Lumbrokinase Attenuates Side-Stream-Smoke-Induced Apoptosis and

Apr 5, 2013 - Graduate Institute of Chinese Medicine, China Medical University, Taichung, Taiwan. ▽ Department of Healthcare Administration, Asia Un...
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Lumbrokinase Attenuates Side-Stream-Smoke-Induced Apoptosis and Autophagy in Young Hamster Hippocampus: Correlated with eNOS Induction and NFκB/iNOS/COX‑2 Signaling Suppression Chih-Yang Huang,† Wei-Wen Kuo,‡ Hung-En Liao,§ Yueh-Min Lin,∥ Chia-Hua Kuo,⊥ Fuu-Jen Tsai,# Chang-Hai Tsai,▽ Jia-Long Chen,‡ and Jing-Ying Lin*,○ †

Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan Department of Biological Science and Technology, China Medical University, Taichung, Taiwan § Department of Healthcare Administration, Asia University, Taichung, Taiwan ∥ Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan ⊥ Laboratory of Exercise Biochemistry, Taipei Physical Education College, Taipei, Taiwan # Graduate Institute of Chinese Medicine, China Medical University, Taichung, Taiwan ▽ Department of Healthcare Administration, Asia University, Taichung, Taiwan ○ Department of Medical Imaging and Radiological Science, Central Taiwan University of Science and Technology, Taichung, Taiwan ‡

ABSTRACT: Recent studies have found that cigarette smoke is epidemiologically linked to an increased risk for impaired cognitive development in adolescents. This study evaluated the influence of side stream smoke (SSS) exposure on hippocampal apoptosis and of the lumbrokinase (LK) effects on SSS induced apoptosis in young hamster hippocampus. Twenty male hamsters at six weeks of age were randomly divided into control group, SSS group (exposed to tobacco cigarettes smoke at doses of 10 cigarettes for 30 min twice a day for 1 month), and SSS hamsters with LK treatment (1.2 mg/kg, ip) for twice a week for 1 month. TUNEL assay and Western blotting were performed. The TUNEL-positive apoptotic cells, as well as Fas-dependent activity and mitochondria-dependent apoptotic pathways, such as Fas, FADD, activated caspase-8, t-Bid, activated caspase-9, and activated caspase-3, were significantly increased in the SSS-exposed hippocampus compared to the control and highly attenuated in the LK treatment group. Additionally, SSS exposure significantly increased the autophagy marker proteins, Beclin-1, ATG7, and LC3-II levels, in the hippocampus compared to those in the control group and obviously attenuated after LK treatment. LK also reduced hippocampus injury by enhancing eNOS expression and remarkably inhibited the proinflammatory NFκB/iNOS/COX-2 signaling activity. We found that the detrimental effects of SSS on the hippocampus are truly mediated by cell apoptosis and autophagy. However, LK reduced the hippocampus apoptosis and autophagy related injuries induced by SSS in a widespread manner. We suggest that LK presents protective effects on hippocampus apoptosis and has therapeutic potential against abnormal hippocampal function.

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radiation, and oxidative stress.4 In fact, it has been suggested that apoptosis plays a crucial role in the pathogenesis of human diseases, including neurodegenerative diseases such as cerebral ischemia and Alzheimer’s and Parkinson’s diseases.5 Therefore, apoptosis process evaluation could be an excellent way of predicting the development of neuropathy by tobacco smoke exposure. Both Fas-dependent and mitochondria-dependent apoptotic pathways are considered major pathways directly causing neuronal apoptosis. The extrinsic Fas-receptor-dependent pathway is initiated by binding the Fas ligand to the Fas receptor. After ligand binding, Fas-receptor oligomerization

INTRODUCTION Tobacco smoke exposure has been implicated as a major risk factor for pulmonary diseases, cardio and cerebrovascular diseases, cancers, and other diseases. 1 Epidemiologic and experimental animal studies have recently showed that tobacco smoke exposure affects adolescent brain function.2,3 However, the precise mechanisms through which tobacco smoking elicits the deleterious effects are not fully elucidated. Among various dysfunctional changes in the central nervous system, uncontrolled apoptosis plays a significant role in the pathological changes. Apoptosis, a physiological program of cellular death, has a critical role during normal development and in cellular homeostasis. Apoptosis may occur spontaneously or be induced by a variety of specific stimuli including toxic chemicals, © 2013 American Chemical Society

