TDAG8 activation attenuates cerebral ischaemia-reperfusion injury via Akt signalling in rats

Accepted Manuscript TDAG8 activation attenuates cerebral ischaemia-reperfusion injury via Akt signalling in rats

X.D. Ma, L.H. Hang, D.H. Shao, W.W. Shu, X.L. Hu, H. Luo PII: DOI: Reference:

S0014-4886(17)30083-3 doi: 10.1016/j.expneurol.2017.03.023 YEXNR 12508

To appear in:

Experimental Neurology

Received date: Revised date: Accepted date:

15 November 2016 8 March 2017 28 March 2017

Please cite this article as: X.D. Ma, L.H. Hang, D.H. Shao, W.W. Shu, X.L. Hu, H. Luo , TDAG8 activation attenuates cerebral ischaemia-reperfusion injury via Akt signalling in rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yexnr(2017), doi: 10.1016/j.expneurol.2017.03.023

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TDAG8 activation attenuates cerebral ischaemia-reperfusion injury via Akt signalling in rats X.D. Ma1,L.H. Hang1, D.H. Shao1*,W.W. Shu, X.L. Hu, H. Luo

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Department of Anaesthesiology, Affiliated People’s Hospital of Jiangsu University, Zhenjiang 212002, China * Corresponding author. E-mail: [email protected]

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Background T-cell death-associated gene 8(TDAG8),a member of the proton-sensitive G-protein-coupled receptor (GPCR) class with an immune-specific expression profile,was recently shown to be expressed in the rat brain; however, its role in ischaemic stroke remains unknown. Methods We initially confirmed the time-dependent expression of TDAG8 in rat brain tissue after ischaemic stroke and reperfusion. Further evaluations were performed to increase TDAG8 expression six hours prior to middle cerebral artery occlusion (MCAO)by injecting a specific agonist, BTB09089,into the lateral ventricle to increase TDAG8 expression.Twenty-four hours before MCAO,a specific small interfering RNA (siRNA) was introduced. The infarction volume, neurological deficit score and cleaved caspase-3 and Bcl-2 expression were used to assess the effects of TDAG8 on ischaemic stroke.Finally, the effects of TDAG8 on the development of primary cortical neurons exposed to oxygen-glucose deprivation (OGD)were investigated. Results TDAG8 expression increased both in vivo and in vitro. Pretreatment with BTB09089 up-regulated TDAG8 and Bcl-2 expression and down-regulated cleaved caspase-3 expression, while the infarction volume was reduced,and neurological deficits were ameliorated 24 and 72 h after MCAO. However, the protective effects of TDAG8 were reversed when its level was reduced in TDAG8-deficient rats.More importantly, these findings are consistent with data from neurons subjected to OGD. Conclusions TDAG8 plays an important neuroprotective role through inhibition of neuronal apoptosis and alleviation of neurological deficits by activating the Akt signalling pathway in rats. Key Words: TDAG8, BTB09089, OGD, MCAO, Neuroprotection, Apoptosis Introduction Stroke is the most serious and potentially deadly neurological disease and the most important factor leading to permanent disability in adults,significantly affecting the quality of life of stroke patients. The main type of stroke is ischaemic stroke, which is predominately a result of middle cerebral artery occlusion (MCAO)(Langhorne et al., 2011).In recent decades, basic studies and clinical trials have been designed to determine a practical method for treating ischaemic stroke. Only thrombolytic therapy has been approved by the United States Food and Drug Administration, using the recombinant

ACCEPTED MANUSCRIPT tissue plasminogen activator (rt-PA)(Lo et al., 2003);however,the short time window for treatment limits the broad application of this method for acute ischaemic stroke(Brainin et al., 2007).Therefore, there is a need to further investigate the pathogenesis of stroke.

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T-cell death-associated gene 8 (TDAG8) is an acid-and psychosine-sensitive G-protein-coupled receptor(GPCR)that was first identified in immature thymocytes during apoptosis in mice(Choi et al., 1996).TDAG8 expression was recently identified in the brain, particularly in limbic regions of the forebrain(McGuire et al., 2009).In addition, TDAG8 promotes Bcl-xl and Bcl-2 expression via MEK/ERK signalling(Ryder et al., 2012) and acts as a negative regulator of the neutrophilic inflammatory response, partly by inhibiting chemokine production (Tsurumaki et al., 2015).Moreover,BTB09089 has been demonstrated to be a TDAG8-specific agonist(Onozawa et al., 2012).Neuronal apoptosis is a significant pathophysiological process during ischaemic stroke and determines the prognosis of stroke;inflammation after ischaemia is another major pathophysiological characteristic of ischaemic stroke(Lindsberg and Grau, 2003).However, the role of TDAG8 in ischaemic stroke remains unknown. Thus, the present study was performed to assess whether TDAG8 exerts a neuroprotective effect and to determine the potential molecular mechanism.

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Methods Animals and transient MCAO model All experimental procedures were approved by theAnimal Care and Use Committee ofthe Affiliated People’s Hospital of Jiangsu University(Zhenjiang, China).

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Adult male SD rats (270to 280 g) were subjected to MCAO via filamentous occlusion of the left MCA as previously described(Guo et al., 2014).After fasting, the rats were anaesthetised with pentobarbital (45 mg/kgi.p).A separate warming lamp was used to maintain the rectal temperature at 36.0-37.5°C. Then, a midline neck incision was made, and the left carotid arteries were carefully dissected. A dedicated MCAO suture coated with silicone rubber was applied to induce the MCAO model through insertion into the left internal carotid artery and across to the origin of the middle carotid artery.After 2 h of MCAO, the intraluminal suture was withdrawn to restore ICA-MCA blood flow and induce reperfusion.The sham-treated rats were subjected to the same procedures without the suture.Standardised neurobehavioural tests were conducted to verify the existence of advanced neurological deficit. Intracerebroventricular injection Four different formats of TDAG8-small interfering RNAs (siRNAs) (Genepharma,Shanghai, China) or BTB09089 (Maybridge,NorthCornwall,UK) were applied via intracerebroventricular (IV) injection at 24 h or 6 h, respectively,before MCAO,as described previously(Liang et al., 2014).Briefly, rats were anesthetised with sodium pentobarbital (40 mg/kgi.p); a midline scalp incision was made, and a burr hole was drilled into the skull above the left hemisphere (approximately 1.6mm on the lateral side of the bregma and 1.0 mm on the posterior side). The TDAG8-siRNA-pool (4 μg/8 μL),

