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Akt signaling pathway
G Model BIOPHA 4537 No. of Pages 5
Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
Available online at
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Original article
Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway Shujie Jiaoa , Hongcan Zhua , Ping Heb , Junfang Tenga,* a b
Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
A R T I C L E I N F O
Article history: Received 12 September 2016 Received in revised form 22 October 2016 Accepted 8 November 2016 Keywords: Betulinic acid (BA) Cerebral ischemia/reperfusion (I/R) Oxidative stress Apoptosis
A B S T R A C T
Betulinic acid (BA), a naturally occurring pentacyclic lupane group triterpenoid, has been demonstrated to protect against ischemia/reperfusion-induced renal damage. However, the effects of BA on cerebral ischemia/reperfusion (I/R) injury remain unclear. Hence, this study was to investigate the effects of BA on oxygen and glucose deprivation/reperfusion (OGD/R) induced neuronal injury in rat hippocampal neurons. Our results showed that BA pretreatment greatly attenuated OGD/R-induced neuronal injury. BA also inhibited OGD/R-induced intracellular ROS production and MDA level in rat hippocampal neurons. Furthermore, the down-regulation of Bcl-2, up-regulation of Bax and the consequent activation of caspase-3 induced by OGD/R were reversed by BA pretreatment. Mechanistic studies demonstrated that BA pretreatment up-regulated the expression levels of p-PI3 K and p-Akt in hippocampal neurons induced by OGD/R. Taken together, these data suggested that BA inhibits OGD/R-induced neuronal injury in rat hippocampal neurons through the activation of PI3 K/Akt signaling pathway. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Cerebral ischemia is among the death-related brain diseases with high morbidity and high mortality in adults [1]. It is characterized by oxidative stress, hypoxia, inflammation, and glutamate excitotoxicity, eventually leading to cell death [2]. Currently, restoration of blood supply, called reperfusion, is still the standard therapy for ischemia [3]. However, reperfusion also contributes to delayed secondary brain injury, because of the sudden recovery of blood supply which will produce excessive reactive species (ROS), leading to mitochondrial dysfunction, oxidative stress and finally cell death [4]. Hence, it is urgently needed to explore the precise mechanism of ischemia-induced neuron death. Betulinic acid (BA), (3b-hydroxy-lup-20(29)-en-28-oic acid), is a naturally occurring pentacyclic lupane group triterpenoid. Increasing evidences have reported that BA possesses a number of biological activities such as an anti-cancer, anti-oxidant, antiinflammatory and anti-HIV [5–8]. In addition, BA was found to protect against I/R injury [9,10]. For example, one study reported that BA attenuates I/R-induced renal damage through regulating
the apoptotic function of leukocytes and inhibiting neutrophil infiltration [9]. However, the effects of BA on cerebral ischemia/ reperfusion (I/R) injury remain unclear. Hence, this study was to investigate the effects of BA on oxygen and glucose deprivation/ reperfusion (OGD/R) induced neuronal injury in primary rat hippocampal neurons cells. Our results demonstrated that BA protects against OGD/R-induced neuronal injury, at least in part, through the activation of PI3 K/Akt signaling pathway. 2. Materials and methods 2.1. Cell culture Primary hippocampal neuronal cells were prepared from neonatal SD rats [11]. In brief, the hippocampi tissues were dissected and dissociated in 0.25% trypsin-EDTA (Sigma, St. Louis, USA), and primary hippocampal neurons were maintained in neurobasal medium supplemented with B-27, glutamine (0.5 mM), glutamate (25 mM), and 1% penicillin/streptomycin. The cultures were kept in a humidified incubator that was maintained at 37 C and supplied with 5% CO2 and 95% air for 14 days. Culture medium was changed two times a week.
* Corresponding author at: Department of Neurology, The First Affiliated Hospital of Zhengzhou University, 1st of Jianshe East Road, Zhengzhou 450000, China. E-mail address: [email protected] (J. Teng). http://dx.doi.org/10.1016/j.biopha.2016.11.028 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: S. Jiao, et al., Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.028
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2.2. Oxygen-glucose deprivation/reperfusion (OGD/R) model and treatment For OGD model construction to mimic ischemia in vitro, hippocampal neurons were pretreated with various concentrations of BA (1–10 mM, 2 h pretreatment; Sigma), and then exposed to DMEM without serum or glucose in a humidified atmosphere containing 95% nitrogen and 5% CO2 for 4 h. Subsequently, the medium was replaced with glucose-containing medium supplemented with 10% FBS again and, cells were subjected to culture under normoxic conditions (95% air, 5% CO2) for 24 h. Cells cultured in growth culture medium under normoxic condition were used as control. 2.3. Cell viability assay Cell viability was detected using the MTT assay. In brief, neurons were seeded into 96-well plates (Corning Inc., Corning, NY, USA) at a density of 5 103 cells/well before treatment. After 24 h of incubation, 100 mL MTT (5 mg/mL; Sigma) was added to each well for 4 h. Then the supernatant was removed and 100 mL DMSO (Sigma) was added to lyse the cells. The absorbance was measured at 490 nm with an enzyme linked immunosorbent assay plate reader (Olympus, Tokyo, Japan). 2.4. Evaluation of reactive oxygen species (ROS) and MDA 20 ,70 -Dichlorodi-hydrofluorescein diacetate (DCFH-DA) was used to evaluate intracellular ROS level. In brief, after treatment, neurons were washed with PBS, and then incubated with 10 mM DCFH-DA in the dark for 30 min at 37 C. The cells were washed with PBS and then observed under a fluorescence microscope. The fluorescence intensity was measured by a fluorospectrophotometer at excitation/emission maxima of 485/525 nm. The MDA level was detected using the commercial kit (Beyotime, Haimen, Jiangsu, China). In brief, after treatment, the supernatant of neurons was collected, then the level of MDA was measured by a method based on a reaction with thiobarbituric acid. The optical density at 532 nm was measured with a microplate reader (Molecular Device, Spectra Max 190, USA). 2.5. Caspase-3 activity detection The supernatant of the treated cells was collected, then the activity of caspase-3 was examined using the colorimetric caspase3 assay kit (Beyotime, Jiangsu, China) according to the manufacturer’s instructions.
