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【摘要】 目的: 探讨外源性一氧化碳释放分子对脓毒症炎症反应的抑制作用及可能的机制。方法: 应用盲肠结扎及穿孔脓毒症小鼠模型,使用外源性一氧化碳释放分子(CORM2, 8 mg/kg 体质量,尾静脉注射)进行干预。检测肝、肺脏髓过氧化物酶(MPO) 活性。应用内毒素(LPS,10 g/ml)刺激的人脐静脉内皮细胞炎症模型,使用外源性一氧化碳释放分子( CORM2,10~100 mol/L)进行干预。检测核因子κB (NFκB)活性, 内皮细胞黏附分子的表达,氧化产物、NO产物以及多形核白细胞对内皮细胞的黏附作用。 结果: 盲肠结扎及穿孔脓毒症小鼠模型使用外源性一氧化碳释放分子干预后肝、肺组织MPO活性明显下降。CORM2 抑制了LPS刺激导致的 NFκB活性上调。 同时,NO产物下降,内皮细胞ICAM1的表达抑制,白细胞对内皮细胞的黏附作用明显抑制。结论: 外源性一氧化碳释放分子通过抑制NFκB 活性,抑制ICAM1 蛋白和NO的表达,抑制白细胞对内皮细胞的黏附作用,进而有效抑制脓毒症炎症反应。
【关键词】 一氧化碳; 盲肠结扎及穿孔; 炎症反应; 核因子κB
CLP (cecal ligation and puncture) may induce the activation of an inflammatory cascade, cause damage to multiple organs distant from the original burn wound and may lead to sepsis and multiple organ failure[1].There have been several reports indicating that the inflammatory response syndrome, which contributes to oxidative cell/tissue damage, might frequently be accompanied by leukocyte sequestration in many important organ systems in the body[2].The increase of production of proinflammatory mediators such as interleukin (IL)1β and tumor necrosis factor (TNF)α is closely associated with activation of leukocytes and macrophages which were sequestrated in the tissue[3,4].
Leukocytes sequestration and their subsequent infiltration in organ tissue can cause leukocyte activation and contribute to vascular damage and the development of systemic inflammatory reaction.As the prerequisite, activation of leukocytes and endothelial cells results in aggregation of leukocytes, platelets and erythrocytes in vivo.This may favor disseminated intravasal coagulation and further multiple organ failure.
Carbon monoxide (CO) has long been known in biology and medicine as a toxic compound, due to its ability to bind hemoglobin with a much higher affinity than oxygen[5].Evidence accumulated to date suggests that endogenous carbon monoxide (CO), a biproduct of inducible heme oxygenase (HO1) can modulate inflammation, inhibits lipopolysaccharide (LPS)induced production of cytokines both in vivo and in vitro, and consequently exhibits important cytoprotective function and antiinflammatory properties that are beneficial for the resolution of acute inflammation[6-8].Inhaled CO at concentrations of 250~500 parts per million (ppm) has also been shown to be beneficial in a number of lung injury models, including hyperoxic injury[9,10] allergeninduced inflammation[11].
Recently, transitional metal carbonyls have been identified as potential COreleasing molecules (CORMs) with the potential to facilitate the pharmaceutical use of CO by delivering it to tissues and organs[12].CORMs have been shown to act pharmacologically in rat aortic and cardiac tissue where liberation of CO produced vasorelaxant effects[13-16] and decreased myocardial ischemiareperfusion damage[17,18] in the absence of dramatic changes in blood carboxyhemoglobin (COHb) levels.
On the basis of these data, the present study was, therefore, designed as a prospective laboratory experiment to investigate the effects of tricarbonyldichlororuthenium (Ⅱ) dimer (CORM2), one of the novel group of CORMs, on attenuation of leukocyte sequestration and decrease of inflammatory responses and oxidative stress in the organs of CLPinduced mice and LPSinduced HUVEC (human umbilical vein endothelial cell), and discussed the possible molecular mechanisms.
1 Material and methods
1.1 Materials
Medium 199 (M199), fetal calf serum (FCS), penicillin, and streptomycin were purchased from GIBCO BRL (Gland Island, NY).Tricarbonyldichlororuthenium(II) dimer (CORM2) was obtained from Sigma Aldrich and solubilized in dimethyl sulfoxide (DMSO) to obtain a 10 mmol/L stock.LPS(Escherichia coli serotype 055:B5) was purchased from Sigma.AntiICAM1 polyclonal antibody was purchased from Transduction Laboratories (Lexington, KY).Antimouse IgG conjugated to horseradish peroxidase was purchased from Kirkegaard and Perry Laboratories (Gaithersburg, MD).
