多西环素对哮喘大鼠气道炎症及气道重塑的影响
发布时间：2018-09-07 15:38
【摘要】：目的：本实验通过建立哮喘大鼠模型，以血清IL-5、IL-13、TNF-α为气道炎性指标，以α-SMA为气道重塑指标，以此来观察多西环素干预治疗后MMP-9、IL-5、IL-13、TNF-α、α-SMA的变化，从而探讨多西环素对哮喘大鼠气道炎症及重塑的预防作用及可能机制，为临床哮喘的防治提供新的途径。 方法：健康清洁SD雄性大鼠33只，6周龄，体重50-100g之间，随机分为正常对照组、哮喘模型组、多西环素干预组，每组11只。哮喘模型组、多西环素干预组分别于实验第1、8天对所有大鼠腹腔注射OVA100mg和氢氧化铝100mg（混于0.9%生理盐水2ml中）进行致敏，第15天起将大鼠置于自制的非完全密闭雾化吸入箱内，给予OVA5ml雾化吸入30min进行激发，隔日1次，共激发20次，其中多西环素干预组在每次OVA激发前30min按30mg/kg采用口腔灌服的方式给大鼠用药。正常对照组的致敏和激发均以生理盐水代替OVA进行，余同哮喘组。 最后一次激发24小时后，所有大鼠用1%戊巴比妥钠麻醉，收集肺泡灌洗液、血清及肺组织。计数肺泡灌洗液中的细胞数并进行分类；ELISA法检测血清中的IL-5、IL-13的浓度，放免法检测血清中TNF-α的浓度，免疫组化法检测肺组织中MMP-9、α-SMA的表达；肺组织行病理切片，HE染色，采用计算机图像分析技术测定支气管基底膜周径（Pbm）、总管壁面积（WAt）、平滑肌面积（WAm）等反映气道壁厚度的指标。 结果： 1肺泡灌洗液（BALF）中细胞总数及嗜酸性粒细胞计数（×10~6/L）的结果：正常对照组9.42±3.67、1.24±0.72，哮喘组35.53±7.06、4.84±0.65，多西环素干预组14.66±5.81、2.14±0.58。哮喘组与多西环素干预组的细胞总数、嗜酸性粒细胞计数均明显高于正常对照组（F=64.840P＜0.05，F=90.598P＜0.05），其中多西环素干预组上述细胞计数均明显低于哮喘组（P＜0.05）。 2病理结果：哮喘组大鼠的肺组织病理切片可见大量炎性细胞浸润，气道管壁、基底膜及血管壁增厚，血管及平滑肌增生。多西环素干预组的炎性细胞的浸润程度，气道管壁、基底膜及血管壁的增厚程度，血管及平滑肌的增生程度均较哮喘组减轻。 3图像分析支气管管壁厚度、平滑肌层厚度（μm~2/μm）分别为：正常对照组65.47±7.22、20.09±6.09，哮喘组109.95±13.85、56.94±8.23，多西环素干预组86.37±5.85、40.64±4.53。哮喘组与多西环素干预组支气管壁厚度、平滑肌厚度均明显高于正常对照组（F=58.737P＜0.05，F=89.782P＜0.050），其中多西环素干预组上述厚度均低于哮喘组（P＜0.05）。 4ELISA法检测血清IL-5、IL-13的浓度（pg/ml）： 血清IL-5浓度：正常对照组（15.92±3.66），哮喘组（38.63±6.64），多西环素干预组（20.42±4.50）。哮喘组和多西环素干预组血清IL-5的浓度均高于正常对照组（F=61.348，P＜0.05），其中多西环素干预组低于哮喘组（P＜0.05）。 血清IL-13浓度：正常对照组（16.98±7.53），哮喘组（57.58±14.09），多西环素干预组（35.75±8.91）。哮喘组和多西环素干预组血清IL-13的浓度均明显高于正常对照组（F=40.713，P＜0.05），其中多西环素干预组低于哮喘组（P＜0.05）。 5放免法检测血清TNF-α的浓度（ng/ml）：正常对照组（0.43±0.25），哮喘组（1.57±0.20），多西环素干预组（1.20±0.24）。哮喘组和多西环素干预组血清TNF-α的浓度均明显高于正常对照组（F=68.254，P＜0.05），其中多西环素干预组低于哮喘组（P＜0.05）。 6免疫组化法检测肺组织MMP-9、α-SMA的表达： MMP-9的平均光度值：正常对照组（0.24±0.06），哮喘组（0.52±0.06），多西环素干预组（0.39±0.06）。哮喘组和多西环素干预组肺组织MMP-9的表达均高于正常对照组（F=57.966，P＜0.05）；其中多西环素干预组低于哮喘组（P＜0.05）。 α-SMA的平均光度值：正常组对照组（0.20±0.04），哮喘组（0.55±0.06），多西环素干预组（0.35±0.06）。