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scramble-siRNA, vehicle and different doses of BTB09089 were injected into the lateral ventricle. The needle was withdrawn 2 min later,and the burr hole was sealed with bone wax. The incision was subsequently closed, and rats were returned to their separate cages until they had completely recovered. The three TDAG8 siRNA sequences used in these experiments were as follows:TDAG8-siRNA-428:5`-GAAUCCGUCUUUAACUCCATT-3` (forward), 5`-UGGAGUUAAAGACGGAUUCTT-3` (reverse);TDAG8-siRNA-57:5`-CAGUGGUCUACAUAUUUGUTT-3` (forward),5`-ACAAAUAUGUAGACCACUGTT-3` (reverse);TDAG8-siRNA-851:5`-GCCGAUCCAAUUCUCUACUTT-3` (forward),5`-AGUAGAGAAUUGGAUCGGCTT-3` (reverse);TDAG8-siRNA negative control:5`-UUCUCCGAACGUGUCACGUTT-3` (forward),5`-ACGUGACACCUUCGGAGAATT-3` (reverse); and β-actin-siRNA:5`-CUCUGAACCCUAAGGCCAATT-3` (forward),5`-UUGGCCUUAGGGUUCAGAGGG-3` (reverse).

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Neurological scores Neurological deficits were evaluated on a 21-point score at 24 or 72 h after reperfusion by a blinded investigator (Liang et al., 2014).The final score for each rat was the sum of seven individual test scores (spontaneous activity in the cage for 5 min,symmetry of movement of the 4 limbs, symmetry of forelimb outstretching while being held by the tail, climbing the wall of the wire cage, reaction to touch on either side of the trunk, response to touching the vibrissae, and beam walking)(Tab.1). The neurological scores ranged from 2 (most severe deficit) to 21 (normal).

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TTC staining The infarct volume was determined at 24 and 72 h after MCAO.The rats were rapidly decapitated, and their brains were removed and cut into seven 2-mm coronal sections from the frontal pole. The tissues were subsequently steeped in a 2% 2,3,5-triphenyltetrazolium chloride(TTC) (T8877,Sigma,USA) solution for 20 min at 37°C. The infarct volume(white region)of each section was then surveyed using ImageJ (ImageJ, National Institutes of Health), and the final infarct volume (%)was calculated following correction for brain oedema, as described previously(Wang et al., 2013). Primary cortical neuron cultures and OGD model SD foetal rat brains (embryonic day 17-18) were prepared for culturing primary cortical neurons. The cortices were separated in 2 mL of 0.2% papain (Sigma, USA)for 30 min at 37°C,followed by the addition of 200 μL of 0.25% DNaseI for 30 sec. Digestion was terminated by the addition of 10 mL of high-glucose Dulbecco’s modified Eagle’s medium (HG-DMEM, Gibco) containing 10% foetal bovine serum (FBS) (Gibco), followed by filtering through a 200-mm sterile cell strainer and centrifugation for 5 min at 800 rpm.The cells were subsequently resuspended in HG-DMEM containing10% FBS, then plated on six-well plates coated with poly-lysine (0.1 g/mL, Sigma) and incubated at 37°C under 5% CO2. After 4 h of incubation, the HG-DMEM was replaced with neurobasal medium (Gibco)

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containing10% B27 (Gibco).Twelve hours later, non-neuronal cell proliferation was inhibited by the addition of cytosine arabinoside (10 mM, Sigma), followed by incubation for 24 h, and the medium was replaced every 48 h.To clarify the effect of TDAG8 on ischaemic brain injury in vitro, the neuronal cultures were subjected to oxygen and glucose deprivation (OGD) for 60 min and then returned to normal conditions at various time-points. For OGD, glucose-free, serum-free Locke’s buffer (3.6 mM NaHCO3, 154 mM NaCl, 2.3 mM CaCl2,1 mM MgCl2,5.6 mM KCl,5 mg/ml gentamicin,and 5 mM HEPES,pH 7.2) was substituted for the neurobasal medium before initiation, followed by incubation at 37°C under 95% N2 and 5% CO2 in an anaerobic incubator. Control cells were cultured under normal conditions.

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Cell viability and LDH assay A cell counting kit-8 (CCK-8) cytotoxicity assay kit (Dojindo, Japan) was used to examine cell viability according to the manufacturer’s instructions.LDH release, as an indicator of cell membrane integrity, was assessed with an in vitro toxicology assay kit (Sigma,USA).

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Immunofluorescence staining Primary neurons were planted on coverslips and subsequently fixed with 4% paraformaldehyde. The coverslips were then thoroughly rinsed with PBS and blocked with 10% goat serum. One hour later,the coverslips were incubated in the following primary antibodies overnight at 4°C: mouse anti-βIII-tubulin (ab78078; 1:1000; Abcam,USA) and mouse anti-NeuN (MAB377; 1:2000; Millipore, USA). After rinsing, the coverslips were incubated with the fluorescein (FITC)-conjugated goat anti-mouse IgG (H+L) (Jackson Immuno Research,USA) for 1 h at room temperature. Nuclei were then stained with DAPI (Beyotime, China), and microphotographs were captured using a laser scanning confocal microscope (Olympus,Japan).

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Western blotting Rats were rapidly decapitated, and their brains were cut into several coronal sections and stained with TTC. The proteins of the ipsilateral ischaemic penumbra were extracted via homogenisation in RIPA lysis buffer containing 10 mM PMSF, and western blotting was conducted as follows.First, 20 μL of loading buffer containing 60 μg of total protein per well was separated on a 10% SD-PAGE gel and then transferred to PVDF membranes, followed by blocking in 5% non-fat milk in TBST for 1 h at room temperature and incubation with primary antibodies overnight at 4°C. After rinsing and incubation with secondary antibodies, the blot signals were detected with a Bio-Rad Ultraviolet Imaging System, and immunoblotting was quantified with ImageJ software. The primary antibodies were as follows: mouse anti-βIII-tubulin (ab78078;1:1000; Abcam),mouse anti-NeuN(MAB377;1:5000; Millipore),goat anti-TDAG8 (sc-9705;Santa Cruz), rabbit anti-cleaved-caspase 3 (#9664;CST), rabbit anti-Bcl-2 (#2870; CST),rabbit anti-caspase3(sc-7148;Santa Cruz), rabbit anti-pAkt(#13038; CST),rabbit anti-Akt (#4691;CST),rabbit anti-CREB (ab32096; Abcam),and rabbit anti-pCREB (ab32515; Abcam);mouse anti-β-actin (sc-47778; Santa Cruz) served as the internal control. The secondary antibodies were as follows: donkey anti-mouse IgG-HRP (sc-2314; Santa

ACCEPTED MANUSCRIPT Cruz),donkey anti-goat IgG-HRP (sc-2020; Santa Cruz), and goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz).