1.2 Animals
The C57BL/6 mice[male, N=21; bw (20± 2)g] were fed a standard laboratory diet and water ad libitum.Mice were assigned to three groups in three respective experiments.In each experiment, mice in sham group (n=7) were underwent sham procedure, whereas mice in CLP group (n=7) received cecal ligation and puncture and mice in CORM2 group (n=7) underwent the same injury with immediate administration of CORM2 (8 mg/kg, i.v.).The concentration of CORM2 used in the present study was based on a previous report in of the use of this compound in mice[19] and the preliminary experiments in our lab by measuring dynamic COHb levels and peak levels which did not averaged 15%±5% above normal levels.The experimental protocol was approved by The Council on Animal Care at Jiangsu University on the protection and the welfare of animals and met National Institutes of Health guidelines for the care and use of experimental animals.
1.2.1 CLP Mice were anesthetized with 2% isoflurane in oxygen via a facemask.A 1to 2 cm midline incision was made through the abdominal wall; the cecum was identified and ligated with a 3-0 silk tie 1 cm from the tip.Care was taken not to cause bowel obstruction.A single puncture of the cecal wall was performed with a 20gauge needle.The cecum was lightly squeezed to express a small amount of stool from the puncture site to assure a fullthickness perforation.Great care was taken to preserve continuity of flow between the small and large bowels.Inspection of mice at various intervals after CLP did not reveal evidence of bowel obstruction.The cecum was returned to the abdominal cavity, and the incision was closed with surgiclips.Sham mice underwent anesthesia and midline laparotomy; the cecum was exteriorized and returned to the abdomen, and the wound was closed with surgiclips.Mice received injection of CORM2 (8.0 mg/kg, i.v.) immediately after CLP.Control mice received 160 μl 0.5% DMSOnormal saline in the same regimen.Measurement of MPO in the liver was performed at 24 h after CLP.
1.2.2 MPO activity MPO activity as an assessment of neutrophil influx was measured according to established protocols[20].In brief, tissue was homogenized in 0.5 ml of 50 mmol/L potassium phosphate buffer (pH 7.4) and centrifuged at 10,000 r/min at 4℃ for 30 min.The remaining pellet was resuspended in 0.5 ml of 50 mmol/L potassium buffer pH 6.0 with 0.5% hexadecyltrimethylammonium bromide, sonicated on ice, and then centrifuged at 12,000 r/min at 4℃ for 10 min.Supernatants were then assayed at a 1∶20 dilution in reaction buffer containing 50 mmol/L PB, 530 mmol/L odianisidine, and 20 mmol/L H2O2 solution.One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance measured at 460 nm for 3 min.MPO activity was expressed as U/g tissue.
1.3 Cells
1.3.1 Isolation and culture of human umbilical vein endothelial cells (HUVEC) Human umbilical vein endothelial cells (HUVEC) were harvested from the fresh human umbilical vein of newborns by collagenase treatment (Worthington Biochem, Freehold, NJ) as previously described[21].The cells were grown in medium 199(M199; GIBCO, Burlington, Canada) supplemented with 10% heatinactivated FCS (Intergen, Purchase, NY), 2.4 mg/L thymidine (Sigma Chemical, Oakville, Canada), 10 IU/ml heparin sodium, antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; GIBCO), 1.5 μg/ml fungizone (GIBCO),and 80 μg/ml endothelial mitogen (Biomedical chnologies, Stoughten, MA).The cell cultures were incubated in room air with 5% CO2 at 37℃ and 95% humidity and were expanded by brief trypsinization with 0.25% trypsin in PBS containing 0.025% EDTA.The experiments were conducted on passage 3 HUVEC.After 18 h, the medium was changed to 500 μl of fresh complete M199.Cells were stimulated with LPS (10 g/ml).After treatment for 4 h, the cells and medium were harvested separately.
1.3.2 PMN adhesion assays Human neutrophilic PMN were isolated from the venous blood of healthy adults using standard dextran sedimentation and gradient separation on Histopaque1077.This procedure yields a PMN population that is 95%~98% viable (trypan blue exclusion) and 98% pure (acetic acidcrystal violet staining).
For the static adhesion assay, isolated neutrophils were suspended in PBS buffer and radiolabeled by incubating the cells at 5×107 cells/ml with 50 μCi Na51CrO4/ml PMN suspension at 37℃ for 60 min.The cells were then washed with cold PBS to remove unincorporated radioactivity.Radiolabeled PMN (5×105/well) were added to HUVEC monolayers grown in 48well plates (Costar), and 30 min later the percentage of added PMN that remained adherent after a wash procedure was quantitated as follows: %PMN adherence = lysate (cpm)/[supernatant (cpm) + wash (cpm) + lysate (cpm)], where cpm is counts per minute.
1.3.3 Oxidant production Oxidant production within HUVEC was assessed by measuring the oxidation of intracellular dihydrorhodamine 123 (DHR 123; Molecular Probes, Inc.), an oxidantsensitive fluorochrome, as described previously[22].Briefly, the cells were treated with DHR 123 (5 mmol/L) for 1 h before being subjected to LPS stimulation.After LPS stimulation the cells were washed with PBS, lysed, and DHR 123 oxidation was assessed spectrofluorometrically at excitation and emission wavelengths of 502 and 523 nm, respectively.