哮喘组、多西环素干预组肺组织α-SMA的表达均高于正常对照组（F=112.663，P＜0.05）；其中多西环素干预组低于哮喘组（P＜0.05）。 7直线相关性分析结果：血清IL-5、IL-13的浓度与BALF中嗜酸性粒细胞计数呈正相关（r=0.817，P＜0.05；r=0.739，P＜0.05），血清TNF-α的浓度与BALF中细胞总数呈正相关（r=0.807，P＜0.05），肺组织MMP-9的表达与BALF中细胞总数呈正相关（r=0.738，P＜0.05），肺组织MMP-9的表达与血清TNF-α浓度呈正相关（r=0.793，P＜0.05），肺组织α-SMA的表达与平滑肌层厚度呈正相关（r=0.841，P＜0.05），肺组织MMP-9的表达与支气管壁厚度、平滑肌层厚度、肺组织α-SMA的表达呈正相关（r=0.819，P＜0.05；r=0.883，P＜0.05；r=0.841，P＜0.05）。 结论： 1本实验通过抗原致敏和反复雾化激发，成功建立了以气道炎症和气道重塑为特征的哮喘大鼠模型。 2哮喘组大鼠的肺组织病理可见大量炎性细胞浸润，肺泡灌洗液中的细胞总数和嗜酸性粒细胞计数均明显高于正常对照组，血清IL-5、IL-13、TNF-α的浓度也明显高于正常对照组。多西环素干预组大鼠的肺组织病理中炎性细胞的浸润程度，肺泡灌洗液中细胞总数和嗜酸性粒细胞计数以及血清IL-5、IL-13、TNF-α的浓度均低于哮喘组。且血清IL-5、IL-13的浓度与BALF中嗜酸性粒细胞计数呈正相关，血清TNF-α的浓度与BALF中细胞总数呈正相关，提示多西环素可以影响炎症介质的生成从而减轻哮喘的气道炎症。 3哮喘组大鼠肺组织MMP-9的表达较正常对照组明显升高，多西环素干预组肺组织MMP-9的表达较哮喘组减少，且肺组织MMP-9的表达与BALF中炎性细胞总数及血清TNF-α的浓度呈正相关，提示MMP-9可以影响气道炎症，多西环素可以通过抑制MMP-9的活性减轻哮喘的气道炎症。 4哮喘组大鼠的支气管管壁厚度、平滑肌层厚度明显高于正常对照组，肺组织α-SMA的表达明显高于正常对照组。多西环素干预组大鼠的支气管管壁厚度、平滑肌层厚度均明显低于哮喘组，，肺组织α-SMA的表达也低于哮喘组。相关分析显示肺组织MMP-9的表达与支气管管壁厚度、平滑肌层厚度及肺组织α-SMA的表达呈正相关，提示MMP-9参与了哮喘的气道重塑，多西环素可以通过抑制MMP-9的活性减轻哮喘的气道重塑。
[Abstract]:AIM: To investigate the effects of doxycycline on airway inflammation and remodeling in asthmatic rats by establishing an asthmatic rat model with serum IL-5, IL-13, TNF-a as inflammatory markers and alpha-SMA as airway remodeling markers. It provides a new way for the prevention and treatment of clinical asthma.
Methods: Thirty-three healthy and clean SD male rats, aged 6 weeks and weighing 50-100g, were randomly divided into normal control group, asthma model group and doxycycline intervention group with 11 rats in each group. From the 15th day, rats were exposed to a self-made incomplete airtight nebulization inhalation chamber. OVA 5 ml was inhaled for 30 minutes, once every other day for 20 times. Doxycycline was administered orally 30 mg/kg 30 minutes before each OVA challenge in the intervention group. Water instead of OVA was used in asthma group.
