[Edited by Professor KB Choo, Aug 9, 2014]
Cardio Protective Effectsof Lumbrokinase and Dilong on
theSecond-HandSmoke-Induced Apoptotic Signaling in Ratthe Heart of a Rat Model
Hung-En Liao1*, Chao-Hung Lai2*, Tsung-Jung Ho3*,Yu-Lan Yeh4,5, Gwo-Ping Jong2,Wu-Hsien Kuo6,Li-Chin Chung7, Pei-yingPai 8, Chang-Hai Tsai9,
Chih-Yang Huang9,10,11
1Department of Healthcare Administration, Asia University,41354, Taichung, Taiwan 2Division of Cardiology, Department of Internal Medicine, Armed Force Taichung
General Hospital, 41168,Taichung, Taiwan
3Chinese Medicine Department, China Medical University, Beigang Hospital,
65152,Yunlin,Taiwan
4 Department of pathology, Changhua Christian Hospital, 50006 ,Changhua, Taiwan. 5 Jen-Teh Junior College of Medicine, Nursing and Management, 35664,Miaoli, Taiwan. 6Division of Gastroenterology, Department of Internal Medicine, Armed-Force, Taichung
General Hospital, 41354, Taichung, Taiwan
7 Department of Hospital and Health Care Administration, Chia Nan University of
Pharmacy & Science, 71710,Tainan, Taiwan
8 Division of Cardiology, China Medical University Hospital,40402, Taichung,
Taiwan9Department of Health and Nutrition Biotechnology, Asia University,41354,
Taichung, Taiwan
10Graduate Institute of Chinese Medical Science, China Medical
University,40402,Taichung, Taiwan
11Graduate Institute of Basic Medical Science, China Medical
University,40402,Taichung, Taiwan *These authors share equal contribution
Author for correspondence: Chih-Yang Huang PhD
Address: Graduate Institute of Basic Medical Science, Graduate Institute of Chinese Medical Science, China Medical University and Hospital, No. 91, Hsueh-Shih Road, Taichung, 404, Taiwan
Phone number: 886-4-2205-3366 ext. 3313 FAX number: 886-4-2207-0465
Email: cyhuang@mail.cmu.edu.tw
Abstract
Exposure to second-hand tobacco smoke (SHS) has been epidemiologically linked to heart disease among non-smokers. However, the molecular mechanism behind SHS-inducedcardiac disease is not well known. This study found that SD rats exposed to cigarette smoke at a dose of 10 cigarettes for 30 min twice a day for 1 month had a reducedleft ventricle-to-tibia length ratio (mg/mm), increased cardiomyocyte apoptosis by TUNEL assay and a wider interstitial space by H&E staining. However, Lmbrokinase and Dilong both reversed the effects of SHS.Western blotting demonstrated significantly increased expression of the pro-apoptotic protein caspase-3 in ratthe hearts of the rats
exposed to SHS. Elevated protein expression levels of Fas, FADD and the apoptotic initiator activated caspase-8, a moleculein the death-receptor-dependent pathway, coupled with increased t-Bid andapoptotic initiator activated caspase-9 were found. Molecules in the mitochondria-dependent pathway, which disrupts mitochondrial membrane potential, were also found in rats exposed to second-hand smokeSHS. These factors indicatemyocardial apoptosis. However, treatment with Lumbrokinase and Dilong inhibited SHS-induced apoptosis. Regarding regulation of the survival pathway, using western blotting,we found in western blot analysis that cardiac protein expression of pAkt, Bcl2, and Bcl-xL was significantly decreased down-regulated in rats exposed to SHS. These effects were reversed with Lumbrokinase and Dilongtreatment. We found thatThe effects of SHS on cardiomyocytesarewere also found to be mediated by the Fas deathreceptor-dependent apoptotic pathway, an unbalanced mitochondria membrane potential and decreased survival signaling. However, treatment with both Lumbrokinase
and Dilong inhibited the effects of SHS. We believeOur data
suggestthatLumbrokinaseandDilongmightmay prevent heart disease in SHS-exposed non-smokers.