Received: October 19, 2012 Published: April 5, 2013 654

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growth factor-mediated neurite outgrowth from PC12 cells and expressions of growth-associated protein 43 and synapsin 1.15 Earthworm extracts also stimulate RSC96 Schwann cell proliferation and survival through the PI3K/Akt system mediated by IGF-1.16 It induced G1 phase cell cycle progression by altering the expression of the proteins that control the cell cycle, resulting in the upregulation of the antiapoptotic Bcl-2 proteins.12 Moreover, earthworm extracts stimulate RSC96 Schwann cell migration and upregulate PA and MMP2/9 expression mediated through the MAPK pathways.17 Lumbrokinase (LK) extracted from earthworms has been used to treat stroke and cardiovascular diseases.18 LK is a group of proteolytic enzymes with molecular weights from 25 to 32 kDa that include a plasminogen activator and plasmin.19 LK can dissolve fibrin directly and also activate plasminogen.18 In healthy human volunteers, orally administered earthworm powder increased levels of tissue plasminogen activator and fibrinolytic activity.20 Oral LK improved regional myocardial perfusion in patients with stable angina LK. A recent study has shown the anti-ischemic action of LK in the brain.21 It has been shown to possess antiplatelet activity by elevating the cAMP level and attenuating calcium release from platelet calcium stores. This antithrombosis action is due to inhibiting ICAM-1 expression in endothelial cells and preventing ischemic injury by activating JAK1/STAT1 expression to inhibit apoptosis.21 However, the molecular mechanisms underlying such neuroprotective actions are not known with certainty. Therefore, this study was aimed at determining the role of LK against extreme SHS-induced NOS expression and apoptosis in young hamster hippocampus. We examined Fas-dependent and mitochondria-dependent apoptosis and also the autophagy-related pathway in the hippocampus from side-stream-smoke (SSS) exposure in young hamsters.

results in the recruitment of the Fas-associated death domain (FADD) adaptor protein and caspase-8, which lead to the activation of caspase-8. Activated caspase-8 cleaves pro-caspase3, which then undergoes autocatalysis to form active caspase-3, a principal effector caspase of apoptosis.5 Activated caspase-8 can also cleave Bcl-2 homology domain 3 (BH3)-interfering domain death agonist (Bid). The cleaved Bid then causes the release of mitochondrial cytochrome c, leading to pro-caspase-9 activation, which can activate pro-caspase-3. Mitochondrial dependent cell death is mediated by internal factors, especially in the mitochondria, which is the main action site for apoptosis regulating proteins such as the Bcl-2 family. Commitment to apoptosis is typically governed by opposing factions of the Bcl2 family, including pro-apoptotic vs antiapoptotic family members. Bcl-2 is an antiapoptotic protein that prevents cytochrome c release, whereas Bax and Bad, pro-apoptotic proteins, enhance cytochrome c release from mitochondria into cytosol, which is responsible for activating caspase-9 and caspase-3, and executes the apoptotic program.5 Our recent study indicated that diabetes causes Fas receptor-dependent and mitochondria-dependent apoptosis in the cerebral cortex tissues of rat brains (Huang et al., unpublished data). Emerging studies also suggest that autophagy plays a pivotal role in the cellular response to stress conditions.6 Impaired autophagy related pathways in the central nervous system tissue cause massive loss of neurons, resulting in neurodegeneration.7 Beclin-1 is involved in class III phosphoinositide 3-kinase protein complexes that regulate the initial autophagy steps and vesicle nucleation. Beclin-1 levels were also used to evaluate the autophagy levels. Atg7 and Atg12 are essential components of the core autophagy machinery that regulates the vesicle elongation process. A microtubule-associated protein light chain 3 (LC3) is the specific marker of phagophores and autophagosomes.6 Apoptosis can be regulated by several signaling molecules including nitric oxide (NO).8 NO is synthesized from Larginine by different isoforms of NO synthases, namely, endothelial (eNOS), neuronal (nNOS), and an isoform expressed during inflammatory reactions (iNOS). Endothelium-derived NO mediates endothelium-dependent vasodilation. In vitro exposure to cigarette smoke extract resulted in endothelial apoptosis. Preactivation of eNOS offers significant protection against cigarette smoke extract-induced endothelial apoptosis.9 In addition, a moderate basal NO production may protect cells from free radical-mediated cytoxicity by scavenging superoxide anions and terminating radical mediated lipid peroxidation propagation.10 However, excessive NO generation by iNOS may be harmful to the CNS. Indeed, huge accumulation of NO and overexpression of iNOS have been observed in epilepsy, ischemia, and degenerative disorders including Alzheimer’s and Parkinson’s diseases.11 Once iNOS is up-regulated, large amounts of oxygen and nitrogen reactive species are formed, which may account for the oxidation, ultimately leading to neuronal cell loss. The SHS effect on eNOS and iNOS expression in vivo and in the brain has not been reported. The Dilong earthworm is well known for its use in traditional Chinese medicine.12 Previous earthworm studies have shown its antimicrobial, hepatoprotective, anti-inflammatory, antipyretic, and scar wound-healing characteristics.13 In vivo experiments have shown that earthworm extracts promote axonal sprouting in peripheral nerve injury.14 In the in vitro studies, earthworm extracts caused a marked enhancement of nerve