24 hours after the last challenge, all rats were anesthetized with 1% sodium pentobarbital to collect alveolar lavage fluid, serum and lung tissue, count and classify the number of cells in alveolar lavage fluid, detect the concentration of IL-5 and IL-13 in serum by ELISA, detect the concentration of TNF-a by radioimmunoassay, and detect the concentration of MMP-9 and alpha-SMA in lung tissue by immunohistochemistry. The lung tissues were stained with HE, and the bronchial basement membrane circumference (Pbm), common duct wall area (WAt) and smooth muscle area (WAm) were measured by computer image analysis.
Result:
1. The total cell count and eosinophil count in BALF were 9.42 [3.67, 1.24] 0.72 in normal control group, 35.53 [7.06, 4.84] 0.65 in asthma group, 14.66 [5.81, 2.14] 0.58] in Doxycycline intervention group and asthma group, respectively. Group A (F = 64.840 P < 0.05, F = 90.598 P < 0.05), and the above cell counts in the doxycycline intervention group were significantly lower than those in the asthma group (P < 0.05).
2. Pathological results: Inflammatory cell infiltration, thickening of airway wall, basement membrane and vascular wall, proliferation of blood vessels and smooth muscle were observed in the lung tissue of asthmatic rats. The group was relieved.
3. The thickness of bronchial wall and smooth muscle layer (micron~2/micron) were 65.47 (+ 7.22) and 20.09 (+ 6.09) in normal control group, 109.95 (+ 13.85) and 56.94 (+ 8.23) in asthma group, and 86.37 (+ 5.85) and 40.64 (+ 4.53) in Doxycycline intervention group, respectively. 37P < 0.05, F=89.782P < 0.050). The thickness of doxycycline intervention group was lower than that of asthma group (P < 0.05).
4ELISA method was used to detect the concentration of IL-5 and IL-13 in serum (pg/ml):
Serum IL-5 levels were higher in asthma group and doxycycline intervention group than in normal control group (F = 61.348, P < 0.05), and lower in Doxycycline intervention group than in asthma group (P < 0.05).
Serum IL-13 concentration: normal control group (16.98+7.53), asthma group (57.58+14.09), doxycycline intervention group (35.75+8.91). The serum IL-13 concentration of asthma group and doxycycline intervention group was significantly higher than that of normal control group (F = 40.713, P < 0.05), and the doxycycline intervention group was lower than that of asthma group (P < 0.05).
5 Radioimmunoassay was used to detect the serum TNF-alpha concentration (ng/ml): normal control group (0.43.25), asthma group (1.57.20), doxycycline intervention group (1.20.24). The serum TNF-alpha concentration of asthma group and doxycycline intervention group was significantly higher than that of normal control group (F = 68.254, P < 0.05), and the doxycycline intervention group was lower than that of asthma group (P < 0.05).
6 immunohistochemical method was used to detect the expression of MMP-9 and -SMA in lung tissue.
The expression of MMP-9 in lung tissue of asthma group and doxycycline intervention group was higher than that of normal control group (F = 57.966, P < 0.05), and that of doxycycline intervention group was lower than that of asthma group (P < 0.05).
The average luminosity of alpha-SMA was 0.20 (+ 0.04) in normal control group, 0.55 (+ 0.06) in asthma group, and 0.35 (+ 0.06) in Doxycycline intervention group.
Linear correlation analysis showed that the concentration of serum IL-5 and IL-13 was positively correlated with eosinophil count in BALF (r = 0.817, P < 0.05; r = 0.739, P < 0.05), the concentration of serum TNF-a was positively correlated with the total number of cells in BALF (r = 0.807, P < 0.05), and the expression of MMP-9 in lung tissue was positively correlated with the total number of cells in BALF (r = 0.738, P < 0.05). MMP-9 expression was positively correlated with serum TNF-a concentration (r = 0.793, P < 0.05), lung tissue alpha-SMA expression was positively correlated with smooth muscle layer thickness (r = 0.841, P < 0.05), lung tissue MMP-9 expression was positively correlated with bronchial wall thickness, smooth muscle layer thickness, lung tissue alpha-SMA expression (r = 0.819, P < 0.05; r = 0.883, P < 0.05; r = 0.841, P < 0.05).
