Bene?t of combined therapy with nicorandil and colchicine in preventing monocrotaline-induced rat pulmonary arterial
Fan-Yen Lee a ,Hung-I Lu a ,1,Yen-Yi Zhen b ,Steve Leu c ,Yung-Lung Chen b ,Tzu-Hsien Tsai b ,Sheng-Ying Chung b ,Sarah Chua b ,c ,Jiunn-Jye Sheu a ,Shu-Yuan Hsu d ,Hsueh-Wen Chang e ,Cheuk-Kwan Sun f ,g ,2,Hon-Kan Yip b ,c ,?
Division of Thoracic and Cardiovascular Surgery,Department of Surgery,Kaohsiung Chang Gung Memorial Hospital,Chang Gung University College of Medicine,Kaohsiung,Taiwan b
Division of Cardiology,Department of Internal Medicine,Kaohsiung Chang Gung Memorial Hospital,Chang Gung University College of Medicine,Kaohsiung,Taiwan c
Center for Translational Research in Biomedical Sciences,Kaohsiung Chang Gung Memorial Hospital,Chang Gung University College of Medicine,Kaohsiung,Taiwan d
Department of Anatomy,Graduate Institute of Biomedical Sciences,Chang Gung University Medical College,Taoyuan,Taiwan e
Department of Biological Sciences,National Sun Yat-Sen University,Kaohsiung,Taiwan f
Department of Emergency Medicine,E-Da Hospital,I-Shou University,Kaohsiung,Taiwan g
This study tested the hypothesis that combined therapy with nicorandil and colchicine is superior to either alone in attenuating monocrotaline (MCT)-induced rat pulmonary arterial hypertension (PAH).Adult male Sprague–Dawley rats (n =50)were equally randomized into group 1(sham control),group 2[MCT (60mg/kg i.p.)],group 3[MCT–Nicorandil (5.0mg/kg/day)],group 4[MCT-Colchicine (1.0mg/kg/day)],and group 5(MCT–Nicorandil–Colchicine).Drugs were given on day 5.All animals were sacri-?ced on day 90after MCT administration.Right ventricular systolic blood pressure (RVSBP)and RV weight were increased in group 2compared to group 1,reduced in groups 3and 4compared to group 2,and further reduced in group 5,whereas arterial-oxygen saturation showed an opposite pattern (all p <0.001).Pulmonary damage severity (thickened alveolar septum and pulmonary arteriolar wall,decreased alveolar-sac numbers),number of CD3+cells,and protein expressions of in?ammatory (MMP-9,NF-j B,VCAM-1,angiotensin II-receptor),apoptotic (Bax,caspase 3,cleaved PARP),and ?brotic (TGF-b ,Smad3)biomarkers showed an identical pattern compared to that of RVSBP,whereas pulmonary expressions of anti-apoptotic (Bcl-2)and anti-?brotic (BMP-2,Smad1/5)biomarkers displayed a reverse pattern (all p <0.01).The protein expressions of RV damage markers (BNP,caspase 3)were increased,whereas expression of biomarker for RV functional preservation (Cx43)was reduced in group 2compared with group 1,elevated in groups 3and 4compared to group 2,and further increased in group 5(all p <0.01).Combined therapy with nicorandil and colchicine is superior to either alone in attenuating MCT-induced PAH in rats.
ó2013Elsevier B.V.All rights reserved.
Pulmonary arterial hypertension (PAH)is a syndrome resulting from pulmonary arterial obstruction,restricted pulmonary arterial blood ?ow,and increased pulmonary vascular resistance (PVR)that ultimately lead to ventricular failure (McLaughlin et al.,2009;Voelkel et al.,2006).Despite the state-of-the-art therapy,the prognosis of PAH remains poor (Archer and Rich,2000;Humbert et al.,2004),with an approximately 15%mortality within one year (Archer and Rich,2000).
Studies have revealed that PAH is a panvasculopathy predomi-nantly affecting small pulmonary arteries (i.e.,‘‘resistance arter-ies’’)that play an essential role in regulating regional blood ?ow in lung parenchyma (Humbert et al.,2004;McLaughlin et al.,2009).Histopathological studies have further shown that PAH is characterized by a variety of arterial abnormalities,including inti-mal hyperplasia,medial hypertrophy,adventitial proliferation,thrombosis in situ,as well as varying degrees of in?ammation and plexiform arteriopathy (Eddahibi et al.,2002;Farber and Loscalzo,2004;McLaughlin et al.,2009;Pietra et al.,1989;Yuan
0928-0987/$-see front matter ó2013Elsevier B.V.All rights reserved.https://www.wendangku.net/doc/3915143682.html,/10.1016/j.ejps.2013.08.004
Medicine,Kaohsiung Chang Gung Memorial Hospital,123Dapi Road,Niao Sung Dist,Kaohsiung 83301,Taiwan.Tel.:+88677317123x2363;fax:+88677322402.
E-mail address:han.gung@https://www.wendangku.net/doc/3915143682.html, (H.-K.Yip).1
Equal contribution in this study compared with the corresponding author.
and Rubin,2005).Additionally,the key pathological mechanisms of PAH have been proposed to be an initial endothelial dysfunction, followed by an excessive proliferation and reduced apoptosis in pulmonary artery smooth muscle cells(PASMCs)(Farber and Loscalzo,2004;Pietra et al.,1989).
In vascular smooth muscle cells,the opening of adenosine tri-phosphate-sensitive potassium(K ATP)channels causes vasodilata-tion under physiological and pathophysiological conditions(Peng et al.,1996).PASMCs express several distinct types of K+channels, including ATP-sensitive K+(K ATP),Ca2+-activated K+(BKCa),and voltage-dependent K+(Kv)channels(Brayden,2002;Nelson, 1993;Yuan et al.,1998).A rise in[Ca2+]cyt in PASMCs due to de-creased activities of certain K+channel(e.g.,Kv1.5and Kv2.1), membrane depolarization(Bonnet et al.,2006;Yuan,1995;Yuan et al.,1998),and de novo over-expression of the anti-apoptotic protein survivin play a crucial role in causing excessive PASMC proliferation,thereby accelerating vascular remodeling and increasing PVR(Pietra et al.,1989;Yuan,1995;Yuan et al., 1998).Therefore,increasing K+channel activity for suppressing PASMC proliferation and enhancing PASMC apoptosis appear to be of therapeutic potential in inhibiting propagation of PAH.