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EXPERIMENTAL PROCEDURES

Animal Models and Treatment. Male hamsters at 6 weeks of age were obtained from the National Center for Experimental Animals (National Science Council, Taiwan, Republic of China). All animals were kept in a temperature- and light-controlled environment with a 12-h light/12-h dark cycle. All protocols were approved by the Institutional Animal Care and Use Committee of China Medical University, Taichung, Taiwan. The principles of laboratory animal care (NIH publications) were followed. All animals were allowed to adapt to the environment for 2 weeks after their arrival before the experiment started. Hamsters were exposed to cigarettes from 8 weeks to 12 weeks, the approximate age range for adolescent and young hamsters, particularly neurochemical patterns, were compared to those at adulthood. Young hamsters were divided into three groups, i.e., control group, side-stream-smoke group (SSS), and side-streamsmoke hamster with LK treatment group (SSS + LK). Exposure SSS procedures were based on previously described methods.22 The animals were placed in whole-body exposure chambers (40 × 40 × 39 cm3), which contained an air exchange fan of about 2 cm in diameter. Animals could move freely while exposed to side stream 0, 10 cigarettes smoke (New Paradise, Taiwan, contains nicotine, 0.4 mg/ cigarette, and tar, 5 mg/cigarette), representing control and SSS, respectively. The cigarettes were fixed and lighted in another chamber (40 × 40 × 39 cm3), which also contained an air exchange fan of about 2 cm in diameter. Smoke from lighted cigarettes was sucked into the animal chamber at a low rate of 200 L/min with the help of a tube and an aerator. The animals were exposed to smoke for 30 min, twice a day, 6 days/week for 1 month. The hamsters in the SSS + LK group were injected with LK (1.2 mg/kg, ip) twice a week for 1 month. The LK used in this study was obtained from Harbin Jiuxin Science & Technology Industrial Co., Ltd. The LK batch number was 20050818. 655