2.7. Statistics analysis Data are expressed as the mean standard deviation (SD). Statistical significance was analyzed with the one-way analysis of variance (ANOVA) or the Student’s two-tailed t-test. The p value less than 0.05 was considered statistically significant difference. 3. Results 3.1. BA ameliorates OGD/R-induced neuron injury First, we examined the effect of BA on OGD/R-induced cellular viability in hippocampal neurons using the MTT assay. As shown in Fig. 1, compared to the control group, the cell viability was obviously reduced by the OGD/R treatment. However, pretreatment with BA significantly increased cell viability in a dosedependent manner, as compared with the OGD/R group. 3.2. BA ameliorates OGD/R-induced oxidative stress in hippocampal neurons Oxidative stress was involved in the development of cerebral I/R injury. Thus, we examined the effect of BA in on OGD/R-induced oxidative stress in hippocampal neurons. As shown in Fig. 2A, OGD/ R exposure caused a significant increase of intracellular ROS generation in hippocampal neurons, compared with that in control group. However, pretreatment with BA markedly decreased ROS level in hippocampal neurons. Similarly, BA pretreatment also suppressed OGD/R-induced MDA level in hippocampal neurons (Fig. 2B). 3.3. BA ameliorates OGD/R-induced cell apoptosis in hippocampal neurons Next, we tested the effect of BA on hippocampal neuron apoptosis induced by OGD/R. As shown in Fig. 3A, neurons subjected to OGD/R showed markedly increased caspase-3 activity. In contrast, BA pretreatment significantly suppressed caspase-3 activity, compared with OGD/R group. Furthermore, we investigated the effects of BA on Bax and Bcl-2 protein levels in OGD/R-treated hippocampal neurons. The results indicated that the protein expression level of Bcl-2 was substantially downregulated in OGD/R-induced hippocampal neurons compared with the control, whereas an enhanced expression of Bax was observed.
2.6. Western blotting The proteins were extracted from neurons using RIPA lysis buffer (Beyotime, Nantong, China). The protein concentration in the lysates was determined by BCA protein assay kit (Beyotime, Nantong, China). The proteins (30 mg/lane) were subjected to 10% SDS-PAGE and electrophoretically transferred to Immobilon P Millipore (Bedford, MA, USA). The blots were blocked in 5% nonfat milk in TBST buffer (5 mM Tris-HCl, pH 7.4, 136 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Then, the membrane was incubated overnight at 4 C with primary antibodies (dilution, 1:1,000) targeting p-PI3 K, PI3 K, p-Akt, Akt and GAPDH (all from Santa Cruz Biotechnology, CA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the immunoreactive protein bands were visualized using an enhanced chemiluminescence detection system (Amersham, Little Chalfont, UK).
Fig. 1. BA ameliorates OGD/R-induced neuron injury. Primary hippocampal neurons were pretreated with indicated concentration of BA (1–10 mM, 2 h pretreatment), and were maintained under OGD for 4 h, followed by 24 h of reoxygenation. Cell viability was determined by the MTT assay. *P < 0.05 vs. control group. #P < 0.05 vs.OGD/R group.
Please cite this article in press as: S. Jiao, et al., Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.028
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Fig. 2. BA ameliorates OGD/R-induced oxidative stress in hippocampal neurons. Primary hippocampal neurons were pretreated with indicated concentration of BA (1–10 mM, 2 h pretreatment), and were maintained under OGD for 4 h, followed by 24 h of re-oxygenation. A, 20 ,70 -dichlorodi-hydrofluorescein diacetate (DCFH-DA) was used to evaluate intracellular ROS level. B, The MDA level was examined using the commercial kit. *P < 0.05 vs. control group. #P < 0.05 vs.OGD/R group.
In addition, pretreatment with BA greatly up-regulated the protein expression of Bcl-2 and reduced the protein expression of Bax, as compared with the OGD/R group (Fig. 3B).
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Fig. 3. BA ameliorates OGD/R-induced cell apoptosis in hippocampal neurons. Primary hippocampal neurons were pretreated with indicated concentration of BA (1–10 mM, 2 h pretreatment), and were maintained under OGD for 4 h, followed by 24 h of re-oxygenation. A, The activity of caspase-3 was examined using the colorimetric caspase-3 assay kit. B, The protein expression levels of Bax and Bcl-2 were detected by the western blotting. *P < 0.05 vs. control group. #P < 0.05 vs.OGD/ R group.