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Quantitative real-time PCR Total RNA was extracted from the ischaemic penumbra using the TRIzol reagent (Life) and reversed transcribed into cDNA using the PrimeScriptTM RT reagent kit (Takara). Quantitative real-time PCR (qRT-PCR) analysis was performed using the SYBR®Premix Ex TaqTM (Takara), and β-actin was used to normalise the relative expression of the target mRNAs.The qRT-PCR conditions were as follows: 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 30 s (plate reading); 95°C for 10 s; melting curve analysis at 65°C to 95°C with increments of 0.5°C and plate reading; final extension at 72°C for 10 min. TDAG8: 5`-CAGCAGCACGGCCTTCCTCACTT-3` (forward),5`-CGACACTTGTTTCGTCTTCCCAC-3` (reverse); MCP-1: 5`-CCATTCCTTATTGGGGTCAG-3` (forward),5`-TCACCTGCTGCTACTCATTCA-3` (reverse); IL-1β: 5`-TCGTTGCTTGTCTCTCCTTG-3` (forward),5`-AAAAATGCCTCGTGCTGTCT-3` (forward); TNF-α: 5`-GAAGAGAACCTGGGAGTAGATAAGG-3` (forward),5`-GTCGTAGCAAACCACCAAGC-3` (forward); β-actin: 5`-CCCATCTATGAGGGTTACGC-3` (forward),5`-TTTAATGTCACGCACGAT TTC-3` (forward).

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Statistical analysis SPSS16.0 software was used to conduct statistical analyses,and the data were expressed as the mean±SEM. One-way ANOVA was applied to examine the differences between groups, followed by the post hoc Tukey`s test. Two-way ANOVA was applied to examine the differences between groups in data from characterisation of primary cortical neurons , followed by the post hoc Tukey`s test. P<0.05 was considered statistically significant.

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Results Characterisation of primary cortical neurons We determined the optimal time-points during culture at which the primary cortical neurons were best suited for the experiments. The expression of the neuronal marker proteins βIII-tubulin and NeuN was detected via western blotting (Fig. 1A). Significantly increased βIII-tubulin and NeuN expression was detected on day 5 (DIV 5,Fig. 1C) and day 3 (Day3,Fig. 1B),respectively. Immunofluorescence staining on day 5 indicated that nearly all cells were primary cortical neurons (Fig. 1D). Therefore, cultured primary cortical neurons on day 5 were used for the subsequent experiments. TDAG8 expression is up-regulated after MCAO To understand whether TDAG8 is involved in ischaemic brain injury, we initially investigated TDAG8 expression in an ischaemic stroke model in rats by occluding the left MCA.Western blotting indicated that TDAG8 expression was markedly up-regulatedin a

ACCEPTED MANUSCRIPT time-dependent manner compared with the sham group at the specified time-points (Fig. 2A,B).This phenomenon was in accordance with the OGD model. Primary cortical neurons were subjected to OGD for 60 min, followed by reoxygenation and harvesting at the indicated times. Surprisingly, a similar trend of TDAG8 expression was identified in the in vitro model (Fig. 2C,D). Thus, neuronal TDAG8 may be involved in cerebral ischaemia-reperfusion injury.

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TDAG8 mitigates brain injury induced by I/R TDAG8 expression was substantially increased in the MCAO and OGD models. Hence, the roles of TDAG8 in ischaemic stroke were assessed using the specific TDAG8 agonist BTB09089. Western blotting demonstrated that TDAG8 expression was significantly increased in BTB09089-pretreated rats compared with control rats. BTB09089-pretreated rats and control rats were subsequently subjected to MCAO, and following reperfusion, the primary outcomes were analysed. TTC staining indicated that the infarct volume in the BTB09089-pretreated rats was markedly smaller than in the control rats at both 24 h (Fig. 3A,B) and 72 h (Fig. 3D,E) after reperfusion. Moreover, the neurological scores were improved in the BTB09089-pretreated rats at both 24 h (Fig. 3C) and 72 h (Fig. 3F) after reperfusion. To further assess the effect of TDAG8 on promoting ischaemia progression, specific siRNA was used to inhibit TDAG8 expression to confirm the effect of TDAG8 down-regulation on ischaemic stroke. The results of TTC staining and the neurological scores indicated that the infarct volume was enlarged, and the neurological scores were exacerbated to a greater degree in the siRNA-pretreated rats than in the control rats at both 24 h (Fig. 3G,H,I) and 72 h (Fig. 3J,K,L) after reperfusion. In order to validate TDAG8 knock-down by specific siRNA, TDAG8 expression was assessed during rats were pretreated with TDAG8-siRNA. Western blotting and qRT-PCR showed that TDAG8 expression was obviously down-regulated (Fig. 3M,N,O). Therefore, these data demonstrated that TDAG8 may exert a neuroprotective effect on cerebral ischaemia-reperfusion injury.

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TDAG8 inhibits neuronal apoptosis induced by ischaemic stroke Ischaemic stroke outcomes are mainly determined by neuronal apoptosis, which is a significant pathological and physiological process in cerebral ischaemia-reperfusion injury. Hence, we investigated whether TDAG8 attenuated reperfusion injury by regulating neuronal apoptosis. Western blotting (Fig. 4A)demonstrated that the expression of the pro-apoptotic factor cleaved caspase-3(Fig. 4C) was substantially down-regulated, while that of the anti-apoptotic factor Bcl-2(Fig. 4B) was markedly up-regulated in the brains of BTB09089-pretreated rats compared with control rats; however, opposite results were obtained in siRNA-pretreated rats following ischaemic stroke (Fig. 4E,F,G).Furthermore, in accordance with the previous results, in vitro experiments also indicated that the cell viability of neurons was increased and that LDH secretion was decreased in BTB09089-pretreated neurons exposed to OGD (Fig. 4I,K). In contrast, the opposite effect was observed following siRNA pretreatment (Fig. 4J,L). These data illustrated that TDAG8 inhibits neuronal apoptosis after ischaemia stroke.

