Acta Pharmacologica Sinica (2018) 39: 24–34 © 2018 CPS and SIMM All rights reserved 1671-4083/18 www.nature.com/aps Article Carnosine suppresses oxygen-glucose deprivation/ recovery-induced proliferation and migration of reactive astrocytes of rats in vitro Li OU-YANG1, Yuan LIU1, Bing-yu WANG1, Pei CAO1, Jing-jing ZHANG1, Yu-yan HUANG1, Yao SHEN1, *, Jian-xin LYU1, 2 1Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life sciences, Wenzhou Medical University, Wenzhou 325035, China; 2Laboratory Medicine College, Hangzhou Medical College, Hangzhou 310053, China Abstract Glial scar formation resulted from excessive astrogliosis limits axonal regeneration and impairs recovery of function, thus an intervention to ameliorate excessive astrogliosis is crucial for the recovery of neurological function after cerebral ischemia. In this study we investigated the effects of carnosine, an endogenous water-soluble dipeptide (β-alanyl-L-histidine), on astrogliosis of cells exposed to oxygen-glucose deprivation/recovery (OGD/R) in vitro. Primary cultured rat astrocytes exhibited a significant increase in proliferation at 24 h recovery after OGD for 2 h. Pretreatment with carnosine (5 mmol/L) caused G1 arrest of reactive astrocytes, significantly attenuated OGD/R-induced increase in cyclin D1 protein expression and suppressed OGD/R-induced proliferation of reactive astrocytes. Carnosine treatment also reversed glycolysis and ATP production, which was elevated at 24 h recovery after OGD. A marked increase in migration of reactive astrocytes was observed at 24 h after OGD, whereas carnosine treatment reversed the expression levels of MMP-9 and suppressed the migration of astrocytes. Furthermore, carnosine also improved neurite growth of cortical neurons co-cultured with astrocytes under ischemic conditions. These results demonstrate that carnosine may be a promising candidate for inhibiting astrogliosis and promoting neurological function recovery after ischemic stroke. Keywords: cerebral ischemia; astrogliosis; carnosine; oxygen-glucose deprivation/recovery (OGD/R); cell cycle; energy metabolism; neurite growth Acta Pharmacologica Sinica (2018) 39: 24–34; doi: 10.1038/aps.2017.126; published online 21 Sep 2017 Introduction ultimately limits axonal regeneration and impairs recovery of Cerebral ischemic stroke is a highly disabling and deadly dis- function[7, 8]. In this regard, manipulation of the glial scar may ease worldwide[1]. Although a portion of patients with stroke be favorable for neuronal regeneration and functional recov- can exhibit long-term survival, they will have lifelong disabil- ery. ity owing to the low axonal regenerative capacity of the adult L-Carnosine (β-alanyl-L-histidine) is an endogenous water- central nervous system (CNS)[2]. The lack of axonal regenera- soluble dipeptide that can be transported from the plasma to tion during the chronic phase following cerebral ischemia is the cerebrospinal fluid through a proton-coupled oligopeptide due not to the intrinsic incapacity of the neuron to regenerate transporter known as PEPT2[9, 10]. In the CNS of vertebrates, but rather to the presence of an adverse environment in the carnosine is mainly distributed in glial cells, especially in injury[3]. astrocytes[11]. Carnosine is a very versatile dipeptide, and it Glial scars predominately consist of reactive astrocytes, has been postulated to have numerous biological roles such microglia/macrophages and extracellular matrix molecules as free radical scavenger, protein glycosylation inhibitor, anti- that are known to be secreted by reactive astrocytes[4, 5]. Glial inflammatory agent and pH buffer[12]. However, so far, the scar formation shows polarity towards the injury[6] and is physiological activity of carnosine in the brain remains to be beneficial in limiting the expansion of injury. However, it elucidated. Recently, data obtained in several independent laboratories suggested that carnosine can provide neuropro- tection against ischemic injury in the acute phase of stroke[13-15]. *To whom correspondence should be addressed. However, the role of carnosine in glial scar formation follow- E-mail [email protected] ing cerebral ischemia remains unclear. Recent studies from Received 2017-02-24 Accepted 2017-06-05 our group and other laboratories have revealed that by inhib- www.chinaphar.com Ou-Yang L et al 25 iting bioenergy production, carnosine inhibits the proliferation CO2 incubator (5% CO2, 95% air, 37 °C). After 48 h in vitro, of several cancer cell lines[16, 17]. In addition, we found that car- the culture medium was half replaced with special culture nosine has dual effects on the energy metabolism of cultured medium supplemented with B-27. The medium was changed astrocytes under physiological and ischemic conditions[18, 19]. every 2–3 d. After 10–12 d, the neurons in these cultures sat Thus, we note that carnosine may have an influence on glial on the top of a confluent monolayer of astrocytes. The experi- scar formation in response to cerebral ischemia by affecting ments were