[1] 18w (S)-ZJM-289 Preconditioning Induces a Late Phase Protection Against Nervous Injury Induced by Transient Cerebral Ischemia and Oxygen-Glucose Deprivation

[1] 256w S)-ZJM-289, a novel nitric oxide (NO)-releasing derivative of 3-n-butylphthalide, induces the neuroprotection in a rat model of focal cerebral ischemia/reperfusion (I/R). However, much is unknown about the late phase effect in the neuroprotection of (S)-ZJM-289 preconditioning. The purpose of this study is to explore the late phase neuroprotection of (S)-ZJM-289 preconditioning, as well as underlying mechanisms involved. Preconditioning with 40-160 mg/kg, (S)-ZJM-289 significantly reduces brain damage after I/R. (S)-ZJM-289 preconditioning is effective when applied 1-3 days before I/R. Moreover, the degrees of neuroprotection offered by (S)-ZJM-289 preconditioning and ischemic preconditioning are virtually identical. (S)-ZJM-289 preconditioning also protects primary cultured cortical neurons against oxygen-glucose deprivation and recovery-induced cytotoxicity in vitro. (S)-ZJM-289 preconditioning significantly increases the generation of NO, but has no effect on the nitric oxide synthase activities. Additionally, (S)-ZJM-289 preconditioning promotes the dissociation between nuclearfactor-E2-related factor (Nrf2) and kelch-like ECH-associated protein 1, and induces Nrf2 nuclear localization. The neuroprotection of (S)-ZJM-289 preconditioning is blocked by Nrf2-siRNA in vitro. (S)-ZJM-289 preconditioning up-regulates antioxidant enzymes against nervous injury. (S)-ZJM-289 preconditioning significantly activates extracellular regulated protein kinases (ERK) and inhibits c-Jun N-terminal kinases signaling cascade. The neuroprotection is abolished by the ERK inhibitor PD98059 in vitro. Subsequently, (S)-ZJM-289 preconditioning increases the levels of anti-apoptotic protein B cell lymphoma 2 (Bcl-2) and inhibited the translocation of Bcl-2 associated X to the mitochondria, thus attenuating the release of cytochrome c from the mitochondria and the activation of downstream caspase. These results suggest that (S)-ZJM-289 preconditioning exerts the late phase protection against nervous injury induced by transient cerebral ischemia and oxygen-glucose deprivation.

[1] 73w Cerebral ischemia is the chief cause of handicap and one of the most common cause of death worldwide following cardiovascular diseases and cancer, and its incidence rises with the increase of age (Margaill et al. 2005;Simerabet et al. 2008). The therapeutic goal in treating cerebral ischemia is to reduce the extent of brain injury and thus minimize neurological impairment. Strategies to prevent cerebral ischemia/reperfusion (I/R) injury have become the focus of extensive research.

[2] 150w Ischemic preconditioning (IPC) modulates a powerful endogenous protection present in the brain and other organs (Joo et al. 2006;Liu et al. 2009). This consists of a single or a series of brief, non-lethal ischemic periods which condition the tissue to resist against significant cell death when subsequently challenged by a normally lethal ischemia. Unlike the early phase, neuroprotection induced by IPC, which lasts 2-3 h and protects against infarction but not against stunning, the late phase neuroprotection induced by IPC lasts 3-4 days and protects against both infarction and stunning, suggesting that it may have greater clinical relevance (Bolli 2000). However, although effective, IPC is unable in many clinical settings because of safety concerns (Freiberger et al. 2006). Therefore, finding a safer preconditioning stimulus that is both practical and effective or a biological agent that is able to induce neuroprotection pharmacologically may be a reasonable choice for treating cerebral I/R.

[3] 96w (S)-ZJM-289, a novel nitric oxide (NO)-releasing derivative of 3-n-butylphthalide (NBP) (Fig. 1a), requires enzymatic hydrolysis or non-enzymatic pathway to durably liberate low level of NO at a constant rate (Wang et al. 2011). Recent studies show that (S)-ZJM-289 induces the neuroprotection in a rat model of focal cerebral I/R (Zhao et al. 2012;Wang et al. 2012). Moreover, ZJM-289 (its enantiomers) preconditioning induces early phase neuroprotection against infarction by I/R (administration 1 h before I/R) (Zhuang et al. 2010). However, much is still unknown about the late phase effect in the neuroprotection of (S)-ZJM-289 preconditioning. Neurotox Res

[4] 22w Thus, in this study, we investigated whether (S)-ZJM-289 preconditioning could induce a late phase neuroprotection, as well as the relevant mechanisms involved.

[1] 119w To measure NO levels and NOS activities affected by (S)-ZJM-289 preconditioning, rats in were intragastrically administrated with 20, 40, 80, and 160 mg/kg (S)-ZJM-289, 23 mg/kg NBP (equivalent on molar basis to (S)-ZJM-289), 29 mg/kg SNAP (equivalent on molar basis to (S)-ZJM-289), or the vehicle (normal saline), respectively (Supplemental Fig. 1c). Three days after preconditioning, rats were anesthetized and their brains were removed. NO production was evaluated indirectly by measuring nitrite/nitrate (NO X ), the stable metabolites of NO. NO X content in brain was measured using colorimetric total NO assay kit (Jiancheng Bioengineering Institute, Nanjing, China) as described previously (Wang et al. 2012). The NO content was expressed as fold change based on comparison with the sham group.

