椒目油防治哮喘气道重建机制的研究
摘要
目的与意义
哮喘气道重建(remodeling)又称气道重塑或重构,是支气管哮喘(哮喘)一大重要病理特征。气道重建主要表现为气道平滑肌增生、基底膜增厚、腺体增生及上皮下纤维增殖、胶原沉积等,从而造成气道管壁增厚、管腔狭窄。气道重建可导致气流不可逆阻塞和持续性气道高反应性,并能成为顽固性哮喘反复发作的病理学基础,研究它有利于哮喘的早期防治和阻止哮喘病情的进一步恶化。目前评价任何一个防治哮喘的药物不仅要看它的平喘作用,而且还要看它能否具有防治气道重建的作用。
中药椒目治疗支气管哮喘历史悠久,疗效显著,目前国内许多单位相继开展了椒目治疗哮喘研究,取得丰硕成果[1-2],国内大量的文献报道以及我们自己的研究成果,无论从分子生物学的角度或是从临床的角度都说明了椒目能有效治疗哮喘。但是椒目能否有效地防止哮喘气道重建(气道重塑或重构),目前还未见到文献报道。本实验研究选择与气道重建密切相关的几种因素通过复制大鼠哮喘气道重建模型,初步揭示椒目防治哮喘气道重建的机制,为更好的应用椒目防治哮喘提供理论依据。
研究内容:
1.探讨椒目油对哮喘大鼠气道重建模型Ⅲ型、V型胶原的影响。
2.探讨椒目油对哮喘大鼠气道重建模型中TGF-β1表达的影响。
3.探讨椒目油对哮喘大鼠气道重建模型气道平滑肌PCNA表达的影响。
材料与方法:
健康SD大鼠30只,体重200-250g,将30只SD大鼠随机分为3组:哮喘组(10只),椒目油治疗组(10只),正常对照组(10只)。参考文献[3]用l% OVA和1%氢氧化铝溶液0.5 ml大鼠腹腔注射,第8 d重复注射1次,至15d开始吸入1% OVA溶液激发5 min。l% OVA溶液需要每天配制,雾化时将大鼠置于特制塑料容器内雾化吸人。治疗组在雾化激发前1h给予椒目油和熟精油的混合物(按1:1混合),以3mL/kg灌胃,对照组以生理盐水代替OVA致敏和雾化。3组大鼠在激发3个月后处死,经右主支气管注入10%中性甲醛溶液至肺适度膨胀,将右肺连同右主支气管剪下放入10%中性甲醛固定一周。从右侧下肺叶开口处以水平位取材,制成石蜡切片,行HE染色作病理观察。以磷酸盐缓冲液(PBS)代替一抗作为空白对照,用ABC法,组织切片按序以PB漂洗,30%羊血清,一抗、二抗孵育后浸入ABC液中1 h,取出经PBS、Tris-HC1缓冲液漂洗,再以DAB显色,苏木精复染,常规脱水,透明、封盖。每例标本取2张切片,每张切片随机选取小支气管5支,应用计算机灰度分析测定粘膜下层吸光度确定Ⅲ型、V型胶原以及TGF-β1的含量。实验数据用?x±s表示,应用SPSS 11.0软件进行t检验分析,p<0.05为差别有统计学意义。
结果:
1肺组织HE染色
哮喘气道重建组大鼠肺组织切片HE染色可见支气管上皮细胞肿胀、脱落,管壁增厚,管腔变窄,管壁内及管周有大量炎性细胞浸润,以EOS和淋巴细胞为主;椒目油治疗组大鼠的肺组织可见支气管上皮细胞的炎症反应较哮喘组为轻,支气管壁及管腔内的炎症细胞也大为减少;对照组大鼠的肺组织则无上述改变。而且可观察到哮喘组大鼠气道壁厚度较对照组明显增加,椒目油治疗组大鼠气道壁厚度较哮喘气道重建组明显降低。
2镜下观察免疫组化切片的变化
Ⅲ型、V型胶原染色呈棕黄色,Ⅲ型胶原主要在气道壁粘膜下层表达,V型胶原表达主要位于基底膜上;测定Ⅲ型、V型胶原阳性表达的计算机灰度分析:哮喘气道重建组明显高于对照组;椒目油治疗组低于哮喘气道重建组(P均<0.05)而略高于对照组( p<0.05)。
TGF-β1主要表达在气道粘膜上皮层,经ABC染色成棕黄色。灰度分析结果显示:哮喘气道重建组明显高于对照组;椒目油治疗组低于哮喘气道重建组(P均<0.05)略高于对照组( p<0.05)。
PCNA免疫组化染色呈棕黑色圆形颗粒,位于上皮下基底膜、平滑肌细胞及外周成纤维细胞。哮喘气道重建组阳性表达明显高于对照组;椒目油治疗组明显低于哮喘气道重建组;略高于对照组。
结论:
1、椒目油可以降低哮喘大鼠气道重建气道壁炎症反应及气道壁厚度。
2、椒目油可降低哮喘大鼠气道重建气道壁Ⅲ、V型胶原的含量。
3、椒目油可降低哮喘大鼠气道重建气道壁TGF-β1在气道壁的表达。
4、椒目油可以降低PCNA在大鼠哮喘重建模型气道壁表达。以上四点说明椒目不仅可以有效地治疗哮喘,而且可以有效地防治气道重建。
Aim and significance
Asthmatic airway remodeling is the most important pathologic charater of brochial asthma. The process of airway remodeling included airway smoothm muscle hyperplasia, basement membrane thickness, gland hyperplasia, subepithelial fiber hyperplasia and collagen deposition and so on, which could cause airway wall thickness and airway lumina stenosis.Airway remodeling could cause inreversible airflow blockage and persistent airway hyperresponsiveness, which may be the pathologic basis of repeated attack of refractoriness asthma, and study them could be in favour of early stage of asthma prevention and curation, and aggravation of asthma. Nowadays we evaluate the effect of any drug preventing and treating asthma not only by its effcacy of preventing wheeze,but also by the effect in preventing airway remodeling.