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After 1 month, the animals were sacrificed under deep anesthesia. Brain tissue was carefully excised. The hippocampus was dissected and used for further analysis. TUNEL Staining. Cell apoptosis was detected using TUNEL staining. Briefly, the hippocampus sections were incubated with proteinase K, washed in PBS, incubated with permeabilization solution, blocking buffer, and then washed two times with PBS. The sections were incubated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and fluorescein isothiocyanatedeoxyuridine 5-triphosphate for 1 h at 37 °C using an apoptosis detection kit (Roche Molecular Biochemicals) for detection. Sections were then rinsed with PBS and stained with 4,6-diamidino-2phenylindole (DAPI; Life Technologies) to detect the cell nucleus using UV light at 340/380 nm under microscopic observation. TUNEL-positive nuclei (fragmented DNA) were fluoresced bright green at 450−500 nm. The number of TUNEL-positive cells was determined by counting 300 cells by at least two independent individuals in a blind manner. Western Blotting. The hippocampus was homogenized in ice-cold lysis buffer for 1 min. The homogenates were centrifuged at 12,000g for 40 min twice. The supernatant was collected and stored at −70 °C for further experiments. Protein concentration of tissue extracts was determined by the Bradford method (Bio-Rad Protein Assay, Hercules, CA). Protein homogenates were separated on a 10% SDS−PAGE with a constant voltage of 75 V. Electrophoresed proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, 0.45 μm pore size) with a transfer apparatus (Bio-Rad). PVDF membranes were incubated in 5% nonfat milk in TBS buffer at room temperature for 1 h. Primary antibodies including Fas ligand, Fas receptor, FADD, Bcl-2, Bax, Bad, Bid, caspase-8, caspase-9, caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA), and α-tubulin (Neo Markers, Fremont, CA) were diluted in antibody binding buffer overnight at 4 °C. The blots were washed 3 times in TBS buffer for 10 min and then immersed in the second antibody solution containing goat antimouse IgG-HRP, goat antirabbit IgGHRP (Santa Cruz) for 2 h, and diluted in TBS buffer. The membranes were washed 3 times for 10 min in TBS buffer. The immunoblotted proteins were visualized using an ECL Western blotting luminal reagent and quantified using a Fujifilm LAS-3000 chemiluminescence detection system (Tokyo, Japan). Statistical Analyses. The mean and SE of each treatment group were calculated for all experiments with statistical analysis. The number of samples is indicated in the description of each experiment. All statistical tests were two sided. P < 0.05 was considered statistically significant. In all experiments, variance was analyzed by Student’s t test.

Figure 1. Lumbrokinase reduces SSS-induced cell apoptosis in the hippocampus. (A) Stained apoptotic cells of the dentate molecular layer of the hippocampus in control, SSS (SSS), and SSS with lumbrokinase treatment (SSS + LK) hamsters were measured by staining with the TUNEL assay (green spots). The relative background tissues were stained with DAPI (blue spots). Bar = 20 μm. (B) Bars present the percentage of TUNEL-positive cells relative to DAPI-positive cells. Values are the means ± SD (n = 3 in each group). ***P < 0.001, significant differences from the control group. ## P < 0.01, significant differences from the SSS group.

pathways including the protein levels of the Fas ligand, Fas, and FADD from all three groups. Compared with control animals, the protein levels of the Fas ligand, Fas, and FADD significantly increased in the SSS group. In contrast, significantly reduced Fas ligand, Fas, and FADD were detected in the hippocampus of hamsters from the LK treated group compared to that in the SSS group (Figure 2A). To identify the downstream

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RESULTS Lumbrokinase Reduced the TUNEL-Positive Apoptotic Cells in SSS Hamster Hippocampus. To investigate the LK effect on SSS-induced apoptosis in the hippocampus cells, we examined the apoptosis positive cells in the dentate molecular layer of the hippocampus from control, SSS, and SSS treated with LK (SSS + LK) groups using the TUNEL assay. We observed that the hippocampus of the SSS group stained with the TUNNEL assay showed around 40% significantly increased TUNEL-positive cells compared to that in the control group (Figure 1A). Significantly reduced TUNEL-positive cells, around 25%, were found in the hippocampus of young hamsters from the LK treatment group compared to that in the SSS group (Figure 1A). Activation of the Fas Receptor-Dependent Apoptotic Pathways in SSS Hamster Hippocampus Was Reduced by Lumbrokinase. The upstream and downstream components of the Fas-dependent apoptotic signaling pathways in the hippocampus of hamsters from control, SSS, and SSS treated with LK (SSS + LK) groups were investigated. We measured the upstream components of the Fas-dependent apoptotic

Figure 2. LK inhibited upstream components of the Fas-dependent apoptotic pathway activation after SSS exposure. (A) The representative protein levels of the Fas ligand, Fas, and Fas-associated death domain (FADD) prepared from hippocampi in the control, SSS, and SSS + LK hamsters were measured using Western blotting analysis. (B) Bars represent the relative protein quantification of the Fas ligand, Fas, and FADD on the basis of α-tubulin and indicate the mean values ± SD (n = 3 in each group). ***P < 0.001, significant differences from the control group. ###P < 0.001, significant differences from the SSS group.

components of the Fas receptor, caspase 8 and Bid cleavage (t-Bid), a mediator that connects the Fas receptor-dependent to the mitochondria-dependent apoptotic pathway, were used. We further examined the protein levels of pro-caspase-8, activated caspase-8, Bid, and t-Bid in the hippocampus. Western blot analysis revealed that the levels of pro-caspase-8, activated 656

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caspase-8, Bid, and t-Bid were significantly higher in SSS animals than those in the control animals (Figure 3); LK treatment significantly reduced pro-caspase-8, activated caspase-8, Bid, and t-Bid levels (Figure 3).

