國⽴立臺灣師範⼤大學⽣生命科學系碩⼠士論⽂文
⾎血栓素及⾎血栓素受體訊息在短期與⾧長期 被動吸菸造成動脈⾎血栓性疾病及肺部損傷
之⾓角⾊色
The Role of Thromboxane A2/Thromboxane Receptor Signaling in Acute and Prolonged Passive Smoking Induced Arterial Thrombotic
Disease and Pulmonary Injuries 研究⽣生：宋伊婷
Yi-Ting Sung
指導教授：鄭劍廷 博⼠士 Chiang-Ting Chien, Ph.D.
中華民國 105 年 01 ⽉月 08 ⽇日
⽬目錄
I. 中⽂文摘要 4
II. ABSTRACT 6
III. ABBREVIATIONS 8
IV. INTRODUCTION 12
1.THE ENVIRONMENTAL TOBACCO SMOKE AND THIRDHAND SMOKE 12 1.1MAINSTREAM AND SIDESTREAM SMOKE OF ENVIRONMENTAL TOBACCO SMOKE 12
1.2THIRDHAND SMOKE 12
2.CIGARETTE SMOKE (CS) EXPOSURE INCREASES THE RISK OF THROMBOTIC
CARDIOVASCULAR DISEASE 13
3.CIGARETTE SMOKE EXPOSURE LEADS TO PULMONARY INJURY 14 4.THE ASSOCIATION OF TXA2-TXAS-TP SIGNALING AND THE PATHOPHYSIOLOGY OF
THORMBOSIS AND PULMONARY INJURY 15
4.1.SMOKING INCREASES PLATELET ACTIVATION AND THROMBOSIS PARTIALLY THROUGH
ACTIVATING TXAS-TXA2-TP SIGNALING PATHWAY 16
4.2SMOKING LEADS TO PULMONARY INJURY PARTIALLY THROUGH TXAS-TXA2-TP
SIGNALING PATHWAY 17
5.USING TXAS OR TP GENE KNOCKOUT MICE FOR EXPLORING TXAS-TXA2-TP
SIGNALING PATHWAY 17
6.AIM AND PURPOSE 18
V. MATERIALS AND METHODS 19
1.ANIMALS 19
1.1TXAS GENE KNOCKOUT MICE 19
1.2TP GENE KNOCKOUT MICE 19
1.3TXAS AND TP DOUBLE KNOCKOUT MICE: 20
1.4GROUPING 20
1.5SURGICAL PREPARATION 21
2.FECL3-INDUCED ACUTE ARTERIAL THROMBOSIS 21
3.CIGARETTE SMOKE EXPOSURE PROTOCOL 22
4.WHOLE BLOOD REACTIVE OXYGEN SPECIES DETECTION 22 5.BRONCHOALVEOLAR LAVAGE FLUID REACTIVE OXYGEN SPECIES DETECTION 22
6.IMMUNOHISTOCHEMISTRY (IHC) 23
7.TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTPNICK END LABELING STAIN 24
8.WESTERN BLOT 26
9.PLATELET ADHESIVENESS DETECTION 27
10.MALONDIALDEHYDE DETECTION ASSAY 28
VI. RESULTS 30
1.ACUTE ARTERIAL THROMBOSIS MODEL OF TIME TO OCCLUSION 30 2.PLATELET ADHESIVENESS DETECTION IN MESENTERIC ARTERIES 30 3.WHOLE BLOOD REATVIE OXYGEN SPECIES DETECTION 31 4.BRONCHOALVEOLAR LAVAGE FLUID OF REACTIVE OXYGEN SPECIES 31
5.MALONDIALDEHYDE CONCENTRATION DETECTION 32
6.HEMATOXYLIN AND EOSIN (H&E) STAIN 33
7.IMMUNOHISTOCHEMISTRY ANALYSIS 34
8.TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTPNICK END LABELING STAIN 37
9.WESTERN BLOT 37
VII. DISCUSSION 43
1. CIGARETTE SMOKE EXPOSURE PROMOTE THROMBOSIS AND PULMONARY INJURIES 43 2.THE RELATIONSHIP BETWEEN CIGARETTE SMOKE EXPOSURE AND TXAS-TXA2-TP
SIGNALING PATHWAY IN THROMBOSIS 43
3. THE RELATIONSHIP BETWEEN CIGARETTE SMOKE EXPOSURE AND TXAS-TXA2-TP
SIGNALING IN PULMONARY INJURIES 44
VIII. CONCLUSION 49
IX. REFERENCES 50
X. FIGURES AND TABLES 55
TABLE.1TIME TO OCCLUSION OF CAROTID ARTERY IN FOUR GENOTYPE MICE WITH FOUR
DIFFERENT PERIODS OF CIGARETTE SMOKE EXPOSURE 55
FIGURE.1WHOLE-BODY SMOKE EXPOSURE MODEL 56
FIGURE.2THE CAROTID ARTERY BLOOD FLOW OF CONTROL GROUP IN THE FOUR
GENOTYPES MICE 57
FIGURE.3THE CAROTID ARTERY BLOOD FLOW OF ACE GROUP IN THE FOUR GENOTYPES
MICE 58
FIGURE.4THE CAROTID ARTERY BLOOD FLOW OF PCE5 TREATED GROUP IN THE FOUR
GENOTYPES MICE 59
FIGURE.5THE CAROTID ARTERY BLOOD FLOW OF PCE8 TREATED GROUP IN THE FOUR
GENOTYPES MICE 60
FIGURE.6COMPARISONS OF TIME TO OCCLUSION OF CAROTID ARTERY IN MICE BASED ON CIGARRETTE SMOKE EXPOSURE PERIODS AND GENOTYPES 61 FIGURE.7PLATELET ADHESIVENESS IN MESENTARIC ARTERY OF DIFFERENT GENOTYPE
MICE 64
FIGURE.8COMPARISONS OF LUMINOL-INDUCED BLOOD H2O2 ACTIVITES IN MICE BASED ON
CIGARETTE SMOKE EXPOSURE PERIODS AND GENOTYPES 65
FIGURE.9REACTIVE OXYGEN SPECIES LEVELS IN BRONCHOALVEOLAR LAVAGE FLUID 66
FIGURE.10PLASMA MDA CONCENTRATION 67
FIGURE.11PULMONARY ARTERY THICKNESS HISTOLOGICAL FETURE AND RELATIVE
THICKNESS 68
FIGURE.12HISTOLOGICAL FETURE OF LUNG 71
FIGURE.13IMMUNOHISTOCHEMISTRY OF 4-HNE 72
FIGURE.14IMMUNOHISTOCHEMISTRY OF VWF 74
FIGURE.15IMMUNOHISTOCHEMISTRY OF IL-1Β 76
FIGURE.16IMMUNOHISTOCHEMISTRY OF PARP-1 78