Key words: cardiac survival signaling, caspases, death-receptor-dependent pathway,
Introduction
Second-hand smoke (SHS) is a mixture of smog from mainstream smoke exhaled from smokers and side stream smoke emitted from the tip of a burning cigarette. Exposure to SHS, also known as environmental tobacco smoke (ETS), has been epidemiologically linked to coronary heart disease (CHD)(2, 9, 6, 8, 16, 17, 25, 26).Cardiovascular disease is the leading global cause of death, accounting for 30% of deaths worldwide. Tobacco use is a major cause of cardiovascular disease(26).Passive smoke exposure is associated with a 25-30% increased risk of coronary heart disease.of 25-30%.SHS contains more than 7,000 chemicals.At least 250 are known to be harmful, and 69 are toxic chemicals that cause cancer. Actually,Epidemiological studies have
indeed shown that SHS increases the incidence of cardiac disease by 4-fold.The mortality from heart failure among passive smokers is 38% higher than non-smokers or those who are not exposed to SHS(1). Pope et al.found that a 2-hour exposure to SHS significantly destroyed cardiac autonomic function based on decreased heart rate variability in 16 adult non-smokers (21).Second-hand smoke also increases platelet activity, accelerates
atherosclerotic lesions and increases tissue damage following ischemia (insufficient oxygen delivery to the heart or brain)or myocardial infarction.(heart attack).Increased platelet activity is associated with the formation of blood clots and atherosclerosis,
(hardening of the arteries),both of which are predisposing factors for a myocardial infarction. The principal mechanisms by which environmental tobacco smokeSHS affects the cardiovascular system include reduced oxygen delivery to the myocardium and increased platelet activity. Further, DNA adducts formed due to excessive stress,
generated due to cigarette smoke, is also known to bring outresult in mutations that bring cause abnormalities in tissue proliferation (4).
Apoptosis is a recognized mechanism for eliminating redundant cells, butapoptosis may also inhibit cell proliferation. In fact,Apoptosis has been suggested to play a critical role in the pathogenesis of human diseases, including heart disease (8). Apoptosis has been reported to contribute to the loss of cardiomyocytes, after which collagen secretion by fibroblasts replaces the damaged cardiomyocytes,leadingto cardiomyopathy. Hence, the fibrosis following apoptosis is a predictor of adverse outcomes in patients with cardiomyopathy (14, 19). Therefore, evaluating the process of apoptosis and/or fibrosis could be an excellent way to predict the development of cardiomyopathy dueto SHS; however, the specificity of the signaling pathways related to the development of apoptosis and/or fibrosis also needs to be identified.
The induction of apoptosis is associated with the activation of aspartate-specific cysteine proteases, including caspase-3 (6). Several studies have demonstrated that mitochondria may play an important role in apoptosis by releasing cytochrome c and activating caspase-9, which activates caspase-3, the molecule responsible for
DNAcleavage (18, 22). In addition, the deathreceptor-induced apoptotic pathway has been reported to beinvolved in the pathogenesis of heart diseasespathogenesis(23). This pathway is initiated by death-receptor agonists, including the Fas ligand. After ligand binding, Fas receptor oligomerization results in the activation of caspase-8, which isacts
upstream of caspase-3, causing activation of apoptosis (8). Therefore, caspase-3, as a common component of apoptotic signaling, caspase-3mediates both mitochondria-dependent and deathreceptor-mitochondria-dependent apoptosis pathways. The down-regulation of a
survival pathway is another possible factor thatpromotes cell apoptosis. In
cardiomyocytes, insulin-like growth factor-1 (IGF-1), the survival factor through which IGF-1 receptor (IGF-1R) activates phosphatidylinositol-3 kinase/protein kinase B (20), should be considered for preventing cardiomyocyte apoptosis (11). In particular,
activated PI3K enhances Akt phosphorylation (24), which regulates Bad and Bcl2 activity
to control cardiomyocyte apoptosis (3).
To understand whether the SHS effects on rat the heart are mediated through the activationof apoptotic pathways, including the mitochondria-dependent and Fas death-receptor-dependent signaling, or through suppressing survival pathways, we examined in a rat model in this study the myocardial expression of signaling proteins in these
pathways using western blotting. We usedThese results were used to explore the molecular pathogenesis of heart disease induced by cigarette smoke.