1.3.4 Nitric oxide production NO production by HUVEC was assessed by measuring the fluorescence of 4amino5methylamino2′,7′difluorofluorescein diacetate (DAFFM diacetate), a specific NO probe (Molecular Probes, Inc.)[23].Briefly, DAFFM diacetate (10 mmol/L) in M199 was added to the HUVEC 1h before the LPS stimulation.After LPS stimulation, the HUVEC and supernatants were collected and analyzed spectrofluorometrically at excitation and emission wavelengths of 495 nm and 515 nm, respectively.
1.3.5 SDSpolyacrylamide gel electrophoresis and Western blotting SDSpolyacrylamide gel electrophoresis and Western blotting were performed as described previously[24].Samples (10 μg of protein) were subjected to electrophoresis on 7% (for ICAM1) SDSpolyacrylamide gels, with the use of the discontinuous system and transferred onto nitrocellulose membranes.The membranes were probed with antiICAM1 monoclonal antibody (1∶2 500).Antimouse IgG conjugated to horseradish peroxidase (1∶2 500) was used as a secondary antibody.The bands were visualized by the use of ECL reagent and Hyperfilm ECL (Amersham, Arlington Heights, IL) as described by the manufacturer.Films were scanned using a flatbed scanner and the bands were quantified using Basic Quantifier software (Bio Image, Ann Arbor, MI), an image analysis program, on computer.
1.3.6 HUVEC nuclear protein extraction and Electrophoretic mobility shift assay(EMSA) Nuclear protein was extracted from HUVEC as previously described[25].Cells were grown to confluence in Petridish, scraped, washed with cold PBS, and incubated in 150 μl of buffer E(+) (0.3% Nonidet P40, 10 mmol/L Tris (pH 8.0), 60 mmol/L NaCl, 1 mmol/L EDTA, 0.5 mmol/L dithiothreitol (DTT), 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride ) for 5 min on ice.Samples were centrifuged at 4℃ for 5 min at 500 r/min.The supernatant was then removed, and the pellets (nuclei) were resuspended in 150 μl of buffer E (10 mmol/L Tris (pH 8.0), 60 mmol/L NaCl, 1 mmol/L EDTA, and 0.5 mmol/L DTT) and centrifuged at 500 r/min for 5 min at 4℃.The nuclei were then extracted in 30~50 μl of buffer E(c)(20 mmol/L HEPES, 0.75 mmol/L spermidine, 0.15 mmol/L spermine, 0.2 mmol/L EDTA, 2 mmol/L EGTA, 2 mmol/L DTT, 20% glycerol, and 1 mmol/L PMSF (4 ℃) in the presence of 0.4 mol/L NaCl) and were incubated on ice for 20 min.Finally, the samples were centrifuged for 10 min at 500 r/min (4℃), and the supernatants were collected and saved as the nuclear protein fraction.Samples were stored at -80℃.
The doublestranded oligonucleotide containing consensus (58AGGGACTTCCGCTGGGGACTTTCC38) binding sites for NFκB (synthesized on site; BeckmanOligo 1 000 mol/L DNA synthesizer) were endlabeled with[γ-32P]ATP (Amersham) by using T4polynucleotide kinase (MBI Fermentas, Flamborough, ON), as described previously[25] .One picomole of the labeled oligonucleotide was incubated with 5 μg of nuclear extract protein in the presence or absence of 50×excess of cold oligonucleotide.Samples were incubated for 30 min at room temperature and then run through a 4% nondenaturing polyacrylamide gel at 280 V for 45~60 min.The gel was dried and then exposed to Xray film (Kodak) in cassettes for 2~4 h at -80℃ with intensifying screens.
1.3.7 Cell ELISA For assessment of ICAM1 surface expression level, an ELISA was performed[26] on HUVEC grown in 96well cell culture plates (Corning).HUVEC were fixed in 4% paraformaldehyde at 4℃ for 30 min.The cells were then washed two times with cold PBS and were incubated with the mouse primary monoclonal antibody (MAb) against human ICAM1 (Dako) at a concentration of 10 μg/ml for 1 h at room temperature.After this treatment, immunocytochemical staining of HUVEC monolayers was performed using an avidinbiotinconjugated peroxidase mouse IgG staining kit (Vectastain), and MAb binding was subsequently quantified with a microplate reader (model 3550UV; BioRad) at 450 nm wavelength.
