neuroprotective effects of a novel peptide fk18 under oxygen glucose deprivation in sh sy5y cells and retinal ischemia in rats via the akt pathway

Accepted Manuscript Neuroprotective effects of a novel peptide, FK18, under oxygen-glucose deprivation in SH-SY5Y cells and retinal ischemia in rats via the Akt pathway Shuyu Xiong, Yupeng Xu, Mingming Ma, Haiyan Wang, Fang Wei, Qing Gu, Xun Xu PII: S0197-0186(16)30365-5 DOI: 10.1016/j.neuint.2017.02.015 Reference: NCI 4018 To appear in: Neurochemistry International Received Date: October 2016 Revised Date: February 2017 Accepted Date: 27 February 2017 Please cite this article as: Xiong, S., Xu, Y., Ma, M., Wang, H., Wei, F., Gu, Q., Xu, X., Neuroprotective effects of a novel peptide, FK18, under oxygen-glucose deprivation in SH-SY5Y cells and retinal ischemia in rats via the Akt pathway, Neurochemistry International (2017), doi: 10.1016/ j.neuint.2017.02.015 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Neuroprotective Effects of a Novel Peptide, FK18, under Oxygen-Glucose Deprivation in SH-SY5Y Cells and Retinal Ischemia in Rats via the Akt pathway Shuyu Xionga,b, Yupeng Xua,b, Mingming Maa,b, Haiyan Wanga,b, Fang Weia,b, Qing Gua,b, Xun RI PT Xu* a,b a Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai, 200080, China SC b Shanghai Key Laboratory of Fundus Disease, Shanghai, 200080, China M AN U *Corresponding authors: Xun Xu Mailing address: No 100 Haining Road, Shanghai 200080, PR China Phone number: +86 13386259538 Fax: +86 21 63240090 TE D E-mail address: drxuxun@sjtu.edu.cn AC C Abbreviations EP Running Title: Neuroprotective effect of peptide FK18 bFGF, basic fibroblast growth factor; OGD, oxygen–glucose deprivation; I/R, ischemia-reperfusion; PBS, phosphate-buffered saline; IOP, intraocular pressure; RGCs, retinal ganglion cells; IPL, inner plexiform layer (IPL); INL, inner nuclear layer; ORL, outer retinal layer; GCL, ganglion cell layer; TUNEL, TdT-Mediated dUTP Nick-End Labeling; FG, Fluoro-Gold; ERG, Electroretinogram; PI3K, phosphatidylinositol 3-kinase ACCEPTED MANUSCRIPT Neuroprotective Effects of a Novel Peptide, FK18, under Oxygen-Glucose Deprivation in SH-SY5Y Cells and Retinal Ischemia in Rats via the Akt pathway RI PT Abstract Ischemic neuronal injury is associated with several life- and vision-threatening diseases Neuroprotection is essential in the treatment of these diseases Here, we identified and SC characterized a novel peptide, FK18, from basic fibroblast growth factor (bFGF) We further M AN U assessed the neuroprotective effects of this peptide and its potential mechanisms using the in vitro oxygen–glucose deprivation (OGD) model in SH-SY5Y cells and the in vivo retinal ischemia-reperfusion (I/R) injury model to mimic ischemic neuronal injury Our results suggested that FK18 significantly increased the viability of and attenuated the apoptosis of SH-SY5Y cells TE D It also markedly alleviated I/R-induced retinal neuronal apoptosis, damage to retinal ganglion cells (RGCs), and morphological and functional damage to the retina Moreover, FK18 increased Akt phosphorylation under both normoxic and OGD conditions, attenuated mitochondrial EP translocation of the proapoptotic protein Bad, up-regulated the expression of Bcl-2/Bax, and AC C inhibited the release of cytochrome c from the mitochondria into the cytoplasm These results suggested that FK18 is a novel neuroprotective agent that may serve as a prototype for neuroprotective drug development Keywords peptide; neuronal ischemia; neuroprotection; Akt ACCEPTED MANUSCRIPT Introduction Cerebral ischemic injury, which occurs when the cerebral blood supply is cut off, resulting in oxygen and glucose deprivation in brain tissues, is one of the leading causes of death and RI PT long-term disability worldwide (Mozaffarian et al., 2016) As part of the central nervous system, the visual system, including retinal ganglion cells (RGCs) and other neurons, is also susceptible to SC ischemic injury Retinal ischemia plays important roles in the pathogenesis of several vision-threatening diseases, including retinal artery occlusion, diabetic retinopathy, glaucoma, and M AN U optic neuropathy (Osborne et al., 2004) These diseases are major causes of blindness worldwide and usually