Oil from bunge pricklyash seed has a long histry of treating asthma with good therapeutic effect. Nowadays many national institutions successively start to study the effect of bunge pricklyash seed oil treating asthma, and had got a lot of achivements[1-2]. A lot national reports and ourselves' study outcomes confirmed the effect of bunge pricklyash seed oil in treating asthma not only in molecular biological aspect but also cilinic aspect.But so far no study had reported bunge pricklyash seed oil could prevend the airway remodeling. We choose some factors associating with airway remodeling to build rat models of asthma, and try to reveal the underlined mechanism of bunge pricklyash seed oil preventing airway remodeling in asthma,in order to supply the theoretial basis of better usage of bunge pricklyash seed oil in preventing asthma.
Content
1. Study the effect of bunge pricklyash seed oil to III and V type collagen in airway-remodel rat models of asthma.
2. Study the effect of bunge pricklyash seed oil to PCNA in airway smooth muscle in airway-remodel rat models of asthma.
3. Study the effect of bunge pricklyash seed oil to the expression of TGF-β1 in airway-remodel rat models of asthma.
Material and methods
Healthy SD rat(n=30),200-250g.All animals were randomly divided into 3 groups:asthma group(A group, 10),treat group(B group, 10)and control group(C group, 10). All animals were subcutaneous injected in forelegs with 0.5ml 1% OVA 0.5ml 1% AL(0H)3, and repeat it again on day 8.On day15 all animals inhaled with 1% OVA for 5min.We prepared OVA ereryday and put rats in a container to aerosol rebreath. The rats from treatment group were gave 3ml/kg mixture of bunge pricklyash seed oil and essential oil(1:1) by intragastric administration one hour before aerosol rebreath. Rats of control group were gave NS instead of OVA. All animals treated to death 3 monthes after provocation.
Results
1.Lung tissue HE stain: Section preparations of asthmatic rat showed that epithelium of bronchi were engorged and ablated, and the wall of airway was thickened and calibre was stenosis. Airway wall and surronding tissues were infaltrated with inflammatory cells, of which EOS and lymphocyte take the advantage.Lung tissue of rats of treatment group showed that less severe inflammation in bronchial epithelium,and the inflammatory cells in airway wall were also reduced.Rat of control group had no such apparance .
2.Changes in thickness of rat airway smooth muscle :the results of image analysis showed:section preparations was amplified 100 times.