1.4 Statistical Analysis
All of the values are presented as means ± SE.Statistical analysis was performed with the use of ANOVA and Student′s ttest for the comparisons.A value of P
2 Results
2.1 Effect of CORM2 on MPO activity in lung and liver of CLPchallenged mice
To determine whether CLPinduced increase in PMN accumulation in the lung and liver was effectively prevented by CORM2, the activity of MPO, an enzyme in azurophilic granules of neutrophils, was assessed.Extracts of the organs samples were examined for content of MPO at 24 h after CLP injury.MPO activity in organs obtained from CLPinduced mice was markedly increased compared to sham (P< 0.01), while it significantly decreased by treatment with CORM2 (Fig.1).
Fig 1 Effects of CORM2 on MPO activity in the
lung and liver of CLPchallenged mice
Mice were injected (i.v.) with CORM2 (8 mg/kg) immediately after CLP challenging.Sham mice received 160 μl bolus injection of 0.5% DMSO/saline.MPO activities in the lung and liver were assessed 24 h following CLP.Results are mean ± SE of three experiments (three mice per group), *P
2.2 Effect of CORM2 on expression of ICAM1 in the lung and liver of CLPchallenged mice
At 24 h after CLP induction, the expression of ICAM1 in lung and liver tissues significantly increased compared to the sham animals.In vivo administration of CORM2 (8 mg/kg, i.v.), expression of ICAM1 was significantly decreased (Fig.2)
Fig 2 Effects of CORM2 on protein expression of ICAM1
in the liver and lung of CLPinduced mice
Mice were challenged with CLP and treated with CORM2 as described in Fig.1.Protein expression of ICAM1 was performed by western blotting at 24 h after CLP.Results are mean ± SE.*P
2.3 Effect of CORM2 on activities of NFκB in lung and liver tissue of CLPchallenged mice
Binding activities of nuclear protein to the radiolabeled consensus binding sequences of NFκB was assessed by EMSA.At 24 h after CLP induction, the NFκB activation in lung and liver was markedly increased and this activity was inhibited by in vivo administration of CORM2 (8 mg/kg,i.v.) (Fig.3).
Fig 3 Effects of CORM2 on NFκB activation in the lung
and liver of CLPinduced mice (EMSA)
Mice were challenged with CLP and treated with CORM2 as described in Fig.1.Measurement of NFκB activity was performed by mobility shift assay (EMSA) with 32Plabeled NFκB probe and 5 μg of nuclear extract from liver of sham, CLP and CLP+CORM2 mice at 24 h after CLP.
2.4 Effect of CORM2 on intracellular production of ROS and NO in HUVEC stimulated by LPS
As shown in Fig.4a, the LPS stimulation resulted in a significant increase in HUVEC oxidant production.Treated HUVEC with different concentration of CORM2 induced less DHR oxidation compared to the LPS with a concentrationdependent manner.As shown in Fig.4b, HUVEC produced significantly more NO during the LPS stimulation as compared to control.HUVEC significantly decreased their NO production after treatment with CORM2 as a concentrationdependent manner.
Fig 4 Effects of CORM2 on intracellular production of
ROS and NO in LPSstimulated HUVEC
HUVEC were grown to confluence in 48well cell culture plates and loaded with dihydrorhodamine 123 (DHR 123) or diaminofluoresceinFM (DAFFM) for 1 h.Subsequently, HUVEC were stimulated with LPS (10 μg/ml) for 4 h in the presence or absence of CORM2 (10, 50, 100 μmol/L).Oxidative stress (DHR123 oxidation) (a) and NO production (DAFFM nitration) (b) were assessed.All values are expressed as mean ± SE (n=4).*P
2.5 Effect of CORM2 on ICAM1 expression in LPSstimulated HUVEC (cell ELISA and Western blotting)
At 24 h after LPS stimulation, the expression of ICAM1 in HUVEC significantly increased compared to the control.At the present of CORM2 (10,50 and 100 μmol/L), expression of ICAM1 (Fig.5a, ELISA and 5b, Western blotting) was significantly decreased.
All values are expressed as mean ± SE (n=4).*P
Fig 5 Effects of CORM2 on ICAM1 expression in LPS
stimulated HUVEC (cell ELISA and Western blotting)
2.6 Effect of CORM2 on activities of NFκB in LPSchallenged HUVEC
Binding activities of nuclear protein to the radiolabeled consensus binding sequences of NFκB was assessed by EMSA.At 4 h after LPS stimulation, the NFκB activation in HUVEC was markedly increased and this activity was inhibited by administration of CORM2 with a concentrationdependent manner (Fig.6).
Fig 6 Effects of CORM2 on NFκB activation in LPS
stimulatedHUVEC (EMSA)
At 4 h after LPS stimulation, the NFκB activation in HUVEC was markedly increased and this activity was inhibited by administration of CORM2 with a concentrationdependent manner.
2.7 Effect of CORM2 on PMN adhesion to HUVEC stimulated by LPS
As shown in Fig.7, adhesion of PMN to HUVEC is low in control.After monolayer of endothelial cells were stimulated by LPS for 4 h, adhesion of PMN to HUVEC significantly increased (P
Confluent monolayers of HUVEC were incubated with LPS (10 μg/ml) in the presence or absence of CORM2 (10, 50 and 100 μmol/L) for 4 h followed by PMN addition in the well.PMN adherence was determined.All values are expressed as means ± SE (n=3).*P< 0.01 compared to control; ﹟P< 0.05 compared to LPS.