Previous study has revealed that nicorandil,a unique hybrid vasodilator,achieves vasodilatation through its dual action of reg-ulating the ATP-sensitive K+(K ATP)channel opening(Sato et al., 2000;Taira,1989)and nitric oxide(NO)release(Taira,1989).Be-sides,evidence has also suggested that nicorandil is bene?cial to the cardiovascular system in several ways(Sato et al.,2000).How-ever,its potential pharmacological action of reducing PASMC pro-liferation in the setting of PAH has not been fully investigated (Hongo et al.,2005).Colchicine,a traditional medication com-monly used for pain relief in patients with acute gouty attacks, mainly targets at b-tubulin with which it forms a complex that pre-vents microtubule polymerization.At high concentrations,colchi-cine causes microtubule depolymerization(Terkeltaub,2009) that can result in cell-cycle arrest and ultimately programmed cell death(i.e.,apoptosis).However,whether colchicine therapy can inhibit PASMC proliferation has not been investigated.Therefore, using a monocrotaline(MCT)-induced rat PAH model,the present study tested the hypothesis that treatment with nicorandil and colchicine can effectively prevent MCT-induced rat PAH through inhibiting PASMC proliferation and enhancing apoptosis of hyper-trophic PASMC,respectively.This study further evaluated the ther-apeutic potential of combining nicorandil with colchicine in attenuating MCT-induced rat PAH in comparison with either treat-ment option alone.
All animal experimental procedures were approved by the Insti-tute of Animal Care and Use Committee at Chang Gung Memorial Hospital–Kaohsiung Medical Center(Af?davit of Approval of Ani-mal Use Protocol No.2011092603)and performed in accordance with the ARRIVE guidelines for the Care and Use of Laboratory Ani-mals(NIH publication No.85-23,National Academy Press,Wash-ington,DC,USA,revised1996).The authors of this manuscript have certi?ed that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology(Coats and Shewan,2011).
On day0,40pathogen-free,adult male Sprague–Dawley(SD) rats,weighing300–320g(Charles River Technology,BioLASCO Co.,Ltd.,Taiwan),were given one subcutaneous injection of MCT (60mg/kg;Sigma,St.Louis,MO).These MCT-treated animals were then assigned to four groups:group2(MCT alone,n=10),group3 (MCT+nicorandil,n=10),group4[MCT+colchicine,n=10], group5(MCT+nicorandil and colchicine,n=10).Another group of ten SD rats(group1,n=10)receiving only subcutaneous injec-tion of3mL normal saline served as sham controls.
The dosages of MCT(60mg/kg)and nicorandil(5.0mg/kg/day) were based on our previous studies(Sun et al.,2009;Yen et al., 2010)and an investigation by other authors(Hongo et al.,2005), respectively.In addition,to assess the safety and effectiveness of colchicine treatment against MCT-induced rat PAH,two dosages of colchicine(i.e.,1.0mg and2.0mg/kg/day)were tested in four rats(two in each dosage group)by oral administration on day5 after MCT induction for5days.On day28,the right ventricular sys-tolic blood pressure(RVSBP)was measured in all four rats.The re-sults of that pilot study showed no signi?cant difference between the two dosages on RVSP(36mmHg vs.38mmHg).Therefore,to avoid the potential side effect of the higher dosage of colchicine, the lower dosage of1.0mg/kg/day was utilized in the present study.These two drugs were administered orally on day5after MCT injection based on our previous study(Sun et al.,2009)that demonstrated the effectiveness of early treatment in reducing MCT-induced rat PAH.
2.3.In vitro study of the pharmacobiological effects of colchicine and nicorandil on smooth muscle cells(SMCs)
To determine the effects of colchicine on inhibiting in?amma-tion,different concentrations of colchicine(0,5,and10l M)were used for co-culturing with SMCs of rat thoracic aorta(A7R5cells, a SMC cell line from the Food Industry Research and Development Institute,Taiwan)with a cell number of5?104for each study for 72h.After3h of colchicine co-culturing,lipopolysaccharide(LPS) (25ng/mL)was then added and the co-culture continued for 69h.Additionally,to evaluate the effect of colchicine on inhibition of SMC growth,colchicine(10l M)was only used for co-culturing with the SMCs for96h.
Similarly,to determine the in?uence of nicorandil on SMCs in term of anti-in?ammation,A7R5cells were co-cultured with dif-ferent concentrations of nicorandil(0,25,and50l M)for72h. LPS(25ng/mL)was then added3h after co-culturing SMCs with nicorandil and the cultivation continued for69h.Additionally,to measure the impact of nicorandil on inhibiting SMC growth,nico-randil(50l M)were only used for co-culturing with the SMCs for 96h.
To determine the effect of colchicine on arresting SMC cycle, SMCs were co-cultured with colchicine(10l M)for36h with and without LPS(25ng/mL)stimulation.Flow cytometry was uti-lized for analyze cells in G0/G1phase,S-phase,and G2/M phase.
The protocol and procedure of hemodynamic measurement were previously described(Sun et al.,2009;Yen et al.,2010).On day90after MCT treatment,the rats were anesthetized by inhala-tion of2.0%iso?urane.Blood(1.0mL)was sampled from the tail artery for measuring arterial oxygen saturation.After being shaved on the chest,each animal was endotracheally intubated with posi-tive-pressure ventilation(180mL/min)with room air using a small animal ventilator(SAR-830/A,CWE Inc.,USA).The heart was ex-posed by left thoracotomy.A sterile20-gauge,soft-plastic coated needle was inserted into the right ventricle and femoral artery of each rat to measure the RVSBP and arterial systolic blood pressure, respectively,when heart rate>310beat/min.The pressure signals were?rst transmitted to a pressure transducer(UFI,model1050,
CA,USA)and then exported to a bridge ampli?er (ML866PowerLab 4/30Data Acquisition Systems.ADInstruments Pty Ltd.,Castle Hill,NSW,Australia)where the signals were ampli?ed and digitized.The data were recorded and later analyzed with the Labchart soft-ware (ADInstrument).After hemodynamic measurements,the rats were euthanized with the hearts and lungs harvested.For each ani-mal,the whole heart weight,right ventricular weight,the lung weight and body weight were recorded from which the ratio of right ventricle-to-whole heart weight and that of right ventricle-to-interventricular septal plus left ventricular weight were calcu-lated.The preparation of lung specimens for morphometric analy-ses were based on our recent reports (Sun et al.,2009;Yen et al.,2010).Brie?y,the left lung were in?ated at a constant airway pres-sure (15–20mmHg)and ?xed with OCT (Tissue-Tek)for immuno-histochemical staining.The right lung was then cut into pieces that were either ?xed in 4%paraformaldehyde/0.1%glutaradehyde PBS solution before being embedded in paraf?n blocks for hematoxy-lin–eosin staining or stored at à80°C for protein and mRNA analyses.