4. Discussion 3.4. BA activates the PI3K/Akt signaling pathway in hippocampal neurons induced by OGD/R PI3K/Akt signaling pathway plays important role in the development of cerebral I/R injury. To further clarity the mechanism underlying the neuroprotective activity of BA, we detected the expression of p-PI3K and p-Akt using western blotting. As shown in Fig. 4A, the protein expression levels of pPI3K and p-Akt were significantly suppressed by OGD/R in hippocampal neurons, compared with the control group. However, BA pretreatment significantly enhanced the expression levels of pPI3K and p-Akt in hippocampal neurons. To further confirm the role of PI3K/Akt pathway in BA-induced neuroprotection, hippocampal neurons were treated with inhibitor of PI3K (LY294002). We found that treatment with LY294002 decreased BA-mediated survival to 63% (Fig. 4C). In addition, treatment with LY294002 significantly increased caspase-3 activity, compared with OGD/R plus BA group (Fig. 4D).
To our knowledge, this is the first report of the effects of BA on OGD/R induced neuronal injury in rat hippocampal neurons. Our results showed that BA pretreatment greatly attenuated OGD/Rinduced neuronal injury. BA also inhibited OGD/R-induced intracellular ROS production and MDA level in rat hippocampal neurons. Furthermore, the down-regulation of Bcl-2, up-regulation of Bax and the consequent activation of caspase-3 induced by OGD/ R were reversed by BA pretreatment. Mechanistic studies demonstrated that BA pretreatment up-regulated the expression levels of p-PI3K and p-Akt in hippocampal neurons induced by OGD/R. OGD/R model was constructed to mimic cerebral I/R injury [12–14]. In the present study, an in vitro OGD/R model was established in hippocampal neuronal cells to examine the neuroprotective effects of BA on cerebral I/R injury. Our results demonstrated that OGD/R treatment significantly reduced neuron viability. However, pretreatment with BA significantly increased
Please cite this article in press as: S. Jiao, et al., Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.028
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Fig. 4. BA activates the PI3 K/Akt signaling pathway in hippocampal neurons induced by OGD/R. Primary hippocampal neurons were pretreated with indicated concentration of BA (1–10 mM, 2 h pretreatment), and were maintained under OGD for 4 h, followed by 24 h of re-oxygenation. A, The protein expression levels of p-PI3 K, PI3 K, p-Akt and Akt were detected by the western blotting. B, Quantification analysis was performed using Gel-Pro Analyzer version 4.0 software. C, Cell viability was determined by the MTT assay following treatment with LY294002 (20 mM). D, The activity of caspase-3 was examined using the colorimetric caspase-3 assay kit in hippocampal neurons treated with LY294002 after exposure to OGD/R for 1 h *P < 0.05 vs. control group. #P < 0.05 vs. OGD/R group, & P < 0.05 vs. OGD/R + BA group.
cell viability in a dose-dependent manner. These data suggest that BA protects against OGD-induced cell injury in hippocampal neurons. Increasing evidences have reported that the high production of ROS cause oxidative stress, which may contribute to the progression of cerebral I/R injury [15–17]. In addition, ROS has been linked to high levels of MDA and claimed to cause lipid peroxidation [18]. Recently, it was reported that BA attenuated macrophage-derived lipoprotein lipase expression and activity induced by oxidative stress, as well as decreased cellular lipid accumulation [19]. Herein, our studies observed that pretreatment with BA markedly decreased OGD/R-induced ROS generation and MDA level in hippocampal neurons. These data suggest that BA may protect against cerebral I/R injury by down-regulating the levels of MDA and SOD in hippocampal neurons after OGD/R. Apoptosis also plays an important role in the process of neuronal death after cerebral ischemia [20]. Bcl-2 is an antiapoptotic factor and important for cell survival while Bax promotes apoptosis [21]. Caspase-3, a key factor in apoptosis, eventually
leads to DNA fragmentation and triggers apoptosis during neuronal development and under pathological conditions including cerebral ischemia [22]. In this study, we observed that OGD/R treatment notably up-regulated the expression of Bax and down-regulated the expression of Bcl-2, and subsequent activation of caspase-3. However, these effects were greatly inhibited suppressed by BA pretreatment. These data suggest that BA protects against OGDinduced injury in hippocampal neurons by inhibiting the activation of mitochondria-mediated apoptotic pathway. Numerous studies have shown that PI3K/Akt signaling pathway plays a crucial role in various physiological processes, such as cell proliferation, apoptosis and oxidative stress [23–25]. Activation of Akt was found to be involved in neuronal survival after cerebral I/R injury [26,27]. Ohba et al. confirmed that transgenic mice overexpressing Akt1 significantly reduced infarct volume after middle cerebral artery occlusion [28]. Akt also exerts antiapoptotic effects through inactivating pro-apoptotic proteins such as Bad and caspase-9 [29]. In this study, we observed that BA pretreatment up-regulated the expression levels of p-PI3K and p-