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TDAG8 attenuates post-ischaemic inflammatory responses in ischaemic stroke Inflammatory responses are another important pathological characteristic of ischaemic stroke after reperfusion. Thus, we investigated whether TDAG8 inhibited the inflammatory response by reducing pro-inflammatory cytokine release following brain injury. Several pro-inflammatory cytokines (IL-1β,MCP-1 and TNF-α)are acutely expressed in ischaemic stroke(Lindsberg and Grau, 2003).qRT-PCR analysis indicated that BTB09089 pretreatment inhibited the expression of the above pro-inflammatory cytokines at 24 h following reperfusion (Fig. 5A),and this effect was reversed by siRNA treatment(Fig. 5B).These findings indicated that TDAG8 mitigated inflammation after ischaemic stroke.

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TDAG8 reduces cerebral ischaemia-reperfusion injury via the Akt signalling pathway Based on the above results, we determined that TDAG8 maybe a potent regulator of ischaemic stroke via regulation of neuronal apoptosis and the post-ischaemia inflammatory response, although the specific mechanism is still unclear. However, the Akt signalling pathway is known to play an important role during ischaemic brain injury(Chan, 2004).As a primary nuclear transcription factor downstream of Akt, CREB is activated upon phosphorylation by Akt, which leads to up-regulation of the expression of Bcl-2 and BDNF(CREB target genes). In this study, we illustrated that Akt and downstream CREB phosphorylation was up-regulated in the brains of rats pretreated with BTB09089 relative to control rats (Fig. 6B,C); however, this up-regulation was completely reversed in TDAG8-siRNA-pretreated rats compared with control rats exposed to MCAO and reperfusion (Fig. 6E,F). These findings indicated that Akt/CREB signalling may regulate the roles of TDAG8 in the pathological and physiological processes involved in cerebral ischaemia-reperfusion injury.

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Discussion Neuronal degeneration, inflammatory responses, cellular apoptosis and oxidative stress are important pathological processes during cerebral ischaemia-reperfusion injury(Brouns and De Deyn, 2009).These pathological processes are responsible for the dysfunction of the internal environment after ischaemic stroke. Neuronal apoptosis and post-ischaemic inflammatory responses may predict the outcome of ischaemic stroke in patients and inanimal models of stroke. Thus, novel modulators of the relevant cell signalling pathway need to be studied,and the potential molecular mechanisms elucidated. However, whether TDAG8 plays a role during ischaemic stroke is still unclear. In this study, we demonstrated that activating TDAG8 with its specific agonist BTB09089 reduced the infarct volume and improved neurological deficit scores via inhibition of the inflammation and neuronal apoptosis induced by ischaemic stroke. In contrast,suppression of TDAG8 using siRNA resulted in more serious ischaemic brain injury. We also found that cleaved caspase-3 expression was down-regulated correlated with BTB09089 treatment increased p-Akt and was up-regulated associated with TDAG8-siRNA treatment inhibited p-Akt. These observations may provide evidence that TDAG8 is a novel molecular target for regulating cerebral ischaemia-reperfusion injury.

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TDAG8 (also referred to as GPR65) is a proton-sensing GPCR belonging to the ovarian cancer GPCR subfamily 1 with a reported immune-specific expression profile(Tomura et al., 2005).Recently, TDAG8 expression was detected in the rat brain, particularly in such forebrain limbic regions as the frontal cortex, hippocampus,amygdala,hypothalamus and striatum(McGuire et al., 2009).However, the effect of TDAG8 in brain injury induced via ischaemia-reperfusion injury is still unclear. In this study, we demonstrated that TDAG8 expression was apparently increased in the brain tissues of rats subjected to MCAO. Primary cortical neurons that were subjected to OGD yielded similar results. Western blotting and qRT-PCR analyses indicated that TDAG8 expression was substantially up-regulated in both in vitro and in vivo acute ischaemic stroke models. This substantial change in TDAG8 expression demonstrated that TDAG8 may play a significant role in the pathophysiological process of ischaemic stroke injury.

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Neuronal apoptosis is an important decisive factor in the outcome of strokes, primarily when occurring in the ischaemic penumbra or around the infarct zone within minutes of the onset of ischaemic stroke and continuing for several days(Brouns and De Deyn, 2009). Thus, targeting and preventing neuronal apoptosis in and around the infarct zone or penumbra to reduce the infarct volume may represent a promising treatment strategy for mitigating ischaemic stroke injury.Previous studies have demonstrated that multiple Bcl-2 family members are regulated to inhibit cellular apoptosis through TDAG8 proton sensing via MEK/ERK signalling in an extracellular acidosis microenvironment(Ryder et al., 2012).In addition, TDAG8 enhances Lewis lung carcinoma development by promoting tumour cell survival/proliferation through adaptation to the acidic environment in mice(Ihara et al., 2010).Moreover, the expression of caspase-3, 8 and 9 ismarkedly increased in theTDAG8-deficient thymocytes in mice stimulated with dexamethasone(Tosa et al., 2003).In the present study, TDAG8 inhibited neuronal apoptosis in both in vitro and in vivo stroke models. Accordingly,changes in the expression of apoptosis-related molecules, such as cleaved caspase-3, caspase-3 and Bcl-2, were identified, which were induced by activation or inhibition of TDAG8 in both in vivo and in vitro cerebral ischaemia-reperfusion injury models.

