The objectives of my project are to examine the potential anti-inflammatory effects of Rip-2 siRNA, RPS-3siRNA, and fisetin, a bioactive flavonol in mouse asthma model or TNF-α stimulate
Trang 12 Rationale and objective
Trang 21 Rational and objective
Globally 300 million people suffer from asthma and the prevalence of asthma still continues
to increase (Pawankar et al., 2012) Asthma is a multifactorial disease, involving a complex network of molecular and cellular interaction In addition to genetic predisposition, environmental factors also mediates the initiation and development of asthma (von Mutius, 2009)
Current therapies for asthma generally rely on SABAs or LABAs and inhaled corticosteroids Although these current therapies are relatively effective at controlling symptoms, these therapies do not change the chronic course of disease In addition, major concerns about the systemic effects of inhaled corticosteroids remains Furthermore, around 15% of asthmatic patients who suffer from uncontrollable asthmatic symptoms fail to respond well to inhaled corticosteroids Currently, there is no established method to prevent asthma Therefore, the major unmet needs of this area include better management of the severe forms of the disease and the developments of curative therapies (Akdis, 2012) Consequently, much research has been done to better understand the pathophysiology of asthma and to explore novel therapies for this asthma
One attractive target for therapeutic intervention would be the NF-κB signaling pathway, which plays an important role in Th2-mediated inflammation (Edwards et al., 2009) Thus, the development of specific inhibitors targeting NF-κB signaling pathway is promising for attenuating allergic airway inflammation Nonetheless, direct inhibition of NF-κB may not be
a safe approach because NF-κB plays a pivotal role in numerous normal biological functions and pathological conditions Therefore, a safer and yet effective anti-inflammatory approach for attenuation of allergic airway inflammation would be through appropriate and specific inhibition of signaling molecules which regulate the activation of NF-κB (Uwe, 2008)
Rip-2 has been shown to mediate the formation of functional IKK complex, which is essential for NF-κB activation; while RPS-3 has been demonstrated to enhance the binding of NF-κB
Trang 3to the κB sites of selected NF-κB targets Fisetin, on the other hand, is a natural product that has been reported to interfere with the activity of NF-κB pathway The objectives of my project are to examine the potential anti-inflammatory effects of Rip-2 siRNA, RPS-3siRNA, and fisetin, a bioactive flavonol in mouse asthma model or TNF-α stimulated lung cell lines and investigate their effects in the regulation of NF-κB pathway Future aims include developing efficacious therapies which are easy to comply with and have minimal systemic side effects
Trang 43 Materials and methods
Trang 53.1 Materials and Reagents
Drugs and chemicals used in this Ph.D project are as follows: Bicinchonic acid (BCA) protein assay kit, calf bovine serum (CBS); custom control siRNA; fetal bovine serum (FBS); M-PER Mammalian Protein Extraction Reagent containing phosphatase inhibitor; ON-
TARGETplus Receptor interacting protein (Rip)-2 small interfering RNA (siRNA); TARGETplus Rip-2 siRNA with in vivo processing; ON-TARGETplus Ribosomal protein
ON-(RP)S3 siRNA, RestoreTM PLUS western blot stripping buffer; and 2 X Sybrgreen master mix were obtained from Thermo Scientific, Waltham, MA, USA Acetyl-β-methylcholine chloride; aluminium hydroxide (Al(OH)3; bovine serum albumin (BSA); chicken ovalbumin (OVA), dimethyl sulfoxide (DMSO); eosin Y, fisetin; G418; harris hematoxylin solution; 10% neutral buffered formalin; skim milk powder; tissue culture grade 10 X phosphate buffered saline (PBS); tween-20 were obtained from Sigma-Aldrich, St Louis, MO, USA Ammonium chloride (NH4Cl) was obtained from BDH Laboratory Supplies, Poole, England Agarose; alkaline phosphatise (AP) conjugated substrate kit; blotting paper, polyvinylidene difluoride (PVDF) membrane; tetramethylbenzidine (TMB) substrate kit; tetramethylethylenediamine (TEMED); and 3,3’,5,5’- Triton X-100 were obtained from Bio-Rad laboratories, Hercules,