FIGURE.17IMMUNOHISTOCHEMISTRY OF BECLIN-1 80 FIGURE.18TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTPNICK END LABELING
STAIN 82
FIGURE.19WESTERN BLOT OF PARP-1 84
FIGURE.20WESTERN BLOT OF BCL-2 AND BAX 86
FIGURE.21WESTERN BLOT OF LC3 Β 87
FIGURE.22WESTERN BLOT OF BECLIN-1 88
FIGURE.23WESTERN BLOT OF 4-HNE 89
FIGURE.24WESTERN BLOT OF VWF 90
FIGURE.25WESTERN BLOT OF NF-ΚB 91
FIGURE.26WESTERN BLOT OF ATF-6 92
FIGURE.27WESTERN BLOT OF P-JNK 93
I. 中⽂文摘要
被動菸害，包含⼆二⼿手菸害及三⼿手菸害，其中⼆二⼿手菸害⼜又分成主流菸煙 及側流菸煙， 被動菸害與主動菸害皆會造成⼼心⾎血管疾病及肺部疾病，本 研究主要利⽤用側流菸煙及三⼿手菸探討⼼心⾎血管及肺部發炎性相關疾病。︒｡
⾹香菸中的活性氧化物質會導致⾎血⼩小板過度活化，促進⾎血栓形成，⽽而花
⽣生四烯酸經⾎血栓素合成酶 (Thromboxane A2 synthase, TXAS) 合成⾎血栓 素 A2 (Thromboxane A2, TXA2) ，與⾎血栓素受體 (Thromboxane prostanoid
receptor, TP receptor) 結合會引起⾎血⼩小板活化，⽽而⾎血⼩小板過度活化或內⽪皮 細 胞 受 損 ， ⼜又 會 導 致 TXA2 濃 度 增 加 ， 然 ⽽而 過 去 對 於 ⾹香 菸 及
TXAS-TXA2-TP 訊息傳遞路徑的調控仍未釐清。︒｡
本研究利⽤用 TXAS、︑､TP 基因剔除⼩小⿏鼠及兩者基因皆剔除之⼩小⿏鼠，吸
⼊入側流菸煙及三⼿手菸煙與動脈⾎血栓性疾病的模式，探討被動菸害對動脈
⾎血栓形成及肺部發炎性變化。︒｡實驗分成 TXAS+/+TP+/+、︑､TXAS-/-TP+/+、︑､
TXAS+/+TP-/- 及 TXAS-/-TP-/-⼩小⿏鼠並給予無菸煙暴露，菸煙暴露 1 週，菸煙 暴露 5 週及菸煙暴露 8 週處理，共 16 組 （各組 n=6），並利⽤用氯化鐵誘 發急性動脈⾎血管栓塞以檢測⾎血栓形成所需時間，再藉由病⽣生理及分⽣生結
果了解⾹香菸及 TXAS-TXA2-TP引起肺部發炎性之關聯。︒｡我們利⽤用菸煙暴
露刺激以評估肺損傷與氯化鐵誘發之急性動脈⾎血管栓塞在四種⼩小⿏鼠之作
⽤用。︒｡我們以化學發光放⼤大⽅方式偵測活性氧數量，以西⽅方墨點和免疫染⾊色 法探究氧化壓⼒力、︑､細胞凋亡、︑､細胞⾃自噬、︑､發炎性細胞凋亡和發炎之分⽣生 機轉。︒｡
研究結果指出利⽤用氯化鐵誘發之急性動脈⾎血管栓塞，其⾎血栓形成時 間之結果在不同基因⽼老⿏鼠與菸煙處理之間，並無顯著關聯性，表⽰示各基 因對於菸煙暴露時間的反應具有⼀一致性，但菸害仍會影響⾎血栓形成，因
⽽而利⽤用⾎血⼩小板吸附模式探討不同基因⿏鼠的結果，在無菸害暴露的⽼老⿏鼠裡
發現 TXAS+/+TP+/+⿏鼠之⾎血⼩小板吸附性顯著顯著⾼高於其他三個品系品的⽼老
⿏鼠，未來會再加⼊入抽煙處理的組別以繼續探討;在肺部發炎性疾病⽅方⾯面，
由病⽣生理結果發現，不同基因⿏鼠與菸煙處理之間具顯著關聯，表⽰示菸煙
會藉由 TXAS-TXA2-TP訊息傳遞路徑引起肺部發炎性疾病。︒｡
關鍵字：⼆二⼿手菸、︑､三⼿手菸、︑､⾎血管栓塞、︑､肺部損傷、︑､⾎血栓素、︑､⾎血栓素受體
II. Abstract
Passive smoking contains second-hand smoke (SHS) and third-hand smoke (THS). SHS can be divided into mainstream smoke and sidestream smoke. Both active and passive smoking would cause cardiovascular disease (CVD) and pulmonary injuries. We used sidestream smoke and THS as our cigarette smoke (CS) model.
Oxidants in cigarettes activate platelets consequently induce thrombus formation. It is well known that thromboxane A2 (TXA2) through its receptor—thromboxane prostanoid (TP) receptor — would activate platelets, and the overactivation of platelets or endothelial cells injury would further induce TXA2 generation. The relationship between CS and thromboxane A2 synthase (TXAS)-TXA2-TP signaling is still ambiguous.
In order to investigate the influence of CS on TXAS-TXA2-TP signaling in thrombosis and pulmonary injuries, we used TXAS and TP gene knockout mice, inhaled sidestream and THS as whole body CS exposure model. Mice were divided into TXAS+/+TP+/+, TXAS-/-TP+/+, TXAS+/+TP-/- and TXAS-/-TP-/- treated with non-CS exposure (i.e. Control), acute cigarette smoke exposure group (ACE), prolonged cigarette smoke exposure 5 weeks group (PCE 5) and prolonged cigarette smoke exposure 8 weeks group (PCE 8) (16 groups, n=6 in each group). We evaluated the effects of CS exposure on lung injuries and FeCl3-induced thrombus formation in these four groups. We utilized the chemiluminescence amplification method to measure the amount of reactive oxygen species (ROS), western blot and
immunohistochemistry to explore the underlying mechanisms including oxidative stress, apoptosis, autophagy, pyroptosis and inflammation in these animals.
Our results showed CS exposure triggered the formation of thrombus by the index of time to occlusion (TTO). The response of CS shortened TTO was attenuated by blocking TXAS or TP receptors. CS exposure induced oxidative stress would cause pulmonary injuries including endothelial dysfunction, endoplasmic reticulum (ER) stress, inflammation, cell apoptosis, autophagy and pyroptosis.
In conclusion, TXAS-TXA2-TP signaling plays an important role in CS induced pulmonary injury and FeCl3-induced thrombus formation.