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In addition to neuronal apoptosis, the post-ischaemic inflammatory response is another significant pathophysiological process in acute ischaemic brain injury. Previous studies have illustrated that TDAG8 inhibits the acute lung injury inflammatory response induced by LPS(Tsurumaki et al., 2015).As a proton-sensing GPCR, the activation of TDAG8 may inhibit the production of pro-inflammatory cytokines, including TNF-α and IL-6 (as well as their gene transcription)via a TDAG8-dependent pathway in macrophages(Mogi et al., 2009),superoxide anion (O2-) induced by formyl-Met-Leu-Phe or C5a in a cyclic adenosine monophosphate (cAMP)-independent manner in neutrophils(Murata et al., 2009) and IL-2 in splenocytes via BTB09089 in a dose-dependent manner(Onozawa et al., 2012)in addition to regulating allergen-induced airway eosinophilia(Kottyan et al., 2009). In addition, we found that activates TDAG8 inhibits IL-1β production induced by MCAO in

ACCEPTED MANUSCRIPT this study and this is consistent with the findings of the study that activation of TDAG8 reduced IL-1β production induced by LPS via inhibition of ERK and JNK activity in mouse microglia (Jin et al.,2014). However,TDAG8 deficiency was founded that associated with reduced proinflammatory cytokines IL-1β within the subfornical organ (Vollmer et al.,2016). Therefore, further studies are needed to confirm the possible association between TDAG8 and IL-1β.Consistent with these findings, inhibition of the inflammatory response was observed in rat brains or primary cortical neurons pretreated with BTB09089 after

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ischaemia-reperfusion surgery or OGD exposure, respectively, whereas the inflammatory

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response was promoted following preconditioning with TDAG8-siRNA.

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Akt, also known asserine/threonine protein kinase B, controls survival and apoptosis and is typically phosphorylated in non-ischaemic brain tissue,and this phosphorylation is further enhanced following ischaemic stroke(Osuka et al., 2004).Moreover, the application of exogenous growth factors may protect against ischaemic brain injury by activating Akt signalling(Jin et al., 2000).In addition to this influence on apoptosis, the activation of Akt and its downstream proteins, such as mTOR and FoxO1, potently suppresses inflammatory genes(Brown et al., 2011).However, a previous study demonstrated that TDAG8 activation, coupled with the Gi protein, inhibits pro-inflammatory cytokine production induced by extracellular acidification via the Gs protein/cAMP/PKA signalling pathway in mouse macrophages to inhibit target gene transcription (Mogi et al., 2009).In mouse microglia, an acidic extracellular pH suppresses LPS-induced IL-11β production via the TDAG8/cAMP/PKA pathway through inhibition of ERK and JNK activity(Jin et al., 2014).Furthermore, cancer cells may adopt a pro-survival Bcl-2 expression pattern in response to external proton-dependent signals in a TDAG8-dependent manner via the MEK/ERK pathway(Ryder et al., 2012).TDAG8 may also activate G12/13 and then subsequently activate RhoA signalling and further regulate the expression of serum response element (SRE)(Siehler, 2009).In the present study, Akt and CREB phosphorylation was found to be up-regulated in the brains of rats pretreated with BTB09089 and down-regulated in rats preconditioned with TDAG8-siRNA in an ischaemic stroke model. These findings indicate that TDAG8 attenuates ischaemic brain injury via Akt signalling. In summary,this study provides convincing evidence that TDAG8 regulates ischaemic brain injury by inhibition of the inflammatory response and neuronal apoptosis. Akt signalling may mediate the physiological functions of TDAG8 in ischaemic stroke model rats. Therefore, we propose that targetingTDAG8 may lead to novel and promising strategies for attenuating ischaemia-reperfusion injury in stroke patients. Author contributions X.D.M. was responsible for the study design,conducting the experiments, and writing the paper. D.H.S. and L.H.H.were responsible for the study design and mentoring. D.H.S.was responsible for providing funding. W.W.S, H.L. and X.L.H.helped performthe experiments

ACCEPTED MANUSCRIPT and data collection and analysis. Conflict of interest None declared. Funding This study was supported by the Zhenjiang Social Development Subjects (SH2015039).

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References

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Brainin, M., Teuschl, Y., Kalra, L., 2007. Acute treatment and long-term management of stroke in developing countries. Lancet Neurol.6, 553-561. Brouns, R., De Deyn, P.P., 2009. The complexity of neurobiological processes in acute ischemic stroke. Clin. Neurol. Neurosurg.111, 483-495. Brown, J., Wang, H., Suttles, J., Graves, D.T., Martin, M., 2011. Mammalian target of rapamycin complex 2 (mTORC2) negatively regulates toll-like receptor 4-mediated inflammatory response via FoxO1. J. Biol. Chem.286, 44295-44305. Chan, P.H., 2004. Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia. Neurochem. Res.29, 1943-1949. Choi, J.W., Lee, S.Y., Choi, Y., 1996. Identification of a putative G protein-coupled receptor induced during activation-induced apoptosis of T cells. Cell. Immunol.168, 78-84. Guo, S., Li, Z.Z., Jiang, D.S., Lu, Y.Y., Liu, Y., Gao, L., Zhang, S.M., Lei, H., Zhu, L.H., Zhang, X.D., Liu, D.P., Li, H., 2014. IRF4 is a novel mediator for neuronal survival in ischaemic stroke. Cell Death Differ.21, 888–903. Ihara, Y., Kihara, Y., Hamano, F., Yanagida, K., Morishita, Y., Kunita, A., Yamori, T., Fukayama, M., Aburatani, H., Shimizu, T., Ishii, S., 2010. The G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) facilitates tumor development by serving as an extracellular pH sensor. Proc. Nalt Acad. Sci.107, 17309-17314. Jin, K.L., Mao, X.O., Greenberg, D.A., 2000. Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc. Natl Acad. Sci. U. S. A.97, 10242-10247. Jin, Y., Sato, K., Tobo, A., Mogi, C., Tobo, M., Murata, N., Ishii, S., Im, D.S., Okajima, F., 2014. Inhibition of interleukin-1β production by extracellular acidification through the TDAG8/cAMP pathway in mouse microglia. J. Neurochem.129, 683-695. Kottyan, L.C., Collier, A.R., Cao, K.H., Niese, K.A., Hedgebeth, M., Radu, C.G., Witte, O.N., Khurana Hershey, G.K., Rothenberg, M.E., Zimmermann, N., 2009. Eosinophil viability is increased by acidic pH in a cAMP- and GPR65-dependent manner. Blood114, 2774-2782. Langhorne, P., Bernhardt, J., Kwakkel, G., 2011. Stroke rehabilitation. Lancet377, 1693-1702. Liang, X., Hu, Q., Li, B., McBride, D., Bian, H., Spagnoli, P., Chen, D., Tang, J., Zhang, J.H., 2014. Follistatin-like 1 attenuates apoptosis via disco-interacting Protein 2 homolog A/Akt pathway after middle cerebral artery occlusion in Rats. Stroke45, 3048-3054.