result in vision loss due to irreversible damage to retinal neurons Neuroprotection is necessary in the treatment of ischemic neuronal injury to optimize patient TE D outcome, in addition to therapeutic strategies primarily directed at the vasculature to promote prompt restoration of the blood supply or to maintain circulatory patency (Ginsberg, 2008; Osborne et al., 2004) At present, chemicals, such as calcium channel blockers, glutamate EP antagonists, and antioxidant/radical scavenging agents, are primarily used to provide AC C pharmacological neuroprotection (Gwag et al., 2007; Maniskas et al., 2016; Sakata et al., 2010; Sun et al., 2015; Wang et al., 2016; Weng and Kriz, 2007) Recombinant protein therapeutics is another popular approach for neuroprotection, and it involves the use of neurotrophic factors, growth factors, erythropoietin (EPO), etc (Jiang et al., 2011; Junk et al., 2002; Larpthaveesarp et al., 2016; Li and Stephenson, 2002; Tabakman et al., 2005; Zhao et al., 2016) Nevertheless, the drawbacks of small-molecule chemicals (e.g low target specificity and unexpected side effects) and protein therapeutics (e.g poor bioavailability, low membrane permeability, and a long C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT manufacturing cycle) impede their effective application (Craik et al., 2013) Therefore, small-molecule bioactive peptides that combine the advantages of chemical and protein therapeutics but overcome their disadvantages will likely become a new hot area of investigation RI PT for researchers To this end, we identified a novel 18-amino acid peptide, FK18, from basic fibroblast growth SC factor (bFGF), one of the 22 members of the FGF family that share an internal core region of ~120 amino acids, with ~30%-60% identical residues (Itoh and Ornitz, 2004) Then, we assessed its M AN U neuroprotective effects and potential mechanisms using the well-established in vitro oxygen– glucose deprivation (OGD) model in neuronal SH-SY5Y cells and the highly reproducible in vivo retinal ischemia-reperfusion (I/R) injury model to mimic ischemic insult to neurons TE D Materials and methods 2.1 Preparation of peptides EP A sequence segment of 18 amino acids (FFFERLESNNYNTYRSRK, molecular weight: 2371.6 AC C Da) was selected based on combined analysis of the active binding domain of bFGF (the loop between β10-β11 and the regions involving β8-β9) and the conserved amino acid sequence among members of the FGF family and among different species (Baird et al., 1988; Eriksson et al., 1991; Itoh and Ornitz, 2004, 2008; Plotnikov et al., 1999; Zhu et al., 1991) The structure of the peptide is presented in Fig To determine whether the neuroprotective effect of FK18 is sequence dependent, a scrambled peptide (SCpep) was simultaneously synthesized and used as a negative control in the following in vitro and in vivo experiments Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT FK18 and SCpep were synthesized by China Peptides Co., Ltd (Shanghai, PR China) with a high-efficiency solid-phase method utilizing an automatic peptide synthesizer (Symphony; Protein Technologies, Tucson, AZ) Characterization of the purified peptides was performed by analytical RI PT high-performance liquid chromatography (HPLC, analytical; Shimadzu, Kyoto, Japan) and mass spectrometry (MS, Finnigan TSQ 7000; Thermo, Waltham, MA) after synthesis and purification, M AN U 2.2 Stability assay of peptides in aqueous solutions SC and all peptides were freeze-dried and stored at -20°C until use The stability of peptides in various aqueous solutions was investigated, as described in previous study.