Key words: Second-hand smoke, third-hand smoke, thrombosis,
pulmonary injury, thromboxane A
2, thromboxane
prostanoid receptor
III. Abbreviations
TXAS Thromboxane A2 synthase
TXA2 Thromboxane A2
TP receptor Thromboxane prostanoid receptor
SHS Second-hand smoke
THS Third-hand smoke
CVD Cardiovascular disease
CS Cigarette smoke
CON Control
ACE Acute cigarette smoke exposure
PCE 5 Prolonged cigarette smoke exposure 5 weeks PCE 8 Prolonged cigarette smoke exposure 8 weeks
ER Endoplasmic reticulum
ETS Environmental tobacco smoke
ROS Reactive oxygen species
CO Carbon monoxide
BALF Bronchoalveolar lavage fluid
NF-κB Nuclear factor kappa B
PGK-HPRT Phosphoribosyltransferase
ES Embryonic stem
KO Knockout
PGK Phosphoglycerate kinase
i.p. Intraperitoneal
KCl Potassium choloride
PE-60 Polyethylene 60
FeCl3 Ferric chloride
TTO Time to occlusion
CL Chemiluminescence
PBS Phosphate buffer saline
H2O2 Hydrogen peroxide
IHC Immunohistochemistry
BSA Bovine serum albumin
PARP-1 Poly (ADP-ribose) polymerase-1
VWF Von Willebrand factor
4-HNE 4-Hydroxynonenal
IL-1β Interleukin-1 beta
IgG Immunoglobulin G
PBST Phosphate buffer saline tween-20
ddH2O Double-distilled water
TUNEL Terminal deoxynucleotide transferase dUTP Nick End Labeling
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
PVDF Polyvinylidene difluoride
HRP Horseradish peroxidase
LC3 β Microtubule-associated protein light chain 3
Bcl-2 B cell lymphoma-2
Bax Bcl-2-associated X-protein
ATF-6 Activating transcription factor 6 p-JNK Phospho-c-Jun N-terminal kinases
PGI2 Prostacylin
NaCl Sodium chloride
Na2HPO4 Sodium hydrogen phosphate
NaHCO3 Sodium hydrogen carbonate
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
AM Acetoxymethyl
FP Fluorescently-labeled platelets
MDA Malondialdehyde
TBA Thiobarbituric Acid
H2SO4 Sulfuric acid
rcf Relative centrifugal field
BHT Butylated hydroxytoluene
H&E Hematoxylin and eosin
GPIIb-IIIa Glycoprotein IIb/IIIa
CHOP CCAAT/enhancer-binding protein (C/EBP)
homologous protein
PKB, Akt Protein kinase B
Bcl-xL B-cell lymphoma-extralarge
SEM Standard error of the mean
IV. Introduction
1. The Environmental Tobacco Smoke and Thirdhand Smoke
1.1 Mainstream and sidestream smoke of environmental tobacco smoke
Environmental tobacco smoke (ETS) is one of the hazardous pollutants in the air. For a non-smoker living with a smoker, the exposure tobacco smoke is about 1% of active smoke with 20 cigarettes per day (Law and Hackshaw 1996). Cigarettes in burning situation will emit two types of ETS: the mainstream smoke and the sidestream smoke. The mainstream smoke is the smoke directly inhaled into smokers’ lungs and sooner exhaled, while the sidestream smoke means the smoke emit into the air from the burning cigarettes. ETS accounts for 15% mainstream smoke and 85% sidestream smoke. Comparing two types of ETS, the sidestream smoke is much more harmful with more toxicants than the mainstream smoke is (Taylor, Johnson et al. 1992). Based on previous research, we took advantage of sidestream smoke as one of the cigarette smoke (CS) exposure donors in our research.
1.2 Thirdhand smoke
After the cigarette is extinguished, the remaining pollutants from tobacco smoke indoor is called the third-hand smoke (THS) (Winickoff, Friebely et al. 2009). THS is the pollutant remains on any surface of visible and invisible substances after cigarettes extinguished, reemitting
into the gas-phase, or reacting with other compounds in the environment that form secondary pollutants (Ferrante, Simoni et al. 2013). The main components of THS are nicotine, phenol, 3-ethenylpyridine, formaldehyde, cresols, tobacco-specific nitrosamines and naphthalene (Matt 2013). The association of exposure of THS pollutants and related diseases has not been thoroughly studied. However, through previously known THS compounds, nicotine reveals a harmful role in carcinogenesis, vascular system and may promote inflammation through oxidative stress.
Moreover, oxidant gases may cause oxidant damage and inflammation through the production of reactive oxygen species (ROS) (Matt 2013).
Hence, we used THS as the other CS exposure donors in our research.
2. Cigarette smoke (CS) exposure increases the risk of thrombotic cardiovascular disease
Cigarette smoke (CS) exposure leads to an increased risk of thrombosis, which plays a major part in the pathogenesis of smoke-induced cardiovascular disease (CVD). Nicotine, carbon monoxide (CO), and oxidant gases are recognized as three major constituents of CS that contributes to CVD through increasing lipid peroxidation and activating platelets (Ambrose and Barua 2004). The thrombosis occurs when fibrinogen is converted to fibrin and interacts of platelets, blood-borne proteins, endothelial cells, and subendothelial vascular tissue (Control and Prevention 2010, Health and Services 2010). Exposure to CS may alter the hemostatic process through multiple mechanisms, including changing
function in platelets, endothelial cells, coagulation factors and fibrinogen.
Besides, studies have shown either active or passive cigarette smoking can cause platelets activation (Rubenstein, Jesty et al. 2004), rapid deterioration in endothelial function, promotion of atherosclerotic plaque development (Brook, Franklin et al. 2004), and lead to an imbalance of antithrombotic/prothrombotic factors, which support the initiation and propagation of thrombosis (Barua and Ambrose 2013).
3. Cigarette smoke exposure leads to pulmonary injury
CS exposure has been demonstrated to induce inflammation in the lung, including recruitment of neutrophils to the site of inflammation (Chalmers, MacLeod et al. 2001), lung endothelial cells dysfunction and emphysema.
Study has shown lung weights in CS-induced emphysema increased and was dependent on CS exposure duration (Awji, Seagrave et al. 2015).
Comparing to air exposure groups, mice treated with active smoking (250mg TPM/m3) 300 hours, 480 hours and 660 hours in total CS exposure duration expressed higher inflammatory cells, neutrophils and macrophages in bronchoalveolar lavage fluid (BALF), which did not indicate a dose dependent manner in durations (Awji, Seagrave et al. 2015). These evidences reveal that short term CS exposure may have a reverse impact on pulmonary disease thus we focused on short CS exposure durations in passive smoking.
4. The association of TXA
2-TXAS-TP signaling and the pathophysiology of thormbosis and pulmonary injury
There is scarce understanding in the relationship between thromboxane A2 (TXA2)- TXA2 synthase (TXAS)- thromboxane prostanoid (TP) receptor signaling and thrombotic disease or pulmonary injuries. TXA2 is an unstable arachidonic acid metabolite generated through TXAS. TXA2 triggers multiple biological responses via its cell surface receptor-TP to mediate pathophysiologic conditions (Nakahata 2008, Wilson, Cavanagh et al. 2009).
Various kinds of cells including neural cells, endothelial cells, cardiac myocytes, epithelial cells, smooth muscle cells, glomerular mesangial cells, and Kupffer cells have TP receptors on their membranes. The wide distribution of TP receptors in different organs suggests that TXA2 is involved in a wide range of pathophysiological conditions. Nakahata showed the possible signaling pathways of TXAS-TXA2-TP in 2008.
The TP receptors and TP ligands, including TXA2, isoprostanes and prostaglandin H2, are elevated in vascular and atherothrombotic diseases
(Zhang, Song et al. 2011).