The earthworm (Lumbricusrubellus), in the form of a raw animal ‘‘herb,’’ has been used as traditional medicine for several thousand years.The extract from earthworms has been used empirically in Asia to treat vascular disorders. Recent clinical trials have confirmed its clinical efficacy in treating coronary artery disease(CAD)and thrombotic cerebral infarcts, albeit publications related to the former have been largely confined to the Chinese literature. Lumbrokinase, the aterm given to the group of enzymes extracted from the earthworm, possesses plasminogen-activating and direct fibrinolytic
properties(13).Various concentrations of earthworm extracts ranging between 80 to 300 mg/kg have been successfully used for various treatments. High amounts of earthworm extracts were found to be non-toxic and have shown higher therapeutic potentials(5). A
treatment. The anti-fibrotic effects of 100 mg/kg of dilong extract show that it is one of the highly efficient earthworm extracts. The chemical structures and amino acid
sequences of each of the six isoenzymesfromlumbrokinase with their respective fibrinolytic activities were previously identified and characterized(13, 29).Oral
Lumbrokinase improves regional myocardial perfusion in patients with stable angina(13).This study was designed to investigate the effects of
Materials and Methods
DilongExtraction
Powdered earthworm was extracted with 70% absolute ethanol at room temperature for 24 h, and the extract was collected after centrifugation for 5 minutesmin at 2,000g, and was maintained in a water bath at 37 °C for 4 h to remove the alcohol.
Animal Model and Exposure to Cigarette Smoke Exposure
We purchased male SD rats (6 weeks of age; body weight, 300 ± 30g) from the National Science Council Animal Center, Taipei, Taiwan. The animals were housed three per cage in an environment-controlled animal room with water provided ad libitum. All
of theanimals were handled according to the guidelines of the Taiwan Society for Laboratory Animals Sciences for the care and use of laboratory animals
(Institute of Laboratory Animal Resources 1996). We divided 24Twenty-four ratswere divided into four groups, six rats per group. The rats were placed in whole-body exposure chambers and exposed to 0 or 10 cigarettes (Gentle, Taiwan), representing the control and smoke groups, respectively. The smoke group was further subdivided into the smoke
group with Lumbrokinase(1.2 mg/kg/day, twiceweekly) treatment, or smoke group with Dilong(100 mg/kg/day, twiceweekly) treatment[To authors: Please check if it is necessary to capitalized lumbrokinase and dilong; in some places in the text, they are not capitalized. Please be consistent …]. Filtered air was introduced into the chamber at a flow rate of 200L/min. The rats were exposed to the cigarette smoke for 30 min, twice a day, 6 days/week for 1 month. The room temperature was maintained at
22-25℃, and the relative humidity was approximately 40%. The Lumbrokinase and
Dilongwere administered by intraperitoneal injection.After 1 month, the rats were weighed and sacrificed. After removing the thorax, the hearts were cleaned with double-distilled water and dried before weighing. The left and right atria and the right ventricle were then removed, and the left ventricle was weighed. We calculatedThe ratios of the total heart weight and left ventricular weight to the tibia length (mg/mm) were calculated.
Hematoxylin-Eosin Staining
The hearts were fixed in formalin, embedded in paraffin and sectioned. The slides were hydrated through a series of ethanol concentrations (100, 95 and 75%) for 15 min each. The slides were then stained with hematoxylin and eosin (H&E). After gently rinsing with water, the slides were dehydrated through serial ethanol concentrations for 15 min each, cleanedwith xylene, and cover slipped. Photomicrographs were obtained using a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany).
Tissue Extraction
The heart tissue extracts were obtained by homogenizing the left ventricle samples in phosphate-buffered saline (PBS; 0.14 M NaCl, 3 mMKCl, 1.4 mM KH2PO4, 14mM K2HPO4) at a concentration of 1 mg tissue/10 μL PBS for 5 min. The homogenates were placed on ice for 10 min and then centrifuged at 12,000 rpm for 30 min. The supernatant was collected and stored at -80℃ for future experiments.