Figure 4. LK inhibited mitochondria-dependent apoptotic pathway activation after SSS exposure. (A) The representative protein levels of Bax, cytochrome c, caspase-9, and activated caspase-9 prepared from hippocampi in control, SSS, and SSS + LK hamsters were measured using Western blotting analysis. (B) Bars represent the relative protein quantification of Bax, cytochrome c, caspase-9, and activated caspase-9 on the basis of α-tubulin and indicate the mean values ± SD (n = 3 in each group). *P < 0.05, **P < 0.01, and ***P < 0.001, significant differences from the control group. ###P < 0.001, significant differences from the SSS group.

Figure 3. LK prevented downstream components of the Fasdependent apoptotic pathway activation after SSS exposure. (A) The representative protein levels of caspase-8, activated caspase-8, BID, and t-BID prepared from hippocampi in control, SSS, and SSS + LK hamsters were measured using Western blotting analysis. (B) Bars represent the relative protein quantification of caspase-8, activated caspase-8, BID, and t-BID on the basis of α-tubulin and indicate the mean values ± SD (n = 3 in each group). *P < 0.05 and **P < 0.01, significant differences from the control group. ##P < 0.01, significant differences from the SSS group.

Activation of the Mitochondria-Dependent Apoptotic Pathways in SSS Hamster Hippocampus Was Reduced by Lumbrokinase. We examined the Bax, cytochrome c, caspase-9, and activated caspase-9 protein levels in the hippocampus of hamsters from the control, SSS, and SSS + LK groups to reveal changes in the mitochondria-dependent apoptotic pathway. The Bax, cytochrome c, caspase-9, and activated caspase-9 protein levels in the hippocampus were significantly higher in SSS hamsters than in control animals (Figure 4). LK treatment significantly reduced Bax, cytochrome c, caspase-9, and activated caspase-9 levels (Figure 4). Moreover, the presence of executive apoptotic protein caspase-3 and activated caspase-3 were analyzed. The hippocampus obtained from SSS hamsters showed the significant caspase-3 activation; however, this upregulation in caspase-3 activation in the hippocampus of SSS hamster was efficiently abolished by LK treatment (Figure 5). Lumbrokinase Ameliorated the Upregulated Autophagy-Related Pathways. The levels of the autophagy-related proteins, Beclin-1, ATG7, LC3-I, and LC3-II, were measured to investigate the changes in autophagy in the hippocampus of hamsters from control, SSS, and SSS + LK hamster groups. As shown in Figure 6, exposure to SSS resulted in an increase in these autophagy-related proteins levels when compare to those in the control group, and this biochemical sign of increased autophagy was markedly reduced by LK treatment. Lumbrokinase Suppressed SSS Upregulated ProInflammatory NFκB/iNOS/COX-2 Signaling and Enhanced SSS Downregulated eNOS Expression. We examined the protein levels of NFκB, p-NFκB, COX-2, iNOS, and eNOS in the hippocampus of hamsters from the control, SSS, and SSS + LK using Western blotting to identify the LK effects on stress-related proinflammatory proteins. The

Figure 5. LK inhibited caspase-3 activation after SSS exposure. (A) The representative protein levels of caspase-3 and activated caspase-3 prepared from hippocampi in control, SSS, and SSS + LK hamsters groups were measured using Western blotting analysis. (B) Bars represent the relative protein quantification of caspase-3 and activated caspase-3 on the basis of α-tubulin and indicate the mean values ± SD (n = 3 in each group). ***P < 0.001, significant differences from the control group. ###P < 0.001, significant differences from the SSS group.