CA, USA) NF-κB/Secreted alkaline phosphatase (SEAP) gene reporter assay kit was obtained from Imgenex, San Diego, CA, USA Anti-β-actin monoclonal antibody, anti-mouse Rip-2 monoclonal antibody, anti-human RPS-3 monoclonal antibody were obtained from Abcam, Cambridge, UK Diethylpyrocarbonate (DEPC)-treated water; Dulbecco’s modified Eagle medium (DMEM) with 4.5 g/L glucose; Lipofectamine 2000; optimum I reduced serum medium; Recombinant human tumor necrosis factor (TNF)-α; Roswell Park Memorial Institute (RPMI) Medium; RNAlater; TRIzol; trypan blue were obtained from Invitrogen, Carlsbad, CA, USA Anesthetic mixture (10 µl/g ketamine: medetomidine: H2O = 3:4:3); Sterile NaCl 0.9% were obtained from Animal Holding Unit AHU (NUS, Singapore) Aerosolised isofluorane was obtained from Halocarbon, NJ, USA Fetal bovine serum (FBS) was obtained from Hyclone Laboratories, South Logan, Utah, USA) Sodium dodecyl sulfate
Trang 6(SDS) and tris-acetate-EDTA (TAE) were obtained from 1st BASE, Singapore) Anti-mouse IL-13 monoclonal antibody; biotinylated anti-mouse IL-13 antibody; recombinant murine IL-
13, anti-mouse eotaxin; biotinylated anti-mouse eotaxin; and recombinant mouse eotaxin (R&D Systems, Minneapolis, MN, USA), Avidin-horseradish peroxidise (HRP); biotinylated anti-mouse IgE; biotinylated anti-mouse OptEIA™ mouse IFN-γ set, biotinylated anti-mouse OptEIA™ IgG1; biotinylated antimouse IgG2aOptEIA™ mouse; total IgE set; OptEIA™ mouse IL-4 set; OptEIA™ mouse IL-5 set, OptEIA™ mouse IL-33 set, OptEIA™ human IL-
6 set; and OptEIA™ human IL-8 set were obtained from BD Biosciences Pharmingen, San
Diego, CA, USA Fluoresave was obtained from Calbiochem, Billerica, MA Sodium
carbonate (Na2CO3) was obtained from Kanto Chemical, Tokyo, Japan Absolute ethanol; isopropanol; methanol; 1% periodic acid were obtained from Merck, Darmstadt, Germany HistoClear, and Histomount were obtained from National Diagnostics, Atlanta, GA, USA); Avian myeloblastosis virus (AMV) reverse transcriptase; dNTP mix; polymerase chain
reaction (PCR) master mix; and ribonuclease inhibitor were obtained from Promega, Madison,
WI, USA Anti-IκBα monoclonal antibody; anti-p65 monoclonal antibody were obtained from Cell Signaling Technology, Beverly, MA, USA Enhanced chemiluminescent (ECL) western blotting detection reagents; and hyperfilm were obtained from GE Healthcare, Piscataway, NJ, USA HRP-conjugated anti-mouse Ig; HRP-conjugated anti-rabbit Ig; AP-conjugated anti-mouse Ig; and AP-conjugated anti-rabbit Ig were obtained from Dako, Glostrup, Denmark Human MUC5AC ELISA Kit was obtained from USCN, Houston, Texas, USA Nuclear extract kit and TransAM™ NF-κB p65 Kit were obtained from ActiveMotif,
Carlsbad, CA, USA
Trang 73.2 siRNA preparation
3.2.1 ON-TARGETplus siRNA without in vivo processing
ON-TARGETplus siRNA complementary to mouse Rip-2 or RPS-3 mRNA were purchased
from Thermo Scientific (Waltham, MA) Table 3.1 shows the sequences of siRNA used A stock solution of siRNA (20 µM) was prepared by dissolving every 5 nmole of siRNA in 250
µl of 1 x PBS Briefly, the resuspended siRNA was pipette up and down for three to five times Extra care was taken to avoid introduction of bubbles The siRNA was then vortex for
30 mins at room temperature Following that, the dissolved siRNA was aliquoted into small volumes and stored at -30oC The aliquots were limited to freeze-thaw events of no more than
five
3.2.2 ON-TARGETplus siRNA with in vivo processing
ON-TARGETplus siRNA with in vivo processing complementary to mouse Rip-2 with
sequence S2 (Table 3.1) was dissolved in tissue culture grade 1 X PBS (Sigma-Aldrich, St Louis, MO) Every 5 nmole of siRNA was dissolved in 30 µl of tissue culture grade 1 X PBS
Following steps taken to dissolve the siRNA is similar to that listed for ON-TARGETplus
siRNA without in vivo processing
3.3 siRNA transfection
The mouse macrophage cell line RAW264.7, mouse fibroblast cell line NIH/3T3 (American Type Culture Collection, Rockville, MD) and NF-κB/ secreted alkaline phosphatase (SEAP) human embryonic kidney (HEK)293 cell line (IImgenex, San Diego, CA) were maintained in Dulbecco's modified Eagle medium (DMEM) with 4.5 g/L glucose (Invitrogen, Carlsbad, CA) In order to maintain the selection of SEAP transfected HEK293, selection agent G418 (Invitrogen, Carlsbad, CA) was added to the maintaining medium The human bronchial epithelial cell line BEAS2B and human pulmonary mucoepidermoid carcinoma cell line NCI-
Trang 8siRNA Sequence