[2] 110w NOS activities were determined as described previously (Luo et al. 2005). Briefly, NOS activities in the brain were measured spectrophotometrically using a commercially available kit (Jiancheng Bioengineering Institue, Nanjing, China) that is based on the oxidation of oxyhaemoglobin to methaemoglobin by NO. iNOS (inducible nitric oxide synthase) activity was measured by adding ethyleneglycolbis (beta-aminoethylether)-N, N 0 -tetraacetic acid (EGTA) at 3 mM to chelate free Ca 2? from the reaction mixture. cNOS (constitutive nitric oxide synthase) activity was computed by subtracting the Ca 2? -independent NOS activity from the total NOS activities. Total NOS, iNOS, and cNOS activities were expressed as fold change based on comparison with the sham group.

[3] 150w The neuroprotective effect of (S)-ZJM-289 preconditioning depended on the concentrations of (S)-ZJM-289 and the time intervals between the ischemia and the administration of (S)-ZJM-289. Compared with MCAO group, exposing rats to 40-160 mg/kg (S)-ZJM-289 1 day before I/R offered protection against the brain damage by MCAO. In addition, 80 mg/kg (S)-ZJM-289 preconditioning proved to be more effective in reducing brain damage. However, 20 mg/kg (S)-ZJM-289 preconditioning exerted no protective effect (Fig. 1b). To determine the optimal ischemia-administration interval, 80 mg/kg (S)-ZJM-289 was selected as the concentration of preconditioning. The maximal neuroprotection was induced with 3 days interval between ischemia and the administration of (S)-ZJM-289, and 1 day or 2 days interval also led to statistically significant effect (Fig. 1c). Above all, (S)-ZJM-289 preconditioning could induces a late phase neuroprotection against I/R injury. 80 mg/kg (S)-ZJM-289 and 3 days interval between ischemia and the administration were selected for the subsequent study.

[4] 297w The Comparison of (S)-ZJM-289 Preconditioning and Ischemic Preconditioning on Brain Injury Following Cerebral Ischemia/Reperfusion To compare the neuroprotective effects of (S)-ZJM-289 preconditioning with IPC, rats were exposed to 10 min IPC, followed 3 days later by 2 h MCAO, reperfused for 24 h and then subjected to brain damage evaluation. IPC or (S)-ZJM-289 preconditioning significantly reduced the I/R-induced brain infraction by 31 % or 40 % (Fig. 2a). The water content was remarkably increased in the MCAO group (84.51 %) versus sham (78.93 %), representing a severe I/R injury (Fig. 2b). IPC or (S)-ZJM-289 preconditioning decreased the brain water content after I/R, with the percent 82.23 % or 82.63 %, respectively. The result of HE and examined under light microscope (magnification 9400) after I/R. e Representative microphotographs (magnification 9400) of Nissl staining in the ischemic cortex after I/R. # p \ 0.05, compared with the sham group; * p \ 0.05, as compared with MCAO group, using one-way ANOVA followed by Tukey's multiple comparison test HE staining was performed to determine the neuroprotective effects of (S)-ZJM-289 preconditioning and IPC after I/R. As shown in Fig. 2d, the normal neurons in the prefrontal cortex of the sham group were packed tightly and orderly with clear nuclei. However, there were many unhealthy neurons with the characteristic histopathological features of I/R damage, nucleus shrinkage, cytoplasm with increased eosinophilia, and cellular edema in the MCAO Importantly, the histopathological abnormalities were attenuated in S-ZJM-289 preconditioning and IPC group. In parallel with the HE staining, (S)-ZJM-289 preconditioning or IPC significantly increased the intact neuron in the cortex, compared with MCAO group (Nissl staining, Fig. 2e). However, there was no significant difference between the two groups. Together, these results suggested that the degrees of neuroprotection offered by IPC and (S)-ZJM-289 preconditioning were virtually identical.

[5] 70w To investigate the involvement of apoptosis in the delayed death of cortical neurons after I/R, TUNEL staining was carried out after I/R. As shown in Fig. 3a, TUNEL-positive cells did not appear in the sham group, whereas a large number of cells with green fluorescent (TUNEL-positive) were detected in the MCAO group. (S)-ZJM-289 preconditioning or IPC significantly reduced the number of TUNEL-positive cells, indicating that (S)-ZJM-289 preconditioning reduced I/R-induced apoptosis.

[6] 153w The proto-oncoproteins (Bcl-2 and Bax) are the powerful regulator of the mitochondrial apoptotic pathway initiated by a variety of extracellular and intracellular stresses (Yang et al. 1997). The I/R injury is accompanied by the imbalance between pro-apoptotic Bax and antiapoptotic Bcl-2, the translocation of Bax to the mitochondria, the release of cytochrome c and the reduction in the mitochondrial membrane potential in vitro (Qian et al. 2011). As shown in Fig. 3b-c, (S)-ZJM-289 preconditioning and IPC obviously increased the ratio of Bcl-2/Bax. In addition, (S)-ZJM-289 preconditioning and IPC significantly increased the expression of Bcl-2 in the mitochondria and inhibited the translocation of Bax to the mitochondria. Consistently, the expression of cytochrome c in the cytosolic fraction was significantly increased in the MCAO group. (S)-ZJM-289 preconditioning or IPC significantly attenuated the release of cytochrome c from mitochondria to cytosolic (Fig. 3d-e). These observations showed that (S)-ZJM-289 preconditioning inhibited the mitochondrial apoptotic pathway after I/R.

[7] 93w Caspases are widely expressed in an inactive proenzyme form in normal cells. Active/cleaved caspases can often activate other pro-caspases, allowing initiation of a protease cascade in apoptotic cells (Cohen 1997). Caspase 9 and caspase 3, the marker of mitochondrial apoptotic pathways, were selected to evaluate the effect of (S)-ZJM-289 preconditioning on the caspase pathway. As shown in Fig. 3f-g, caspase 9 and caspase 3 were activated in the MCAO group. However, both (S)-ZJM-289 preconditioning and IPC effectively suppressed I/R-induced caspase activation as the cleaved bands were further faded compared with the MCAO group.