Please cite this article in press as: S. Jiao, et al., Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.028
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Akt in hippocampal neurons induced by OGD/R. These data suggest that BA protects against OGD-induced injury in hippocampal neurons by the activation of the PI3K/Akt signaling pathway. In conclusion, our results demonstrated that treatment with BA reduced cerebral injury in hippocampal neurons exposed to OGD/ R, and this was mediated by the activation of the PI3 K/Akt signaling pathway. This study provides evidence that BA may serve as a potential therapeutic agent for treatment of cerebral ischemia. Conflict of interest statement The authors declare that they have no conflict of interest. References [1] T. Borsello, P.G. Clarke, L. Hirt, A. Vercelli, M. Repici, D.F. Schorderet, et al., A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia, Nat. Med. 9 (2003) 1–1. [2] V. Janardhan, A.I. Qureshi, Mechanisms of ischemic brain injury, Curr. Cardiol. Rep. 6 (2004) 117–123. [3] B. Schaller, R. Graf, Cerebral ischemia and reperfusion: the pathophysiologic concept as a basis for clinical therapy, J. Cereb. Blood Flow Metab. 24 (2004) 351–371. [4] B.C. White, J.M. Sullivan, D.J. Degracia, B.J. O’Neil, R.W. Neumar, L.I. Grossman, et al., Brain ischemia and reperfusion: molecular mechanisms of neuronal injury, J. Neurol. Sci. 179 (2000) 1–33. [5] D. Dutta, B. Chakraborty, A. Sarkar, C. Chowdhury, P. Das, A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells, BMC Cancer 16 (2016) 1–19. [6] A.M.O. Amoussa, L. Lagnika, M. Bourjot, C. Vonthron-Senecheau, A. Sanni, Triterpenoids from Acacia ataxacantha DC: antimicrobial and antioxidant activities, BMC Complement. Altern. Med. 16 (2016) 284. [7] J. Wang, A. Chen, Q. Wu, X. Li, H. Li, Betulinic acid inhibits IL-1b-induced inflammation by activating PPAR-g in human osteoarthritis chondrocytes, Int. Immunopharmacol. 29 (2015) 687–692. [8] J. Li, M. Goto, X. Yang, S.L. Morris-Natschke, H. Li, C.H. Chen, et al., Fluorinated betulinic acid derivatives and evaluation of their anti-HIV activity, Bioorg. Med. Chem. Lett. 26 (2015) 68–71. lu-Demiralp, E.R. Kardaş, S. Ozgül, T. Yag ci, H. Bilgin, O. Sehirli, F. [9] E. Ekşiog Ercan, G. Sener, Betulinic acid protects against ischemia/reperfusion-induced renal damage and inhibits leukocyte apoptosis, Phytother. Res. 24 (2010) 325–332. [10] A. Xia, Z. Xue, Y. Li, W. Wang, J. Xia, T. Wei, et al., Cardioprotective effect of betulinic Acid on myocardial ischemia reperfusion injury in rats, Evid-Based Compl. Alt. Med. 2014 (2014) 743–767. [11] A.J. Krohn, E. Preis, J.H. Prehn, Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-1-like proteases as upstream initiators and increased production of superoxide as a main downstream effector, J. Neurosci. 18 (1998) 8186–8197.
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[12] X. Tao, X. Sun, L. Yin, H. Xu, L. Xu, Q. Yan, et al., Dioscin ameliorates cerebral ischemia/reperfusion injury through the downregulation of TLR4 signaling via HMGB-1 inhibition, Free Radic. Biol. Med. 84 (2015) 103–115. [13] Y. Cao, L. Zhang, S. Sun, Z. Yi, X. Jiang, D. Jia, Neuroprotective effects of syringic acid against OGD/R-induced injury in cultured hippocampal neuronal cells, Int. J. Mol. Med. 38 (2016) 567–573. [14] Z. Mo, Y. Fang, Y. He, S. Zhang, b-Asarone protects PC12 cells against OGD/Rinduced injury via attenuating Beclin-1-dependent autophagy, Acta Pharmacol. Sin. 33 (2012) 737–742. [15] J. Rodrigo, A.P. Fernández, J. Serrano, M.A. Peinado, A. Martínez, The role of free radicals in cerebral hypoxia and ischemia, Free Radic. Biol. Med. 39 (2005) 26–50. [16] C.A. Brüning, M. Prigol, C. Luchese, C.R. Jesse, M.M.M.F. Duarte, S.S. Roman, et al., Protective effect of diphenyl diselenide on ischemia and reperfusioninduced cerebral injury: involvement of oxidative stress and proinflammatory cytokines, Neurochem. Res. 37 (2012) 2249–2258. [17] P. Liu, H. Zhao, R. Wang, P. Wang, Z. Tao, L. Gao, et al., MicroRNA-424 protects against focal cerebral ischemia and reperfusion injury in mice by suppressing oxidative stress, Stroke 46 (2015) 513–519. [18] S. Leutner, A. Eckert, W.E. Müller, ROS generation, lipid peroxidation and antioxidant enzyme activities in the aging brain, J. Neural Transm. 108 (2001) 955–967. [19] J. Peng, Y.C. Lv, P.P. He, Y.Y. Tang, X. Wei, X.Y. Liu, et al., Betulinic acid downregulates expression of oxidative stress-induced lipoprotein lipase via the PKC/ERK/c-Fos pathway in RAW264.7 macrophages, Biochimie 119 (2015) 192–203. [20] A.N. Miles, N.W. Knuckey, Apoptotic neuronal death following cerebral ischaemia, J. Clin. Neurosci. 