performed using these cultures. cell proliferation. Therefore, the current study was designed to explore Oxygen-glucose deprivation and carnosine treatment whether carnosine could inhibit gliosis and promote neuronal The cells were washed twice and incubated in glucose-free regeneration after ischemia in an in vitro ischemic model and DMEM. Then, the cells were transferred into an anaerobic the possible underlying mechanisms. chamber filled with a gas mixture of 95% N2 and 5% CO2 at 37 °C. At the end of OGD, the cells were replaced with normal Materials and methods culture medium and returned to the normal culture condition Materials for an additional 24, 48 or 72 h. In each experiment, cultures Carnosine (β-alanyl-L-histidine), bovine serum albumin, Tri- exposed to OGD were compared with normoxic controls sup- ton X-100, 5-bromodeoxyuridine, propidium iodide, RNase, plied with DMEM containing glucose and maintained in stan- sodium pyruvate, rotenone, oligomycin, and carbonyl cyanide dard incubation conditions. Carnosine at a concentration of 1 p-trifluoromethoxyphenylhydrazone (FCCP) were obtained or 5 mmol/L was supplied 30 min before OGD and was pres- from Sigma (St Louis, MO, USA). Dulbecco's modified Eagle's ent throughout the OGD and recovery process. medium (DMEM), glucose-free DMEM, fetal bovine serum, horse serum, and B27 were from GIBCO-BRL (Grand Island, Lactate dehydrogenase (LDH) assay protocol NY, USA). Trypsin, poly-D-lysine, penicillin, streptomycin, Relative cell proliferation was determined by comparing the L-glutamine, lactate dehydrogenase kit, paraformaldehyde, total amount of LDH present in cell lysates after 2- or 4-h and BCA Protein Assay Kit were purchased from Beyotime OGD followed by 24-, 48- or 72-h recovery to LDH present in Institute of Biotechnology (Nanjing, China). XF assay medium cell lysates of controls as described previously[20]. Total LDH and XF calibrant solution were purchased from Seahorse Bio- present in cell lysates was measured with an LDH kit accord- science. A rat interleukin 1β (IL-1β) ELISA kit was purchased ing to the manufacturer’s instructions. LDH metabolism was from Nanjing Jiancheng Bioengineering Institute (China). quantified by measuring absorbance at 490 nm and 600 nm in a Thermo Scientific Varioskan Flash. Primary cortical astrocyte-enriched culture All experiments using animals were performed in accordance BrdU immunofluorescent staining with the National Institutes of Health Guide for the Care and To further assess proliferation of glial cells in culture, cells Use of Laboratory Animals. Primary cultures of cortical astro- were incubated with 30 µmol/L 5-bromodeoxyuridine (BrdU) cytes were prepared from the cortices of neonatal Sprague- after OGD. After the experiments, the cells were fixed with Dawley rats as described previously[18]. In brief, cortices were 4% paraformaldehyde for 10 min and then were washed in dissected from the brains under sterile conditions, digested PBS for 3 min, and the DNA was denatured by incubating in in 0.25% trypsin for 20 min at 37 °C, and then the dissociated 2 mol/L HCl for 0.5 h. After being washed twice in 0.1 mol/L cells were seeded in high glucose (4.5 g/L) DMEM supple- borate buffer (pH 8.5) and three times in PBS, cells were mented with 10% fetal bovine serum, 2 mmol/L glutamine, blocked with 3% BSA for 1 h. Then, cells were incubated in 100 U/mL penicillin and 100 µg/mL streptomycin. The cul- mouse monoclonal antibody against BrdU (1:600, CST, USA) tures were maintained at 37 °C in a humidified atmosphere overnight. After being washed twice with PBS, cells were then of 5% CO2/95% air. On d 10–11, the confluent cultures were incubated with goat anti-mouse IgG (1:300, Invitrogen, USA) shaken overnight to minimize microglia contamination. More for 0.5 h. After further washing in PBS, cells were mounted than 95% of the cultured cells were astrocytes as identified by and observed under fluorescence microscopy (Nikon Eclipse immunofluorescent staining for GFAP. TI, Japan). Cell number was counted by NIH ImageJ software. Neuron/astrocyte co-cultures Immunocytochemistry Neuron/astrocyte co-cultures were prepared from 1-d-old Immunostaining was also performed in cultured astrocytes Sprague-Dawley rat cortices. In brief, the cerebral cortices and neurons. Cells seeded on coverslips were fixed with 4% were digested with 0.125% trypsin for 10 min at 37 °C, and the paraformaldehyde for 10 min and incubated in 3% bovine dissociated cells were seeded at a density of 0.5×105 cells/cm2 serum albumin containing 0.1% Triton X-100 for 1 h. Then, in 96-well plates or 25 cm2 flasks previously coated with poly- primary antibodies in 3% BSA were applied for 1 h or over- D-lysine. Cells were cultured in high glucose (4.5 g/L) DMEM night. For astrocytes and neurons, mouse anti-GFAP (1:300, supplemented with 7.5% fetal bovine serum, 7.5% horse CST, USA), rabbit anti-Neu (1:200, Abcam, USA), and rabbit serum, 2 mmol/L glutamine, 100 units/mL penicillin and 100 anti-beta III Tubulin (1:200, Abcam, USA) were used.