Fig 7 Effect of CORM2 on PMN adhesion to
LPSstimulated HUVEC
3 Discussion
Sepsis is a common and serious medical condition caused by a severe systemic infection leading to a systemic inflammatory response, which frequently occurs after hemorrhage, trauma, burn, or abdominal surgery.It is a leading cause of morbidity and mortality in severely ill patients[27].Although some information has been generated from the LPS injection studies, LPS injection is an adequate model of endotoxemia and can not precisely mimic the changes observed during sepsis.On the other hand, cecal ligation and puncture (CLP) model seems to resemble qualitatively as well as quantitatively the clinical observations of vascular reactivity and inflammation during polymicrobial peritonitis, bacteremia, and systemic sepsis[28].Therefore, the aim of this study is to evaluate the possible role of CORMderived CO in CLPinduced sepsis.
Many experimental studies have highlighted the specific and independent role of exogenous CO in the modulation of inflammation[29, 30].As a new metal carbonylbased compounds, COreleasing moleculars (CORMs) have the ability to release CO in biological systems.The vasoactive, antihypertensive and antirejection effects of CORMs have been demonstrated to be due to the CO liberated by the compounds.CORM2, a DMSOsoluble CORM, also has exhibited antiinflammatory actions in an in vitro model of LPSstimulated macrophages[31].Therefore, the aim of this study was to investigate the effects of CORM2 on the dynamics of leukocytes sequestration in the lung and liver, and on the decrease of ROS and NO production in the LPSstimulated HUVEC.
Leukocytes sequestration and their subsequent infiltration in lung and liver tissues can cause leukocyte activation and contribute to vascular damage and the development of systemic inflammatory reaction.Myeloperoxidase (MPO) is an enzyme that is found predominantly in the azurophilic granules of polymorphonuclear leukocytes (PMN).Tissue MPO activity is frequently utilized to estimate tissue PMN accumulation in inflamed tissues and correlates significantly with the number of PMN determined histochemically in tissues[32] .In the present study, we found that tissue MPO activity was markedly elevated after CLP and in vivo administration of CORM2 led to the significantly downregulation of MPO activity.This indicated that CORM2 effectively prevents PMN chemotaxis and infiltration in the tissue after CLP, consequently decreased the production of oxidants, reduced tissue oxidative injury.
The direct cause of leukocytes sequestration after CLP is considered to be the more expression of adhesion molecule (ICAM1).ICAM1 activates leukocytes and endothelial cells (ECs), which in turn prompt the release of various inflammatory mediators, resulting in systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS) and multiorgan dysfunction syndrome (MODS)[33-35].The present results showed that at 24 h postCLP, the expression levels of ICAM1 in lung and liver tissue were markedly upregulated.In vivo administration of CORM2 was able to inhibit the upregulation of ICAM1 induced by CLP.In addition, CORM2 also inhibits the increase of ICAM1 expression in LPSstimulated HUVEC.In parallel, the results of in vitro experiments showed that LPS stimulation caused significant increase of PMNs adhesion to HUVEC, CORM2 treatment effectively prevented this increase.
There is no doubt that the nuclear factor
κB(NFκB) is a ubiquitous, rapidly acting transcription factor involved in immune and inflammatory reactions, it exerts its immune and inflammatory response by regulating expression of cytokines, chemokines, cell adhesion molecules, and growth factors[36,37].In this study, NFκB activities in lung and liver tissue and in LPSstimulated HUVEC were elevated by CLP while it was markedly inhibited by administration of CORM2.These data showed that CORM2 plays a pivotal role in inhibition of NFκB activity, subsequently decreased the expression of cellular adhesion molecules (ICAM1) and CLPinduced proinflammatory mediators.Therefore, an effective therapeutic strategy that inhibit this transcription factor would be expected to improve organ functions after CLP.
In summary, the present studies serve to clarify the role of CORM2, one of the novel COreleasing molecules, on the mechanisms of attenuation of leukocyte sequestration.Application of CORM2 on CLP mice attenuated PMNs accumulation, prevented activation of NFκB, and subsequently decreased the production of inflammatory mediators in the lung and liver.This was accompanied by a decrease of the expression of ICAM1.In parallel, expression of ICAM1, PMN adhesion to HUVEC stimulated by LPS were markedly decreased after CORM2treatment.Taken together these findings indicate that CORMreleased CO attenuates leukocytes sequestration and inflammatory responses by interfering with NFκB activation, protein expression of ICAM1 and therefore suppressing endothelial cells proadhesive phenotype.