Conclusion:
1 The asthmatic rat model characterized by airway inflammation and airway remodeling was successfully established by antigen sensitization and repeated atomization.
The total number of cells and eosinophils in the alveolar lavage fluid were significantly higher than those in the normal control group. The concentrations of serum IL-5, IL-13 and TNF-a were also significantly higher than those in the normal control group. The concentration of serum IL-5, IL-13 and TNF-a was positively correlated with the eosinophil count in BALF, and the concentration of serum TNF-a was positively correlated with the total number of cells in BALF, suggesting that doxycycline could affect the production of inflammatory mediators. Reduce airway inflammation in asthma.
The expression of MMP-9 in lung tissue of asthmatic rats was significantly higher than that of normal control group. The expression of MMP-9 in lung tissue of doxycycline intervention group was lower than that of asthmatic rats. The expression of MMP-9 in lung tissue was positively correlated with the total number of inflammatory cells in BALF and the concentration of TNF-a in serum, suggesting that MMP-9 could affect airway inflammation and doxycycline could inhibit the expression of MMP-9. Activity attenuates airway inflammation in asthma.
The thickness of bronchial wall and smooth muscle layer in asthmatic rats were significantly higher than that in normal control group, and the expression of alpha-SMA in lung tissue was significantly higher than that in normal control group. The expression of MMP-9 was positively correlated with the thickness of bronchial wall, smooth muscle layer and the expression of alpha-SMA in lung tissue, suggesting that MMP-9 was involved in airway remodeling in asthma. Doxycycline can reduce airway remodeling in asthma by inhibiting the activity of MMP-9.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R965;R562.25
本文编号：2228679
[Abstract]:AIM: To investigate the effects of doxycycline on airway inflammation and remodeling in asthmatic rats by establishing an asthmatic rat model with serum IL-5, IL-13, TNF-a as inflammatory markers and alpha-SMA as airway remodeling markers. It provides a new way for the prevention and treatment of clinical asthma.
Methods: Thirty-three healthy and clean SD male rats, aged 6 weeks and weighing 50-100g, were randomly divided into normal control group, asthma model group and doxycycline intervention group with 11 rats in each group. From the 15th day, rats were exposed to a self-made incomplete airtight nebulization inhalation chamber. OVA 5 ml was inhaled for 30 minutes, once every other day for 20 times. Doxycycline was administered orally 30 mg/kg 30 minutes before each OVA challenge in the intervention group. Water instead of OVA was used in asthma group.
24 hours after the last challenge, all rats were anesthetized with 1% sodium pentobarbital to collect alveolar lavage fluid, serum and lung tissue, count and classify the number of cells in alveolar lavage fluid, detect the concentration of IL-5 and IL-13 in serum by ELISA, detect the concentration of TNF-a by radioimmunoassay, and detect the concentration of MMP-9 and alpha-SMA in lung tissue by immunohistochemistry. The lung tissues were stained with HE, and the bronchial basement membrane circumference (Pbm), common duct wall area (WAt) and smooth muscle area (WAm) were measured by computer image analysis.
Result:
1. The total cell count and eosinophil count in BALF were 9.42 [3.67, 1.24] 0.72 in normal control group, 35.53 [7.06, 4.84] 0.65 in asthma group, 14.66 [5.81, 2.14] 0.58] in Doxycycline intervention group and asthma group, respectively. Group A (F = 64.840 P < 0.05, F = 90.598 P < 0.05), and the above cell counts in the doxycycline intervention group were significantly lower than those in the asthma group (P < 0.05).
2. Pathological results: Inflammatory cell infiltration, thickening of airway wall, basement membrane and vascular wall, proliferation of blood vessels and smooth muscle were observed in the lung tissue of asthmatic rats. The group was relieved.
3. The thickness of bronchial wall and smooth muscle layer (micron~2/micron) were 65.47 (+ 7.22) and 20.09 (+ 6.09) in normal control group, 109.95 (+ 13.85) and 56.94 (+ 8.23) in asthma group, and 86.37 (+ 5.85) and 40.64 (+ 4.53) in Doxycycline intervention group, respectively. 37P < 0.05, F=89.782P < 0.050). The thickness of doxycycline intervention group was lower than that of asthma group (P < 0.05).