(Staes et al., 2010; Xu et al., 2010) Lyophilized FK18 and SCpep were dissolved in water or in buffer at a concentration of 250 µg/ml Buffers included: 1) sodium citrate buffer solution pH 4; TE D 2) sodium citrate buffer solution pH 6; 3) Hanks balanced salt solution (HBSS) PH 7.4; 4) phosphate buffer saline (PBS) pH 7.4; and 5) BSS PLUSTM sterile intraocular irrigating solution (Alcon, Inc.; Hünenberg, Switzerland) BSS PLUSTM is an intraocular irrigating solution for use EP during all intraocular surgical procedures (used to imitate the intraocular condition) The peptide AC C solutions were incubated at 4°C or 37°C for 4, 24, or 48 h, then stored at -80°C The solutions without incubation and immediately frozen at -80°C were served as controls The stability of peptides was evaluated by HPLC Samples were thawed at room temperature for before testing 2.3 Cell culture and treatment The human neuroblastoma cell line SH-SY5Y, which has similar morphological, neurochemical Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT and electrophysiological properties to neurons, was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences Cells were maintained in DMED/F-12 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Carlsbad, CA) and were kept in a RI PT humidified atmosphere with 5% carbon dioxide at 37°C The SH-SY5Y cells were passaged by trypsinization every to d The cells were subjected to OGD as previously reported(Marutani et al., 2012) Briefly, they were washed twice with phosphate-buffered saline (PBS), and the medium SC was replaced with glucose-free RPMI 1640 containing L-glutamine (Gibco, Carlsbad, CA) that M AN U had been deoxygenated with an anaerobic gas mixture (95% N2-5% CO2) for 30 before use The cells were then placed in a hypoxia chamber, flushed with the anaerobic gas mixture (95% N2-5% CO2) and incubated at 37°C After different lengths of time (1-12 h), the medium was replaced with DMEM/F-12, and the cells were incubated for 12 h in 95% air/5% CO2 in a TE D humidified incubator Different concentrations of FK18 (0.1, 1, 10, 50, and 100 µg/ml), SCpep (10 µg/ml) and recombinant human bFGF (100 ng/ml, R&D) were each added to separate aliquots of EP cells h prior to the induction of OGD FK18, SCpep and bFGF were dissolved in PBS For investigation of the effect of FK18 on Akt phosphorylation, a phosphatidylinositol 3-kinase (PI3K) AC C inhibitor, LY294002 (20 µM, Beyotime Institute of Biotechnology, Shanghai, China), was added h prior to the addition of FK18 The control cells were subjected to the same experimental procedures with vehicle only and without exposure to the glucose-free medium or anoxia 2.4 Animals Adult male Wistar rats weighing 180-200 g were purchased from Shanghai Laboratory Animal Center, Chinese Academy Sciences and used for all the experiments All animals were fed with a Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT standard diet ad libitum and maintained under a 12-h light/12-h dark photoperiod All procedures involving animals conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research All types of surgery and manipulation were performed in Shanghai Key analysis was n = for each intervention and group SC 2.5 Retinal ischemia RI PT Laboratory of Fundus Disease The number of animals used for morphological and functional M AN U Before initiation of the experiment, the rats were deeply anesthetized by intraperitoneal injection of 10% (w/v) chloral hydrate One drop of 0.4% oxybuprocaine hydrochloride was administered for corneal anesthesia, and one drop of 5% tropicamide was applied for pupil dilation Thirty minutes prior to the onset of retinal ischemia, the rats were randomized to receive one of the TE D following treatments using a computer-generated list: µg/eye FK18, 20 µg/eye FK18, 20 µg/eye SCpep, 20 µg/eye bFGF or normal PBS These injections were performed intravitreally by puncturing the eye with a 30-gauge needle at the corneal-scleral junction For the induction of EP retinal ischemia, the anterior chamber was cannulated with a 30-gauge needle connected to a AC C container of sterile saline The container was elevated to raise the intraocular pressure (IOP) above the systolic blood pressure (150 mmHg) for 60 Retinal ischemia was confirmed by observing whitening of the iris and loss of the red reflex of the retina Then, the pressure was returned to normal, and the eye was examined to ensure that retinal blood flow had been reestablished Rats with lens injuries and vitreous hemorrhages were excluded from the investigation A sham procedure was performed on control eyes without elevation of the container Body temperature was maintained at 37°C with a heating blanket from the time of the induction of anesthesia until Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT the animals had recovered from the anesthesia The injections were repeated every days for one week