We determinedThe protein content of heart tissue extractswas determined using the Bradford protein assay and a protein-dye kit (Bio-Rad, Richmond, CA, USA). We used ACommercially availableBovine serum albumin (Sigma Chemical, St. Louis, MO, USA)
was used as a standard. Changes in optical density were measured at 595 nm.
Electrophoresis and Western Blot
We prepared theTissue extract sampleswere prepared as described above. SDS-PAGE was performed with 10% polyacrylamide gels. The samples were electrophoresed at 100V for 3.5 h and equilibrated for 15 min in 25 mMTris-HCl, pH 8.3, containing 192 mM glycine and 20% (vol/vol) methanol. The electrophoresed proteins were transferred to a polyvinylidenedifluoride (PVDF) membrane at 100V for 70 min. We incubatedThe PVDF membranewas incubated ina blocking buffer for 1 h at room temperature.
Monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted 1:1,000 in an antibody binding buffer containing 100 mMTris-HCl, pH 7.5, 0.9%
(wt/vol)NaCl, and 0.1% (vol/vol) Tween 20. The membranes were incubated at
4℃overnight. We washedThe immunoblotswere washed three times in 10 mL blotting buffer for 10 min and then immersedthem in the secondary antibody solution containing horseradish peroxidase goat anti-rabbit IgG (Santa Cruz Biotechnology) Santa Cruz, CA, USA) diluted 2,000-fold in binding buffer for 1 h. The filters were then washed three times in blotting buffer for 10 min.three times.The immunoblotted proteins were visualized using an ECL western blot analysis luminal reagent and quantified using a Fujifilm LAS-3000 chemiluminescence detection system (Fujifilm, Tokyo, Japan).
Statistical Analysis
We compared theData between groups of animals were compared using one-way analysis of variance. We usedThe Scheffe testwas used to determine differences.
Results
LumbrokinaseandDilongReversed SHS-Induced CardiomyopathicChanges in Rats
After 1-month of SHS, all of the rats generally appeared healthy. Then, theyThe rats
were then weighed and sacrificed, andthe hearts were removed and weighed. The ratio of the left ventricular weight to the tibia length (mg/mm) wassignificantly reduced in the SHS group. Feeding the rats with lumbrokinase or dilongfeeding reversed the effect of SHS (Fig. 1A). To understand the mechanism of decreased heart weight, we
analyzedventricular tissues were stained with H&E. The ventricular myocardium fromthe
healthy controls had a normal architecture andan organizedmyocyte alignment with minimal interstitial spacing at 400X magnification. In contrast, disarray with markedly enlarged interstitiumwas evident in thecardiomyocytes from the SHS group. The SHS effect was attenuated in the LumbrokinaseandDilong groups (Fig.
1B).Additionally,LumbrokinaseandDilong inhibited SHS-induced cardiomyocyte apoptosis based on a TUNEL assays (Fig. 1C).
LumbrokinaseandDilongDecreased Caspase-3Activation inRats Exposed to SHS SHS can lead to cardiomyocyteapoptosis-related heart failure. To assess forthe effects on apoptosis, we measured the proteinexpression levels of the apoptotic protein caspase-3 was measured, and was found to bewhich was higher in the SHS groups than the controls, and was lower in theLumbrokinaseandDilong groups than the SHS untreated
LumbrokinaseandDilongInhibitComponents oftheFas-Receptor-Dependent Apoptotic Pathway in the Hearts From ofRats Exposed to SHS
To further understand the upstream signaling pathways associated with caspase-3 activation, we examined expression of components of the Fas death receptor-dependent
(FADD) apoptotic pathway. Compared to the control animals, Fas and FADD protein expression was up-regulatedincreased in the SHS group. In addition, the SHS group had significantly higher expression levels of active caspase-8 than the control group;
however, theLumbrokinaseandDilonginhibited the apoptotic signaling induced by SHS (Figs. 3A& 3B). There was a significant increase in t-Bid and active caspase-9 protein expression in the SHS group. However, there was a significant decrease in proteinthe
expression of these proteins in the theLumbrokinaseandDilonggroups, which maintained the mitochondrial membrane potential (Figs. 3C&3D).