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言与文互融就是语言文字和人文思想的和谐统一。这就需要教师在章句和清议之间寻求平衡点和切入点,通过语言文字去体会文中的人文内涵,通过悟人文内涵促语言文字训练的夯实,让言与文互融,语言和人文共生。
一、炼言促文
文言文音韵优美,行文简练,可毕竟是一种同实际语言脱离得越来越远的特殊的书面语言,因此学生的阅读障碍比较多。其中影响学生解“文”的最主要障碍是词汇的理解。但如果教学中只注重“言的解读”这一点,那么其中蕴含的古典美、人文美学生就很难领略到了。因此在教学中既要重视学生掌握一些基本的古汉语字词句的知识,又要注重文本的整体把握,关注文章的篇章结构和表达方法,同时强调文本的思想情感和古代文化的传承和熏陶。这也就是说,要在理解言的基础上,结合文章语境积累文言,进而正确把握文本,步入作者的感情世界,与作者达成感情共鸣,使文言不分,文言并重。
1.正言通文路。《语文课程标准》指出:“诵读古代诗词,有意识地在积累、感悟和运用中提高自己的欣赏品位和审美情趣。”记诵诗词散文名句佳篇,对于陶冶和丰富学生的人文精神有着重要作用,同时,它也能体现一个人的语文功底和精神底蕴。因此,文言文教学首要任务是在读准言、读通文中体味古文的韵味,不能未解其义、未悟其文就让学生死记硬背。必须着力于记诵积累以悟文,要在反复、变化的诵读中调动学生的兴趣和情感,在触摸语言韵味中与文本对话,逐渐进入文本的语境。第一,读准字音,解决文字障碍。文言文中有些字的读音异于现在的字音,但字音的确定还是有一些方法,这些方法需要传授。有些字是通假字,那就要读本字字音,有些字要根据文意来确定读音,还有些特殊称谓的读音,就要读它自己特定的读音。第二,读准句读,读顺文意。读清句读、正确停顿是诵读文言文的要求之一,更是文言文阅读能力的重要体现。正确的停顿是为了准确、清楚地表达文章的思想内容,如果把握不好句子的停顿,很容易会错文意。依句停顿:文言文中有些句子和现代文的句子构成的要素是一样的,停顿时就可移用现代文朗读的方法,如“梁国/杨氏子/九岁”;据文停顿:在朗读文言文时,要根据语境理解文意,只有确切理解了文意,才能准确停顿,不能只看句子的外部特征,如“未闻/孔雀/是夫子家/禽”。语文学习是在认知基础上体验熏陶的过程,学生“活生生的体验”及“自我的精神体验”不是靠讲解翻译来代替完成的,而是靠反复的读来沉潜涵泳。这样课文的文意、文情、文趣都可以在铿锵的声调中体现出来。
2.析言知文意。小学作为文言文阅读的启蒙阶段,虽然所选的都是一些浅显、有趣、有味的小短文,但也偶有古今词义、语法表达不同之处,易和学生的原认知产生冲突,是难点所在。教师要善于言文融合,把这些冲突点转化成探究点,帮助学生理解文意。如《杨氏之子》一文,稍一梳理不难发现,文中同一人物的不同称呼和“家禽”是学生理解的难点所在。如文中的家禽与学生生活经验中的家禽并不一致,在教学时让学生先借助注释尝试读懂课文,用讲故事的方式反馈学生对文本内容的把握,在交流中点拨设问:“孔雀是家禽吗?”引发学生思考讨论,明晰家禽的意思及在朗读时两字之间需停顿。在这一过程中教师摈弃简单的字义记录、对等翻译的机械教学方式,运用讲故事的形式充分暴露学生对字、词、句、文的理解障碍所在,继而把难解的字词还原到句子、文章里,在语境中让他们自我解疑,既着眼于对言的理解,又着力在学生对文中语言内化后的自我表达,把言和文很好地杂糅在一起。
3.辨言明文旨。在文言文中,影响学生成长的决不是哪个文言词或者句子的解释翻译,而是文章蕴含的深邃思想和语言的魅力,即古人读书定要“其义自见”的东西。文言文字少意深,所要“见”的往往就藏于一字微言之中,需要教师慧眼识金,引导学生发现、比辨,体味其深藏的妙处与蕴意。如学生初读《杨氏之子》,都会为杨小儿“以其人之道还治其人”的反击而击掌叫好。如果到此为止,那教学只关注了语言的艺术,却忽视了文本的价值取向,有违文之本意。为让学生读透这语言艺术背后的真意,教师先出示两句话:①未闻孔雀是夫子家禽。②孔雀是夫子家禽。再让学生读一读,说说体会到了有什么不同。学生有从语气上体会到第一句比较委婉,更有学生体会到第一句的意思并非是肯定的,而第二句的意思是肯定的……这时,教师顺势引导:你们能联系孔君平的话再来品一品杨小儿的话到底是什么意思吗?把你的理解用“如果……那么……”的句式写下来。在交流中学生体味到了杨小儿的话中之意是:如果我和杨梅是一家子,那么夫子和孔雀也是一家子,反之亦然。至于事实是否如此全在于孔的认为,他把皮球踢回给了孔君平,不但有智而且有礼,实乃聪且慧。以上教学层层激疑,有意引导学生去比较、区分这些不同的精妙之处,在探究性阅读和创造性阅读中拓展思维空间,不但领略了文中语言的魅力,更正确把握了文章的主旨,提升了文言文教学的内涵。