For the evaluation of the effect of FK18 on Akt phosphorylation in vivo, 20 µg/eye FK18 was injected intravitreally without the induction of retinal ischemia RI PT 2.6 Cell viability assay Cell viability was quantitatively evaluated at h, h, h, and 12 h of OGD and at 12 h of SC re-oxygenation with and without pretreatment with different concentrations of FK18, SCpep or M AN U bFGF using an MTS kit (Promega, Madison, WI, USA) Briefly, 20 µl of an MTS solution was added to each well of a 96-well microtiter plate, and the plate was incubated for h at 37°C Absorbance was measured at 490 nm using a microtiter plate reader Cell viability was expressed as a percentage of the optical density measured in the control group TE D 2.7 Annexin V staining and flow cytometry The percentage of apoptotic cells following OGD insult was determined by flow cytometry using EP an Annexin V-FITC/PI Detection Kit (BD Pharmingen, San Diego, CA, USA), according to the AC C manufacturer’s instructions Briefly, cells were digested with 0.05% trypsin for min, and neurons were collected by centrifugation (1000 × g for min) The pellets were washed twice with cold PBS and then resuspended in binding buffer at a concentration of 1×106 cells/ml A 100 àl volume of the cell suspension (1ì105 cells) was then transferred to a ml culture tube with the addition of µl FITC Annexin V and µl PI After incubation of the cells in the dark at room temperature for 15 min, they were analyzed by flow cytometry (FACS Caliber, Becton Dickinson, Heidelberg, Germany) Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 2.8 Histological examination The rats were euthanized on day of reperfusion for morphometric analysis The eyes were enucleated and fixed for 24 h at 4°C After fixation, the anterior segment was removed from each RI PT eye The posterior eyecup was dehydrated and embedded in paraffin Retinal cross sections (5 µm thick) were then cut and stained with hematoxylin and eosin (Sigma, MO, USA) The sections SC were photographed and measured at approximately to disc diameters from the optic nerve using a microscope (Olympus BX53; Olympus, Tokyo, Japan) The thicknesses of the inner M AN U plexiform layer (IPL), inner nuclear layer (INL), and outer retinal layer (ORL, the part of the retina from the outer plexiform layer to the retinal pigment epithelial cell layer) were measured The number of cells in the ganglion cell layer (GCL) was determined by cells counts over a distance scale of 200 µm In each eye, the retinal thickness and cell number were calculated as the TE D mean values of at least three measurements in adjacent sections The thickness of retinal layers and the number of cells were determined in blinded fashion by an independent scientist EP 2.9 TdT-Mediated dUTP Nick-End Labeling (TUNEL) assay AC C For the in vitro experiments, after OGD insult, SH-SY5Y cells were fixed with 4% paraformaldehyde (PFA) in PBS for 25 at 4°C, and they were then permeabilized with 0.2% Triton X-100 for For the in vivo experiments, at 24 h after reperfusion (n = in each group), paraffin-embedded retinal tissue sections, prepared as mentioned above, were deparaffinized, rehydrated, fixed with 4% PFA for 15 at 4°C, and then subjected to enzymatic digestion with 20 µg/ml proteinase K for 8-10 at room temperature Induction of apoptosis was examined by TUNEL assay using a DeadEndTM Fluorometric TUNEL System (Promega, Madison, WI, USA) Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT inhibition and transcriptional activation Exp Eye Res 94, 98-108 Zhao, Y.Z., Lin, M., Lin, Q., Yang, W., Yu, X.C., Tian, F.R., Mao, K.L., Yang, J.J., Lu, RI PT C.T., Wong, H.L., 2016 Intranasal delivery of bFGF with nanoliposomes enhances in vivo neuroprotection and neural injury recovery in a rodent stroke model J Control SC Release 224, 165-175 M AN U Zhu, X., Komiya, H., Chirino, A., Faham, S., Fox, G.M., Arakawa, T., Hsu, B.T., Rees, D.C., 1991 Three-dimensional structures of acidic and basic fibroblast growth factors AC C EP TE D Science 251, 90-93 36 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT Legends Fig 1: The peptide FK18 derived from human bFGF (Protein Data Bank accession no 1BAS) bFGF is a single-stranded 146-amino acid polypeptide that adopts a β-trefoil