5 (1998) 125–145. [21] A. Gross, J.M. Mcdonnell, S.J. Korsmeyer, BCL-2 family members and the mitochondria in apoptosis, Genes Dev. 13 (1999) 1899–1911. [22] D.A. Le, Y. Wu, Z. Huang, K. Matsushita, N. Plesnila, J.C. Augustinack, et al., Caspase activation and neuroprotection in caspase-3- deficient mice after in vivo cerebral ischemia and in vitro oxygen glucose deprivation, Proc. Natl. Acad. Sci. 99 (2002) 15188–15193. [23] T. Ishrat, I. Sayeed, F. Atif, H. Fang, D.G. Stein, Progesterone is neuroprotective against ischemic brain injury through its effects on the PI3K/Akt signaling pathway, Neuroscience 210 (2012) 442–450. [24] J. Zhou, T. Du, B. Li, Y. Rong, A. Verkhratsky, L. Peng, Crosstalk between MAPK/ ERK and PI3 K/AKT signal pathways during brain Ischemia/Reperfusion, ASN Neuro (2015) 7. [25] T. Jover, Acute estradiol protects CA1 neurons from ischemia-induced apoptotic cell death via the PI3K/Akt pathway, Brain Res. 1321 (2010) 1–12. [26] S. Janelidze, B.R. Hu, P. Siesjö, P. Bo, Alterations of Akt1 (PKBa) and p70 S6 K in transient focal ischemia, Neurobiol. Dis. 8 (2001) 147–154. [27] M. Shibata, T. Yamawaki, T. Sasaki, H. Hattori, J. Hamada, Y. Fukuuchi, et al., Upregulation of Akt phosphorylation at the early stage of middle cerebral artery occlusion in mice, Brain Res. 942 (2002) 1–10. [28] N. Ohba, S. Kiryu-Seo, M. Maeda, M. Muraoka, M. Ishii, H. Kiyama, Transgenic mouse overexpressing the Akt reduced the volume of infarct area after middle cerebral artery occlusion, Neurosci. Lett. 359 (2004) 159–162. [29] D.P. Brazil, Z.Z. Yang, B.A. Hemmings, Advances in protein kinase B signalling: aKTion on multiple fronts, Trends Biochem. Sci. 29 (2004) 233–242.
Please cite this article in press as: S. Jiao, et al., Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.028
Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway Shujie Jiaoa , Hongcan Zhua , Ping Heb , Junfang Tenga,* a b
Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
A R T I C L E I N F O
Article history: Received 12 September 2016 Received in revised form 22 October 2016 Accepted 8 November 2016 Keywords: Betulinic acid (BA) Cerebral ischemia/reperfusion (I/R) Oxidative stress Apoptosis
A B S T R A C T
Betulinic acid (BA), a naturally occurring pentacyclic lupane group triterpenoid, has been demonstrated to protect against ischemia/reperfusion-induced renal damage. However, the effects of BA on cerebral ischemia/reperfusion (I/R) injury remain unclear. Hence, this study was to investigate the effects of BA on oxygen and glucose deprivation/reperfusion (OGD/R) induced neuronal injury in rat hippocampal neurons. Our results showed that BA pretreatment greatly attenuated OGD/R-induced neuronal injury. BA also inhibited OGD/R-induced intracellular ROS production and MDA level in rat hippocampal neurons. Furthermore, the down-regulation of Bcl-2, up-regulation of Bax and the consequent activation of caspase-3 induced by OGD/R were reversed by BA pretreatment. Mechanistic studies demonstrated that BA pretreatment up-regulated the expression levels of p-PI3 K and p-Akt in hippocampal neurons induced by OGD/R. Taken together, these data suggested that BA inhibits OGD/R-induced neuronal injury in rat hippocampal neurons through the activation of PI3 K/Akt signaling pathway. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Cerebral ischemia is among the death-related brain diseases with high morbidity and high mortality in adults [1]. It is characterized by oxidative stress, hypoxia, inflammation, and glutamate excitotoxicity, eventually leading to cell death [2]. Currently, restoration of blood supply, called reperfusion, is still the standard therapy for ischemia [3]. However, reperfusion also contributes to delayed secondary brain injury, because of the sudden recovery of blood supply which will produce excessive reactive species (ROS), leading to mitochondrial dysfunction, oxidative stress and finally cell death [4]. Hence, it is urgently needed to explore the precise mechanism of ischemia-induced neuron death. Betulinic acid (BA), (3b-hydroxy-lup-20(29)-en-28-oic acid), is a naturally occurring pentacyclic lupane group triterpenoid. Increasing evidences have reported that BA possesses a number of biological activities such as an anti-cancer, anti-oxidant, antiinflammatory and anti-HIV [5–8]. In addition, BA was found to protect against I/R injury [9,10]. For example, one study reported that BA attenuates I/R-induced renal damage through regulating
the apoptotic function of leukocytes and inhibiting neutrophil infiltration [9]. However, the effects of BA on cerebral ischemia/ reperfusion (I/R) injury remain unclear. Hence, this study was to investigate the effects of BA on oxygen and glucose deprivation/ reperfusion (OGD/R) induced neuronal injury in primary rat hippocampal neurons cells. Our results demonstrated that BA protects against OGD/R-induced neuronal injury, at least in part, through the activation of PI3 K/Akt signaling pathway. 2. Materials and methods 2.1. Cell culture Primary hippocampal neuronal cells were prepared from neonatal SD rats [11]. In brief, the hippocampi tissues were dissected and dissociated in 0.25% trypsin-EDTA (Sigma, St. Louis, USA), and primary hippocampal neurons were maintained in neurobasal medium supplemented with B-27, glutamine (0.5 mM), glutamate (25 mM), and 1% penicillin/streptomycin. The cultures were kept in a humidified incubator that was maintained at 37 C and supplied with 5% CO2 and 95% air for 14 days. Culture medium was changed two times a week.