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Lindsberg, P.J., Grau, A.J., 2003. Inflammation and infections as risk factors for ischemic stroke. Stroke34, 2518-2532. Lo, E.H., Dalkara, T., Moskowitz, M.A., 2003. Mechanisms, challenges and opportunities in stroke. Nat. Rev. Neurosci.4, 399-415. McGuire, J., Herman, J.P., Ghosal, S., Eaton, K., Sallee, F.R., Sah, R., 2009. Acid-sensing by the T cell death-associated gene 8 (TDAG8) receptor cloned from rat brain. Biochem. Biophys. Res. Commun.386, 420-425. Mogi, C., Tobo, M., Tomura, H., Murata, N., He, X.D., Sato, K., Kimura, T., Ishizuka, T., Sasaki, T., Sato, T., Kihara, Y., Ishii, S., Harada, A., Okajima, F., 2009. Involvement of proton-sensing TDAG8 in extracellular acidification-induced inhibition of proinflammatory cytokine production in peritoneal macrophages. J. Immunol.182, 3243-3251. Murata, N., Mogi, C., Tobo, M., Nakakura, T., Sato, K., Tomura, H., Okajima, F., 2009. Inhibition of superoxide anion production by extracellular acidification in neutrophils. Cell. Immunol.259, 21-26. Onozawa, Y., Fujita, Y., Kuwabara, H., Nagasaki, M., Komai, T., Oda, T., 2012. Activation of T cell death-associated gene 8 regulates the cytokine production of T cells and macrophages invitro. Eur. J. Pharmacol.683, 325-331. Osuka, K., Watanabe, Y., Usuda, N., Nakazawa, A., Tokuda, M., Yoshida, J., 2004. Modification of endothelial NO synthase through protein phosphorylation after forebrain cerebral ischemia/reperfusion. Stroke35, 2582-2586. Ryder, C., McColl, K., Zhong, F., Distelhorst, C.W., 2012. Acidosis promotes bcl-2 family-mediated evasion of apoptosis: involvement of acid-sensing G protein-coupled receptor GPR65 signaling to MEK/ERK. J. Biol. Chem.287, 27863-27875. Siehler, S., 2009. Regulation of RhoGEF proteins by G12/13-coupled receptors. Br. J. Pharmacol.158, 41-49. Tomura, H., Mogi, C., Sato, K., Okajima, F., 2005. Proton-sensing and lysolipid-sensitive G-protein-coupled receptors: a novel type of multi-functional receptors. Cell. Signal.17, 1466-1476. Tosa, N., Murakami, M., Jia, W.Y., Yokoyama, M., Masunaga, T., Iwabuchi, C., Inobe, M., Iwabuchi, K., Miyazaki, T., Onoe, K., Iwata, M., Uede, T., 2003. Critical function of T cell death-associated gene 8 in glucocorticoid-induced thymocyte apoptosis. Int. Immunol.15, 741-749. Tsurumaki, H., Mogi, C., Aoki-Saito, H., Tobo, M., Kamide, Y., Yatomi, M., Sato, K., Dobashi, K., Ishizuka, T., Hisada, T., Yamada, M., Okajima, F., 2015. Protective role of Proton-sensing TDAG8 in lipopolysaccharide-induced acute lung injury. Int. J. Mol. Sci.16, 28931-28942. Vollmer, L. L., Ghosal, S., McGuire, J. L., Ahlbrand, R. L., Li, K. Y., Santin, J. M., Ratliff-Rang, C. A., Patrone, L. G., Rush, J., Lewkowich, I. P., Herman, J. P., Putnam, R. W., and Sah, R., 2016. Microglial Acid Sensing Regulates Carbon Dioxide-Evoked Fear. Biol Psychiatry 80, 541-551.

ACCEPTED MANUSCRIPT Wang, L., Lu, Y., Zhang, X., Zhang, Y., Jiang, D., Dong, X., Deng, S., Yang, L., Guan, Y., Zhu, L., Zhou, Y., Zhang, X., Li, H., 2013. Mindin is a critical mediator of ischemic brain injury in an experimental stroke model. Exp. Neurol.247, 506-516.

Tab.1 Neurological evaluation after middle cerebral artery occlusion in SD rats Score Test 0

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2

3

Moves but does not

Moves and approaches at least

Spontaneous activity (in No movement

Barely moves

approach at least three sides of cage

three sides of cage

Symmetry of

Left side: no

Left side: light

Left side: movement

Both sides: movement

movements(four limbs)

movement

movement

slowly

symmetrically

Symmetry of

Left side: no

Left side: light

Left side: movement

forelimbs(outstretching

movement, no

movement to

and outstretches

while held by tail)

outstretching

outstretch

less than right side

Climbing wall of wire cage

…

Fails to clmb

Left side is weak

Normal climbing

No response to the

Weak reponse on

Symmetrical

left side

left side

response

No response to the

Weak reponse on

Symmetrical

left side

left side

response

Spins on beam >60

Balances with steady

seconds

posture

…

either side of trunk Response to vibrissae

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…

touch

Falls off, no attempt to

Attempts to balance

balance or hang onto

on beam but falls

beam <20 seconds

off >40 seconds

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Beam walking

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Reaction to touch on

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cage for 5 min)

Symmetrical outstretch

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Figure 1:Characterisation of primary cortical neurons. Western blot detection of the neuronal marker proteins βIII-tubulin and NeuN during cell culture of primary cortical neurons on days 1, 3, 5, 7, 9 and 11. (A) Immunoblotting of NeuN and βIII-tubulin at the indicated time-points during cell culture. (B-C) The relative of protein expression levels in (A) were

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standardised using β-actin. The values are presented as the mean±SEM. Main effect of between time, F(5,30)=96.476,p<0.001(B);for effect of between groups,F(10,30)=0.039,p=0.999(B).Main effect of between time,F(5,30)=1470.74,p<0.001(C);for effect of between group,F(10,30)=0.609,p=0.794(C).*P<0.001 vs. day 1 via two-way

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ANOVA, followed by the post hoc Tukey`s test. Three independent experiments were performed. (D)Immunofluorescence

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staining on day 5 indicated that nearly all of cells were primary cortical neurons. Bar=100μm.