Equal amounts (50l g)of protein extracts were loaded and sep-arated by SDS–PAGE using acrylamide gradients.After electropho-resis,the separated proteins were transferred electrophoretically to a polyvinylidene di?uoride (PVDF)membrane (Amersham
Biosciences).Nonspeci?c sites were blocked by incubation of the membrane in blocking buffer [5%nonfat dry milk in T-TBS (TBS containing 0.05%Tween 20)]overnight.The membranes were incubated with the indicated primary antibodies [vascular cell adhesion molecule (VCAM)-1(1:100,Abcam),tumor necrosis fac-tor (TNF)-a (1:1000,Cell Signaling),nuclear factor (NF)-j B (1:1000,Abcam),Bax (1:1000,Abcam),caspase 3(1:1000,Cell Sig-naling),poly(ADP-ribose)polymerase (PARP)(1:1000,Cell Signal-ing),protein expressions of Bcl-2(1:200,Abcam),phosphorylated (p)-Smad3(1:1000,Cell Signaling),p-Smad1/5(1:1000,Cell Sig-naling),bone morphogenic protein (BMP)2(1:5000,Abcam)trans-forming growth factor (TGF)-b (1:500,Abcam),brain natriuretic peptide (BNP)(1:500,Abcam),Connexin (Cx)43(1:2000,Millipore)and Actin (1:10,000,Chemicon)]for 1h at room temperature.Horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (1:2000,Cell Signaling)was used as a secondary antibody for 1h at room temperature.The washing procedure was repeated eight times within 1h,and immunoreactive bands were visualized by enhanced chemiluminescence (ECL;Amersham Biosciences)and exposed to Biomax L ?lm (Kodak).For purposes of quanti?ca-tion,ECL signals were digitized using Labwork software (UVP).2.6.Distribution of alveolar sacs,crowded score,and arterial muscularization in lung parenchyma
Left lung specimens from all animals were ?xed in 10%buffered formalin before being embedded in paraf?n and sectioned at 5l
studies of A7R5cell proliferation,cell-cycle arrest,and immuno?uorescent (IF)staining.(A)Time courses of smooth muscle cell (SMC)proliferation control,SMC growth was signi?cantly inhibited by nicorandil at a concentration of 25l M and further signi?cantly inhibited by colchicine vs.àvs.–,p <0.01;(2) vs.àvs.–,p <0.001;(3) vs.àvs.–,p <0.0001.(B)Flow cytometric analysis showed an elevation of A7R5cell population following LPS treatment.However,the number of apoptotic cells (sub-G1phase)was not increased after LPS treatment.Additionally,further culturing colchicine for 36h revealed not only remarkable G2/M phase cell-cycle arrest,but also an increment of cellular apoptosis (sub-G1phase).distortion of tubulin structure and actin integrity (i.e.,cell cytoskeleton)in low-dose colchicine (10l M)treatment group compared to the
for light microscopic analysis.Hematoxylin and eosin (H&E)stain-ing was performed to determine the number of alveolar sacs according to our recent study (Sun et al.,2009)in a blind fashion.Three lung sections from each rat were analyzed and three ran-domly selected HPFs (100?)were examined in each section.The number of alveolar sacs was recorded for each animal.The mean number per HPF for each animal was then determined by summa-tion of all numbers divided by 9.
The percentage of crowded area (de?ned as septal thickness associated with partial or complete collapse of alveoli)in lung parenchyma was determined using H&E staining in a blind fashion and scored as follows:0=no detectable crowded area;1615%
and nicorandil on in?ammatory reactions in smooth muscle cell (SMC)(A7R5cell line)with and protein expressions of tumor necrosis factor (TNF)-a (A)and nuclear factor (NF)-j B (B)were signi?cantly stimulation.*vs.other groups with different symbols (*, ,à),p <0.001.(C and D)The protein expressions nicorandil treatment with or without LPS stimulation.*vs.other groups with different symbols (*, ,à),p
Summarized data of body weight,survival rate,RV weight,ratio of RV to LV &septal weight,and hemodynamics in ?ve groups of animals on day 42after monocrotaline administration.Variables
Group 1àGroup 2àGroup 3àGroup 4àGroup 5àp Value *Body weight (g)
545±44a 398±46b 435±47b 443±50b 454±30b <0.0001Mortality rate (number)0%(0)
30%(3)
30%(3)
10%(1)
0%(0)
0.144Total heart weight (g) 1.62±0.19 1.67±0.19 1.55±0.11 1.61±0.25 1.58±0.210.893RV weigh (g)
0.25±0.04a 0.54±0.08b 0.41±0.08c 0.42±0.07c 0.34±0.04a,c <0.0001LV +septum weight (g)** 1.17±0.17a 0.94±0.07b 0.94±0.04b 0.94±0.11b 0.95±0.11b 0.010Ratio of RV to LV +septum 0.22±0.04a 0.59±0.07b 0.44±0.09c 0.45±0.08c 0.35±0.08d <0.0001Sat O 2(%)
99.4±1.4a 71.3±3.9b 81.4±1.8c 80.7±4.1c 87.8±3.5d <0.0001RVSBP (mmHg)24.1±2.5a 62.7±7.4b 48.8±8.9c 49.4±8.9c 39.2±3.5d <0.0001FASBP (mmHg)
132.7±12.6
124.4±20.4
126.1±13.8
130.1±13.7
128.9±14.6
0.887
Different letters (a,b,c,d)being used for grouping,showing signi?cant difference (at level 0.05)among different groups by Tukey’s multiple comparison procedure.
RV =right ventricular;LV =left ventricular;Sat O 2=arterial oxygen saturation;RVSBP =right ventricular systolic blood pressure;FASBP =femoral arterial systolic blood pressure.*
By one-way ANOVA.**
Analyzed using log transform method to improve normality.
n =7in each group.à
Group 1=sham control;group 2=monocrotaline (MCT)treated only;group 3=MCT +nicorandil;group 4=MCT +colchicine;group 5=MCT +nicorandil +colchicine.
crowded area;2=15–25%of crowded area;3=25–50%of crowded area;4=50–75%of crowded area;5P 75%-100%of crowded area/per high-power ?eld (100?)].The procedure and protocol were based on our recent report (Chen et al.,2011).