The combination of TP receptors and its ligands activates several signaling mechanisms which regulates endothelial cell activation (i.e. adhesion molecules expression), vascular smooth muscle cell contraction and platelet aggregation, thereby accelerating progression of atherosclerotic lesions (Giannarelli, Zafar et al. 2010). Research showed
administration of the TP receptors antagonist (terutroban) improved endothelial function in high-cardiovascular-risk patients with atherosclerosis (Lesault, Boyer et al. 2011). However, the significance of TXAS-TXA2-TP signaling in the smoking related CVD and pulmonary injuries are not fully understood.4.1. Smoking increases platelet activation and thrombosis partially through activating TXAS-TXA
2-TP signaling pathway
Platelets from smokers demonstrate a dose-dependent increase in activities and adhesiveness that rapidly decrease with smoking abstinence (Health and Services 2010). Furthermore, evidence indicates cessation of smoking for two weeks may reverse platelet aggregability and decrease oxidative stress (Morita, Ikeda et al. 2005). Oxidative stress would lead to inflammation, platelet activation, endothelial dysfunction (Santilli, Guagnano et al. 2015) and promote the formation of TP receptors agonists which initiate atherogenesis and thrombosis (Capra, Bäck et al.
2014), indicating CS-induced thrombosis may be affected through TXAS-TXA2-TP signaling pathway.
4.2 Smoking leads to pulmonary injury partially through TXAS-TXA
2-TP signaling pathway
Cigarette smoking is a major risk factor in the development and progression of lung cancer and pulmonary injuries (Alavanja, Field et al.
2001). Cigarette carcinogen has been reported to increase the expressions of TXAS and TP receptors in lung (Ge, Xu et al. 2015). Research displayed that TXAS would activate TP receptors signaling pathway and induce cell proliferation and invasion in lung disease (Iorio-Morin, Germain et al. 2012). As a result of DNA damage, lung was severely impaired. Nuclear factor kappa B (NF-κB), a DNA damage marker, activates TXAS-TXA2
-TP signaling pathway and contributes to the
development of smoking-associated lung injuries (Ge, Xu et al. 2015).5. Using TXAS or TP gene knockout mice for exploring TXAS-TXA
2-TP signaling pathway
To investigate the role of TXAS-TXA2-TP in smoking associated acute arterial thrombotic diseases and pulmonary injuries, we used three types of target gene knocked out mice, TXAS-/-TP+/+, TXAS+/+TP-/-, and TXAS-/-TP-/-. The developed TXAS-/-TP+/+ mice display normal bone marrow megakaryocytes, blood platelet counts, and CD4 and CD8 lymphocyte counts in thymus and spleen but fail to aggregate or generate TXA2 in response to arachidonic acid stimulation. The developed TXAS+/+TP-/- mice display similar as TXAS-/-TP+/+ mice but are lack of TP receptors. The TXAS-/-TP-/- mice fail to generate TXA2 and are lack of TP receptors. These
three groups of mice are able to maintain normal mean arterial blood pressure and protect against arachidonate-induced shock and death (Yu, Lin et al. 2004).
6. Aim and purpose
This study aimed to investigate the role of TXAS-TXA2-TP signaling pathway in sidestream smoke and THS related thrombosis and pulmonary injury including endothelial dysfunction, oxidative damage, inflammation, ER stress, cell apoptosis, cells autophagy and cell pyroptosis.
V. Materials and methods
1. Animals
1.1 TXAS gene knockout mice
The Tbxas1 knockout mice are generated by Dr. Shu-Wha Lin’s group. A targeting vector is designed to replace the distal portion exon 9 of the endogenous gene with a phosphoglycerate kinase-hypoxanthine phosphoribosyltransferase (PGK-HPRT) cassette. This construct is electroporated into E14TG2a embryonic stem (ES) cells. Correct targeted ES cells are injected into C57BL/6J (i.e. wild type and TXAS
+/+TP+/+) blastocysts. Chimeric mice are bred with C57BL/6J for 10 generations, the homozygous Tbxas1 knockout (KO) mutant mice were obtained by intercrossing the heterozygous Tbxas1 KO mice.
1.2 TP gene knockout mice
The Tbxa2r knockout mice are generated by Dr. Thomas M.
Coffman’s group (Thomas, Mannon et al. 1998). A neomycin resistance gene driven by the phosphoglycerate kinase (PGK) promoter is inserted into a unique SfiI site near the proximal end of exon 2. This insertion targeting vector disrupts the coding sequence of the TP gene in the third transmembrane domain. This construct is electroporated into E14TG2a ES cells. Correctly targeted ES cells are injected into C57BL/6J blastocysts. Chimeric mice are bred with C57BL/6J for 10 generations, the homozygous Tbxa2r KO mutant mice are obtained by intercrossing the heterozygous Tbxa2r KO mice.
1.3 TXAS and TP double knockout mice:
Tbxas1 KO and Tbxa2r KO mouse in the C57BL/6 background are crossbred to generate Tbxas1-Tbxa2r double knockout mice. Male mice including TXAS+/+TP+/+ (wild type), TXAS-/-TP+/+, TXAS+/+TP-/- and TXAS-/-TP-/- mice were provided from National Taiwan University core laboratory. The mice were housed at the Experimental Animal Center of National Taiwan Normal University College of Medicine, with a temperature- and humidity-regulated environment (22±2°C, 55±5% RH) and a consistent light cycle (light from 07:00 to 18:00 o'clock). Food and tap water were provided ad libitum. All surgical and experimental procedures were approved by Institutional Animal Care and Use Committee of National Taiwan Normal University and in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997). After experiment, all the animals were sacrificed with overdose of sodium pentobarbital (50 mg/kg, i.p.) or potassium choloride (KCl).
1.4 Grouping
TXAS+/+TP+/+, TXAS-/-TP+/+, TXAS+/+TP-/- and TXAS-/-TP-/- (n=24, 6 for control, CON; 6 for acute cigarette smoke exposure, ACE; 6 for 5 weeks prolonged cigarette smoke exposure, PCE 5 and 6 for 8 weeks prolonged cigarette smoke exposure, PCE 8) were performed as described below.
1.5 Surgical preparation
On the experimental day, the animals were anesthetized with urethane (1500 mg/kg; Sigma, Missouri, USA) injected intraperitoneally (i.p.). The body temperature was kept at 36.5-37.0°C by an infrared light. The tracheas were exposed via a midline cervical incision and were intubated with polyethylene 60 (PE-60).
2. FeCl
3-induced acute arterial thrombosis
The mice model of topical FeCl3-induced carotid artery or femoral artery thrombosis has been used extensively to assess the antithrombotic activities of test agents (Robinson, Welsh et al. 2003). To easily monitor blood flow and obtain larger tissue sampling, we selected carotid artery thrombosis model in this study. We placed a flow probe (Probe# 0.5VBB517, Transonic Systems, Inc., Ithaca, NY) in right carotid artery for measuring the arterial blood flow in anesthetized mice. We dropped 15% FeCl3 solution (Ferric chloride, Sigma, St. Louis, MO, USA) on the right carotid artery. The adventitial FeCl3 solution easily diffused into the arterial tissue, inducing a Fenton reaction-mediated injury (impairment of nitric oxide release) and subsequently reduced arterial blood flow within minutes. Periadvential FeCl3 treatment may affect endothelium, media and intima in the carotid artery. The flow rate was continuously recorded. The required time to induce complete arterial occlusion (arterial blood flow decreases to zero) was defined as time to occlusion (TTO).