* Corresponding author at: Department of Neurology, The First Affiliated Hospital of Zhengzhou University, 1st of Jianshe East Road, Zhengzhou 450000, China. E-mail address: [email protected] (J. Teng). http://dx.doi.org/10.1016/j.biopha.2016.11.028 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: S. Jiao, et al., Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.028
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2.2. Oxygen-glucose deprivation/reperfusion (OGD/R) model and treatment For OGD model construction to mimic ischemia in vitro, hippocampal neurons were pretreated with various concentrations of BA (1–10 mM, 2 h pretreatment; Sigma), and then exposed to DMEM without serum or glucose in a humidified atmosphere containing 95% nitrogen and 5% CO2 for 4 h. Subsequently, the medium was replaced with glucose-containing medium supplemented with 10% FBS again and, cells were subjected to culture under normoxic conditions (95% air, 5% CO2) for 24 h. Cells cultured in growth culture medium under normoxic condition were used as control. 2.3. Cell viability assay Cell viability was detected using the MTT assay. In brief, neurons were seeded into 96-well plates (Corning Inc., Corning, NY, USA) at a density of 5 103 cells/well before treatment. After 24 h of incubation, 100 mL MTT (5 mg/mL; Sigma) was added to each well for 4 h. Then the supernatant was removed and 100 mL DMSO (Sigma) was added to lyse the cells. The absorbance was measured at 490 nm with an enzyme linked immunosorbent assay plate reader (Olympus, Tokyo, Japan). 2.4. Evaluation of reactive oxygen species (ROS) and MDA 20 ,70 -Dichlorodi-hydrofluorescein diacetate (DCFH-DA) was used to evaluate intracellular ROS level. In brief, after treatment, neurons were washed with PBS, and then incubated with 10 mM DCFH-DA in the dark for 30 min at 37 C. The cells were washed with PBS and then observed under a fluorescence microscope. The fluorescence intensity was measured by a fluorospectrophotometer at excitation/emission maxima of 485/525 nm. The MDA level was detected using the commercial kit (Beyotime, Haimen, Jiangsu, China). In brief, after treatment, the supernatant of neurons was collected, then the level of MDA was measured by a method based on a reaction with thiobarbituric acid. The optical density at 532 nm was measured with a microplate reader (Molecular Device, Spectra Max 190, USA). 2.5. Caspase-3 activity detection The supernatant of the treated cells was collected, then the activity of caspase-3 was examined using the colorimetric caspase3 assay kit (Beyotime, Jiangsu, China) according to the manufacturer’s instructions.
2.7. Statistics analysis Data are expressed as the mean standard deviation (SD). Statistical significance was analyzed with the one-way analysis of variance (ANOVA) or the Student’s two-tailed t-test. The p value less than 0.05 was considered statistically significant difference. 3. Results 3.1. BA ameliorates OGD/R-induced neuron injury First, we examined the effect of BA on OGD/R-induced cellular viability in hippocampal neurons using the MTT assay. As shown in Fig. 1, compared to the control group, the cell viability was obviously reduced by the OGD/R treatment. However, pretreatment with BA significantly increased cell viability in a dosedependent manner, as compared with the OGD/R group. 3.2. BA ameliorates OGD/R-induced oxidative stress in hippocampal neurons Oxidative stress was involved in the development of cerebral I/R injury. Thus, we examined the effect of BA in on OGD/R-induced oxidative stress in hippocampal neurons. As shown in Fig. 2A, OGD/ R exposure caused a significant increase of intracellular ROS generation in hippocampal neurons, compared with that in control group. However, pretreatment with BA markedly decreased ROS level in hippocampal neurons. Similarly, BA pretreatment also suppressed OGD/R-induced MDA level in hippocampal neurons (Fig. 2B). 3.3. BA ameliorates OGD/R-induced cell apoptosis in hippocampal neurons Next, we tested the effect of BA on hippocampal neuron apoptosis induced by OGD/R. As shown in Fig. 3A, neurons subjected to OGD/R showed markedly increased caspase-3 activity. In contrast, BA pretreatment significantly suppressed caspase-3 activity, compared with OGD/R group. Furthermore, we investigated the effects of BA on Bax and Bcl-2 protein levels in OGD/R-treated hippocampal neurons. The results indicated that the protein expression level of Bcl-2 was substantially downregulated in OGD/R-induced hippocampal neurons compared with the control, whereas an enhanced expression of Bax was observed.