The detailed procedure and protocol for determining muscular-ization (i.e.,an index of vascular remodeling)of pulmonary arteri-oles was described in details in our recent report (Yen et al.,2010).Brie?y,three measurements were taken for the thickness of pul-monary arterioles.Muscularization of the arterial medial layer in lung parenchyma was de?ned as a mean thickness of vessel wall greater than 50%of the lumen diameter in a vessel of diameter >30l m.Measurement of arteriolar diameter and wall thickness was achieved using the Image-J software (NIH,Maryland,USA).
2.7.Distribution of small vessels in lung parenchyma
The detailed procedure and protocol have been described in our recent reports (Chen et al.,2011;Sun et al.,2009;Yen et al.,2010).Brie?y,immunohistochemical staining of a -smooth muscle actin using the appropriate antibodies (Sigma)was performed to iden-tify pulmonary arterioles according to manufacturer’s instructions.Three lung sections from each rat were analyzed and three ran-domly selected HPFs (100?)were analyzed in each section.The
mean number per HPF for each animal was then determined by summation of all numbers divided by 9.
2.8.Immunohistochemical (IHC)staining of CD3+cell
Serial 4l m-thick cryosections of lung parenchyma with an average distance of 5l m apart were ?xed in acetone for 15min at à20°C.IHC staining was performed by incubating the tissue sections with an anti-CD3+primary antibody (1:100)at room tem-perature for 1h,followed by washing with PBS thrice.Ten minutes after addition of the anti-mouse-HRP conjugated secondary anti-body,the tissue sections were washed with PBS thrice again.Then 3,30diaminobenzidine (DAB)(0.7g/tablet)(Sigma)was used for colorizing signals,followed by counter-staining with hematoxylin.2.9.Statistical analysis
Quantitative data are expressed as means ±SD.Statistical anal-ysis was adequately performed by ANOVA followed by Bonferroni multiple-comparison post hoc test.Statistical analysis was per-formed using SAS statistical software for Windows version 8.2(SAS institute,Cary,NC).A probability value <0.05was considered statistically
signi?cant.
(IHC)staining of lung tissue on day 90after monocrotaline (MCT)treatment (n =7).IHC staining (a -smooth muscle (<100l m)(red arrows)microscopically (100?)in (A)sham control,(B)monocrotaline (MCT)treatment,MCT +Nico +Col on day 90following MCT treatment.(F)*vs.other groups with different symbols (*, ,àby Bonferroni multiple comparison post hoc test.Symbols (*, ,à,§)indicate signi?cance (at 0.05level).high-power ?led.n =7per each group.
3.Results
3.1.Colchicine treatment inhibited smc proliferation and in?ammation,and arrested SMC cycle (Figs.1and 2)
As expected,proliferation of SMCs (i.e.,A7R5cells)was signi?-cantly inhibited by colchicine treatment compared to that in nor-mal controls and nicorandil-treated groups,especially at 72h of cell culturing (Fig.1-A).
Flow cytometry-based cell cycle analysis (Fig.1-B)showed an ele-vation in the population of S and G2/M phase cells following LPS treatment,indicating an increased cellular proliferation.However,the number of apoptotic cells (sub-G1phase)was not increased after LPS treatment.To determine the ef?cacy of colchicine in triggering cell-cycle arrest in smooth muscle cells,LPS-treated A7R5cells were further cultured with colchicine for 36h.The results demonstrated that an additional treatment with colchicine not only signi?cantly induced G2/M phase cell-cycle arrest,but also resulted in an incre-ment of cellular apoptosis (sub-G1phase).
Additionally,immuno?uorescent staining identi?ed marked distortion of tubulin structure and actin integrity (i.e.,cytoskele-ton)in low-dose (10l M)colchicine treatment group (Fig.1-C)than in controls at 72h of cell culturing.
Western blot analysis (Fig.2)on cultured SMCs revealed that the protein expressions of TNF-a (Fig.2-A)and NF-j B (Fig.2-B),two indexes of in?ammation,were signi?cantly reduced with pro-gressive increase in colchicine concentration at 72h of cell cultur-ing.The expressions of these two parameters were also signi?cantly suppressed by colchicine treatment even in the
presence of LPS stimulation.The results of these in vitro studies suggest that colchicine therapy not only inhibited cell growth and in?ammation,but also arrested cell cycle of SMC.
3.2.Inhibition of in?ammatory reaction and SMC proliferation after nicorandil treatment (Figs.1and 2)
Similar to the action of colchicine,the proliferation of SMC was also signi?cantly inhibited by nicorandil treatment compared to that in the control group,especially at 72h of cell culturing (Fig.1-A).Moreover,the protein expressions of TNF-a (Fig.2-C)and NF-j B (Fig.2-D)were signi?cantly decreased in SMCs with progressive increase in nicorandil concentration at 72h of cell cul-turing.The expressions of these two in?ammation biomarkers were also signi?cantly suppressed by nicorandil treatment even in the presence of LPS stimulation.These ?ndings suggest that nic-orandil treatment attenuated in?ammation and SMC proliferation.3.3.Survival rate,arterial oxygen saturation,hemodynamics,and ratio of RV weight to LV +septal weight by day 90after MCT administration (Table 1)
During the follow-up of 90days,8of the 60animals having re-ceived MCT died,including 0in group 1(sham control),3in group 2(MCT only),3in group 3(MCT +nicorandil 5mg/kg/day),2in group 4(MCT +colchicine 1mg/kg/day),and 0in group 5(MCT +nicorandil +colchicine).However,statistical analysis dem-onstrated no signi?cant difference in mortality rate among the ?ve groups (p =0.114)
.