[8] 108w To examine the neuroprotective effects of (S)-ZJM-289 against OGD/R-induced cytotoxicity, various concentrations (0.1-10 lM) of (S)-ZJM-289 were added to the culture medium 24 h before OGD/R treatment. As shown in Fig. 4a, the viability of the cells exposed to OGD/R was reduced compared with the control. (S)-ZJM-289 preconditioning significantly attenuated OGD/R-induced cell death. Moreover, (S)-ZJM-289 preconditioning also abolished the increased LDH release caused by the OGD/R treatment (Fig. 4b). The cytoprotective effects of (S)-ZJM-289 were also confirmed by AV/PI staining (Fig. 4c). Cells exposed to OGD/R showed significant damage with the apoptotic rate of 45.7 %. (S)-ZJM-289 preconditioning at 10 lM decreased the apoptosis rate to 18.3 %.

[9] 74w The association of the neuroprotective effect of (S)-ZJM-289 preconditioning with NO was investigated, by assessing activity of two isoforms of NOS and measuring stable products of NO. As shown in Fig. 5a, (S)-ZJM-289 preconditioning significantly increased the generation of NO, as compared with vehicle. However, NBP had no effect on NO production. Moreover, (S)-ZJM-289 preconditioning did not generate more NO compared with SNAP, a classical NO donor with neuroprotective action (Um et al. 2011).

[10] 195w iNOS and cNOS activity were significantly increased in SNAP group. Indeed, (S)-ZJM-289 preconditioning showed no statistical difference, similar to the effect of NBP treatment, on iNOS and cNOS activity (Fig. 5b). It could be due to the different mechanism between (S)-ZJM-289 and SNAP. As shown in our results, (S)-ZJM-289 preconditioning increased the production of NO, but had no effect on the NOS activities. It is well established that, after cerebral I/R, the ROS production is dramatically increased and overwhelms endogenous antioxidant systems, leading to brain injury (Madamanchi et al. 2005;Schreibelt et al. 2007). As shown in Fig. 6a, a significant increase in the ROS production was observed in the MCAO group after I/R. The elevated level of ROS generation brought down notably following (S)-ZJM-289 preconditioning. Moreover, (S)-ZJM-289 preconditioning protected primary cultured cortical neurons against OGD/R-induced oxidative stress (Fig. 6b). Lipid peroxidation is the hallmark of ROS-induced brain injury. In this study, MDA was used as a marker of lipid peroxidation. The content of MDA was significantly increased in the MCAO group after I/R. However, there were no significant differences in the content of MDA between the two preconditioning groups and the MCAO group (Fig. 6c).

[11] 295w Part of IPC induced protection stems from its ability to upregulate the cellular antioxidant defense system. Nuclearfactor-E2-related factor (Nrf2) is a key transcription factor that regulates antioxidant genes as an adaptive response to oxidative stress or pharmacological stimuli (Thompson et al. 2012). To investigate the effect of (S)-ZJM-289 preconditioning on the Nrf2 signaling, the cellular locations of Nrf2 and kelch-like ECH-associated protein 1 (Keap-1) were elevated after I/R. Immunoblot analysis revealed that the expression of Nrf2 in the nuclear fraction was significantly higher in (S)-ZJM-289 preconditioning and IPC groups than in the MCAO group. On the contrary, Keap-1 expression in the cytosolic fraction decreased in (S)-ZJM-289 preconditioning and IPC groups compared with the MCAO group (Fig. 6d-e). Moreover, (S)-ZJM-289 preconditioning also reinforced the translocation of Nrf2 to the nucleus compared with the OGD/R group in vitro (Fig. 6f-g). These results demonstrated that (S)-ZJM-289 preconditioning promoted the dissociation between Nrf2 and Keap-1 and induced Nrf2 nuclear localization. In order to better clarify the mechanistic link between (S)-ZJM-289 preconditioning and the translocation of Nrf2, cells were transfected with Nrf2-siRNA. Fig. 6h represented the effective down-regulation of Nrf2 obtained with siRNA in cultured cortical neurons. Moreover, Nrf2 down regulation led to a marked decrease of cell viability elicited by (S)-ZJM-289 preconditioning (Fig. 6i). were detected by staining with Annexin V and PI. # p \ 0.05, compared with the control group. * p \ 0.05 and ** p \ 0.01, as compared with OGD/R group by one-way ANOVA followed by Tukey's multiple comparison test Fig. 5 Effects of (S)-ZJM-289 preconditioning on NO content and NOS activities. a The NO content. b The activities of NOS. * p \ 0.05 and ** p \ 0.01, as compared with vehicle group by one-way ANOVA followed by Tukey's multiple comparison test

[12] 181w The regulation of most of the antioxidant occurs through the Nrf2 nuclear localization (Schulke et al. 2012). Such antioxidant enzymes as SOD, GSH-PX, and HO-1, have a key role in the resistance of the neuronal cells to ROS-induced cell death. To evaluate the effects of (S)-ZJM-289 preconditioning on antioxidant enzymes, the Nrf2 and Keap1 in neurons after OGD/R. g Statistical results from the densitometric measurements after normalization against b-actin or Lamin B were calculated as the mean ± SEM. h Neurons were transfected with control siRNA (Ctrl siRNA) or Nrf2 siRNA B (siNrf2) for 24 h, and then cells were harvested to detect Nrf2 protein levels by Western blot. i Neurons were transfected with siNrf2 or Ctrl siRNA for 24 h. The transfected cells were treated with (S)-ZJM-289 for 24 h before OGD/R treatment. Cell viability was measured by MTT assay. # p \ 0.05, compared with the sham group or control group. * p \ 0.05 and ** p \ 0.01, as compared with MCAO group or OGD/R group by one-way ANOVA followed by Tukey's multiple comparison test Neurotox Res