structure containing RI PT four-stranded β-sheets arranged in a triangular array The sequence of FK18 is presented as a stick structure in magenta, and the remainder of bFGF is presented in cartoon form in wheat This SC image was created with a PyMOL Molecular Graphic system (Delano Scientific) M AN U Fig 2: The effect of FK18 on cell viability after OGD-induced injury determined by MTS assay (A) The viability of SH-SY5Y cells after OGD for h, h, h or 12 h, followed by re-oxygenation for 12 h The insult of h of OGD, with a decrease in cell viability of 48%, was chosen in the present study (B) The viability of SH-SY5Y cells incubated with different TE D concentrations of FK18 (0.1, 1, 10, 50, and 100 µg/ml), SCpep (10 µg/ml) and recombinant human bFGF (100 ng/ml) h prior to the induction of OGD for h, followed by re-oxygenation for 12 h The peak effect was observed with 10 µg/ml FK18, and it was selected to examine the EP protective effects in the following assays The data are expressed as the mean ± SD **p < 0.01 AC C compared with the control group; #p < 0.05 and ##p < 0.01 compared with the OGD group Fig 3: The effect of FK18 on cell apoptosis after OGD-induced injury (A) Apoptotic cells were assessed by flow cytometry (B) Representative immunofluorescence images of TUNEL-positive (green) SH-SY5Y cells Scale bar, 50 µm (C) Histogram indicating the average numbers of Annexin V-positive cells following various treatments (D) Histogram showing the percentages of TUNEL-positive cells relative to the total number of neurons following various treatments Six fields were randomly selected to count positive signals in each experimental group The data are 37 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT expressed as the mean ± SD **p < 0.01 compared with the control group; #p < 0.05 and ##p < 0.01 compared with the OGD group Fig 4: The neuroprotective effects of FK18 against retinal I/R injury (A) Representative RI PT photomicrographs of retinal cross sections showing the effects of FK18 on retinal morphological changes at d after I/R Scale bar, 50 µm (B, C) Histogram showing cell body counts from the SC retinal ganglion cell layer (GCL) over 200 µm distances and the thicknesses of the retinal layers, including the inner plexiform layer (IPL), inner nuclear layer (INL), and outer retinal layer (ORL) M AN U (D) Representative immunofluorescence images of TUNEL-positive (green) retinal cells at 24 h after I/R Scale bar, 50 µm (E) Histogram showing the numbers of TUNEL-positive cells in the GCL and INL within mm of the optic disk (F) Representative photomicrographs of Fluoro-Gold-labeled RGCs showing the effect of FK18 on RGCs at d after I/R Scale bar, 50 µm TE D (G) Histogram showing the density of RGCs (H) Individual typical electroretinogram (ERG) recordings of the five groups at d after I/R (I) Histogram showing the relative a- and b-wave EP ratios The data are expressed as the mean ± SD, n = **p < 0.01 compared with the control AC C group; #p < 0.05 and ##p < 0.01 compared with the I/R-plus-vehicle group Fig 5: The effect of FK18 on Akt phosphorylation (A, B) Representative western blot analysis showing the effect of FK18 on p-FGFR1 and p-Akt expression after incubation with FK18 for different durations under normoxic conditions (C) Representative western blot image showing the effect of FK18 on p-Akt expression after induction of OGD in the presence or absence of LY294002 (D) Quantification of p-FGFR1 expression The p-FGFR1 level is expressed as the relative signal ratio of p-FGFR1/total FGFR1 (E, F) Quantification of p-Akt expression The 38 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT p-Akt level is expressed as the relative signal ratio of p-Akt/total Akt (G) The viability of SH-SY5Y cells treated in the same manner as in (C) Cell viability was determined by MTS assay (H) Representative western blot image showing the effect of FK18 on p-Akt expression in vivo (I) RI PT Quantification of p-Akt expression in vivo The data are expressed as the mean ± SD **p < 0.01 compared with the control group; ##p < 0.01 compared with the OGD group; $$p < 0.01 compared with the OGD and FK18 group; NS*, no significance compared with the control group, NS#, no SC significance compared with the