Histopathological changes of lung parenchyma on day 90after monocrotaline (MCT)treatment (n =7)(A–E)Number of alveolar sacs under microscope treatment (n =7).Markedly reduced number of alveolar sacs in MCT-treated group compared with other groups (H&E).More compact septum,remarkably thickened pulmonary arterioles (PA),and also notably increased number of muscularized PA in MCT-treated group lower corners represent 100l m.(F)Statistical analysis of number of alveolar sacs among ?ve groups.*vs.other groups with different Statistical analysis of crowded score among ?ve groups.*vs.other groups with different symbols (*, ,à,§),p <0.0001.H&I)Semi-quantitative (H)and number of muscularized PA (I)under higher magni?cations [(a),(c),(e),(g),and (i)being magni?ed images of (b),and I)*vs.other groups with different symbols (*, ,à,§),all p <0.0001.Scale bars in right lower corners represent 100l m.All statistical followed by Bonferroni multiple comparison post hoc test.Symbols (*, ,à,§)indicate signi?cance (at 0.05level).Nico =nicorandil,
The ?nal body weight was signi?cantly higher in group 1than in groups 2to 5that showed no difference among themselves.In addition,the total heart weight did not differ among the ?ve groups.On the other hand,the RV weight was highest in group 2,signi?cantly higher in groups 3and 4than in group 1,but showed no difference among groups 3,4,and 5,or between groups 1and 5.The left ventricular-septal weight was signi?cantly higher in group 1than in groups 2–5,but it was similar among these four groups.Consistently,the ratio of RV weight to left ventricular-sep-tal weight was lowest in group 1and highest in group 2,signi?-cantly higher in groups 3and 4than in group 5,but there was no difference between groups 3and 4.The percentage of arterial oxygen saturation (Sat O 2,%)was highest in group 1,signi?cantly higher in group 5than that in groups 2,3and 4,and signi?cantly higher in groups 3and 4than that in group 2,but it showed no dif-ference between groups 3and 4at day 90after MCT-induced PAH.By contrast,RVSBP showed opposite changes compared with that of Sat O 2among the ?ve groups.However,the femoral arterial sys-tolic blood pressure did not differ among all groups,suggesting that nicorandil treatment had no signi?cant impact on arterial blood pressure.
3.4.Histopathological ?ndings:numbers of small arteriolar density,alveolar sacs,muscularized arterioles,and crowed score in lung parenchyma by day 90after MCT administration (Figs.3and 4)Immunohistochemical staining for a -smooth muscle actin dem-onstrated that the number of arterioles in lung parenchyma was lowest in group 2and highest in group 1,signi?cantly lower in groups 3and 4than in group 5,but no signi?cant difference be-tween groups 3and 4(Fig.3).
The number of alveolar sacs was highest in group 2than in other groups,signi?cantly increased in groups 3and 4than in groups 1and 5,and signi?cantly higher in group 5than in group 1,but no difference was noted between groups 3and 4(Fig.4-F).Conversely,the thickness of alveolar septum showed an opposite pattern among the ?ve groups (Fig 4A–E).Microscopically,the lung parenchyma was most crowded in group 2,signi?cantly crowed in groups 3and 4than that in groups 1and 5,and signi?cantly cro-wed in group 5than that in group 1,but no signi?cant difference was noted between groups 3and 4(Fig.4-G).Moreover,histopa-thological analysis using H&E staining revealed that the percentage of thickened arterioles (Fig.4-H)and the number of
muscularized
Changes in protein expressions of in?ammatory and vasodilatation biomarkers in right ventricle and lung on day 90after monocrotaline (MCT)treatment expressions of (A)tumor necrosis factor (TNF)-a ,and (B)nuclear factor (NF)-j B in right ventricle (RV)were highest in untreated MCT group and lowest signi?cantly higher in MCT +nicorandil (Nico)and MCT +colchicine (Col)groups than in MCT +Nico +Col group;*vs.other groups with different symbols The protein expression of endothelial nitric oxide synthase (eNOS)in RV (C)and in lung (D)was highest in normal controls and lowest in untreated lower in MCT +Nico and MCT +Col groups than in MCT +Nico +Col group;*vs.other groups with different symbols (*, ,à,§),all p <0.001.Protein metalloproteinase (MMP)-9,(F)NF-j B,(G)angiotensin II receptor (Ang II-R),and (H)vascular cell adhesion molecule (VCAM)-1in lungs were MCT group and lowest in normal controls,signi?cantly higher in MCT +Nico and MCT +Col groups than in MCT +Nico +Col group;*vs.other groups à,§,–),all p <0.001.All statistical analyses using one-way ANOVA,followed by Bonferroni multiple comparison post hoc test.Symbols (*, ,à(at 0.05level).
arterioles (Fig.4-I)exhibited an identical pattern compared to that of the crowded score among the ?ve groups.3.5.Protein expressions of in?ammatory and anti-in?ammatory biomarkers in lung and right ventricle at day 90after MCT administration (Figs.5and 6)
The protein expressions of TNF-a (Fig.5-A)and NF-j B (Fig.5-B)in right ventricle and the protein expressions of MMP-9(Fig.5-E),NF-j B (Fig.5-F),and angiotensin II-R (Fig.5-G)in lung paren-chyma,the biomarkers of in?ammation,were highest in group 2and lowest in group 1,and signi?cantly higher in groups 3and 4than that in group 5,but it showed no difference between groups 3and 4(Fig.5).Consistently,the protein expression of VCAM-1(Fig.5-H)in lung parenchyma,another index of in?ammatory mediator,was highest in group 2,lowest in group 1,signi?cantly higher in groups 3and 4than that in group 5,and higher in group 3than 4.By contrast,the protein expression of eNOS (Fig.5C and D),an inhibitor of in?ammation,smooth cell proliferation,and vasoconstriction in both lung parenchyma and right ventricle showed an opposite pattern compared to that of the in?ammatory biomarkers among the ?ve groups.
IHC staining for the number of CD3+cells,an index of in?am-matory cells,in lung parenchyma demonstrated an identical
pattern compared to that of pulmonary in?ammatory protein expressions among the ?ve groups (Fig.6).
3.6.Protein expressions of cleaved caspase 3,BNP,and Cx43in right ventricle and protein expressions of apoptotic and anti-apoptotic biomarkers in lung parenchyma at day 90after MCT administration (Fig.7)
The protein expression of cleaved caspase-3(Fig.7-A),an index of activated apoptosis,and BNP (Fig.7-B),an index of RV pressure overload,were highest in group 2and lowest in group 1,and sig-ni?cantly higher in groups 3and 4than that in group 5,but no sig-ni?cant difference was noted between groups 3and 4.On the other hand,the protein expression of Cx43(Fig.7-C),a component of gap junction responsible for cell-to-cell communication among cardio-myocytes,displayed an inversed pattern compared with that of BNP among the ?ve groups.
The protein expressions of cleaved caspase 3(Fig.7-D)and mitochondrial Bax (Fig.7-E)in lung tissue,two indexes of apopto-sis,were highest in group 2,signi?cantly higher in groups 3and 4than that in groups 1and 5,and signi?cantly higher in group 5than that in group 1,but it exhibited no difference between groups 3and 4.By contrast,the protein expression of Bcl-2(Fig.7-F)in lung parenchyma,an indicator of anti-apoptosis,showed
an
(IHC)staining for pulmonary CD3+cells on day 90after monocrotaline (MCT)administration (n =7).(A–E)in lung parenchyma of the ?ve groups.Scale bars in right lower corner represent 50l m.(F)*vs.other groups using one-way ANOVA,followed by Bonferroni multiple comparison post hoc test.Symbols (*, ,à,§)indicate
opposite manner compared with that of apoptotic biomarkers among the ?ve groups.