[13] 148w activities of SOD, GSH-PX, and HO-1 were after I/R. In response to I/R injury, the activities of SOD and GSH-PX in the MCAO groups were significantly lower. (S)-ZJM-289 preconditioning significantly increased the activities of SOD and GSH-PX (Fig 7a, b). Similarly, the activity of GSH-PX was also obviously up-regulated by IPC treatment. Of note, the HO-1 activity was weakly decreased in the MCAO group after I/R, while (S)-ZJM-289 preconditioning significantly increased by 42 % of the MCAO group (Fig. 7c). However, IPC failed to affect the activity of HO-1. Moreover, (S)-ZJM-289 preconditioning significantly increased the expression of HO-1 (Fig. 7d-e), which was consistent with the result of HO-1 activity. The increase of HO-1 expression was also observed in the IPC group, disagreeing with the result of HO-1 activity (Fig. 7c). Above all, these observations indicated that (S)-ZJM-289 preconditioning activated Nrf2 and up-regulated antioxidant enzymes against I/R-induced nervous injury.

[14] 12w (S)-ZJM-289 Preconditioning Regulating Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway Following Cerebral Ischemia/Reperfusion

[15] 173w The previous studies reported that MAPK signaling pathways were related to cell apoptosis (Nishina et al. 2004;Xia et al. 1995). In order to better clarify the neuroprotective mechanism of (S)-ZJM-289 preconditioning, the activation of three fundamental protein kinases: extracellular regulated protein kinases (ERK), p38, and c-Jun N-terminal kinases (JNK) were evaluated after I/R. The expressions of p-p38 and p-JNK were significantly increased in MCAO group, while (S)-ZJM-289 preconditioning and IPC markedly decreased the expression of p-JNK, whereas the expression of p-p38 was not influenced by (S)-ZJM-289 preconditioning or IPC (Fig. 8a, b). In addition, (S)-ZJM-289 preconditioning upgraded the expression of p-ERK. These results indicated that (S)-ZJM-289 preconditioning regulated the expressions of ERK and JNK to cope with the nervous injury following I/R. We next examined whether ERK pathway participated in the neuroprotective effect of (S)-ZJM-289 in vitro. Consequently, cortical neurons exposed to OGD/R showed a decrease in the expression of phospho-ERK, in agreement with the previous report (Liu et al. 2012). However, (S)-ZJM-289 treatment augmented the reduced phosphorylation of ERK (Fig. 8c, d).

[16] 103w No differences were detected in the total ERK. Moreover, specific inhibitor (PD98059) for ERK activation partially reduced the neuroprotection elicited by (S)-ZJM-289 (Fig. 8e). These results indicated the involvement of activation of ERK signaling pathways in neuroprotective effect of (S)-ZJM-289. measurements after normalization against b-actin were calculated as the mean ± SEM (n = 5 for each group). Values are expressed as a percentage of the corresponding sham value. # p \ 0.05, compared with the sham group. * p \ 0.05 and ** p \ 0.01, as compared with MCAO group by one-way ANOVA followed by Tukey's multiple comparison test Neurotox Res

[1] 308w The algorithm of preconditioning (i.e., interval ischemia and the administration, the concentration) ences the effectiveness of neuroprotection, hence doseresponse and therapeutic time windows studies were designed. Our results showed that 40-160 mg/kg (S)-ZJM-289 preconditioning significantly reduced brain damage after MCAO, while 20 mg/kg (S)-ZJM-289 preconditioning did not exert neuroprotection (Fig. 1b). This phenomenon could be attributed to the low concentration (20 mg/kg) of (S)-ZJM-289 preconditioning made it insensitive to alter protein expression responsible for neuroprotection against I/R. To date, Puisieux et al. (2004) report IPC performed 3 days before focal cerebral ischemia can induce neuroprotection. The time required for the development of the ischemic tolerance strongly suggests that the protection is mediated by the activation of genes encoding for cytoprotective proteins of which up-regulation is activated by trigger mechanisms occurring after IPC. Based on this paradigm, the therapeutic time window of (S)-ZJM-289 preconditioning was assessed. Our study showed that administration of (S)-ZJM-289 performed 3 days before MCAO induced neuroprotection, while neuroprotection was insignificant if the intervention was delayed for 4 days (Fig. 1c). Our study demonstrated that (S)-ZJM-289 preconditioning exerted the late phase neuroprotection against I/R-induced neuronal injury as manifested by the reduction of brain infarction (Fig. 2a), decreased in the water content (Fig. 2b) and reducing neurological deficit (Fig. 2c). HE staining (Fig. 2d) expressions of total and phosphorylated ERK in rat cortical neurons. d Statistical results from the densitometric measurements after normalization against b-actin were calculated as the mean ± SEM. e Neurons were pretreated with ERK inhibitor PD98059 for 1 h in the absence or presence of (S)-ZJM-289. Cell viability was measured by MTT assay. # p \ 0.05, compared with the sham group or control group. * p \ 0.05 and ** p \ 0.01, as compared with MCAO group or OGD/R group by one-way ANOVA followed by Tukey's multiple comparison test Neurotox Res

[2] 52w and Nissl staining (Fig. 2e) also confirmed (S)-ZJM-289 preconditioning and IPC rescued neuronal loss after ischemia. Taken together, the degree of neuroprotection induced by (S)-ZJM-289 preconditioning was equivalent to that of IPC in the same I/R model. In addition, (S)-ZJM-289 preconditioning reversed the OGD/R-induced cultured neurons injury in vitro (Fig. 4a, b).