Mouse Rip-2 Sequence 1 (S1) 5’-GCUCGACAGUGAAAGAAAU-3’ Mouse Rip-2 Sequence 2 (S2) 5’-ACGAGAAGCCGAAAUA UUA-3’ Mouse Rip-2 Sequence 3 (S3) 5’-CAAAUUUCCCUCAGAAUAA-3’
Table 3.1: Sequences of siRNA
Trang 9292 (American Type Culture Collection, Rockville, MD) were maintained in RPMI Aldrich, St Louis, MO) These cell lines were seeded at 60 to 70 % confluency in antibiotics-free media one day before transfection During transfection, the cells were maintained in GIBCO ™ optimum I reduced serum medium (Invitrogen, Carlsbad, CA) The cells were then transfected with 100 nM siRNA or negative siRNA control 4 – 6 h at 37°C in OptiMEM (Invitrogen, Carlsbad, CA) containing Lipofectamine 2000 (Invitrogen, Carlsbad, CA) After transfection, the cells were allowed to recover in complete DMEM or RPMI for 18, 42, or 66
(Sigma-h before t(Sigma-hey were analysed for Rip-2, IL-6, IL-8, MUC5AC, and TSLP mRNA and t(Sigma-heir protein expression
NCI-H292 is a human pulmonary mucoepidermoid carcinoma cell line This cell lineproduces MUC2, MUC4 and MUC5AC, which is a major component of mucin that contributes to the viscosity of mucous Mucin protein synthesis by H292 can be stimulated by TNF-α or EGF Therefore, NCI-H292 is commonly used to study signaling pathway involved
in mucus hypersecretion (Lora et al., 2005; Zhen et al., 2007) and is consequently used in this study On the other hand, BEAS2B is a human bronchial epithelial cell line that is developed
by transformation of normal human bronchial epithelial cells The transformation involved adenovirus 12-simian virus 40 hybrid virus (Ad12SV40) BEAS-2B form tight junctions but retain the ability to undergo squamous differentiation in response to serum BEAS-2B cells have been used to study pulmonary inflammatory response in a large number of studies (Verstraelen et al., 2008) Therefore, BEAS-2B was used in this study
3.4 Mouse asthma model and treatments
Female BALB/c mice, 6-8 wk of age (Interfauna, East Yorkshire, UK) used in this project were ordered from centre for animal resources and housed in plastic cages (maximum 5 mice/cage) in Animal Holding Unit in the National University of Singapore (NUS) according
to the spirit of Good Laboratory Practice Animal experiments were performed according to the Institutional Guidelines for Animal Care and Use Committee of the NUS Briefly, animal
Trang 10rooms were regulated by automatic timers to provide cycles with 12-14 h of light and 10-12 h
of dark The temperature in the animal room ranged from 18 °C to 26 °C with an average temperature of 22 °C Standard diets generally contained 4-5 % fat and 14% protein A minimum of 3 days of acclimatization were given to the mice to adapt to their new surroundings Cage bedding was changed thrice a week Mice were sensitized with 20 µg OVA and 4 mg Al(OH)3 in 0.1 ml saline intraperitoneally on day 0 and day 14 For OVA Challenge, 0.15 g of OVA was dissolved in 15 ml of saline to be aerosolized by a DeVilbiss Ultra-Neb Large-Volume Ultrasonic Nebulizer (Sunrise Medical Respiratory Products, Somerset, PA) (Figure 3.2) Mice were then challenged with 1% OVA aerosol for 30 min in a chamber on days 22, 23 and 24 (Figure 3.2) Average OVA aerosol particle size was less than
5 µm Saline aerosol was used as a negative control
Female BALB/c mouse was used because airway inflammation and AHR are easily induced
in this mouse strain Furthermore, female mice displayed higher serum IgE level and are more susceptible to airway inflammation than male mice (Melgert et al., 2005) Also, OVA rather than HDM model was used at the point of study because of the following reasons: (1) OVA does not occur as an allergen naturally, so it is easy to prevent the mice from OVA exposure prior to sensitisation; (2) OVA model is more well-characterised than HDM model
in terms of the participation of various leukocytes; and (3) OVA model allows the sensitisation period and activation of inflammatory response to be completely discerned (Blanchet et al., 2012; Brewer et al., 1999) Nonetheless, given the model’s growing popularity, it is likely that the detailed knowledge of the immunopathology induced by HDM will soon become equivalent to that of the OVA model (Blanchet et al., 2012)
ON-TARGETplus Rip-2 siRNA or control siRNA were prepared as mentioned in section