NFκB, p-NFκB, COX-2, and iNOS expression levels were significantly increased in the SSS group, whereas eNOS was significantly reduced in the SSS group (Figure 7). In contrast, significantly reduced NFκB, p-NFκB, COX-2, and iNOS and increased eNOS levels were detected in the hippocampus of hamsters from the LK-treated group compared to those in the SSS group (Figure 7).

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DISCUSSION This study first demonstrated the protective effects of LK on hippocampus apoptosis in young hamsters that were exposed to 10 cigarettes for 30 min, twice per day for 1 month. Our findings can be summarized as follows: (1) TUNEL-positive apoptotic cells were increased in the dentate molecular layer of the hippocampus from SSS-exposed young hamsters compared 657

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(4) Autophagy was also enhanced in the hippocampus of SSSexposed young hamsters, as indicated by the increased expression of Beclin-1, ATG7, LC3-I, and LC3-II. However, LK treatment significantly reduced the SSS-induced autophagy effect. (5) The stress sensitive proinflammatory NFκB, COX-2, and iNOS expression are enhanced in the hippocampus of SSSexposed young hamsters, while LK treatment attenuated these expressions. In contrast, eNOS expression is reduced in the SSS-exposed hippocampus, while LK treatment increased eNOS expression. Programmed cell death has a critical role in the neurodegenerative diseases process.23 Several studies have shown that cigarette smoke can induce excessive apoptosis in different tissues.24,25 Studies describing the cigarette smoke effects on nervous tissues focused on brain development.26 The results in this study support the notion that SSS exposure elicits an increase in programmed cell death in the hippocampus of young hamsters. A recent study reported that side stream cigarette smoke exposure induced apoptosis in the cerebral cortex of adult rats.27 Another study indicated that secondhand smoke exposure increased caspase-3 proteolysis of αII-spectrin in adult rat brains.28 Since an increased number of apoptotic cells was detected in rat brains after cigarette smoke or secondhand smoke, the apoptotic pathway induced by smoking is still unclear. In our findings, the Fas-receptor-dependent apoptotic pathway was significantly increased in the hippocampus of young hamsters exposed to SSS (Figure 2). The Fas apoptotic pathway is activated by the Fas ligand, Fas death receptors, caspase-8, and cleaved Bid activation, which in turn induced cell apoptosis. Fuller et al. found dephosphorylation of PEA-15 in secondhand smoke exposed rats, suggesting the activation of the extrinsic apoptotic pathway.28 Our previous study also reported activation of Fas-dependent apoptosis by SSS in cardiac cells.22 The mitochondria-dependent apoptotic pathway is mediated by the Bcl-2 family, such as Bax and Bcl-2. Cytochrome c is released from the mitochondrial membrane after the Bcl-2 family pro-apoptotic proteins open the pores. Cytochrome c then activates caspase-9, which further activates caspase-3, where caspase-3 acts as the final common pathway and executes the apoptotic program. Anbarasi et al. showed brain mitochondrial dysfunction during side stream cigarette smoking. 29 In this study, the mitochondria-dependent apoptotic pathways were also activated in the hippocampus of SSS-exposed young hamsters. Therefore, we suggested that the Fas-dependent and mitochondria-dependent apoptotic pathways play a key role in SSS-exposure-induced hippocampus apoptosis. These results are consistent with previous reports demonstrating increased apoptosis and reduced neuronal and glial densities in the hippocampus due to nicotine exposure in adolescent mice.30 Collectively, data from the present study raise the possibility that secondhand smoke may also induce hippocampus apoptosis and could affect hippocampus function in adolescents.2 Various mechanisms have been reported to be involved in cigarette-smoke-exposure-induced brain apoptosis. Secondhand smoke contains at least 250 chemicals known to be toxic. SSS is the major component of secondhand smoke. Therefore, such SSS exposure is a stressful stimulus that may impact brain cells. Exposure to cigarette smoke can cause substantial adverse effects in the cardiovascular system that include increased platelet activation, endothelial dysfunction, inflammation and infection, atherosclerosis, increased oxidative stress, and decreased energy metabolism.31 Secondhand smoke exposure

Figure 6. LK inhibited autophagy after SSS exposure. (A) The representative protein levels of Beclin-1, ATG 7, LC3 I, and LC3 II prepared from hippocampi in control, SSS, and SSS + LK hamster groups were measured using Western blotting analysis. (B) Bars represent the relative proteins quantification of Beclin-1, ATG 7, and LC3 II on the basis of α-tubulin and indicate the mean values ± SD (n = 3 in each group). ***P < 0.001, significant differences from the control group. ###P < 0.001, significant differences from the SSS group.