Conclusion
1.Bunge pricklyash seed oil could decrease the extent of inflammation and thickness of airway wall in airway-remodeled rats.
2.Bunge pricklyash seed oil could decrease the expression of III and V type collagen in airway-remodeled rats.
3.Bunge pricklyash seed oil could decrease the expression of TGF-β1 in airway-remodeled rats.
4.Bunge pricklyash seed oil could decrease the expression of PCNA in airway-remodeled rats.
All of the four points above indicated that Bunge pricklyash seed oil could not only treat asthma effectively, but also prevent airway remodeling effectively.
哮喘气道重建(remodeling)又称气道重塑或重构,是支气管哮喘(哮喘)一大重要病理特征。气道重建主要表现为气道平滑肌增生、基底膜增厚、腺体增生及上皮下纤维增殖、胶原沉积等,从而造成气道管壁增厚、管腔狭窄。气道重建可导致气流不可逆阻塞和持续性气道高反应性,并能成为顽固性哮喘反复发作的病理学基础,研究它有利于哮喘的早期防治和阻止哮喘病情的进一步恶化。目前评价任何一个防治哮喘的药物不仅要看它的平喘作用,而且还要看它能否具有防治气道重建的作用。
中药椒目治疗支气管哮喘历史悠久,疗效显著,目前国内许多单位相继开展了椒目治疗哮喘研究,取得丰硕成果[1-2],国内大量的文献报道以及我们自己的研究成果,无论从分子生物学的角度或是从临床的角度都说明了椒目能有效治疗哮喘。但是椒目能否有效地防止哮喘气道重建(气道重塑或重构),目前还未见到文献报道。本实验研究选择与气道重建密切相关的几种因素通过复制大鼠哮喘气道重建模型,初步揭示椒目防治哮喘气道重建的机制,为更好的应用椒目防治哮喘提供理论依据。
研究内容:
1.探讨椒目油对哮喘大鼠气道重建模型Ⅲ型、V型胶原的影响。
2.探讨椒目油对哮喘大鼠气道重建模型中TGF-β1表达的影响。
3.探讨椒目油对哮喘大鼠气道重建模型气道平滑肌PCNA表达的影响。
材料与方法:
健康SD大鼠30只,体重200-250g,将30只SD大鼠随机分为3组:哮喘组(10只),椒目油治疗组(10只),正常对照组(10只)。参考文献[3]用l% OVA和1%氢氧化铝溶液0.5 ml大鼠腹腔注射,第8 d重复注射1次,至15d开始吸入1% OVA溶液激发5 min。l% OVA溶液需要每天配制,雾化时将大鼠置于特制塑料容器内雾化吸人。治疗组在雾化激发前1h给予椒目油和熟精油的混合物(按1:1混合),以3mL/kg灌胃,对照组以生理盐水代替OVA致敏和雾化。3组大鼠在激发3个月后处死,经右主支气管注入10%中性甲醛溶液至肺适度膨胀,将右肺连同右主支气管剪下放入10%中性甲醛固定一周。从右侧下肺叶开口处以水平位取材,制成石蜡切片,行HE染色作病理观察。以磷酸盐缓冲液(PBS)代替一抗作为空白对照,用ABC法,组织切片按序以PB漂洗,30%羊血清,一抗、二抗孵育后浸入ABC液中1 h,取出经PBS、Tris-HC1缓冲液漂洗,再以DAB显色,苏木精复染,常规脱水,透明、封盖。每例标本取2张切片,每张切片随机选取小支气管5支,应用计算机灰度分析测定粘膜下层吸光度确定Ⅲ型、V型胶原以及TGF-β1的含量。实验数据用?x±s表示,应用SPSS 11.0软件进行t检验分析,p<0.05为差别有统计学意义。
结果:
1肺组织HE染色
哮喘气道重建组大鼠肺组织切片HE染色可见支气管上皮细胞肿胀、脱落,管壁增厚,管腔变窄,管壁内及管周有大量炎性细胞浸润,以EOS和淋巴细胞为主;椒目油治疗组大鼠的肺组织可见支气管上皮细胞的炎症反应较哮喘组为轻,支气管壁及管腔内的炎症细胞也大为减少;对照组大鼠的肺组织则无上述改变。而且可观察到哮喘组大鼠气道壁厚度较对照组明显增加,椒目油治疗组大鼠气道壁厚度较哮喘气道重建组明显降低。
2镜下观察免疫组化切片的变化
Ⅲ型、V型胶原染色呈棕黄色,Ⅲ型胶原主要在气道壁粘膜下层表达,V型胶原表达主要位于基底膜上;测定Ⅲ型、V型胶原阳性表达的计算机灰度分析:哮喘气道重建组明显高于对照组;椒目油治疗组低于哮喘气道重建组(P均<0.05)而略高于对照组( p<0.05)。
TGF-β1主要表达在气道粘膜上皮层,经ABC染色成棕黄色。灰度分析结果显示:哮喘气道重建组明显高于对照组;椒目油治疗组低于哮喘气道重建组(P均<0.05)略高于对照组( p<0.05)。
PCNA免疫组化染色呈棕黑色圆形颗粒,位于上皮下基底膜、平滑肌细胞及外周成纤维细胞。哮喘气道重建组阳性表达明显高于对照组;椒目油治疗组明显低于哮喘气道重建组;略高于对照组。
结论:
1、椒目油可以降低哮喘大鼠气道重建气道壁炎症反应及气道壁厚度。
2、椒目油可降低哮喘大鼠气道重建气道壁Ⅲ、V型胶原的含量。
3、椒目油可降低哮喘大鼠气道重建气道壁TGF-β1在气道壁的表达。
4、椒目油可以降低PCNA在大鼠哮喘重建模型气道壁表达。以上四点说明椒目不仅可以有效地治疗哮喘,而且可以有效地防治气道重建。
Aim and significance