3.2.2 For drug treatment, Rip-2 siRNA also diluted to 1 nM using 1X PBS (tissue culture grade) The mice were anesthetized with aerosolised isofluorane (Halocarbon, NJ, USA) before Rip-2 siRNA (1 and 5 nmol) or control siRNA in 30 µl 1X PBS was administered
Trang 11intratracheally siRNA was given daily on days 19-21, and 2 h before each OVA aerosol challenge on days 22-24 Fisetin (0.3, 1, and 3 mg/kg; Sigma-Aldrich, St Louis, MO) or vehicle (8 µl DMSO in a total of 20 µl saline) was given by intravenous injection into the tail vein of the mice 2 h before each OVA aerosol challenge on days 22-24 Saline aerosol was used as a negative control Mice were sacrificed 24 h after the last aerosol challenge and various samples such as lung, BALF (BALF) and serum were collected for subsequent studies
3.5 Collection of BALF from mice
BALF was collected 24 h after the last saline or OVA challenge from all four treatment groups of mice Mice were anaesthetized by an intraperitoneal injection of 300 ul of mice anesthetic mixture (ketamine: medetomidine: H2O = 3:4:3) Tracheotomy was carried out and
a cannula (20G) was inserted into the trachea Ice cold 1 X PBS (0.5 ml X 3) was instilled into the lungs and a final volume of approximately 1.3 ml of BALF was retrieved from the lungs
3.6 Preparation of BALF for total and differential cell count
The BALF was centrifuged at 3000 rpm for 5 min at 4 °C Supernatant was collected and stored at -80°C for further analysis The pellet was resuspended in 200 µl of 8.5 mg/ml
NH4Cl (BDH Laboraties supplies, Poole, England) for 5 min at room temperature to remove red blood cells The cell suspension was centrifuged at 3000 rpm for 5 min at 4°C and the
Trang 12Figure 3.1 OVA aerosolization system (Taken from Cheng, 2011) The ultrasonic nebulizer (Sunrise Medical Respiratory Products, Somerset, PA) aerosolizes 1%
OVA into aerosol mist (particle size less than 5 µM) The aerosolized OVA was transferred
into the aerosol chamber through a tube Mice in the chamber were then exposed to the
aerosolized OVA
Trang 13supernatant was discarded The cell pellet was then resuspended in 200 µl of RPMI supplemented with 10 mg/ml bovine serum albumin (BSA) (Sigma-Aldrich,St Louis, MO)
A total number of viable cells was enumerated using a haemocytometer (10 µl cell suspension:
10 µl 0.4% trypan blue) under a light microscope (magnification ×100) Following the total cell count, aliquots (105 cells/150 µl) of the cell suspension were cytospined onto a slide in a Shandon Cytospin 3 (Thermo Electron Corporation, Pittsburgh, PA) at 600 rpm for 10 min at room temperature The BALF cells were stained with Liu stain (modified Wright stain) Briefly, cell smear was stained with 800 µl of Liu A for 30 sec followed by 1600 µl of Liu B for 90 sec Differential cell count was then performed on a minimum of 500 leukocytes under oil immersion lens (magnification ×1000) Four types of inflammatory cells —eosinophils, macrophages, neutrophils, and lymphocytes — were identified and their respective percentage in the total inflammatory cells was enumerated, based on standard morphological criteria and staining (Figure 3.1) The cell count was performed on a single blinded manner
to eliminate bias The absolute number of four types of inflammatory cells was calculated by their percentages and total inflammatory cell count
3.7 Histological examination
Lungs were isolated from the thoracic cavity
24 h after the last OVA or saline challenge The isolated lungs were fixed in 10 % neutral buffered formalin solution for at least 48 h, and processed in a tissue processor (Leica Microsystems, Wetzler, Germany) Briefly, lungs were dehydrated in a serial concentration of ethanol mixtures (70% 80% 90% 100%) 30 min each and 1 h for 100%, and were immersed in xylene for 10.5 h Lungs were then infiltrated with hot paraffin for 3 h and embedded in paraffin wax The specimens were then section into 4 µm sections using a microtome (Leica Microsystems, Wetzler, Germany) The sectioned tissues were then placed and dried on glass slides before staining
Trang 14Figure 3.2 Types of inflammatory cells in mouse BALF (A) Representative diagram of BALF collected from OVA sensitised and saline challenged mice (negative control) (B) Representative diagram of BALF collected from OVA sensitised and OVA challenged mice (positive control)
Abbreviations: Mac, macrophages; Eos, eosinophil; Lym, lymphocyte; Neu, neutrophil
Lym Eos
Neu Mac