Figure 7. LK effects on stress-related inflammatory proteins. (A) The representative protein levels of NFκB, p-NFκB, COX-2, iNOS, and eNOS prepared from hippocampi in the control, SSS, and SSS + LK hamster groups were measured using Western blotting analysis. (B) Bars represent the relative protein quantification of NFκB, p-NFκB, COX-2, iNOS, and eNOS on the basis of α-tubulin and indicate the mean values ± SD (n = 3 in each group). **P < 0.01 and ***P < 0.001, significant differences from the control group. ##P < 0.01 and ### P < 0.001, significant differences from the SSS group.

with those in the control group. LK administration prevented apoptosis during SSS. (2) The key components of the Fasreceptor-dependent apoptotic pathway, such as the Fas ligand, Fas, FADD, activated caspase-8, Bid, and t-Bid, in the hippocampus of SSS-exposed young hamsters were significantly increased compared with those in control animals, and these SSS-induced Fas-receptor-dependent apoptotic pathways were significantly attenuated after LK treatment. (3) The key mitochondria-dependent apoptotic pathway components, such as Bax, cytochrome c, activated caspase-9, and activated caspase3, in the hippocampus of SSS-exposed young hamsters were significantly increased compared with those in control animals, and these SSS-induced mitochondria-dependent apoptotic pathways were significantly attenuated after LK treatment. 658

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increases cerebral blood flow in response to diverse stimuli, alters neuronal signaling, and promotes angiogenesis and neurogenesis.40 Impairment of these eNOS-dependent mechanisms in the presence of cardiovascular risk factors may contribute to reductions in cerebral blood flow, vascular cognitive impairment, and neuronal death.41 The lower levels of eNOS protein were also observed in SSS-exposed hippocampi, suggesting that eNOS was inhibited by SSS exposure. It was previously found that smoke exposure decreased NO production in the aortic endothelium and pulmonary artery endothelium and decreased endothelium-dependent vasodilation.31 The physiological amount of eNOS-derived NO in endothelial cells protects against cigarette-smoke-induced apoptosis.9 Previous studies suggested that targeting eNOS such as statin, cortisosteroids, and thyroid hormone or exercise provides neuroprotection.42 Our finding on the LK effects in enhancing eNOS expression on vessel hippocampi suggests the therapeutic potential of LK in neuroprotection. The levels of iNOS in the CNS are generally fairly low. However, increased iNOS induction in response to diverse stimuli mediated some of the neurotoxic effects of inflammation and reactive oxygen species. COX-2 is a proinflammatory enzyme, and its reaction products are responsible for cytotoxicity in several neurological diseases. The activity and expression of iNOS and COX-2 are mediated by NFκB.43 The transcription factor NFκB is a key regulator of genes involved in cell survival/apoptosis and inflammation. We founded that LK decreased NFκB phosphorylation together with reduced enhancement of NFκB transcriptional target proteins, iNOS/ COX-2, in the hippocampus of SSS-exposed hamsters. This suggested that the LK neuroprotective effects appear to be partially dependent on the NFκB/iNOS/COX-2 signaling pathway inhibition. LK administration resulted in a remarkable decrease in iNOS, thus blunting the formation and deleterious effects of abnormal amounts of peroxynitrite. It was presumed that LK protected against SSS-induced hippocampus injury by decreasing oxidative stress and ROS-induced inflammatory response induced by inducible NO. In conclusion, our findings show that SSS exposure induced hippocampus injury in young hamsters andthat this injury is related to the Fas receptor- and mitochondria-dependent apoptotic and autophagy pathways. eNOS downregulation and iNOS overexpression were observed in SSS-exposed hippocampi. LK treatment significantly inhibited the SSSinduced activation of apoptosis and autophagy. LK also reversed the SSS-induced eNOS/iNOS expression imbalance. Taken together, our data suggests that SSS exposure might cause hippocampus cell death in young animals, which might cause abnormal hippocampal morphology. However, LK could function as a protective agent, with LK administration effective as a preventive and/or therapeutic strategy against abnormal hippocampal function.

