB alleviates inflammation as evidenced by the decrease of TNF-␣ and IL-1 levels in brain tissue. However, this effect was abolished by EX527, indicating that the anti-inflammatory effect of SalB is associated with SIRT1 signaling. In summary, the present study suggested for the first time that SalB has a protective effect against ischemic stroke via the activation of SIRT1. SalB activates SIRT1 signaling, accompanied by reduced expression of Ac-FOXO1, leading to increased Bcl-2 and decreased Bax expression. Taken together, these findings suggest that SalB may be an important therapeutic strategy for attenuating brain injury associated with ischemic stroke.
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Fig. 6. The effects of salvianolic acid B and EX527 on Ac-FOXO1, Bcl-2 and Bax expression in rats subjected to MCAO. The representative images of SIRT1, Ac-FOXO1, Bcl-2, and Bax are shown. The results are expressed as the mean ± S.E.M., n = 10, ** P < 0.05 vs. the MCAO group, ## P < 0.05 vs. the MCAO + SalB group, $$ P < 0.05 vs. the MCAO + SalB + EX527 group.
Conflict of interest There is no conflict of interest. References Brunet, A., Sweeney, L.B., Sturgill, J.F., Chua, K.F., Greer, P.L., Lin, Y., Tran, H., Ross, S.E., Mostoslavsky, R., Cohen, H.Y., Hu, L.S., Cheng, H.L., Jedrychowski, M.P., Gygi, S.P., Sinclair, D.A., Alt, F.W., Greenberg, M.E., 2004. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303, 2011–2015. Donnan, G.A., Fisher, M., Macleod, M., Davis, S.M., 2008. Stroke. Lancet 371, 1612–1623. Hankey, G.J., 2012. Anticoagulant therapy for patients with ischaemic stroke. Nat. Rev. Neurol. 8, 319–328. He, X., Shen, Q., 2014. Salvianolic acid B promotes bone formation by increasing activity of alkaline phosphatase in a rat tibia fracture model: a pilot study. BMC Complement. Altern. Med. 14, 493. Hernandez-Jimenez, M., Hurtado, O., Cuartero, M.I., Ballesteros, I., Moraga, A., Pradillo, J.M., McBurney, M.W., Lizasoain, I., Moro, M.A., 2013. Silent information regulator 1 protects the brain against cerebral ischemic damage. Stroke J. Cereb. Circ. 44, 2333–2337. Herskovits, A.Z., Guarente, L., 2014. SIRT1 in neurodevelopment and brain senescence. Neuron 81, 471–483. Huang, M., Wang, P., Xu, S., Xu, W., Xu, W., Chu, K., Lu, J., 2015. Biological activities of salvianolic acid B from Salvia miltiorrhiza on type 2 diabetes induced by high-fat diet and streptozotocin. Pharm. Biol., 1–8. Ji, X.Y., Tan, B.K., Zhu, Y.Z., 2000. Salvia miltiorrhiza and ischemic diseases. Acta Pharmacol. Sin. 21, 1089–1094.
Kilic, U., Gok, O., Erenberk, U., Dundaroz, M.R., Torun, E., Kucukardali, Y., Elibol-Can, B., Uysal, O., Dundar, T., 2015. A remarkable age-related increase in SIRT1 protein expression against oxidative stress in elderly: SIRT1 gene variants and longevity in human. PLOS ONE 10, e0117954. Kim, D.H., Park, S.J., Kim, J.M., Jeon, S.J., Kim, D.H., Cho, Y.W., Son, K.H., Lee, H.J., Moon, J.H., Cheong, J.H., Ko, K.H., Ryu, J.H., 2011. Cognitive dysfunctions induced by a cholinergic blockade and Abeta 25-35 peptide are attenuated by salvianolic acid B. Neuropharmacology 61, 1432–1440. Lee, Y.W., Kim, D.H., Jeon, S.J., Park, S.J., Kim, J.M., Jung, J.M., Lee, H.E., Bae, S.G., Oh, H.K., Son, K.H., Ryu, J.H., 2013. Neuroprotective effects of salvianolic acid B on an Abeta25-35 peptide-induced mouse model of Alzheimer’s disease. Eur. J. Pharmacol. 704, 70–77. Li, M., Lu, Y., Hu, Y., Zhai, X., Xu, W., Jing, H., Tian, X., Lin, Y., Gao, D., Yao, J., 2014. Salvianolic acid B protects against acute ethanol-induced liver injury through SIRT1-mediated deacetylation of p53 in rats. Toxicol. Lett. 228, 67–74. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke J. Cereb. Circ. 20, 84–91. Mattiasson, G., Shamloo, M., Gido, G., Mathi, K., Tomasevic, G., Yi, S., Warden, C.H., Castilho, R.F., Melcher, T., Gonzalez-Zulueta, M., Nikolich, K., Wieloch, T., 2003. Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma. Nat. Med. 9, 1062–1068. Mohammadi, M.T., Shid-Moosavi, S.M., Dehghani, G.A., 2012. Contribution of nitric oxide synthase (NOS) in blood-brain barrier disruption during acute focal cerebral ischemia in normal rat. Pathophysiol.: Off. J. Int. Soc. Pathophysiol./ISP 19, 13–20. Palencia, G., Medrano, J.A., Ortiz-Plata, A., Farfan, D.J., Sotelo, J., Sanchez, A., TrejoSolis, C., 2015. Anti-apoptotic, anti-oxidant, and anti-inflammatory effects of thalidomide on cerebral ischemia/reperfusion injury in rats. J. Neurol. Sci. 351, 78–87.