OGD group M AN U Fig 6: The effect of FK18 on the mitochondrial apoptosis pathway in SH-SY5Y cells (A) Representative western blots showing cytochrome c expression in the mitochondria and cytoplasm (B, C) Quantification of cytochrome c expression Protein expression in the mitochondria is normalized by COXIV expression, while that in the cytoplasm is normalized by β-actin expression TE D (D, E, F) Representative western blots showing Bad phosphorylation, the expression of Bcl-2, Bax, Bcl-xL after OGD injury with or without FK18 pretreatment (G, H) Quantification of p-Bad and EP Bcl-xL expression The relative protein expression levels are normalized by GAPDH expression AC C (I) The ratio of Bcl-2 to Bax expression after various treatments (J) Representative western blots showing the expression of cleaved caspase-9 and caspase-3 after OGD injury with or without FK18 pretreatment (K, L) Quantification of cleaved caspase-9 and caspase-3 expression The relative protein expression levels are normalized by full length caspase-9 and caspase-3 expression, respectively The data are presented as the mean ± SD **p < 0.01 compared with the control group; #p < 0.05, and ##p < 0.01 compared with the OGD group, $p < 0.05, and $$p < 0.01 compared with the OGD and FK18 group 39 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT Fig 7: Safety evaluation of FK18 (A) MTS assay showed that the viability of SH-SY5Y cells was not affected following incubation with different concentrations of FK18 for 24 h (B) Histologic examination of rat retinas at d after FK18 treatment (20 µg/eye) showed a normal retinal RI PT structure; all layers of the retina were clear and intact, without edema, infiltration, or any other signs of inflammatory or immune reactions Scale bar: 50 µm (C, D, E) Transmission electron micrographs of different cell types in the neuroretina show normal morphologies, with no SC apparent swelling, vacuolization or disruption (C) Photoreceptor cells, (D) bipolar cell, (E) M AN U ganglion cell (arrowhead) with nerve fibers (*) and Müller cells (arrow) (F) ERG recordings from rat eyes at baseline, d and d after intravitreal injection of FK18 No obvious changes in a- and b-wave morphology and amplitudes were observed Fig 8: Schematic mechanism of the neuroprotective effects of FK18 against OGD injury in TE D SH-SY5Y cells After acting on FGFR1, FK18 activates Akt by phosphorylating it at Ser437 In turn, Akt phosphorylates BAD at Ser136, leading to up-regulation of the anti-apoptotic proteins EP Bcl-2/Bcl-xL and down-regulation of the pro-apoptotic protein Bax These events attenuate the AC C release of cytochrome c from the mitochondria into the cytoplasm, resulting in reduced caspase-9 and caspase-3 activities, eventually leading to a significant increase in neuronal survival 40 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT Table Stability of FK18 and SCpep in Different Conditions Mean ± SD percentage of controls Temperature ( ) Time (h) FK18 SCpep sodium citrate 4 sodium citrate 37 sodium citrate sodium citrate 37 PBS 7.4 PBS 7.4 37 HBSS 7.4 HBSS 7.4 37 H2O 4.7 H2O 4.7 37 BSS PLUSTM 7.4 BSS PLUSTM 7.4 24 48 24 48 24 48 24 48 24 48 24 48 24 48 24 48 24 48 24 48 24 48 24 48 100.18 ± 0.22 99.68 ± 0.06 99.44 ± 0.44 99.83 ± 0.07 99.73 ± 0.07 99.38 ± 0.04 100.06 ± 0.07 99.83 ± 0.06 99.21 ± 0.09* 99.94 ± 0.14 98.47 ± 0.27* 97.30 ± 0.15* 100.03 ± 0.09 99.78 ± 0.10 99.74 ± 0.11 99.84 ± 0.05 99.73 ± 0.03 99.70 ± 0.25 99.97 ± 0.03 99.78 ± 0.06 99.57 ± 0.03 99.98 ± 0.14 99.68 ± 0.07 99.48 ± 0.06 100.09 ± 0.15 99.76 ± 0.06 99.66 ± 0.06 100.06 ± 0.07 99.83 ± 0.08 99.43 ± 0.06 100.00 ± 0.15 99.74 ± 0.07 99.42 ± 0.11 99.77± 0.05 99.61 ± 0.06 99.12 ± 0.26 99.86 ± 0.05 99.73 ± 0.05 99.44 ± 0.12 99.82 ± 0.06 99.45 ± 0.09 99.21 ± 0.03 99.89 ± 0.07 99.82 ± 0.04 99.53 ± 0.04* 99.89 ± 0.08 98.44 ± 0.09* 97.70 ± 0.06* 100.01 ± 0.12 99.88 ± 0.05 99.68 ± 0.05 99.90 ± 0.04 99.70 ± 0.03 99.52 ± 0.03 100.07 ± 0.18 99.65 ± 0.13 99.50 ± 0.36 99.85 ± 0.07 99.72 ± 0.09 99.43 ± 0.05 99.90 ± 0.03 99.81 ± 0.07 99.55 ± 0.07 99.86 ± 0.04 99.79 ± 0.03 99.62 ± 0.19 99.99 ± 0.11 99.72 ± 0.09 99.61 ± 0.14 99.85 ± 0.10 99.65 ± 0.07 99.31 ± 0.29 TE D M AN U SC RI PT pH 37 EP Medium AC C No statistical differences unless otherwise stated *P