[3] 180w NO is a free radical gaseous molecule,which regulates several physiologic processes. NO may react with other oxygen species such as O À 2 and hydrogen peroxide to generate radical nitrogen species, such as peroxynitrite (ONOO -). NO can directly regulates certain proteins through S-nitrosylation. NO may plays several separate roles in IPC (Huang 2004). Zhuang et al. (2010) report that ZJM-289 appears to have NO release in vitro. In the present study, (S)-ZJM-289 preconditioning notably increased the NO content in the rat brain (Fig. 5a). Two isoforms of NOS, named cNOS and iNOS, are involved in the mechanisms of IPC (Kapinya et al. 2000;Atochin et al. 2003). However, (S)-ZJM-289 preconditioning had no effect on the iNOS activity and cNOS activity (Fig. 5b). Moreover, the neuroprotective effect of (S)-ZJM-289 preconditioning was stronger than that of NBP or SNAP treatment (data not shown in the text). The ability of (S)-ZJM-289 preconditioning to protect against cerebral I/R was not simply related to NO generation. This phenomenon can be attributed to the multiple mechanism of (S)-ZJM-289, which integrates the function of NBP and NO-donor.

[4] 347w Part of IPC induced protection stems from its ability to upregulate the cellular antioxidant defense system. The regulation of most of these antioxidant enzymes occurs through the activation of Nrf2 (Fisher et al. 2007). The previous studies show that Nrf2 translocates from the cytosolic to the nucleus and bands to the antioxidant responsive element (ARE) (Nguyen et al. 2005), a cis-acting regulatory element or enhancer sequence, found in the promoter region of certain genes, include glutathione synthase, HO-1, and catalase (Schulke et al. 2012). The coordinated action of Nrf2 mediates the response to oxidative stress (Jaiswal 2004). (S)-ZJM-289 preconditioning combated against oxidative stress as evidenced by the suppression of ROS and MDA (Fig. 6a-c). This was the first study to report that (S)-ZJM-289 preconditioning significantly induced the translocation of Nrf2 to the nucleus (Fig. 6d-g) and marked increased the cellular antioxidant defense system (Fig. 7a-e). Under normal conditions, the Nrf2 inhibitor Keap-1 bands and retains Nrf2 in the cytoplasm where it can be targeted for ubiquitinmediated degradation (Cullinan et al. 2004). However, under certain conditions, Keap1 or Nrf2 may be chemically modified through phosphorylation (Motohashi and Yamamoto 2004), deacetylation (Tkachev et al. 2011), and S-nitrosylation (Um et al. 2011). These chemical modifications enhance Nrf2 disassociation from Keap-1, thus facilitating Nrf2 nuclear translocation and subsequent Nrf2-dependent gene expression. In the present study, (S)-ZJM-289 preconditioning decreased the expression of Keap-1 in the cytosolic fraction (Fig. 6d, e). This result demonstrated that the induction of Nrf2 translocation by (S)-ZJM-289 preconditioning might through inhibiting the ubiquitin ligase activity of Keap-1 or promoting the dissociation between Nrf2 and Keap-1. In order to clarify the involvement of Nrf2 in (S)-ZJM-289 preconditioning mediated neuroprotection, we silenced Nrf2 gene expression by siRNA (Fig. 6h). Data obtained confirmed that Nrf2 played a fundamental role in neuroprotection induced by (S)-ZJM-289 preconditioning (Fig. 6i). The major finding of the current study supported the notion of (S)-ZJM-289 preconditioning inducing a signaling mechanism to combat oxidative stress, as evidenced by the ability of (S)-ZJM-289 preconditioning to increase cellular antioxidants and substantial reduction of intracellular ROS production via Keap1-Nrf2 signaling cascade.

[5] 206w In addition, NO is reported to activate MAPK signaling pathway (Um et al. 2011). The current study was the first reports that (S)-ZJM-289 preconditioning regulated MAPK in vivo (Fig. 8a, b). The activation of ERK signaling cascade has previously been shown to confer neuroprotection through an inhibition of apoptotic cell death (Anderson 1997), and plays a prominent role in the neuroprotective signaling of IPC (Gonzalez-Zulueta et al. 2000). The neuroprotective actions of this pathway are mediated in part by the up-regulation of the pro-survival factors Bcl-2 and Bcl-xL (Griner and Kazanietz 2007). Indeed, (S)-ZJM-289 preconditioning significantly increased the expression of phospho-ERK both in vivo and in vitro (Fig. 8a-d). Moreover, specific inhibitor for ERK activation partially reduced the neuroprotection elicited by (S)-ZJM-289 preconditioning (Fig. 8e). These results indicated the involvement of the activation of ERK signaling pathways in neuroprotective effect of (S)-ZJM-289 preconditioning. JNK is activated by many stress stimuli and plays a key role in I/R induced brain injury. Targeted gene disruption studies have established that JNK is required for the stress-induced release of mitochondrial cytochrome c and apoptosis. Moreover, Bax is essential for JNK-dependent apoptosis. JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3, a cytoplasmic anchor of Bax (Tsuruta et al. 2004).

[6] 80w In the present study, we observed that (S)-ZJM-289 preconditioning markedly inhibited JNK signaling cascade (Fig 8a,b). Subsequently, (S)-ZJM-289 preconditioning increased the expression of anti-apoptotic Bcl-2 protein and inhibited the translocation of Bax to the mitochondria (Fig. 3b, c), thus attenuating the release of cytochrome c from the mitochondria (Fig. 3d, e) and the activation of downstream caspase (Fig. 3f, g), thus inhibited apoptosis (Figs. 3a,4c). These results implied that the expression of ERK and JNK to the (S)-ZJM-289 preconditioning-induced neuroprotection.

[7] 70w The activation of multiple pathways by (S)-ZJM-289 preconditioning in the current study highlights the diversity of this novel compound. Unlike other pharmacological agents that rely on receptor-mediated signaling, (S)-ZJM-289 activates multiple pathways simultaneously. An important question that remains unanswered relates to the mechanism by which (S)-ZJM-289 preconditioning induces the nuclear translocation of Nrf2. Whether (S)-ZJM-289 preconditioning alters Keap1 and/or Nrf2 directly or through upstream signaling (i.e. MAPK) requires further study.