2:TDAG8

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after

MCAO.

TDAG8

expression

was

assessed

during

ischaemia-reperfusion. (A) Immunoblotting of TDAG8 expression in the in vivo stroke model at the indicated times. (B) The quantification of TDAG8 was normalised to β-actin.F(4,29)=230.768,p<0.001(B); *P<0.001(B) vs. sham; #P=0.001(B) vs. I/R 3 h; #P<0.001(B) vs. I/R 12 h.(C)The quantitative real time PCR of TDAG8 in vivo stoke model at the indicated times and

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TDAG8 was normalised to β-actin.F(4,29)=21.612,p<0.001(C);*P<0.004(C) vs. sham. #P=0.027(C) vs. I/R 12 h. (D) Immunoblotting of TDAG8 in the in vitro stroke model at the indicated times. (E) The quantification of TDAG8 was standardised based on β-actin.F(4,29)=153.779,p<0.001(E); *P<0.001(E) vs. sham. (F) The quantitative real time PCR of

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TDAG8 in vitro stoke model at the indicated times and TDAG8 was normalised to β-actin.F(4,29)=29.328,p<0.001(F); P<0.001(F) vs. sham.one-way ANOVA, followed by the post hoc Tukey`s test. The data are presented as the mean±SEM.

n=6 per group.

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Figure 3:TDAG8 mitigates brain injury induced by ischaemia-reperfusion. (A and D) Activation of TDAG8 with BTB09089

ACCEPTED MANUSCRIPT reduced the infarct volume (%) and improved neurological function at 24 and 72 h post-MCAO, represented as images of coronal sections stained with 2% TTC. The neurological deficit scores were improved (C and F), and the infarct volumes were reduced (B and E) by activating TDAG8 using higher doses of BTB09089 at both 24 and 72 h after reperfusion. F(4,29)=268.617,p<0.001(B);F(4,29)=194.023,p<0.001(C);F(4,29)=1365,p<0.001(E);F(4,29)=69.315,p<0.001(F); *P<0.001(B,C,E,F) vs. sham; #P<0.001(B,C,E) vs. MCAO; #P=0.001(F) vs. MCAO; #P<0.001(B,C,E,F) vs. BTB 5 μM+MCAO; ##

P<0.001(B,C,E,F) vs. BTB 10 μM+MCAO; **P=0.001(E) vs. MCAO.one-way ANOVA, followed by the post hoc Tukey`s test.

The data are presented as the mean±SEM.n=6 per group.(G and J) Inhibition of TDAG8 with siRNA increased the infarct volume (%) and exacerbated neurological function 24 and 72 h after MCAO. The quantified infarct volume (H and K) and neurological scores (I and L) indicated that TDAG8 inhibition with siRNA increased the infarct volume and exacerbated the scores.F(5,35)=374.306,p<0.001(H);F(5,35)=112.415,p<0.001(I);F(5,35)=252.498,p<0.001(K);F(5,35)=79.057,

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neurological

p<0.001(L); *P<0.001(H,I,K,L) vs. sham; #P<0.001(H,I,K,L) vs. MCAO; #P<0.001(H,I,K,L) vs.vehicle+MCAO; #P<0.001(H,I,K,L) ##

P=0.006(H) vs. siRNA-C+MCAO. one-way ANOVA, followed by the post hoc Tukey`s test. The data

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vs. siRNA-C+ MCAO;

are presented as the mean±SEM. n=6 per group.(M) In order to validate TDAG8 knock-down by specific siRNA,

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Immunoblotting of TDAG8 expression was assessed during rats were pretreated with TDAG8-siRNA. The quantification of TDAG8 was normalised to β-actin in (N), and the quantitative real time PCR of TDAG8 was normalised to β-actin in (O). F(4,14)=28.219,p<0.001(N);F(4,14)=32.775,p<0.001(O);#P<0.001(N,O) vs. normal; #P<0.001(N) vs. vehicle; #P=0.001(O) vs. ##

P<0.001(N,O) vs. vehicle;

##

P<0.001(N,O) vs. siRNA-C;

##

P<0.001(N,O) vs.

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vehicle; #P<0.001(N,O) vs. siRNA-C;

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siRNA.one-way ANOVA, followed by the post hoc Tukey`s test. The data are presented as the mean±SEM. n=3 per group.

Figure 4:TDAG8 inhibits neuronal apoptosis induced by ischaemic stroke. (A) Western blotting of caspase-3, cleaved caspase-3 (CC3) and Bcl-2 expression in the brains of rats pretreated with BTB09089 at 6 h after MCAO.Quantitative immunoblotting of Bcl-2 (B), CC3 (C) and caspase-3 (D) indicated that TDAG8 activation using BTB09089 inhibited neuronal apoptosis

induced

by

MCAO.β-actin

was

used

as

a

loading *

control

in

each #

lane.

F(4,29)=47.77,p<0.001(B);F(4,29)=1208,p<0.001(C);F(4,29)=79.383,p<0.001(D); P<0.001(B,C,D) vs. sham; P=0.024(B) vs.

ACCEPTED MANUSCRIPT MCAO; #P=0.012(B) vs. BTB 5 μM+MCAO; ##P=0.038(B) vs. BTB 10 μM+MCAO; **P<0.001(C) vs. MCAO; #P<0.001(C) vs. MCAO; #P=0.005(C) vs. BTB 5 μM+MCAO; ##P<0.001(C) vs. BTB 10 μM+MCAO; #P=0.001(D) vs. MCAO; #P<0.001(D) vs. BTB 5 μM+MCAO.one-way ANOVA, followed by the post hoc Tukey`s test.The data are presented as the mean±SEM.n=6 per group.(E) Western blotting of caspase-3, CC3 and Bcl-2 expression in the brains of rats pretreated with siRNA at 6 h after MCAO. Quantitative immunoblotting of Bcl-2 (F), CC3 (G) and caspase-3 (H) indicated that inhibition of TDAG8 with siRNA exacerbated neuronal apoptosis induced by MCAO.β-actin was used as the loading control in each lane. F(5,35)=293.197,p<0.001(H);F(5,35)=315.132,p<0.001(I);F(5,35)=184.272,p<0.001(K);*P<0.001(F,G,H) vs. sham; #P<0.001 (F,G,H) vs. MCAO; #P<0.001(F,H) vs. vehicle+MCAO; #P<0.001(F,H) vs. siRNA-C+MCAO; #P=0.001(G) vs. vehicle+MCAO; #

P=0.001(G) vs. siRNA-C+MCAO. one-way ANOVA, followed by the post hoc Tukey`s test. The data are presented as the

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mean±SEM.n=6 per group. (I-L) Primary cortical neurons were pretreated with BTB09089 or siRNA and subsequently exposed to OGD-reoxygenation.Cell viability (I and J) and LDH release (K and L) were subsequently examined.