Asthmatic airway remodeling is the most important pathologic charater of brochial asthma. The process of airway remodeling included airway smoothm muscle hyperplasia, basement membrane thickness, gland hyperplasia, subepithelial fiber hyperplasia and collagen deposition and so on, which could cause airway wall thickness and airway lumina stenosis.Airway remodeling could cause inreversible airflow blockage and persistent airway hyperresponsiveness, which may be the pathologic basis of repeated attack of refractoriness asthma, and study them could be in favour of early stage of asthma prevention and curation, and aggravation of asthma. Nowadays we evaluate the effect of any drug preventing and treating asthma not only by its effcacy of preventing wheeze,but also by the effect in preventing airway remodeling.
Oil from bunge pricklyash seed has a long histry of treating asthma with good therapeutic effect. Nowadays many national institutions successively start to study the effect of bunge pricklyash seed oil treating asthma, and had got a lot of achivements[1-2]. A lot national reports and ourselves' study outcomes confirmed the effect of bunge pricklyash seed oil in treating asthma not only in molecular biological aspect but also cilinic aspect.But so far no study had reported bunge pricklyash seed oil could prevend the airway remodeling. We choose some factors associating with airway remodeling to build rat models of asthma, and try to reveal the underlined mechanism of bunge pricklyash seed oil preventing airway remodeling in asthma,in order to supply the theoretial basis of better usage of bunge pricklyash seed oil in preventing asthma.
Content
1. Study the effect of bunge pricklyash seed oil to III and V type collagen in airway-remodel rat models of asthma.
2. Study the effect of bunge pricklyash seed oil to PCNA in airway smooth muscle in airway-remodel rat models of asthma.
3. Study the effect of bunge pricklyash seed oil to the expression of TGF-β1 in airway-remodel rat models of asthma.
Material and methods
Healthy SD rat(n=30),200-250g.All animals were randomly divided into 3 groups:asthma group(A group, 10),treat group(B group, 10)and control group(C group, 10). All animals were subcutaneous injected in forelegs with 0.5ml 1% OVA 0.5ml 1% AL(0H)3, and repeat it again on day 8.On day15 all animals inhaled with 1% OVA for 5min.We prepared OVA ereryday and put rats in a container to aerosol rebreath. The rats from treatment group were gave 3ml/kg mixture of bunge pricklyash seed oil and essential oil(1:1) by intragastric administration one hour before aerosol rebreath. Rats of control group were gave NS instead of OVA. All animals treated to death 3 monthes after provocation.