[1] 99w In summary, (S)-ZJM-289 preconditioning induced a late phase neuroprotection against cerebral I/R. The degrees of neuroprotection offered by (S)-ZJM-289 preconditioning and IPC were virtually identical. Also, (S)-ZJM-289 preconditioning protected cultured cortical neurons from OGD/R induced injury. The translocation of Nrf2 to the nuclear, accompanied with the up-regulation of endogenous antioxidant systems, was partly responsible for the neuroprotective effect of (S)-ZJM-289 preconditioning. In addition, the protective effects of (S)-ZJM-289 preconditioning might correlate with the modulation of ERK and JNK phosphorylation. Above all, the findings suggested that (S)-ZJM-289 preconditioning had the potential of therapeutic benefit in the setting of cerebral I/R.

[1] 46w All surgical procedures were approved by Ethics Committee for Animal Experimentation and conducted according to the Guidelines for Animal Experimentation of our institutes. All efforts were made to minimize the number of animals used in this study and every effort was taken to reduce animal suffering.

[2] 198w (S)-ZJM-289 (purity [99 %) was synthesized by the Center of Drug Discovery, China Pharmaceutical University, and the molecular structure was shown in Fig. 1a (Wang et al. 2011). The compound was dissolved in normal saline. Nuclear and cytoplasmic protein extraction kit, tissue mitochondria isolation kit, and TUNEL apoptosis detection kit were obtained from Beyotime Institute of Biotechnology (Haimen, China). Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) kit was purchased from BD Biosciences (NJ, USA). Lactate dehydrogenase (LDH) kit was purchased from JianCheng Bioengineering Institute (Nanjing, China). The primary antibodies used were polyclonal antibodies against Bcl-2, Bax, caspase 3, cleaved caspase 3 (Asp175), caspase 9, cleaved caspase 9 (Asp353), HO-1 (P249), Nrf2 (D1C9), b-actin (Bioworld Technology Inc., MN, USA), Lamin B, cytochrome C (136F3), COX IV (3E11) (Santa Cruz Biotechnology, CA, USA), ERK, p-ERK (Thr202/Tyr204), p38, p-p38 (Thr180/ Tyr182), JNK, and p-JNK (Thr183/Tyr185) (Cell Signaling Technology, Beverly, MA, USA). The goat anti-rabbit IgG-HRP secondary antibody was supplied by Bioworld Technology Inc. (MN, USA). ERK1/2 inhibitor PD98059 was purchased from Tocris Bioscience (Tocris Cookson Limited, Bristol, UK). 2,3,5-triphenyltetrazolium chloride (TTC), NBP, S-nitroso-N-acetylpenicillamine (SNAP), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

[3] 49w Adult male Sprague-Dawley rats (57-61 days) weighing 250-280 g were purchased from B&K Universal Group Limited (Shanghai, China). The rats were housed at 22-24 °C with light from 08:00 a.m. to 08:00 p.m. All animals were fed a commercial diet before and free access to water during the experiment.

[4] 40w In the case of preconditioning, the suture was inserted as described above for 10 min. The suture was then removed completely and the wound was sutured. 72 h later, the animals were re-anesthetized and suffered lethal MCAO for 2 h.

[5] 91w (S)-ZJM-289 Preconditioning (S)-ZJM-289 preconditioning experimental protocol was illustrated in Supplemental Fig. 1. To evaluate the effects of different concentrations (Supplemental Fig. 1a), rats were randomly assigned to six groups consisting of MCAO group (Group 1), (S)-ZJM-289 preconditioning groups (20, 40, 80, or 160 mg/kg (S)-ZJM-289, intragastrically administrated), and the vehicle (normal saline, intragastrically administrated), respectively (Groups 2-6, performed 1 day before MCAO). To detect therapeutic time windows for (S)-ZJM-289 (Supplemental Fig. 1b), 80 mg/kg (S)-ZJM-289 or the vehicle (normal saline) were performed at 1, 2, 3, or 4 days before .

[6] 61w Twenty-four hours after MCAO, the evaluation of neurological deficits was tested for sensorimotor performance via a five-point neurological severity scale before termination (Shah et al. 2006). Briefly, neurological deficits were graded by the following scale: 0, no deficit; 1, forelimb weakness; 2, circling to affected side; 3, inability to bear weight on the affected side; and 4, no spontaneous motor activity.

[7] 70w Twenty-four hours after MCAO, rats were anesthetized and their brains were removed and cut into 2 mm coronal Neurotox Res sections, and then stained with 0.5 % TTC at 37 °C for 15 min. Brain slices were scanned individually. The TTCstained sections were photographed with a digital camera and the infarct areas of each section were determined with the analysis of pixel counting by a computer program of IPP 6.0.

[8] 49w Twenty-four hours after MCAO, rats were anesthetized and their brains were removed. The brain samples were weighed immediately after dissection (wet weight) and then dried at 105 °C for 24 h. The percent of water content was calculated with the formula: (wet weight -dry weight)/wet weight 9 100 %.

[9] 122w Twenty-four hours after MCAO, rats were anesthetized with 2 % pentobarbital sodium (50 mg/kg) and then perfused through the left ventricle with 200 ml of ice-cold 0.1 M phosphate buffer solution (PBS, pH 7.4) followed by 400 ml of 4 % paraformaldehyde in PBS. The brains were removed and immersed in the same fixative for 2 days. The brains were embedded in paraffin and cut into sections (7 lm thickness). Brain sections obtained 1.5 mm behind the Bregma in the coronal plane were stained with HE using standard methods (Lin et al. 1993). The normal morphology and the presence and nature of ischemic damage were verified by a neuropathologist who was unaware of the experimental design or results of the TTC assay.