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F(4,29)=164.836,p<0.001(I);F(5,35)=505.803,p<0.001(J);F(4,29)=86.892,p<0.001(K);F(5,35)=60.487,p<0.001(L); *

P<0.001(I,J,K,L) vs. sham; #P<0.001(I,J,K) vs. OGD; #P=0.001(L) vs. OGD; #P<0.001(I,K) vs. BTB 5 μM+OGD; ##P=0.012(I)

vs. BTB 10 μM+OGD; ##P=0.001(K) vs. BTB 10 μM+OGD;

#

P<0.001(J,L) vs. vehicle+OGD; #P<0.001(J) vs. siRNA-C+OGD;

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#

P=0.001(L) vs. siRNA-C+OGD. one-way ANOVA, followed by the post hoc Tukey`s test. The data are presented as the

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mean±SEM. n=6 per group.

Figure 5:TDAG8 attenuated post-MCAO inflammation. IL-1β, MCP-1 and TNF-α mRNA expression was quantified in the brains of rats pretreated with BTB09089 or siRNA using real-timePCR24h after cerebral ischaemia-reperfusion injury. (A)

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Real-time PCR demonstrated that TDAG8 activation with BTB09089 decreased the levels of the pro-inflammatory cytokines IL-1β, MCP-1 and TNF-α, and (B) inhibition of TDAG8 with siRNA increased the levels of the pro-inflammatory cytokines IL-1β, MCP-1 and TNF-α. F(4,29)=119.369,p<0.001(A,TNA-α); F(4,29)=193.28,p<0.001(A,MCP-1); F(4,29)=297.304,p<0.001(A,IL-1

*

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β); F(5,35)=112.759,p<0.001(B,TNA-α); F(5,35)=116.744,p<0.001(B,MCP-1); F(5,35)=586.833,p<0.001(B,IL-1β); P<0.001(A,B) vs. sham; #P<0.001(A. TNF-α) vs. MCAO; #P<0.001(A. TNF-α) vs. BTB 5 μM+MCAO;##P=0.005(A. TNF-α) vs.

BTB 10 μM+MCAO; #P<0.001(A. MCP-1) vs. MCAO; #P<0.001(A. MCP-1) vs. BTB 5 μM+MCAO;##P=0.034(A. MCP-1) vs. BTB 10 μM+MCAO; #P<0.001(A. IL-1β) vs. MCAO; #P<0.001(A. IL-1β) vs. BTB 5 μM+MCAO;##P=0.005(A. IL-1β) vs. BTB 10 μM+MCAO; #P=0.015(B. TNF-α) vs. MCAO; #P=0.002(B. TNF-α) vs. vehicle+MCAO; #P=0.001(B. TNF-α) vs. siRNA-C+MCAO; #P=0.003(B. MCP-1) vs. MCAO; #P=0.002(B. MCP-1) vs. vehicle+MCAO; #P=0.001(B. MCP-1) vs. siRNA-C+MCAO; #P<0.001(B. IL-1β) vs. MCAO; #P<0.001(B. IL-1β) vs. vehicle+MCAO; #P<0.001(B. IL-1β) vs. siRNA-C+MCAO.one-way ANOVA, followed by the post hoc Tukey`s test.The data are presented as the mean±SEM.n=6 per group.

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Figure 6:TDAG8 reduced cerebral ischaemic injury via Akt signalling in rats. The present study indicates that TDAG8 activation prevents neuronal apoptosis via the Akt pathway after MCAO in rats. (A) Western blots of pAkt and pCREB and quantitative immunoblotting of pAkt (B) and pCREB (C)demonstrated that TDAG8 activation promotes Akt and CREB phosphorylation.β-actin was used as theloading control in each lane.

D

F(4,29)=170.912,p<0.001(B);F(4,29)=248.601,p<0.001(C); *P<0.001(B,C) vs. sham; #P=0.004(B) vs. MCAO; #P=0.010(B) vs. BTB 5 μM+MCAO;##P=0.006(B) vs. BTB 10 μM+MCAO;#P=0.005(C) vs. MCAO; #P=0.003(C) vs. BTB 5

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μM+MCAO;##P<0.001 (C) vs. BTB 10 μM+MCAO.one-way ANOVA, followed by the post hoc Tukey`s test.The data are presented as the mean±SEM.n=6 per group.(D) Western blots for pAkt and pCREB and quantitative immunoblotting of pAkt (E) and pCREB (F) demonstrated that TDAG8 activation inhibits Akt and CREB phosphorylation. F(5,35)=99.295,p<0.001(E);F(5,35)=82.611,p<0.001(F);*P<0.001(E,F) vs. sham; #P=0.001(E) vs. MCAO; #P=0.003(E) vs.

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vehicle+MCAO;#P=0.001(E) vs. siRNA-C+MCAO; #P=0.001(F) vs. MCAO; #P<0.001 (F) vs. vehicle+MCAO;#P<0.001 (F) vs.

group.

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siRNA-C+MCAO.one-way ANOVA, followed by the post hoc Tukey`s test.The data are presented as the mean±SEM.n=6 per

ACCEPTED MANUSCRIPT

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Hightlights: -TDAG8 receptor exerts neuroprotective effects in a rat model of ischaemic stroke. -TDAG8 expression was increased following ischaemic stroke and reperfusion. -BTB09089,a TDAG8 agonist, robustly reduced cerebral infarct volume and rescued neurological function in cerebral ischaemia-reperfusion injury model in rats.