4ELISA method was used to detect the concentration of IL-5 and IL-13 in serum (pg/ml):
Serum IL-5 levels were higher in asthma group and doxycycline intervention group than in normal control group (F = 61.348, P < 0.05), and lower in Doxycycline intervention group than in asthma group (P < 0.05).
Serum IL-13 concentration: normal control group (16.98+7.53), asthma group (57.58+14.09), doxycycline intervention group (35.75+8.91). The serum IL-13 concentration of asthma group and doxycycline intervention group was significantly higher than that of normal control group (F = 40.713, P < 0.05), and the doxycycline intervention group was lower than that of asthma group (P < 0.05).
5 Radioimmunoassay was used to detect the serum TNF-alpha concentration (ng/ml): normal control group (0.43.25), asthma group (1.57.20), doxycycline intervention group (1.20.24). The serum TNF-alpha concentration of asthma group and doxycycline intervention group was significantly higher than that of normal control group (F = 68.254, P < 0.05), and the doxycycline intervention group was lower than that of asthma group (P < 0.05).
6 immunohistochemical method was used to detect the expression of MMP-9 and -SMA in lung tissue.
The expression of MMP-9 in lung tissue of asthma group and doxycycline intervention group was higher than that of normal control group (F = 57.966, P < 0.05), and that of doxycycline intervention group was lower than that of asthma group (P < 0.05).
The average luminosity of alpha-SMA was 0.20 (+ 0.04) in normal control group, 0.55 (+ 0.06) in asthma group, and 0.35 (+ 0.06) in Doxycycline intervention group.
Linear correlation analysis showed that the concentration of serum IL-5 and IL-13 was positively correlated with eosinophil count in BALF (r = 0.817, P < 0.05; r = 0.739, P < 0.05), the concentration of serum TNF-a was positively correlated with the total number of cells in BALF (r = 0.807, P < 0.05), and the expression of MMP-9 in lung tissue was positively correlated with the total number of cells in BALF (r = 0.738, P < 0.05). MMP-9 expression was positively correlated with serum TNF-a concentration (r = 0.793, P < 0.05), lung tissue alpha-SMA expression was positively correlated with smooth muscle layer thickness (r = 0.841, P < 0.05), lung tissue MMP-9 expression was positively correlated with bronchial wall thickness, smooth muscle layer thickness, lung tissue alpha-SMA expression (r = 0.819, P < 0.05; r = 0.883, P < 0.05; r = 0.841, P < 0.05).
Conclusion:
1 The asthmatic rat model characterized by airway inflammation and airway remodeling was successfully established by antigen sensitization and repeated atomization.
The total number of cells and eosinophils in the alveolar lavage fluid were significantly higher than those in the normal control group. The concentrations of serum IL-5, IL-13 and TNF-a were also significantly higher than those in the normal control group. The concentration of serum IL-5, IL-13 and TNF-a was positively correlated with the eosinophil count in BALF, and the concentration of serum TNF-a was positively correlated with the total number of cells in BALF, suggesting that doxycycline could affect the production of inflammatory mediators. Reduce airway inflammation in asthma.
The expression of MMP-9 in lung tissue of asthmatic rats was significantly higher than that of normal control group. The expression of MMP-9 in lung tissue of doxycycline intervention group was lower than that of asthmatic rats. The expression of MMP-9 in lung tissue was positively correlated with the total number of inflammatory cells in BALF and the concentration of TNF-a in serum, suggesting that MMP-9 could affect airway inflammation and doxycycline could inhibit the expression of MMP-9. Activity attenuates airway inflammation in asthma.
The thickness of bronchial wall and smooth muscle layer in asthmatic rats were significantly higher than that in normal control group, and the expression of alpha-SMA in lung tissue was significantly higher than that in normal control group. The expression of MMP-9 was positively correlated with the thickness of bronchial wall, smooth muscle layer and the expression of alpha-SMA in lung tissue, suggesting that MMP-9 was involved in airway remodeling in asthma. Doxycycline can reduce airway remodeling in asthma by inhibiting the activity of MMP-9.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R965;R562.25
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