二、赏文习言
教文言文,指导学生理解词句的根本目的在于更准确、深入地把握文意;反过来说,把握了文意也可以更好地理解词句。文章是作者思想情感、道德评价、文化素养、审美趣味等的“集成块”,是一个活的整体,而不是各种语言材料的“堆积物”。文章语言之所以值得揣摩咀嚼,因为它是作者思想情感的载体,如果只着眼于词句本身的学习,而忽视甚至舍弃了它所承载的丰富内容,结果必然连语言本身也不可能真正学好。把文言文作为文章(它本来就是文章)来教,就要遵循教读文章的一般规律,处理好词句和文章整体的关系,这不仅是学习文章的需要,也是更好地理解文言词句的需要。
1.依文悟言,言文合一。品味感悟文章的语言,是引导学生理解作者情感思想最常用的教学方法。不同的文体决定了写作语言的不同特点,不同的文体对写作语言也有不同的要求。如曹丕所言:“奏议宜雅,书论宜理,名诔尚实,诗赋欲丽。”其实优秀的作品在语言创作时,都不自觉地将其文体意义发挥到了极致。从这个意义上说,引导学生根据文体把握文中最显著的语言表达特征,也就是为他们学习语言打开了一条通道。《杨氏之子》和《两小儿辩日》都是描写古时小孩生活的有名篇章。两文对小孩的语言描写用词细腻传神,教师可以围绕对话展开品读教学,为学生展现一幅生动的生活场景,体会文言文的语言魅力。但由于两文的重点又有区别,所以对对话揣摩理解的落脚点也应有一定的区别。《杨氏之子》一文选自《世说新语》。《世说新语》首先是言谈轶事,然后才是小说。这就是说,《世说新语》的着眼点主要是“言语”,为了增强表述的生动性和形象性,才把言语加工成了小说的样式。教学此文,对此文词句的理解应该落在语言之“智”上。而《两小儿辩日》是寓言,运用对话描写的目的是要说明一个道理,所以对文中语言的理解要侧重于“理”。文言文文体的多样性和独特性,赋予了课堂丰富多彩的教育内容和形式。通过不同文体的语言特征的把握,对准确掌握教学的尺度也有一定的指导意义,指导学生针对不同文体特点理解和把握文本,不管是对文章语言的理解,对文章的写法、意蕴的体会,还是对学生阅读能力的培养,都是很有帮助的。
关键词:小古文 目标 趣 教学方法
中图分类号:G623.2 文献标识码:A 文章编号:1004-6097(2012)05-0059-02
作者简介:柯向妹(1977―),女,福建泉州人,本科学历,小学高级教师,福建泉州市泉港庄重文实验小学语文教师。研究方向:阅读与写作。
小古文被称为中华语文的“活化石”,不管是哪个版本的小学语文教材,均选编了经典小古文。根据新课程标准、小学生年龄特征及小古文的特点,激发学生对小古文学习的兴趣就成了小古文教学目标的重中之重。那么,如何在教学过程中落实这一教学目标呢?笔者做了如下尝试:
一、趣读
诵读是小古文教学的命脉。小古文的表达方式与现代文有所不同,更具音乐美、对称美及节奏美,尤其适合诵读。小古文的诵读形式丰富多彩:除常见的分角色朗读、配乐朗读、男女生轮读、创设情境读外,还可古文今文对照读、根据平仄音律读、插图补白读、古文版文本和现代版文本对照读……教师可以根据教学需要自由灵活地选择。
著名特级教师戴建荣老师引导学生按照“平仄”规律吟诵诗文,深受学生欢迎。对于短小精悍的小古文,教者可以让学生先用“―”“|”表示“平”“仄”,遵循音韵规律趣读。如《鹬蚌相争》语句的吟诵指导:
| ― ― | ― | ― ― | | ― ― ― ― |
蚌方出曝,而鹬啄其肉,蚌合而箝其喙。
这样,既朗朗上口,又抑扬顿挫,诗文的节奏之美被淋漓尽致地表现了出来。学生兴致勃勃地尝试,在快乐的诵读体验中亲近小古文。
二、趣品
品味主要是指用比较、揣摩的方法对文本的意蕴和妙处进行语感分析。小古文的词、句、段高度凝练,内涵丰富,皆可纳入品味范畴。
在《鹬蚌相争》一文中通过比较,品味“箝”的精妙:
师:刚才有同学说“箝”就是“夹”的意思,那么直接把“夹”替换进句子,你们觉得怎么样?