3. Cigarette Smoke Exposure Protocol
Mice were exposed to LONFLIFE original (each contains 0.8mg nicotine, 10mg tar; Taiwan Tobacco & Liquor Corporation, Taipei, Taiwan) cigarette smoke using a whole-body smoke exposure model as Figure 1. Mice were exposed to cigarettes for a period of approximately 50 minutes, twice daily, for 4 days (acute CS exposure) or 5 days per week last for 5 and 8 weeks (prolonged CS exposure). This exposure period followed an initial acclimatization period whereby mice move freely within the exposure box and are accustomed to this type of restraint over a 3-day period. Mice were placed into the exposure box for 20 minutes on Day 1, 30 minutes on Day 2 and 50 minutes on Day 3 for. Control animals were exposed to room air only.
4. Whole blood reactive oxygen species detection
Whole blood was collected from mice and blood ROS was measured by the Chemiluminescence Analyzing System (Model CLA-ID3; Tohoku Electronic In Co., Sendai, Japan) detecting Luminol-enhanced chemiluminescence (CL) signal emitted from the sample surface continuously.
5. Bronchoalveolar lavage fluid reactive oxygen species detection
Bronchoalveolar lavage fluid (BALF) of mice was performed after TTO experiment (Li, Tsai et al. 2013). The chest cavity was opened, the main bronchus was ligated, and the lung was lavaged with Ca2+/Mg2+-free phosphate buffer saline (PBS pH 7.4). The volume of buffer was estimated
to 35 ml/kg of body weight (approximately 90% of total lung capacity).
After the buffer was instilled into the lungs 3 times, we collected the solution. BALF were run through a CL Analyzing System (Model CLA-ID3;
Tohoku Electronic In Co., Sendai, Japan) in order to quantify Luminol/Lucigenin-enhanced CL signal emitted from the sample.
6. Immunohistochemistry (IHC)
Tissue sections were deparaffinized in xylene and rehydrated in ethanol.
The slides were immersed in xylene for 10 minutes at room temperature and repeated using fresh xylene for second 10 minutes incubation. The slides were sequentially immersed in 100%, 95%, 75% and 50% ethanol each for 5 minutes at room temperature. Then the tissue sections were submitted to antigen retrieval buffer for 20 minutes at 95oC. The retrieval buffer solution used for heat-induced epitope retrieval was sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0). After 20 minutes, we applied 3%
hydrogen peroxide (H2O2) to clean H2O2 enzyme for 15 minutes at room temperature and then the slides were blocked for non-specific binding with 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at room temperature and incubated with the primary antibodies for 18 hours at 4°C. Primary antibodies included rabbit anti Beclin-1 (1:200;
Proteintech, Chicago, IL, USA), rabbit anti poly (ADP-ribose) polymerase-1 (PARP-1, 1:500; Bioss, Woburn, MA, USA), rabbit anti Von Willebrand factor (VWF, 1:100; Proteintech, Chicago, IL, USA), rabbit anti 4-hydroxynonenal (4-HNE, 1:500; Alpha Diagnostic, San Antonio, TX,
USA) and mouse anti Interleukin-1 beta (IL-1β, 1:1000; Cell Signaling Technology, Denver, MA, USA). Tissue sections were washed with PBST three times and then incubated with secondary antibodies HRP-conjugated rabbit anti-mouse
i
mmunoglobulin G (IgG) or goat anti-rabbit IgG(1:800;both from Sigma Aldrich St. Louis, MO, USA) for 1 hour at room temperature. After washing with phosphate buffer saline tween-20 (PBST) for 3 times, we immersed slides in DAB (ImmPACT DAB Peroxidase Substrate; Vector, Burlingame, California, USA) for 3-5 minutes, washed with double-distilled water (ddH2O), and immersed slides in hematoxylene for 5 minutes. The slides were dehydrated in ethanol series and were mounted in mounting medium (Leica, Wetzlar, Germany).
7. Terminal deoxynucleotide transferase dUTP Nick End Labeling stain
We used BioVision’s Apo-BrdU-IHCTM Kit to performed a two-color TUNEL stain (Terminal deoxynucleotide transferase dUTP Nick End Labeling) (catalog #K403-50; 50 assays; stored at -20oC) assay for labeling DNA breaks to detect apoptotic cells by immunohistochemistry. The slides were immersed in xylene for 10 minutes at room temperature and repeated using fresh xylene for another 10 minutes incubation. The slides were sequentially immersed in 100%, 95%, 75% and 50% ethanol each for 5 minutes at room temperature. We immersed slides into 1X PBST, dried the glass slide and applied 100 µl Proteinase K solution at 1:100 in 10 mM Tris with pH 8 of entire specimen for 20 minutes at room temperature. The slides were then rinsed with 1X PBST, gently tapped off excess liquid and applied100 µl of 3% H2O2 at 30% H2O2 1:10 in methanol for 5 minutes at room temperature. We rinsed the slide with 1X PBST, gently tapped off excess liquid and carefully dried the glass slide then applied entire specimen with 1X Reaction Buffer at 5X diluted Reaction Buffer in ddH2O for 10 to 30 minutes at room temperature. We carefully blotted the 1X Reaction Buffer from the specimen and immediately applied 50 µl of Complete Labeling Reaction Mixture onto each specimen, which were then covered with a piece of Parafilm for 1 to 1.5 hours at 37°C.
After that, we removed Parafilm cover slip, rinsed slide with 1X PBST, gently tapped off excess liquid and carefully dried the glass, then applied 100 µl of Blocking Buffer for 10 minutes at room temperature. We immediately covered specimen with 100 µl of Antibody Solution in the dark for 1-1.5 hours at room temperature after blotted the Blocking Buffer from the specimen. We washed the specimen with 1X PBST and covered the entire specimen with 100 µl of Blocking Buffer and 100 µl of conjugate at 200X Conjugate 1:200 in Blocking Buffer for 30 minutes at room temperature. Five minutes before concluding incubation, we prepared DAB solution by dissolving one tablet of DAB and one tablet of H2O2/Urea in 1 ml of tap H2O and the specimen were tapped off excess liquid, covered with 100 µl of DAB solution for 15 minutes at room temperature. We rinsed slides with ddH2O and immediately covered the entire specimen with 100 µl of Methyl Green Counterstain solution for 2 to 3 minutes at room temperature. At the end, the slides were dehydrated in ethanol series and
were mounted in mounting medium.
8. Western Blot
Tissues were grinded to powders in liquid nitrogen. Then the tissue powders were lysed in Lysis Buffer (Cell Signaling Technology, Denver, MA, USA) supplemented with protease inhibitor (Roche) for 10 minutes at 4℃. The tissue homogenate was centrifuged for 30 minutes at 14000 rpm.