[10] 182w Twenty-four hours after MCAO, rats were anesthetized with 2 % pentobarbital sodium (50 mg/kg) and then perfused through the left ventricle with 200 ml of ice-cold 0.1 M PBS followed by 400 ml of 4 % paraformaldehyde in 0.1 M PBS. The brains were removed and immersed in the same fixative for 2 days. After dehydration in the graded ethanol and xylene, the brain sections were embedded in paraffin. Paraffin Sects. 4 lm in thickness were dewaxed and rehydrated according to standard protocols, and then stained in 1 % toluidine blue at 50 °C for 5 min. After rinsing with double distilled water, the sections were dehydrated in increasing concentrations of ethanol and cleared in xylene, then mounted with permount cover slip and observed under a light microscope. Cells that contained Nissl substance in the cytoplasm, loose chromatin and prominent nucleoli were considered to be normal neurons, and damaged neurons were identified by the loss of Nissl substance, cavitation around nucleus and by the presence of pyknotic homogenous nuclei. Ischemic core and penumbra of the cerebral cortex were photographed in each section.

[11] 118w TDT-Mediated dUTP-Biotin Nick End-Labeling (TUNEL) Analysis Twenty-four hours after MCAO, rats were anesthetized with 2 % pentobarbital sodium (50 mg/kg) and then perfused through the left ventricle with 200 ml of ice-cold 0.1 M PBS followed by 400 ml of 4 % paraformaldehyde in 0.1 M PBS. The brains were removed and immersed in the same fixative for 2 days. TUNEL assay, as determined by DNA laddering using commercial kits, was performed essentially as described previously with a little modification (Zhao et al. 2012). TUNEL positive (green fluorescent) cells/field were counted by fluorescence microscopy from ten randomly selected fields in a single slide using 3 slides/group and were used to evaluate the degree of apoptosis for each treatment.

[12] 146w Primary cultured cortical neurons were isolated and cultured as described in our previous studies (Zhao et al. 2012). Briefly, whole cerebral cortices were dissected, incubated for 15 min in 0.25 % trypsin at 37 °C, and mechanically dissociated using Pasteur pipette. Cells were collected by centrifugation and resuspended in DMEM/F12 (1:1) with 10 % (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were seeded at a density of 1.5 9 10 5 cells/cm 2 onto poly-D-lysine-coated 96-or 6-well plates and were maintained in a humidified incubator in air with 5 % CO 2 (Thermo Scientific 3110, OH, USA). After 24 h, the culture medium was changed to neurobasal medium supplemented with 2 % B27 (Life Technologies, Carlsbad, CA, USA). Medium replacement was performed every 3 days. The neurons were maintained for 7-10 days in primary cultures until used for the following experiments.

[13] 94w Brain homogenate supernatant and cells lysed in lysis buffer (30 mM Tris-Hcl (pH 7.5), 150 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 % Nonidet P-40, 10 % glycerol, and phosphatase and protease inhibitors). Nuclear and mitochondria extracts were prepared according to the manufacturer's protocols. Western blot analysis was performed as described previously (Zhang et al. 2013). Briefly, the proteins were then separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore Corporation, MA, USA). Immunoreactive proteins were visualized using enhanced chemiluminescence (KEN GENE), and CLUBIO 2850 gel catcher.

[14] 37w Data are expressed as mean ± SEM. Statistical analyses were made using one-way ANOVA, followed by a Tukey's multiple comparison test for multiple comparisons. A p value of less than 0.05 was considered to be statistically significant.

[1] 111w Rats were first anesthetized with chloral hydrate sodium (300 mg/kg intraperitoneally). MCAO surgery was performed as described in our previous report (Zhao et al. 2012). Briefly, the common carotid artery (CCA), the external carotid artery (ECA), and the internal carotid artery (ICA) were isolated from connective tissues. A 4-0 mono-filament nylon suture (Beijing Sunbio Biotech Co., Ltd., Beijing, China) with a rounded tip was inserted into the ICA through the ECA stump and gently advanced to occlude the CCA. After 2 h sustained ischemia, blood flow was restored by the withdrawal of the intraluminal suture. Sham-operated rats were performed with the same surgical procedures except that the arteries were not occluded.

[2] 100w Oxidant generation was evaluated in homogenate and cortical neurons using fluorescence microsphere 2 0 ,7 0 -dichlorofluorescin (DCFH), as described previously (Bejma et al. 2000). 50 ll brain homogenate supernatant or 1 9 10 6 cortical neurons were added in assay medium and incubated at 37 °C for 15 min. This allowed DCFH-DA to be cleaved by intracellular esterase to derive free DCFH. The rate of oxidation from DCFH to dichlorofluorescein (DCF) indicative of oxidant production was followed at the excitation wavelength of 488 nm and emission wavelength of 525 nm for 30 min using a Hitachi F-2000 fluorescence spectrometer.

[3] 172w Twenty-four hours after MCAO, rats were sacrificed and the brains were removed. The homogenates extracted from the cortex of ischemic hemisphere of brain were measured. SOD activity was measured based on the extent inhibition of amino blue tetrazolium formazan formation in the mixture of nicotinamide adenine dinucleotide, phenazine methosulphate, and nitroblue tetrazolium (NADH-PMS-NBT), as described previously (Kakkar et al. 1984). Assay mixture contained 0.1 ml of brain homogenate supernatant, 1.2 ml of sodium pyrophosphate buffer (pH 8.3, 0.052 M), 0.1 ml of PMS (186 lM), 0.3 ml of NBT (300 lM), and 0.2 ml of NADH (750 lM). Reaction was started by addition of NADH. After incubation at 30 °C for 90 s, the reaction was stopped by addition of 0.1 ml of glacial acetic acid. Reaction mixture was stirred vigorously with 4.0 ml of n-butanol. Color intensity of the chromogen in butanol was measured spectrophotometrically at 560 nm. The SOD activity was calculated according to the SOD standard control and expressed as fold change based on comparison with the sham group.