2.6. Western blotting The proteins were extracted from neurons using RIPA lysis buffer (Beyotime, Nantong, China). The protein concentration in the lysates was determined by BCA protein assay kit (Beyotime, Nantong, China). The proteins (30 mg/lane) were subjected to 10% SDS-PAGE and electrophoretically transferred to Immobilon P Millipore (Bedford, MA, USA). The blots were blocked in 5% nonfat milk in TBST buffer (5 mM Tris-HCl, pH 7.4, 136 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Then, the membrane was incubated overnight at 4 C with primary antibodies (dilution, 1:1,000) targeting p-PI3 K, PI3 K, p-Akt, Akt and GAPDH (all from Santa Cruz Biotechnology, CA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the immunoreactive protein bands were visualized using an enhanced chemiluminescence detection system (Amersham, Little Chalfont, UK).
Fig. 1. BA ameliorates OGD/R-induced neuron injury. Primary hippocampal neurons were pretreated with indicated concentration of BA (1–10 mM, 2 h pretreatment), and were maintained under OGD for 4 h, followed by 24 h of reoxygenation. Cell viability was determined by the MTT assay. *P < 0.05 vs. control group. #P < 0.05 vs.OGD/R group.
Please cite this article in press as: S. Jiao, et al., Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.028
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Fig. 2. BA ameliorates OGD/R-induced oxidative stress in hippocampal neurons. Primary hippocampal neurons were pretreated with indicated concentration of BA (1–10 mM, 2 h pretreatment), and were maintained under OGD for 4 h, followed by 24 h of re-oxygenation. A, 20 ,70 -dichlorodi-hydrofluorescein diacetate (DCFH-DA) was used to evaluate intracellular ROS level. B, The MDA level was examined using the commercial kit. *P < 0.05 vs. control group. #P < 0.05 vs.OGD/R group.
In addition, pretreatment with BA greatly up-regulated the protein expression of Bcl-2 and reduced the protein expression of Bax, as compared with the OGD/R group (Fig. 3B).
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Fig. 3. BA ameliorates OGD/R-induced cell apoptosis in hippocampal neurons. Primary hippocampal neurons were pretreated with indicated concentration of BA (1–10 mM, 2 h pretreatment), and were maintained under OGD for 4 h, followed by 24 h of re-oxygenation. A, The activity of caspase-3 was examined using the colorimetric caspase-3 assay kit. B, The protein expression levels of Bax and Bcl-2 were detected by the western blotting. *P < 0.05 vs. control group. #P < 0.05 vs.OGD/ R group.
4. Discussion 3.4. BA activates the PI3K/Akt signaling pathway in hippocampal neurons induced by OGD/R PI3K/Akt signaling pathway plays important role in the development of cerebral I/R injury. To further clarity the mechanism underlying the neuroprotective activity of BA, we detected the expression of p-PI3K and p-Akt using western blotting. As shown in Fig. 4A, the protein expression levels of pPI3K and p-Akt were significantly suppressed by OGD/R in hippocampal neurons, compared with the control group. However, BA pretreatment significantly enhanced the expression levels of pPI3K and p-Akt in hippocampal neurons. To further confirm the role of PI3K/Akt pathway in BA-induced neuroprotection, hippocampal neurons were treated with inhibitor of PI3K (LY294002). We found that treatment with LY294002 decreased BA-mediated survival to 63% (Fig. 4C). In addition, treatment with LY294002 significantly increased caspase-3 activity, compared with OGD/R plus BA group (Fig. 4D).
To our knowledge, this is the first report of the effects of BA on OGD/R induced neuronal injury in rat hippocampal neurons. Our results showed that BA pretreatment greatly attenuated OGD/Rinduced neuronal injury. BA also inhibited OGD/R-induced intracellular ROS production and MDA level in rat hippocampal neurons. Furthermore, the down-regulation of Bcl-2, up-regulation of Bax and the consequent activation of caspase-3 induced by OGD/ R were reversed by BA pretreatment. Mechanistic studies demonstrated that BA pretreatment up-regulated the expression levels of p-PI3K and p-Akt in hippocampal neurons induced by OGD/R. OGD/R model was constructed to mimic cerebral I/R injury [12–14]. In the present study, an in vitro OGD/R model was established in hippocampal neuronal cells to examine the neuroprotective effects of BA on cerebral I/R injury. Our results demonstrated that OGD/R treatment significantly reduced neuron viability. However, pretreatment with BA significantly increased
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Fig. 4. BA activates the PI3 K/Akt signaling pathway in hippocampal neurons induced by OGD/R. Primary hippocampal neurons were pretreated with indicated concentration of BA (1–10 mM, 2 h pretreatment), and were maintained under OGD for 4 h, followed by 24 h of re-oxygenation. A, The protein expression levels of p-PI3 K, PI3 K, p-Akt and Akt were detected by the western blotting. B, Quantification analysis was performed using Gel-Pro Analyzer version 4.0 software. C, Cell viability was determined by the MTT assay following treatment with LY294002 (20 mM). D, The activity of caspase-3 was examined using the colorimetric caspase-3 assay kit in hippocampal neurons treated with LY294002 after exposure to OGD/R for 1 h *P < 0.05 vs. control group. #P < 0.05 vs. OGD/R group, & P < 0.05 vs. OGD/R + BA group.