LumbrokinaseandDilongUp-Regulated the Expression of pAkt, Bcl2 and Bcl-xLExpression in Hearts Fromof Rats Exposed to SHS
We also examined the expression of the cell survival-related proteins pAkt, Bcl-2 and Bcl-xL. In the SHS group,pAkt, Bcl-2 and Bcl-xL were more significantly decreased thanin the control group. The dilongandtheLumbrokinaseandDilonggroups exhibited greater increases than in the SHS group (Fig. 4).
Discussion
SHS increases the mortality risk of from ischemic heart disease or myocardial infarction by 30%. Ischemia induces adverse pathological events including reperfusion that gets deteriorates in time. Cigarette smoke triggers platelets through numerous mechanisms such as endothelial dysfunction and oxidative stress by reducing the production of platelet-derived nitric oxide, accelerating the levels of fibrinogen and thromboxane. The effect on platelet activation during when exposed to SHS is
comparatively 96% to that of active smokers. Activation of platelets potentially leads to thrombosis, which is a major risk factor of myocardial infarction and sudden death(7, 15, 28). Afterexposure to 10 cigarettes for 30min, twice per day for 1 month, the rat hearts of the experimental rats in this study showed weight loss and a wider interstitial space. However, theLumbrokinaseandDilonginhibited SHS-induced apoptosis. We
demonstrated that myocardial expression of pAkt, Bcl-2 and Bcl-xL waslower in rats exposed to SHS. However, treatment with theLumbrokinaseandDilongproduced an increasedpAkt, Bcl-2 and Bcl-xl expression (Fig. 5). The normal myocardial architecture became disorganized after SHS in this study. H&E staining showed myofibril disarray in the SHS-exposed heart and wider interstitial spaces (Fig. 1). These results suggest that the development of prematurecardiomyocyte death is characterized by distorted myocardial architecture.
Cardiomyocyte apoptosis has important implications in cardiac dysfunction because of the reduced number of cardiomyocytes per functional units. The weight loss we found in the ventricles of the SHS ratsventriclesmay be associated with the progression of heart
failure similar to that found in DM [To authors: Does DM mean diabetes? Please spell out DM in full …]rats (30) (Fig. 1). The increased caspase-3 expression and activity
shown by western blotting raise the possibility of cardiomyocyte apoptosis (31) (Fig. 2).Actually,Caspase-3 isshown to be an important molecular marker of apoptotic
signaling thatplays a key role in mitochondria-dependent and Fasdeathreceptor-dependent apoptotic pathways (32).Findings of elevated Fas, FADD and caspase-8 levels, coupled with increased t-Bid and caspase-9 activities in the ventricles of the SHS
ratsventriclesfurther confirmswhich the pathway is involved (Fig. 3). These results demonstrate that the Fas-signaling pathway is associated with increased caspase-3 expression, which may indicate apoptosis. MyocardialpAkt, Bcl-2 and Bcl-xL survival signaling was also decreased in the hearts of theSHS-exposed ratshearts(Fig. 4).Notably, SHS led to decreased heart weight and wider interstitial spaces.
SHS activates blood platelets and thereby increases the risk of thrombus formation and damaging the lining of arterieswhich to facilitate the development of atherosclerosis. The inflammatory responses that follow result in
wouldinducecardiomyocyte apoptosis. LumbrokinaseandDilong, with their thombolytic activities, would potentially ameliorate such effects of SHS and provide
cardio-protection. Our findings on the SHS effects in heart tissues include weight loss, altered morphology and apoptosis-related effects dueto caspase-3 activation and
Fasdeathreceptor-pathway-dependent signaling. These molecular mechanisms (Fig. 5) [To authors: There is a Figure 5, but it is not mentioned in the text. Please check if insertion here is appropriate, or insert Figure 5 in the text…]may explain how SHS
leads to the increased risk of cardiac events reported in epidemiological studies. Because cardiomyocyte apoptosis is typically an end-stage condition, earlier intervention is warranted. Therefore,our results suggest that theLumbrokinaseandDilongcould beused as appropriate treatments to block cardiac Fas signaling, which might help to prevent the development of apoptosis-related SHS-induced heart disease.