Results
1.Lung tissue HE stain: Section preparations of asthmatic rat showed that epithelium of bronchi were engorged and ablated, and the wall of airway was thickened and calibre was stenosis. Airway wall and surronding tissues were infaltrated with inflammatory cells, of which EOS and lymphocyte take the advantage.Lung tissue of rats of treatment group showed that less severe inflammation in bronchial epithelium,and the inflammatory cells in airway wall were also reduced.Rat of control group had no such apparance .
2.Changes in thickness of rat airway smooth muscle :the results of image analysis showed:section preparations was amplified 100 times.
Conclusion
1.Bunge pricklyash seed oil could decrease the extent of inflammation and thickness of airway wall in airway-remodeled rats.
2.Bunge pricklyash seed oil could decrease the expression of III and V type collagen in airway-remodeled rats.
3.Bunge pricklyash seed oil could decrease the expression of TGF-β1 in airway-remodeled rats.
4.Bunge pricklyash seed oil could decrease the expression of PCNA in airway-remodeled rats.
All of the four points above indicated that Bunge pricklyash seed oil could not only treat asthma effectively, but also prevent airway remodeling effectively.
引文
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[23] Kuwano K, Bosken CH, Pare PD, Bai TR,Wiggs BR,Hogg JC. Small airways dimensions in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis,1993;148(5): 1220-1224.
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[36] Hoshino M,Nakmura Y,Sim JJ,et a1.Expression of growth fac-tors and remodeling of the airway wall in bronchial asthma.Tho-rax,1998,53:21-27
[37] Takasaki Y,Fishwild D,Tan EM. Characterization of proliferating cell nuclear antigen recognized by autoantibodies in lupus sera. J Exp Med,1984;159:981-92
[38] Pauwds RA.et a1.Alr J Respir Crit Care Med.1997;156(4Pt2); S78-8l
[39] Barbato A. Turato G.. Baraldo S, et al. Airway inflammation in childhood asthma [J]. Am Respir Crit Care Med,2003,168(7):789-803
[40] 朱宏。哮喘患儿转化生长因子β1的作用研究[J].江苏医药杂志,2001,27(8):587-588
[41] 林耀广. 现代哮喘病学 ,中国协和医科大学出版社
[42] 魏永昆. 增殖细胞核抗原与肿瘤研究进展.国外医学肿瘤学分册,1996;23 (增刊): 47-9
[43] 黄杰雄,黄致治。增殖细胞核抗原的研究进展。国外医学生理病理科学与临床册,1994;14(1):9-11
[44] Rochelle L,Garcia MD,Coltrera et al. Analysis of proliferative grade using anti- PCNA/cyclin monoclonal antibodies in fixed,embedded tissues. Am J pathols 1989;134(4):733-9
[45] Takasaki Y,Fishwild D,Tan EM. Characterization of proliferating cell nuclear antigen recognized by autoantibodies in lupus sera. J Exp Med,1984;159:981-92
[46] Pendoeton N,Dixon GR,Durnet He,et a1.Expression of proliferating cell nuclear antigen(PCNA) in dysplasia of the bronchial epithelium,J.Pathol,1993,170:169
[47] Gland P,Degvaef C Cyclin/PCNA immunostaining as an alternative to tritilsted thymidine pulse labelling for making S phase cells in paraffin sections from animal and human tissue. Cell Tissue Kinet,1989,22(5):383
[48] Swlroce O et al.Chest ,1995.108:1228-1234
[49] Laitinen LA.Lairtinen A.J.Allergy Clin lmmunol,1996;97:153-158
[50] Stewart AG,et al.Br J Phurrnacol 1995;116:3219-3226
[51] 庄世宏,李孟楼。花椒籽油的成分分析。西北农业学报,2002,11(2):43-45
[52] 陈孝伯,等。椒目劫喘的临床研究一附 958 例病例分析。中医杂志,1987;28(12):899～901
[53] 潘金友,椒目油丸治疗哮喘的疗效观察。中药材,1999;22(2):103-104
[54] 李时珍,本草纲目。北京:人民卫生出版社,1992:1855
[55] 江苏新医学院编.中药大辞典(下册).上海:上海科学技术出版社,1986.2292～22935
[56] 傅吾儒等。息喘平胶丸治疗老年慢性支气管炎(喘息型)320 例疗效观察。北京中医杂志。1987,(1):23