4℃. After centrifugation, the supernatant was collected into fresh eppendorf.
The concentration of the protein was measured by Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA). 60 µg protein samples were mixed with 1X sample buffer and were boiled for 3 min at 100oC.
Protein samples were resolved in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) for over 18 hours. After transferring, the membrane was blocked with Hyblock (Hycell, Taipei, Taiwan) for 1 minute, and incubated with primary antibodies overnight at 4oC. The membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature.
Detection of signals was performed by Immobilon Western Chemiluminescent HRP Substrate (Millipore). Primary antibodies included microtubule-associated protein light chain 3 (LC3 β, MBL, Naka-ku, Nagoya, Japan), B cell lymphoma-2 (Bcl-2), Bcl-2-associated X-protein (Bax), Beclin-1, PARP-1 (Cell Signaling Technology, Denver, MA, USA),
VWF (Proteintech, Chicago, IL, USA), 4-HNE (Bioss, Woburn, MA, USA), activating transcription factor 6 (ATF-6, Abcam, Cambridge, UK), NF-κB, phospho-c-Jun N-terminal kinases (p-JNK, Santa Cruz, Dallas, TX, USA), and β-actin (1:500; Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies included HRP-conjugated rabbit anti-mouse IgG, HRP-conjugated rabbit anti-goat IgG, and HRP-conjugated goat anti-rabbit IgG (all for 1:10000; all from Sigma Aldrich St. Louis, MO, USA).
9. Platelet adhesiveness detection
Whole blood was collected from dorsal vein and heart by puncture and collected in 1.5-ml polypropylene tubes containing 300 µl of heparin (30 U/ml). Platelet-rich plasma was obtained by centrifugation for 5 min at 1,200 revolutions 4oC. After centrifugation, transferred the plasma and buffy coat containing some RBCs to fresh polypropylene tubes and recentrifuged at 1,200 revolutions. The platelet-rich plasma was transferred to fresh tubes containing 2 µl of prostacylin (PGI2, 2 µg/ml) and incubated for 5 min at 37°C. Then, the pellets were resuspended in Tyrode-HEPES buffer 〔137 mM sodium chloride (NaCl), 0.3 mM sodium hydrogen phosphate (Na2HPO4), 2 mM KCl, 12 mM sodium hydrogen carbonate (NaHCO3), 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5 mM glucose, 0.35% BSA〕containing 2 µl PGI2 and were centrifugated for 5 min at 2,800 revolutions 4oC. After centrifuguration, the pellets were incubated for 5 min at 37oC. In order to remove PGI2, the washing steps would be conducted twice and platelets were fluorescently
labeled with calcein acetoxymethyl (AM) 2.5 µg/ml (catalog #1674595;
sotred at -20oC, Life technologies, Waltham, MA, USA) for 10 minutes at room temperature.
The fluorescently-labeled platelets (FP) were injected to mice through jugular vein with PE-10. 200 seconds after injecting FP, we applied 10µl FeCl3 onto the mesenteric artery (about 200~300µm diameter) to induce acute artery thrombosis and was detected by fluorescent dissecting microscope (Leica DMLFSA; Wetzar, Hessen, Germany).
10. Malondialdehyde detection assay
To explore the relationship between CS exposure and plasma malondialdehyde (MDA), a lipid peroxidation of oxidative stress, we used MDA Colorimetric Assay Kit (catalog #K739-100; 100 assays; stored at -20oC, BioVision, Milpitas, CA, USA). We collected plasma and stored at -80oC for further usage. We prepared thiobarbituric acid (TBA) buffer by adding 7.5 ml glacial acetic acid, then adjusting with ddH2O to a final volume of 25 ml one week before conducting MDA assay. The kit and the plasma samples were cooled down to temperature on ice the day of MDA detection. We diluted 10 µl of the MDA standard with 407 µl ddH2O to generate a 0.1 M MDA solution, then diluted 20 µl of 0.1 M solution with 980 µl ddH2O to prepare a 2 mM MDA solution. We diluted the 2 mM MDA solution with 200 µl ddH2O to prepare 0, 4, 8, 12, 16 and 20 nmol standard solution. We used 10 µl of each plasma samples, added 500 µl of
42 mM sulfuric acid (H2SO4) and 125 µl phosphotungstic acid solution and well mixed. The plasma mixture were incubated for 5 min at room temperature and then centrifuged for 3 min at 13000 relative centrifugal field (rcf). We collected the pellet and resuspended on ice with 100 µl ddH2O containing 2 µl butylated hydroxytoluene (BHT). We added 600 µl of TBA reagent buffer in standard solution and plasma mixtures before incubated for 60 min at 95oC. The mixtures were cooled down to room temperature on ice and we pipetted 200 µl from each standard solution and plasma mixtures into a 96-well microplate and detected the absorbance at 532 nm. The absorbance was then calculated.
VI. Results
1. Acute arterial thrombosis model of time to occlusion
The average TTO in each group are shown in Table 1. The blood flow over time plots are shown in Figure 2~5, representing the TXAS+/+TP+/+, TXAS-/-TP+/+, TXAS+/+TP-/- and TXAS-/-TP-/- group respectively. The comparison of TTO among different groups and exposure treatments are provided in Figure 6.
The mean ± SEM of TTO in minutes of wild type mice are 31.4 ± 4.9 in CON group, 14.2 ± 1.6 in ACE group, 11.6 ± 1.3 in PCE 5 group and 12.3 ± 2.1 in PCE 8 group. Acute CS exposure treatment group comparing to CON group significantly attenuates TTO in wild type mice.
The mean ± SEM of TTO in minutes of TXAS-/-TP+/+ mice are 41.6 ± 5.2 in CON group, 17.3 ± 3.4 in ACE group, 16.7 ± 1.7 in PCE 5 group and 15.8 ± 1.5 in PCE 8 group ;TXAS+/+TP-/- mice are 38.2 ± 4.4 in CON group, 27.3 ± 3.7 in ACE group, 19.0 ± 5.0 in PCE 5 group and 20.0 ± 4.0 in PCE 8 group; TXAS-/-TP-/- mice are 34.6 ± 6.2 in CON group, 16.8 ± 2.7 in ACE group, 17.3 ± 2.1 in PCE 5 group and 12.3 ± 0.9 in PCE 8 group. Acute CS exposure treatment group comparing to CON group significantly attenuates TTO in gene knockout mice as wild type mice. The TTO under same exposure treatment of wild type mice were shorter than the three gene knockout mice.
2. Platelet adhesiveness detection in mesenteric arteries
Fluorescently labeled platelets were used to detect platelet adhesiveness
in mesenteric arteries through Fenton reaction in the four genotypes mice.
The results are shown in Figure 7. The platelet adhesiveness significantly displayed higher fluorescence intensity in wild type mice than that of the other three gene knockout mice.