Acknowledgments
This study was supported by CMU100-ASIA-6 and in part by the Taiwan
Department of Health Clinical Trial andResearch Center of Excellence (DOH101-TD-B-111-004).
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Figure Captions
Figure 1.Lumbrokinase and Dilong improved SHS-induced cardiomyopathic changes in rats.Abbreviations:C, control; S, second-hand smoke(SHS); SL, second-hand smokeSHS rats treated with Lumbrokinase; SD, second-hand smokeSHS rats treated with Dilong. The numbers represent the sample sizes. (A) The left ventricular weight to tibia length ratio (mg/mm) (mean ± SD) was lower in the rats exposed to SHS (at least six rats per group). (B) Histopathologic analysis of heart tissue sections stained with H&E.
Magnification: 400X; bars=100 μm. An enlarged interstitium was observed in the SHS-administered animal hearts, and the arrows indicate the myocardial interstitium. (C) Histopathologic analysis of heart tissue sections stained with TUNEL. Magnification: 400X; bars=100 μm. The TUNEL-positive to DAPI-positive ratio (%) increased in the SHS-administered animal hearts.
Figure 2.Lumbrokinase and Dilong reduced SHS-induced activation of caspase-3 in rat hearts. Abbreviations as in Figure 1.: C, control; S, second-hand smoke; SL, second-hand smoke with Lumbrokinase; SD, second-hand smoke with Dilong.(A) Western blot analysis of activated caspase-3 in the left ventricle. (B) Caspase-3 activity shown as a percent of the control (mean ± SD of three independent experiments). Significant differences compared to the control group: *(P<0.05), ** (P<0.01), and *** (P<0.001).
Figure 3.Lumbrokinase and Dilong attenuated SHS-induced activation of Fas, FADD, caspase-8, tBid and caspase-9 in rat hearts. Abbreviationsas in Figure 1.: C, control; S,
hand smoke; SL, hand smoke with Lumbrokinase; SD, second-handsmoke with Dilong. Fas, FADD, and caspase-8 activation in the left ventricles shown by (A) western blot analysis and (B) quantification of signal intensity for Fas, FADD, and caspase-8 (mean ± SD of three independent experiments, using α-tubulin as a loading control). Activation of tBid and caspase-9 in the left ventricles shown by (C)
western blot analysis and (D) quantification of the signal intensity for tBid and activated caspase-9 (mean ± SD of three independent experiments). Significant differences
compared to the control group: *(P<0.05), ** (P<0.01), and *** (P<0.001).
Figure 4.Lumbrokinase and Dilong restored expression of pAkt, Bcl2 and Bcl-xL in the rat hearts exposed to SHS.Bcl-xL, pAkt, and Bcl-xL in the left ventricles shown by (A)
western blot analysis and (B) quantification of the signal intensity for pAkt, Bcl2, and Bcl-xL (mean ± SD of three independent experiments). *Significant differences
compared to the control group: (P<0.05), ** (P<0.01), and *** (P<0.001).
Figure 5.Proposed mechanism of Lumbrokinase and Dilong inhibition of SHS-induced cardiomyocyte apoptosis. SHS activates Fas, and the receptor recruits the cytosolic adaptor protein Fad-associated death domain(FADD). The adaptor protein transmits a signal from the activated receptor Fas to the initiator caspase-8. Caspase recruitment to the plasma membrane increases the local concentration of the protease and induces auto-cleavage and caspase activation. Activated initiator caspases cleave and activate the effector caspase-3. Once activated, the effector caspases are responsible for the
apoptotic pathway, induced by Fas crosstalk to the intrinsic pathway through caspase-8-mediated cleavage of BID (a BH3-only member of the BCL2 family of proteins), produces the pro-apoptotic tBID fragment. In the mitochondria, tBID disrupts
mitochondrial membrane integrity and activates caspase-9, which cleaves and activates caspase-3. In addition, SHS inhibits myocardial survival signaling, including pAkt, Bcl-2, and Bcl-xL. However, bothLumbrokinaseandDilongboth reversed SHS-induced