could also be a major risk factor for cerebrovascular diseases.32 Cigarette smoke activates blood platelets33 and increases the risk of thrombus formation. Exposure to cigarette smoke can cause an increased level of fibrinogen and thromboxane, which are markers for platelet activation.34,35 Evidence suggests that exposure to cigarette smoke may damage the endothelium of arteries, which facilitates the development of atherosclerosis.31 Rabbits exposed to cigarette smoke decreased endotheliumdependent vasodilation and NO production.36 LK treatment significantly inhibited SSS-induced apoptosis in young hamster hippocampus, as indicated by reducing the upregulation in Fasdependent and mitochondria-dependent apoptotic pathways and lowering the number of TUNEL-positive cells. Many studies showed that LK has potent fibrinolytic activity, decreased fibrinogen, and lower blood viscosity and that it inhibits platelet activation and aggregation.13,18 LK shows thrombolytic activity only in the presence of fibrin; therefore, it has the profound advantage of not causing hemorrhage due to excessive fibrinolysis. Ji et al. found that LK had antiapoptosis action in the brain and antiplatelet activity by reducing calcium release from calcium stores and had antithrombosis action due to the inhibition of the expression of intercellular adhesion molecule-1 in endothelial cells.21 LK has also been reported to be effective in the prevention and treatment of ischemic cerebrovascular diseases and myocardial infarction.18 Secondhand smoke enhanced platelet thrombosis, and hypercoagulation may be an important contributory mechanism in acute coronary and cerebrovascular events in nonsmokers. The protective effects of LK on brain injury from exposure to SSS may be due, at least partially, to the inhibition of platelet aggregation and support of a healthy balance of coagulation and fibrinolysis in the body. Autophagy plays a key role in maintaining neuronal homeostasis because neurons are postmitotic cells and require effective protein degradation to remove toxic aggregated proteins. Although autophagy is beneficial, excessive autophagy may also exert a detrimental role in neurological disease leading to autophagic neuronal death.6,37 This study showed that autophagy was upregulated in the hippocampus of SSS-exposed hamsters, as indicated by Beclin-1, ATG7, and LC3 expression (Figure 6). The excessive autophagy induced by SSS could contribute to SSS-induced hippocampal cell death. Chen and Kim38,39 recently showed that autophagy protein LC3B and Beclin-1 activates extrinsic apoptosis during cigarette-smokeinduced cell death in bronchial epithelial cells. These studies suggested an interdependence between autophagic and apoptogenic signaling in cigarette smoking extract-induced cell death. In the evaluation of LK effects on SSS-induced autophagy in the hippocampus, we found that LK administration prevented the upregulation of autophagy, which implies that autophagy inhibition is likely a new pathway for LK to protect against SSS-induced brain injury. Whether brain NOS activities and expression are also involved in the onset of SSS-exposure-induced brain injury is not known at present. The effects of LK on iNOS and eNOS protein levels in the hippocampus of SSS-exposed young hamsters were determined is this study. As shown in Figure 7, exposure to SSS showed significantly higher levels of iNOS and lower levels of eNOS proteins. LK administration resulted in a remarkable decrease in iNOS and increase in eNOS protein levels in the brain, indicating that the brain tissue was able to cope better with SSS-induced toxic insult. eNOS activation with the production of NO decreases resting vascular tone and

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AUTHOR INFORMATION

Corresponding Author

*Department of Medical Imaging and Radiological Science, Central Taiwan University of Science and Technology, No. 666, Buzih Road, Beitun District, Taichung City 40601, Taiwan (R.O.C.). Tel: 886-4-22391647-7119. Fax: 886-4-22396762. Email: [email protected]. Funding

This work was supported by the Central Taiwan University of Science and Technology (CTU101-P-07) and Taiwan Depart659

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Chemical Research in Toxicology

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ment of Health Clinical Trial and Research Center of Excellence (DOH101-TD-B-111-004), and also supported in part by the (CMU99-ASIA-12) project. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS LK, lumbrokinase; SSS, side-stream smoke; FADD, Fasassociated death domain; eNOS, endothelial NO synthase; iNOS, inducible NO synthase; LC3, microtubule-associated protein light chain 3

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