(学生思考,摇头,但似乎说不出个所以然)
师:伸出你的手,试着做一做这两个动作。
(学生伸出双手,比划着,若有所悟)
师:用一个词来形容,可以说是――
(生的思路顿时打开,答案精彩纷呈:使劲地、用尽全身力气地、牢牢地、竭尽全力、死死地等)
师:同学们都从力度上来回答,那么,从速度上来讲,那是――
(生:迅速地、迅雷不及掩耳之势、闪电般等)
师:孩子们,想想此时此刻鹬蚌的心情,那么,鹬蚌又会怎么“箝”呢?
(生:狠狠地、互不相让、毫不示弱等)
师:是的,在文本中,牢牢地、死死地、费尽九牛二虎之力地、迅速地、互不相让地,这样的“夹”,就可以说是“箝”。一个小小的“箝”字,让我们感受到了鹬蚌相争的激烈程度及二者复杂的内心世界;一个小小的“箝”字,也让我们感受到了古人用字的精准与独到。
通过“箝”和“夹”的比较品味,挖出了“箝”中无限的精彩,激发了学生学习兴趣的同时也带领学生领略了古文所传递的情感和语言的魅力,在不知不觉中落实了教学目标。
三、趣拓
选编入教材的小古文大多出自《论语》《战国策》《韩非子》等经典,所选编的小古文背后或有脍炙人口的故事,或蕴涵着深刻的哲理,或在谋篇布局、细节描写等写作方法方面有精妙之处可挖掘。“教育不是灌满一桶水,而是点燃一团火”(叶芝语),教师要适当取舍,巧妙拓展,开阔学生视野,努力点燃学生学习小古文及课外阅读的兴趣之火。
内容方面的拓展。《鹬蚌相争》一文,学生反复品读感悟后,教师可告诉学生这则小小的寓言曾经制止了一场战争。在学生迫切想深入了解之时,把《鹬蚌相争》在《战国策》中的原文呈现出来,让学生思考:为什么听了苏代的话后,赵惠王就消除了攻打燕国的念头?适当的拓展,让学生进一步体会到寓言的特点及魅力,也体现了古文浓浓的文化味。
写作方法的拓展。著名特级教师林莘老师执教《东施效颦》一文时,抛出“你怎么知道东施长得丑”这一话题,引导学生潜心会文,讨论交流,学习“侧面描写”的方法,并适时引入《陌上桑》内容:“行者见罗敷……但坐观罗敷”,体会侧面描写之精妙。
情感升华的拓展。《伯牙绝弦》一文,学生感受伯牙与钟子期的情谊后,配乐诵读悲痛欲绝的伯牙在子期墓前写下的短歌,学生深深地被这旷世“知音”之情感染,升华了情感,陶冶了情操。
四、趣说
不同版本的教材均为学生了解文言文内容提供了一定的帮助,如北师大版教材直接附译文,人教版教材附注释等,这也为学生的想象提供了很大的空间。教师要善于鼓励学生发挥想象,挑战教材,在理解的基础上,把文言文翻译、创造得更生动有趣,甚至超越教材。如《鹬蚌相争》一文中“蚌方出曝,而鹬啄其肉,蚌合而箝其喙”一句,学生展开丰富的想象进行趣说:“一只河蚌张开壳儿在河滩上懒洋洋地晒着太阳,嘴里还哼着小曲儿,瞧,它多么惬意呀!有只鹬循声望去,一阵激动:啊哈哈,我的美餐!它以迅雷不及掩耳之势把嘴伸到蚌壳儿里去啄肉。歌声戛然而止,突如其来的情况让河蚌猝不及防,它急忙把壳儿合上,牢牢地夹住鹬的嘴不放。”这样的趣说,通过丰富的想象把文本创造性地读“长”了,训练了学生的口语表达能力,同时也培养了学生的创造想象能力。
五、趣演
小古文中人物形象很鲜明,比如《杨氏之子》中聪慧的孩子,《两小儿辩日》中活泼可爱的小儿和谦虚谨慎、实事求是的孔子,《郑人买履》中迂腐的郑人等。教学中引导学生走近文本中的人物,讨论揣摩人物的语言、动作、心理等,试着辩一辩,演一演,激发兴趣,领会主旨。《鹬蚌相争》一文中,鹬蚌相争可谓达到了你死我活的白热化程度。教师让学生组内分好角色,合作表演“鹬蚌相争,渔翁得利”,学生情绪高涨。
表演中,学生感受到强烈的对比:那样你死我活的激烈争斗,到头来却是双双失去性命的可悲下场,这两个场景鲜明而突兀地呈现在眼前,学生情绪被充分调动起来,在哈哈大笑之余触发深深的思考,心灵受到震撼。
六、趣“得意”