3. Whole blood reatvie oxygen species detection
Whole blood ROS was measured by the CL analyzing system detecting Luminol-enhanced CL signal from the sample surface continuously. The values of CL signal are shown in Figure 8. Blood ROS counts were higher in CS exposure treatment groups of wild type mice. The results showed statistical higher in wild type mice under PCE 8 treatment in comparison to CON and a dose-dependent manner. Blood ROS counts were higher in CS exposure treatment groups of the three gene knockout mice and showed a dose-dependent increment in TXAS-/-TP+/+ mice. The blood ROS counts under same exposure treatment of wild type mice were higher than those of the three gene knockout mice.
4. Bronchoalveolar lavage fluid of reactive oxygen species
To quantify CS exposure induced ROS in BALF, we used Lucigenin- and Luminol-amplified CL methods to measure O2-˙and H2O2 activity in BALF.
The CL counts by Lucigenin-amplified method are shown in Figure 9a, 9b.
CS exposure treatment increased superoxide (O2-˙) in BALF of wild type mice. Wild type mice significantly expressed higher O2-˙ counts in PCE 5 and PCE 8 treated group comparing to CON group. CS exposure treatment also increased O2-˙in three gene knockout groups. TXAS-/-TP+/+ mice
significantly expressed higher O2-˙ counts in PCE 8 group in comparison to CON group; TXAS+/+TP-/- and TXAS-/-TP-/- showed no significance in comparison to CON group. The BALF ROS counts under same exposure treatment of wild type mice were higher than those of the three gene knockout mice.
The CL counts by Luminol-amplified method are shown in Figure 9c, 9d.
CS exposure treatment increased H2O2 in BALF of wild type mice. Wild type mice significantly expressed higher H2O2 counts in PCE 8 group comparing to CON group and showed a dose-dependent increment. CS exposure treatment also increased H2O2 in the three gene knockout groups and showed a dose-dependent increment. TXAS-/-TP+/+ mice significantly expressed higher O2-˙ counts in PCE 8 group in comparison to CON group;
TXAS+/+TP-/- and TXAS-/-TP-/- showed no significance in comparison to CON group. The BALF ROS counts under same exposure treatment of wild type mice were higher than those of the three gene knockout mice.
5. Malondialdehyde concentration detection
MDA, an oxidative stress induced metabolites, is shown in Figure 10.
Longer CS exposure treatment, PCE 5 and PCE 8 increased MDA in plasma of wild type mice and no dose-dependent increment in CS exposure treated groups. CS exposure treatment also increased MDA amounts in the three knockout mice. TXAS-/-TP+/+ mice showed dose-dependent increment of MDA concentration in CS exposure treated groups but no significance.
TXAS+/+TP-/- mice showed dose-dependent decrement of MDA
concentration in CS exposure treatment groups but no significance.
TXAS-/-TP-/- mice did not show dose-dependent increment or decrement of MDA concentration. The plasma MDA concentration of all CS exposure treated groups in wild type mice were higher than those of the three gene knockout mice.
6. Hematoxylin and eosin (H&E) stain
In order to examine the tissue abnormality, the histological lung sections and pulmonary arteries were stained with H&E. The histological stains of pulmonary artery are shown in Figure 11a and the pulmonary artery relative thickness is shown in Figure 11b and 11c. CS exposure treatment increased the thickness of artery in wild type mice. Wild type mice significantly expressed higher ratios in PCE 8 treated group than CON treated group.
TXAS-/-TP+/+ and TXAS+/+TP-/- mice showed higher ratios in PCE 5 and PCE 8 treated group but no significance in comparison of CON treated group.
TXAS-/-TP-/- mice significantly showed higher ratios in PCE 8 treated group than CON treated group. The pulmonary artery thickness of PCE 8 treated group in wild type mice was higher than that of the three gene knockout mice and was similar to the other three exposure treatments.
The histological stain of lung sections is shown in Figure 12a, 12b and the leukocytes numbers is shown in Figure 12c and 12d. CS exposure treatment increased septal thickening, leukocytes infiltration and numbers in wild type mice. Wild type mice significantly expressed higher leukocytes numbers in all CS exposure treated groups than CON treated group and
showed a dose-dependent increment of numbers. TXAS-/-TP+/+ mice showed higher ratios in PCE 8 treated group but no significance in comparison of CON treated group. CS exposure treatment also increased septal thickening, leukocytes infiltration and numbers as wild type mice and showed the same trend as wild type mice. The leukocytes numbers in PCE 5 and PCE 8 treated groups in wild type mice were higher than those of the three gene knockout group.
7. Immunohistochemistry analysis
For further understanding of lung injury caused by cigarette smoke exposure, several IHC stains were performed. Tissue sections were focused on oxidative stress, endothelial injury, inflammatory cytokines, apoptosis and autophagy.
Oxidative injury represented by 4-HNE—a product of lipid peroxidation—is shown in Figure 13. CS exposure treatment increased 4-HNE expressions in wild type mice. Wild type mice significantly expressed higher 4-HNE in all CS exposure treated groups than CON treatment group. CS exposure treatment also induced higher 4-HNE expressions in the three gene knockout mice. TXAS-/-TP+/+ and TXAS-/-TP-/- mice significantly expressed higher 4-HNE in all CS exposure treatment group than CON treatment group while in TXAS+/+TP-/- mice, there was significantly higher 4-HNE expressions under PCE 8 treatment. The 4-HNE expressions under PCE 5 and PCE 8 treatment of wild type mice were higher than those of the three gene knockout group.
VWF—a marker of endothelial activation and injury—is shown in Figure 14. CS exposure treatment increased VWF expressions in wild type
mice. Wild type mice significantly expressed higher VWF in PCE 5 and PCE 8 treated groups than CON treated group and showed dose-dependent increments under CS exposure of VWF expressions. CS exposure treatment also induced higher VWF expressions in the three gene knockout group.TXAS-/-TP+/+ mice significantly expressed higher VWF in PCE 8 treated group than CON treated group; TXAS+/+TP-/- mice significantly expressed higher VWF in all CS treated groups than CON treated group and TXAS-/-TP-/- mice significantly expressed higher VWF in PCE 5 and PCE 8 treated groups than CON treated group. The VWF expressions of all CS exposure treated groups in wild type mice were higher than those of the three gene knockout group.
IL-1β—a pro-inflammatory cytokines—is shown in Figure 15. CS
exposure treatment increased IL-1β expressions in wild type mice. Wild type mice significantly expressed higher IL-1β in all CS treated groups than CON treated group. CS exposure treatment also induced higher IL-1β expressions in the three gene knockout group. TXAS-/-TP+/+ mice significantly expressed higher IL-1β in PCE 5 and PCE 8 treated groups than CON treated group and TXAS-/-TP-/- mice significantly expressed higher IL-1β in PCE 5 treated group than CON treated group. The IL-1β expressions of all CS exposure treated groups in wild type mice were higher than those of the three gene knockout group.PARP-1—a family member of PARP, is a nuclear DNA-binding zinc
finger protein that influences DNA repair, DNA replication, modulation of chromatin structure and the most importantly-apoptosis— is shown inFigure 16. CS exposure treatment increased PARP-1 expressions in wild
type mice. Wild type mice significantly expressed higher PARP-1 in the three CS treated group than CON treated group and showed dose-dependent increments under CS exposure of PARP-1 expressions. CS exposure treatment also induced higher PARP-1 expressions in the three gene knockout group. TXAS-/-TP+/+ and TXAS+/+TP-/- mice showed dose-dependent PARP-1 expression increments. TXAS-/-TP-/- mice expressed nearly same expressions of PARP-1 in the three CS exposure treated groups.The PAPR-1 expressions of all CS exposure treated groups in wild type mice were higher than those of the three gene knockout group.