[4] 162w Twenty-four hours after MCAO, rats were sacrificed and the brains were removed. The homogenates extracted from the cortex of ischemic hemisphere of brain were measured. Activities of GSH-PX were determined as representative antioxidant enzyme by following the modified version of the previous method (Flohe and Gunzler 1984). The reaction mixture was prepared by adding 600 ll of potassium phosphate buffer (0.05 M, pH 7.0, 0.1 mM EDTA), 100 ll of 10 mM GSH (reduced form), 100 ll of 1.5 mM NADPH, and 100 ll of brain homogenate supernatant. An amount of 100 ll of brain homogenate supernatant was added to the reaction mixture and incubated at 37 °C for 10 min. To this, 50 ll of 12 mM t-butyl hydroperoxide was added. The change in the absorbance was measured at 340 nm for 1 min. One unit of activity is equal to millimole of NADPH oxidized/mg protein/min. The GSH-Px activity was expressed as fold change based on comparison with the sham group.

[5] 158w Heme Oxygenase-1(HO-1) Activity Assay Twenty-four hours after MCAO, rats were sacrificed and the brains were removed. The homogenates extracted from the cortex of ischemic hemisphere of brain were measured. HO-1 activity was assessed by the protocol described previously with the following modifications (Yang et al. 1999). Aliquots of brain homogenate supernatant were incubated with heme (20 lM), rat liver cytosol (1 mg/ml), MgCl 2 (1 mM), glucose-6-phosphate dehydrogenase (0.2 units), glucose 6-phosphate (2 mM), and NADPH (0.8 mM) in 0.2 mL of 0.1 M potassium phosphate buffer (pH 7.4) in subdued lighting for 15 min at 37 °C. Negative controls (blanks) were prepared as above only without NADPH. The reaction was stopped by snap cooling in ice (0 °C). The amount of bilirubin generated was estimated as the difference in values detected at 463 nm and 520 nm on a microplate reader. The HO-1 activity was expressed as fold change based on the comparison with the sham group.

[6] 192w Twenty-four hours after MCAO, rats were sacrificed and the brains were removed. The homogenates extracted from the cortex of ischemic hemisphere of brain were measured. Lipid peroxidation, a major indicator of oxidative stress, was determined by measuring of the malondialdehyde (MDA) level in tissue homogenates. MDA was an end product of lipid peroxidation and its level was determined spectrophotometrically by use of thiobarbituric acid reactive substances method (Mihara and Uchiyama 1978) with little modification. Briefly, 5 ll supernatant was mixed with 25 ll 3.0 % sodium dodecylsulfate, 75 ll acetic acid buffer (pH 3.5), 75 ll 0.8 % 2-thiobarbituric acid, and 20 ll deionized water, followed by heating in a water bath at 95 °C for 1 h. After cooling, 250 ll n-butanol and pyridine (15:1) was added and the mixture was centrifuged at 1,4009g for 15 min. The organic phase was collected and the fluorescence intensity was monitored with excitation at 515 nm and emission at 553 nm. A standard curve was run simultaneously with each set of samples by using 1,1,3,3-tetramethoxypropane as an external standard. The MDA content was expressed as fold change based on comparison with the sham group.

[7] 152w Oxygen-glucose deprivation was used as an in vitro model of cerebral ischemia and carried out as described previously (Liu et al. 2012). Neurons were rinsed twice and incubated in Earle's solution without glucose containing (in mM) 116 NaCl, 5.4 KCl, 0.8 MgSO 4 , 1.0 NaH 2 PO 4 , 1.8 CaCl 2 , and 26 NaHCO 3 (pH 7.35). Then, the cultures were introduced into a specialized, humidified chamber filled with 95 % N 2 and 5 % CO 2 at 37 °C for 2 h (OGD). At the end of this period, cultures were replaced with the normal DMEM/F12 medium under normoxic conditions for 24 h (recovery, R). (S)-ZJM-289 (0.1, 1 and 10 lM) was applied to cortical cell cultures 24 h before and during OGD/R. Controls were incubated with Earle's solution containing 5.6 mM glucose and maintained in an incubator with 5 % CO 2 atmosphere at 37 °C.

[8] 32w At 24 h post-OGD, neuronal cell viability was measured by the MTT assay and lactate dehydrogenase (LDH) activity in the medium was determined according to our previous studies (Zhao et al. 2012).

[9] 47w For the detection of cell apoptosis, cells were stained with Annexin V-FITC and propidium iodide at room temperature for 15 min. Cells were then analyzed by flow cytometry (488 nm excitation and 600 nm emission filters) using BD FACSCalibur flowcytometer (Becton & Dickinson Company, Franklin Lakes, NJ).

[10] 76w siRNA Transfection Cells were transfected with 50 nM Nrf2-annealed siRNA (small interfering RNA; Genechem, Shanghai, China) by electroporation with a high-efficiency electroporator (type NEPA21, Nepa Gene Co. Ltd., Chiba, Japan) according to the previous study (Aoyagi et al. 2012). The siRNA sequences utilized targeted the following rat Nrf2 coding sequences 5 0 -UGGAGCAAGACUUGGGCCACUUAAA-3 0 and 5 0 -UUUAAGUGGCCCAAGUCUUGCUCCA-3. Control experiments were performed using equivalent amounts of the StealthTM RNAi Negative Control Med GC (Genechem, Shanghai, China).