3.7.Fibrotic,anti-?brotic,and proliferative biomarkers in lung parenchyma at day 42after MCT administration (Figs.8and 9)The protein expressions of TGF-b (Fig.8-A)and Smad3(Fig.8-B),two indicators of ?brosis,and Cx43(Fig.8-C),an indicator of ?bro-blast/smooth muscle cell proliferation,were signi?cantly enhanced in group 2than those in other groups,signi?cantly increased in groups 3and 4than those in groups 1and 5,and signi?cantly up-regulated in group 5than those in group 1,but it showed no difference between groups 3and
4.Conversely,the protein expressions of BMP-2and Smad1/5,two indicators of anti-?brosis,showed a reversed pattern compared to those of apoptotic biomarkers among the ?ve groups (Fig.8D and E).
IHC staining (i.e.,double-staining of Ki67and a -smooth muscle actin)for quantifying smooth muscle cell proliferation in the med-ial layer of larger pulmonary arterioles demonstrated an identical pattern compared to that of Cx43expression in lung parenchyma among the ?ve groups (Fig.9).Additionally,the Ki67+cells in lung parenchyma (i.e.,extra-pulmonary artery)also showed an identi-cal expression of intra-pulmonary arterial Ki67+cells.4.Discussion
This study,which investigated the impact of colchicine and nic-orandil treatment on MCT-induced PAH yielded several striking implications.First,one novel ?nding in the current study is that colchicine alone was effective in reducing MCT-induced PAH.Second,histopathological ?ndings demonstrated that combined
therapy with nicorandil and colchicine offered more bene?ts than either alone in reducing MCT-induced lung parenchymal injury.Third,combined therapy with nicorandil and colchicine was supe-rior to either alone in attenuating the propagations of in?amma-tion,apoptosis,and ?brosis in lung parenchyma as well as the proliferation of PASMCs after MCT administration.Besides,com-bined therapy with these two drugs provided an additional bene?t in reducing cellular apoptosis and BNP secretion as well as in pre-serving the integrity of Cx43gap function in right ventricle.Finally,of importance is the fact that the improvement of molecular-cellu-lar and pathological perturbations was translated into functional improvement (i.e.,reduced RVSBP and increased arterial oxygen saturation)in either monotherapy regimen and was further up-lifted in combined therapy with nicorandil and colchicine.
4.1.Impact of combined therapy with nicorandil and colchicine on functional improvement of lung and right ventricle
The most important ?nding in the present study is that hypox-emia [i.e.,decreased Sat O 2(%)],an index of pulmonary function,was signi?cantly aggravated in MCT-treated animals but was nota-bly alleviated through either nicorandil or colchicine treatment,and further improved by combined treatment with the two drugs.In addition,one distinctive ?nding in the present study is that RVSBP (i.e.,a direct indicator of pulmonary arterial SBP),another index of lung and RV functional integrity,was substantially ele-vated in MCT-treated animals and was remarkably reduced after either nicorandil or colchicine therapy and further reduced by combined treatment with these two drugs.Consistently,two pre-vious studies (Hongo et al.,2005;Sahara et al.,2012)have shown that nicorandil treatment signi?cantly attenuated MCT-induced
rat
Western blot analyses of apoptosis,pressure overload,component of gap-junction in right ventricle and apoptotic biomarkers in lung parenchyma at monocrotaline (MCT)administration (n =7).Protein expressions of (A)cleaved caspase 3(C-Casp3),and (B)brain natriuretic peptide (BNP)in right ventricle MCT group and lowest in sham control group,signi?cantly higher in MCT +nicorandil (Nico)and MCT +colchicine (Col)groups than in MCT +Nico +Col groups with different symbols (*, ,à,§),all p <0.001.The protein expression of connexin43(Cx43)in RV (C)was highest in normal controls and lowest in signi?cantly lower in MCT +Nico and MCT +Col groups than in MCT +Nico +Col group.*vs.other groups with different symbols (*, ,à,§),p <0.0001.of (D)C-Casp3,and (E)mitochondrial Bax in lung were highest in the untreated MCT group and lowest in normal controls,signi?cantly higher in MCT groups than in MCT +Nico +Col group.*vs.other groups with different symbols (*, ,à,§),for (D),p <0.0001;for (E),p <0.001.(F)The protein expression showed an opposite pattern compared to that of (E)and (D)among ?ve groups.*vs.other groups with different symbols (*, ,à,§),p <0.0001.Statistical analysis ANOVA,followed by Bonferroni multiple comparison post hoc test.Symbols (*, ,à,§)indicate signi?cance (at 0.05level).
vascular endothelial damage and PAH.Of interest in the present study is that not only was colchicine treatment alone effective in reducing MCT-induced hypoxemia and PAH,but combined therapy with nicorandil was also found to offer a greater bene?t.Accord-ingly,our ?ndings,in addition to extending those of previous stud-ies (Hongo et al.,2005;Sahara et al.,2012),also highlight the need for prospective clinical investigation into the therapeutic potential of combined therapy with these two drugs in patients with PAH.4.2.Impact of combined therapy with nicorandil and colchicine on lung parenchymal damage
One important ?nding in the current study is that nicorandil treatment remarkably reduced the crowed score,number of mus-cularized PA,and thickness of muscularized pulmonary arteriole and alveolar septum,and also preserved the number of alveoli and small vessels in lung parenchyma.Histopathological ?ndings from previous studies (Hongo et al.,2005;Sahara et al.,2012)have also demonstrated that nicorandil therapy effectively protected lung parenchymal injury from MCT-induced PAH.In this way,our ?ndings reinforced those of these studies (Hongo et al.,2005;Sahara et al.,2012).Interestingly,the therapeutic attempt of using
colchicine in the treatment of lung ?brosis has been studied by previous studies (Douglas et al.,1998;Selman et al.,1998).Surpris-ingly,the therapeutic role of colchicine in the setting of PAH has not been investigated.The novel ?nding of the present study is that the therapeutic action of colchicine was comparable to that of nic-orandil in preserving the number of alveolar sacs and small vessel in lungs,and protecting the lung parenchyma from MCT-induced PAH damage.Of particular importance is that combined therapy with nicorandil and colchicine was superior to either alone in reducing lung parenchymal damage.Therefore,our ?ndings ex-tended those of previous studies (Douglas et al.,1998;Hongo et al.,2005;Sahara et al.,2012;Selman et al.,1998).Furthermore,these could partially explain the most notable improvement in right ventricular and pulmonary functions after combined therapy with nicorandil and colchicine.