36
H. Lv et al. / Brain Research Bulletin 115 (2015) 30–36
Ramis, M.R., Esteban, S., Miralles, A., Tan, D.X., Reiter, R.J., 2015. Caloric restriction, resveratrol and melatonin: role of SIRT1 and implications for aging and relateddiseases. Mech. Ageing Dev. 146–148C, 28–41. Reiter, R.J., Tan, D.X., Manchester, L.C., Tamura, H., 2007. Melatonin defeats neurallyderived free radicals and reduces the associated neuromorphological and neurobehavioral damage. J. Physiol. Pharmacol.: Off. J. Pol. Physiol. Soc. 58 (Suppl. 6), 5–22. Ridder, D.A., Schwaninger, M., 2009. NF-kappaB signaling in cerebral ischemia. Neuroscience 158, 995–1006. Sahota, P., Savitz, S.I., 2011. Investigational therapies for ischemic stroke: neuroprotection and neurorecovery. Neurotherap.: J. Am. Soc. Exp. Neurotherap. 8, 434–451. Shi, C.S., Huang, H.C., Wu, H.L., Kuo, C.H., Chang, B.I., Shiao, M.S., Shi, G.Y., 2007. Salvianolic acid B modulates hemostasis properties of human umbilical vein endothelial cells. Thromb. Res. 119, 769–775. Tang, Y., Jacobi, A., Vater, C., Zou, X., Stiehler, M., 2014. Salvianolic acid B protects human endothelial progenitor cells against oxidative stress-mediated dysfunction by modulating Akt/mTOR/4EBP1, p38 MAPK/ATF2, and ERK1/2 signaling pathways. Biochem. Pharmacol. 90, 34–49. Tatlisumak, T., Carano, R.A., Takano, K., Opgenorth, T.J., Sotak, C.H., Fisher, M., 1998. A novel endothelin antagonist, A-127722, attenuates ischemic lesion size in rats with temporary middle cerebral artery occlusion: a diffusion and perfusion MRI study. Stroke J. Cereb. Circ. 29, 850–857 (discussion 857–858). Thompson, J.W., Dave, K.R., Saul, I., Narayanan, S.V., Perez-Pinzon, M.A., 2013. Epsilon PKC increases brain mitochondrial SIRT1 protein levels via heat shock protein 90 following ischemic preconditioning in rats. PLOS ONE 8, e75753. Thompson, J.W., Narayanan, S.V., Koronowski, K.B., Morris-Blanco, K., Dave, K.R., Perez-Pinzon, M.A., 2015. Signaling pathways leading to ischemic mitochondrial neuroprotection. J. Bioenerg. Biomembr. 47, 101–110.
Tian, J., Fu, F., Li, G., Wang, Y., Gao, Y., Liu, Z., Zhang, S., 2009. SMND-309, a novel derivate of salvianolic acid B, ameliorates cerebral infarction in rats: characterization and role. Brain Res. 1263, 114–121. Wang, S.X., Hu, L.M., Gao, X.M., Guo, H., Fan, G.W., 2010. Anti-inflammatory activity of salvianolic acid B in microglia contributes to its neuroprotective effect. Neurochem. Res. 35, 1029–1037. Wang, Z.S., Luo, P., Dai, S.H., Liu, Z.B., Zheng, X.R., Chen, T., 2013. Salvianolic acid B induces apoptosis in human glioma U87 cells through p38-mediated ROS generation. Cell. Mol. Neurobiol. 33, 921–928. Xu, S., Zhong, A., Bu, X., Ma, H., Li, W., Xu, X., Zhang, J., 2015. Salvianolic acid B inhibits platelets-mediated inflammatory response in vascular endothelial cells. Thromb. Res. 135, 137–145. Yang, Y., Duan, W., Li, Y., Yan, J., Yi, W., Liang, Z., Wang, N., Yi, D., Jin, Z., 2013. New role of silent information regulator 1 in cerebral ischemia. Neurobiol. Aging 34, 2879–2888. Yang, Y., Jiang, S., Dong, Y., Fan, C., Zhao, L., Yang, X., Li, J., Di, S., Yue, L., Liang, G., Reiter, R.J., Qu, Y., 2015. Melatonin prevents cell death and mitochondrial dysfunction via a SIRT1-dependent mechanism during ischemic-stroke in mice. J. Pineal Res. 58, 61–70. Yepes, M., Roussel, B.D., Ali, C., Vivien, D., 2009. Tissue-type plasminogen activator in the ischemic brain: more than a thrombolytic. Trends Neurosci. 32, 48–55. Zeng, G., Tang, T., Wu, H.J., You, W.H., Luo, J.K., Lin, Y., Liang, Q.H., Li, X.Q., Huang, X., Yang, Q.D., 2010. Salvianolic acid B protects SH-SY5Y neuroblastoma cells from 1-methyl-4-phenylpyridinium-induced apoptosis. Biol. Pharm. Bull. 33, 1337–1342. Zhou, L., Zuo, Z., Chow, M.S., 2005. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J. Clin. Pharmacol. 45, 1345–1359. Zhu, H., Zou, L., Tian, J., Du, G., Gao, Y., 2013. SMND-309, a novel derivative of salvianolic acid B, protects rat brains ischemia and reperfusion injury by targeting the JAK2/STAT3 pathway. Eur. J. Pharmacol. 714, 23–31.