An autophagy-related protein, Beclin-1, is shown in Figure 17. CS exposure treatment increased Beclin-1 expressions in wild type mice. Wild type mice significantly expressed higher Beclin-1 in PCE 5 and PCE 8 treated groups than CON treated group and showed a dose-dependent increment. CS exposure treatment also induced higher Beclin-1 expressions in the three gene knockout group. TXAS-/-TP+/+ mice showed dose-dependent increments under CS exposure of Beclin-1 expressions, but no significance in comparison of CON treated group. TXAS+/+TP-/- and TXAS-/-TP-/- mice significantly expressed higher Beclin-1 in PCE 8 treated group than CON treated group and showed dose-dependent increments
under CS exposure of Beclin-1 expressions. The Beclin-1 expressions of all CS exposure treated groups in wild type mice were higher than those of the three gene knockout group.
8. Terminal deoxynucleotide transferase dUTP Nick End Labeling stain
We used TUNEL stain to quantify the apoptotic cells formation and the DNA fragmentation occurred in the nucleus appeared brown color, which is shown in Figure 18. CS exposure treatment increased TUNEL ratios in wild type mice. Wild type mice significantly expressed higher ratios in PCE 8 treated group than CON treated group. TXAS-/-TP+/+ mice showed higher ratios in PCE 8 treated group but no significance in comparison of CON treated group. TXAS+/+TP-/- mice showed higher ratios in PCE 5 treated group but no significance in comparison of CON treated group. TXAS-/-TP-/- mice significantly showed higher ratio in the three CS treated groups than CON treated group and showed dose-dependent increments under CS exposure of the stain ratios. The TUNEL stain ratios of all CS exposure treated groups in wild type mice were higher than those of the three gene knockout group.9. Western Blot
In order to understand the apoptosis pathway induced by CS exposure, we used western blot and focused on several markers, including cleavage PARP-1 (Figure 19), Bax and Bcl-2 (Figure 20). CS exposure treatment increased PARP-1 expressions in wild type mice. Wild type mice expressed higher PARP-1 in the three CS treated groups than CON treated group but
showed no significance. CS exposure treatment also induced higher PARP-1 expressions in TXAS-/-TP+/+ and TXAS+/+TP-/- mice, which showed dose-dependent PARP-1 expression increments but no significance.
TXAS-/-TP-/- mice expressed nearly same expressions of PARP-1 in the four exposure treated groups. The PAPR-1 expressions of PCE 5 and PCE 8 treated groups in wild type mice were higher but not significantly than those in the three gene knockout mice. The western blot result was similar to the IHC stain of PARP-1. CS exposure treatment increased Bax/Bcl-2 ratios in wild type mice. Wild type mice significantly expressed higher ratios in PCE 8 treated group than CON treated group and showed a dose-dependent increment of the ratios. CS exposure treatment also induced higher ratios in the three gene knockoutmice. The three gene knockout mice significantly expressed higher ratios in PCE 8 treated group than CON treated group and showed a dose-dependent increment of ratios. The ratios of all exposure treated groups in wild type mice were similar to the three gene knockout mice.
We focused on LC3 β and Beclin-1 on autophagy pathway to find out the effect of CS exposure (Figure 21 and 22). CS exposure treatment increased LC3 β expressions in wild type mice. Wild type mice significantly expressed higher LC3 β in PCE 5 and PCE 8 treated group than CON treated group and showed dose-dependent increment of LC3 β expressions in CS exposure treatment. CS exposure treatment also induced higher LC3 β expressions in the three gene knockout mice and showed dose-dependent
increment of LC3 β expressions in CS exposure treatment. There were significantly higher LC3 β expressions of PCE 8 treated group in the three gene knockout mice. The LC3 β expressions of CS exposure treated groups in wild type mice were higher than the three gene knockout mice. CS exposure treatment increased Beclin-1 expressions in wild type mice. Wild type mice significantly expressed higher Beclin-1 in PCE 5 and PCE 8 treated group than CON treated group and showed dose-dependent increment of Beclin-1 expressions in CS exposure treatment. CS exposure treatment also induced higher Beclin-1 expressions in the three gene knockout mice and showed dose-dependent increment of Beclin-1 expressions in CS exposure treatment. TXAS+/+TP-/- and TXAS-/-TP-/- mice significantly expressed higher Beclin-1 in PCE 8 treated group than CON treated group. The Beclin-1 expressions of CS exposure treated groups in wild type mice were higher than those in the three gene knockout mice.
The oxidative stress marker, 4-HNE is shown in Figure 23. CS exposure treatment increased 4-HNE expressions in wild type mice but did not show dose-dependent increment. CS exposure treatment also induced higher 4-HNE expressions in the three gene knockout mice. TXAS-/-TP+/+ and TXAS+/+TP-/- mice showed dose-dependent increment of 4-HNE expressions in CS exposure treatment but no significance. TXAS-/-TP-/- mice expressed higher 4-HNE in ACE and PCE 8 treated groups than CON treated group.
The 4-HNE expressions of ACE and PCE 5 treated groups in wild type mice were higher than the three gene knockout mice and of PCE 8 treated group
was lower than TXAS-/-TP+/+ and TXAS+/+TP-/- mice. The result was correlated with IHC of 4-HNE.
VWF, the marker of endothelial injury is shown in Figure 24. CS exposure treatment increased VWF expressions in wild type mice. Wild type mice significantly expressed higher in PCE 5 and PCE 8 treated groups than CON treated group and show dose-dependent increment of VWF expressions. CS exposure treatment also induced higher VWF expressions in the three gene knockout mice. TXAS-/-TP+/+ mice significantly expressed higher in PCE 8 treated group than CON treated group and showed dose-dependent increment of VWF expression in CS exposure treated groups. TXAS+/+TP-/- and TXAS-/-TP-/- mice expressed higher in PCE 5 and PCE 8 treated groups than CON treated group and showed dose-dependent increment of VWF expressions in CS exposure treated groups. The VWF expressions of CS exposure treated groups in wild type mice were higher than those of the three gene knockout mice. The result was comparable with IHC of VWF.
The inflammatory protein, NF-κB is shown in Figure 25. CS exposure treatment increased NF-κB expressions in wild type mice. Wild type mice expressed higher in ACE and PCE 8 treated groups than CON treated group.
CS exposure treatment also induced higher NF-κB expressions in TXAS-/-TP+/+ and TXAS+/+TP-/- mice. TXAS-/-TP+/+ and TXAS+/+TP-/-mice showed dose-dependent increment of NF-κB expression in CS exposure treated groups but no significance. TXAS-/-TP-/- mice expressed higher in