cell viability in a dose-dependent manner. These data suggest that BA protects against OGD-induced cell injury in hippocampal neurons. Increasing evidences have reported that the high production of ROS cause oxidative stress, which may contribute to the progression of cerebral I/R injury [15–17]. In addition, ROS has been linked to high levels of MDA and claimed to cause lipid peroxidation [18]. Recently, it was reported that BA attenuated macrophage-derived lipoprotein lipase expression and activity induced by oxidative stress, as well as decreased cellular lipid accumulation [19]. Herein, our studies observed that pretreatment with BA markedly decreased OGD/R-induced ROS generation and MDA level in hippocampal neurons. These data suggest that BA may protect against cerebral I/R injury by down-regulating the levels of MDA and SOD in hippocampal neurons after OGD/R. Apoptosis also plays an important role in the process of neuronal death after cerebral ischemia [20]. Bcl-2 is an antiapoptotic factor and important for cell survival while Bax promotes apoptosis [21]. Caspase-3, a key factor in apoptosis, eventually
leads to DNA fragmentation and triggers apoptosis during neuronal development and under pathological conditions including cerebral ischemia [22]. In this study, we observed that OGD/R treatment notably up-regulated the expression of Bax and down-regulated the expression of Bcl-2, and subsequent activation of caspase-3. However, these effects were greatly inhibited suppressed by BA pretreatment. These data suggest that BA protects against OGDinduced injury in hippocampal neurons by inhibiting the activation of mitochondria-mediated apoptotic pathway. Numerous studies have shown that PI3K/Akt signaling pathway plays a crucial role in various physiological processes, such as cell proliferation, apoptosis and oxidative stress [23–25]. Activation of Akt was found to be involved in neuronal survival after cerebral I/R injury [26,27]. Ohba et al. confirmed that transgenic mice overexpressing Akt1 significantly reduced infarct volume after middle cerebral artery occlusion [28]. Akt also exerts antiapoptotic effects through inactivating pro-apoptotic proteins such as Bad and caspase-9 [29]. In this study, we observed that BA pretreatment up-regulated the expression levels of p-PI3K and p-
Please cite this article in press as: S. Jiao, et al., Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.028
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Akt in hippocampal neurons induced by OGD/R. These data suggest that BA protects against OGD-induced injury in hippocampal neurons by the activation of the PI3K/Akt signaling pathway. In conclusion, our results demonstrated that treatment with BA reduced cerebral injury in hippocampal neurons exposed to OGD/ R, and this was mediated by the activation of the PI3 K/Akt signaling pathway. This study provides evidence that BA may serve as a potential therapeutic agent for treatment of cerebral ischemia. Conflict of interest statement The authors declare that they have no conflict of interest. References [1] T. Borsello, P.G. Clarke, L. Hirt, A. Vercelli, M. Repici, D.F. Schorderet, et al., A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia, Nat. Med. 9 (2003) 1–1. [2] V. Janardhan, A.I. Qureshi, Mechanisms of ischemic brain injury, Curr. Cardiol. Rep. 6 (2004) 117–123. [3] B. Schaller, R. Graf, Cerebral ischemia and reperfusion: the pathophysiologic concept as a basis for clinical therapy, J. Cereb. Blood Flow Metab. 24 (2004) 351–371. [4] B.C. White, J.M. Sullivan, D.J. Degracia, B.J. O’Neil, R.W. Neumar, L.I. Grossman, et al., Brain ischemia and reperfusion: molecular mechanisms of neuronal injury, J. Neurol. Sci. 179 (2000) 1–33. [5] D. Dutta, B. Chakraborty, A. Sarkar, C. Chowdhury, P. Das, A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells, BMC Cancer 16 (2016) 1–19. [6] A.M.O. Amoussa, L. Lagnika, M. Bourjot, C. Vonthron-Senecheau, A. Sanni, Triterpenoids from Acacia ataxacantha DC: antimicrobial and antioxidant activities, BMC Complement. Altern. Med. 16 (2016) 284. [7] J. Wang, A. Chen, Q. Wu, X. Li, H. Li, Betulinic acid inhibits IL-1b-induced inflammation by activating PPAR-g in human osteoarthritis chondrocytes, Int. Immunopharmacol. 29 (2015) 687–692. [8] J. Li, M. Goto, X. Yang, S.L. Morris-Natschke, H. Li, C.H. Chen, et al., Fluorinated betulinic acid derivatives and evaluation of their anti-HIV activity, Bioorg. Med. Chem. Lett. 26 (2015) 68–71. lu-Demiralp, E.R. Kardaş, S. Ozgül, T. Yag ci, H. Bilgin, O. Sehirli, F. [9] E. Ekşiog Ercan, G. Sener, Betulinic acid protects against ischemia/reperfusion-induced renal damage and inhibits leukocyte apoptosis, Phytother. Res. 24 (2010) 325–332. [10] A. Xia, Z. Xue, Y. Li, W. Wang, J. Xia, T. Wei, et al., Cardioprotective effect of betulinic Acid on myocardial ischemia reperfusion injury in rats, Evid-Based Compl. Alt. Med. 2014 (2014) 743–767. [11] A.J. Krohn, E. Preis, J.H. Prehn, Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-1-like proteases as upstream initiators and increased production of superoxide as a main downstream effector, J. Neurosci. 18 (1998) 8186–8197.
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Please cite this article in press as: S. Jiao, et al., Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.028