4.3.Impact of nicorandil and colchicine on in?ammation and SMC proliferation
Another important ?nding of in vitro study showed that either colchicine or nicorandil treatment inhibited SMC proliferation and in?ammation.Besides,colchicine treatment
induced
?brosis,anti-?brosis,and smooth muscle proliferation (Cx43)biomarkers in lung parenchyma at expressions of (A)transforming growth factor (TGF)-b ,(B)phosphorylated (p)-Smad3,and (C)connexin43(Cx43)controls,signi?cantly higher in MCT +Nico and MCT +Col groups than in MCT +Nico +Col group.*vs.other expressions of bone morphogenic protein (BMP)-2(D)and Smad1/5(E)exhibited a reversed pattern compared with different symbols (*, ,à,§),all p <0.001.Statistical analysis using one-way ANOVA,followed by Bonferroni indicate signi?cance (at 0.05level).
microtubule depolymerization and SMC-cell cycle arrest.In addi-tion,in vivo study demonstrated that either nicorandil or colchicine treatment signi?cantly attenuated in?ammation in lung paren-chyma and also SMC proliferation in pulmonary arterioles.Consis-tently,one recent study has shown that nicorandil therapy suppressed in?ammation and SMC proliferation of pulmonary arterioles in the setting of MCT-induced PAH in rats(Hongo et al.,2005).Furthermore,colchicine has been identi?ed to possess the property of inhibiting innate-mediated in?ammation and the generation of reactive oxygen species(ROS)(Sahara et al.,2012; Terkeltaub,2009).Our?ndings,therefore,corroborate those of previous studies(Hongo et al.,2005;Sahara et al.,2012;Terkel-taub,2009).Of importance is the fact that combined therapy with nicorandil and colchicine offered an additional bene?t compared to either regimen alone in reducing in?ammatory reaction and SMC proliferation.Accordingly,our results strengthened those of the previous studies(Hongo et al.,2005;Sahara et al.,2012;Terkel-taub,2009).Collectively,these?ndings could,at least in part,account for the remarkable reduction in pulmonary parenchymal damage through combined therapy with these two drugs.
4.4.Impact of combined therapy with nicorandil and colchicine on pulmonary apoptosis and?brosis
Apoptosis and?brosis from lung parenchymal damage have been well recognized in the setting of MCT-induced PAH in recent studies(Sun et al.,2009;Yen et al.,2010).An essential?nding in the present study is that the expressions of both apoptotic and?-brotic biomarkers were substantially increased in lung paren-chyma of MCT-treated animals.In this way,our?ndings were consistent with those of the recent studies(Sun et al.,2009;Yen et al.,2010).Of particular importance is that the elevations of these biomarkers were remarkably suppressed in animals treated with nicorandil or colchicine and further signi?cantly reduced following combined treatment.In concert with the results of the current study,two previous studies have reported reduction in pulmonary apoptosis and?brosis in the setting of MCT-induced PAH after nic-orandil treatment(Hongo et al.,2005;Sahara et al.,2012).There-fore,our?ndings not only support those of these studies(Hongo et al.,2005;Sahara et al.,2012),but also explain the preservation of pulmonary function after the combined regimen.
4.5.Impact of combined therapy with nicorandil and colchicine on functional integrity of RV
The link between hypoxia and pressure or volume overload of right ventricle and an increase in circulating level of BNP has been well recognized in previous studies(Arjamaa and Nikinmaa,2011; Cavallero et al.,2007;Irzmanski et al.,2012).Moreover,the down regulation of Cx43gap junction and up-regulation of apoptotic bio-markers in right ventricle have been demonstrated in recent stud-ies in animal model of MCT-induced PAH(Sun et al.,2009,2011; Yen et al.,2010).In concert with the?ndings of previous studies (Sun et al.,2009,2011;Yen et al.,2010),the results of the present study also demonstrated that the protein expression of Cx43was signi?cantly decreased,whereas that of cleaved caspase3and BNP was markedly increased in RV tissue in MCT-treated animals. The expressions of these biomarkers,however,were signi?cantly suppressed in animals treated with either nicorandil or colchicine and were further reduced by combined therapy with these two drugs.Taken together,our?ndings suggest that successful treat-ment of MCT-induced pulmonary damage was re?ected in signi?-cant preservation of RV functional integrity and alleviation of RVSBP.Intriguingly,a recent study has displayed that nicorandil therapy ameliorated PAH-induced RV remodeling in rats not only by attenuating pressure overload in RV but also by inhibiting apop-tosis in cardiomyocytes through the activation of mitochondrial ATP-sensitive K+(K ATP)(Zuo et al.,2012).In this way,our results and the results from this recent study(Zuo et al.,2012)are consistent.
4.6.Study limitation
This study has limitations.First,since the sample size of this study was relatively small,the mortality rate in each group of ani-mals may be over-estimated or vice versa.Second,although the hemodynamic and cellular-molecular biomarkers involved in MCT-induced lung injury were extensively investigated,the exact mechanisms by which MCT induced PAH and lung damage remain uncertain.Third,Studies have previously demonstrated that sus-tained pulmonary vasoconstriction was an essential component of MCT-induced PAH(Casey et al.,2010;Pankey et al.,2012).In the current study,we did not measure the effect of the drug treat-ment on regulating the vasoconstrictor component of the increased
Fig.9.Immunohistochemical(IHC)staining for pulmonary Ki67+cells on day90
after monocrotaline(MCT)administration(n=7)(A–E)IF staining for identi?cation
of accumulated Ki67+cells in pulmonary arterioles(PA)(yellow arrows)(400?
and extra-PA lung tissue(white arrows)among the?ve groups.Dotted-line square
magni?ed into solid-line square.DAPI counter-staining for nuclei(blue).Scale bars
in right lower corners represent20l m.(F)*vs.other groups with different symbols
, ,à,§),p<0.0001.(G)*vs.other groups with different symbols(*, ,à,§),
<0.001.Statistical analysis using one-way ANOVA,followed by Bonferroni
multiple comparison post hoc test.Symbols(*, ,à)indicate signi?cance(at0.05
level).
Sciences50(2013)372–384
pulmonary vascular resistance and PAH.Thus,we did not know whether both of colchicine and nicorandil therapy on reducing PAH was also through the mechanism of reducing pulmonary arte-rial vasoconstriction.5.Conclusion
Our results demonstrated that combined therapy with nicoran-dil and colchicine has an additive therapeutic action against MCT-induced rat PAH.The proposed mechanisms are summarized in Fig.10.Our experimental ?ndings may warrant further investiga-tion into the potential application of this therapeutic strategy in patients with PAH.Acknowledgment
This study was supported by a program Grant from Chang Gung Memorial Hospital,Chang Gung University (Grant Number:CMRPG8A0091).In addition,?nancial support from E-Da Hospital research grants EDPJ100032and EDPJ101033are gratefully acknowledged.References
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Fig.10.Proposed mechanisms underlying the effects of different regimens on monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH)in a rat model based ?ndings of the present study.ROS =reactive oxygen species;TGF-b =transforming growth factor-b ;p =phosphorylated;BMP-2=bone morphogenic protein-2;Cx43=connexin43;TNF-a =tumor necrosis factor-a ;NF-j B =nuclear factor-j B;RV =right ventricle;eNOS =endothelial nitric oxide synthase;VCAM-1=vascular cell adhesion molecule-1;MMP-9=matrix metalloproteinase;PA =pulmonary arterioles;SMC =smooth muscle cell;C-casp3=cleaved caspase 3;Ang II-R =angiotensin receptor;Sat O 2(%)=%of oxygen saturation;RVSB =right ventricular systolic blood pressure;LV =left ventricular.
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四川省绵阳南山中学实验学校2019-2020学年高三10月月考数学(理)试题及答案
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1999-2012年知识产权司考真题及答案解析
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A. 同一电源接入不同的电路电动势会发生改变 B. 电源电动势就是接入电源两极间的电压表测量的电压 C. 电源电动势是表征电源把其它形式的能转化为电能本领的物理量，与是否接外电路无关 D. 电源电动势与电势差是一回事 4.用伏安法测电阻时，若不知Rx 的大概值，为了选择正确的电路以减小误差，可以用图所示的方法判断( ) A ．若安培表的示数有显著变化，K 应接a B ．若安培表的示数有显著变化，K 应接b C ．若伏特表示数变化的差值大于安培表示数变化的差值，K 应接a D ．若伏特表示数有显著变化，K 应接b 5.如图所示，平行板电容器与电动势为E 的直流电源（内阻不计）连接，下极板接地．一 带电油滴位于电容器中的P 点且恰好处于平衡状态．现将平行板电容器的上极板竖直向上 移动一小段距离( ) A ．P 点的电势将降低 B ．带电油滴将沿竖直方向向上运动 C ．带电油滴的电势能将减少 D ．若电容器的电容减小，则极板带电量将增大 6.在某静电场中P 点放一个带负电的检验电荷q ，它所受的电场力为F ，则关于P 点的电场强度E ，正确的说法是( ) A ．E=F/q ，方向与F 相同 B ．若取走q ，P 点的电场强度E=0 C ．若在P 点放一个2q 的检验电荷，则P 点的电场强度将变为原来的2倍 D ． E 的大小与检验电荷无关 7.一负电荷从电场中A 点由静止释放，只受电场力作用，沿电场线运动到B 点，它运动的 速度一时间图象如图甲所示．则A 、B 两点所在区域的电场线分布情况可能是图乙中的( ) t C D B A 乙
2013年全国高考语文古诗鉴赏题汇总
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全国卷2 阅读下面这首宋诗，完成8-9题。 次韵雪后书事二首(其一) 朱熹 饲怅江头几树梅，杖藜行绕去还来。 前时雪压无寻处，昨夜月明依旧开。 折寄遥怜人似玉，相思应恨劫成灰。 沉吟日落寒鸦起，却望柴荆独自回。 8.这首咏梅诗中，作者用什么手法来表现梅花的？请简要分析（5分） 运用了烘托和渲染的手法，全诗几乎未涉及梅花的色香，而注重环境的烘托和情感的渲染，从而表现美化的精神和品格。（烘托、渲染，2分，简要分析3分。） 9诗的最后一联表达了作者什么样的心情？请简要分析。（6分）答：表现了作者落寞惆怅、若有所失的心情，作者将自己复杂的情感投射到梅花上，思绪万端却又无从说起，以至在梅树下沉吟许久，直到日暮才独自离开。（答出心情3分，简要分析3分。） 北京卷 12.阅读下面这首诗，完成题。(7分) 古风(其十)
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Ｄ．通过报刊转载９.王军创作摄影作品一套２０００年５月４日发表，２００２年３月２６日王军因车祸死亡，该套摄影作品的保护期限截止到（）Ａ．２０５０年５月４日Ｂ．２０５２年５月４日Ｃ．２０５０年１２月３１日Ｄ．２０５２年１２月３１日１０.根据我国著作权法著作权的产生是从（）Ａ．作品发表日起Ｂ．版权局审查批准日起Ｃ．作品创作完成日起Ｄ．版权局颁发著作权证书日起１１.我国著作权法不保护的对象是（）Ａ．歌曲Ｂ．地铁Ｃ．建筑Ｄ．法律法规１２.甲计划将乙的作品通过信息网络向公众提供，下列说法正确的是（）Ａ．甲无需取得乙的许可，但应支付报酬Ｂ．甲可以取得乙的许可，并应支付报酬Ｃ．甲应当取得乙的许可，无需支付报酬Ｄ．甲应当取得乙的许可，并应支付报酬１３.以下使用作品的行为，可以不经过著作权人的许可且不必支付报酬的有（）Ａ．将作品经过信息网络免费传播Ｂ．在文艺晚上演唱该某歌曲作品Ｃ．将汉字出版发行翻译成少数名族文字作品Ｄ．将他人的讲义复制卖给学生学习使用１４.画家甲将其新作“冬梅图”送给好友乙，现关于这幅画的说法不正确的是哪些（）Ａ．甲仍享有署名权Ｂ．甲仍享有展览权Ｃ．甲仍享有修改权Ｄ．乙享有展览权１５.依创作的完成自动产生的知识产权是（）Ａ．著作权Ｂ．专利权Ｃ．商标权Ｄ．集成电路布局设计权１６.我国专利法规定的专利权期限的起算点是（）Ａ．申请日Ｂ．授权公告之日Ｃ．专利权被批准之日Ｄ．登记之日１７.下列不属于专利法所保护对象的是（）Ａ．发明
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