五虎汤对RSV诱发幼年大鼠喘气道重塑模型的影响

发布时间：2018-07-04 11:33

本文选题：哮喘气道重塑 + 五虎汤　；参考：《湖南中医药大学》2015年博士论文

【摘要】：目的：本文在建立病毒诱发幼年大鼠哮喘气道重塑模型的基础上,从体内研究五虎汤对哮喘大鼠血清中NGF、肺组织TGF-β1、IL-13及气道重塑的影响；体外研究五虎汤含药血浆对NGF及Sema4D受体Plexin-B1和CD72表达的影响,以及Sema4D与NGF之间的相互关系,从调整NEI网络紊乱达到改善气道重塑,为进一步阐明五虎汤防治病毒诱发儿童哮喘机理提供实验依据。方法：1.建立模型：将30只幼年SD大鼠随机分为正常对照组、哮喘模型组与地塞米松组,每组10只,于实验第1、2天哮喘模型组与地塞米松组以RSV滴鼻联合10%OVA皮下注射,第9天以1%OVA雾化吸入,每次30min,隔日一次,持续2w；正常对照组以Hep-2细胞滴鼻联合生理盐水致敏、激发作为对照。通过测定气道反应性,留取BALF作细胞计数与分类,肺组织HE染色及Masson染色观察其病理变化及胶原纤维沉积情况,验证模型建立成功与否。 2.体内实验：将48只幼年SD大鼠随机分为正常对照组、哮喘模型组、地塞米松组、五虎汤高、中、低剂量组,除正常对照组外,其余各组均建立哮喘气道重塑模型,于实验第15天起,五虎汤高、中、低剂量组分别予4.752g/kg/d、2.376g/kg/d、1.188g/kg/d浓度的五虎汤,每日分2次灌胃,持续2w,正常对照组与哮喘模型组予生理盐水灌胃,地塞米松组予地塞米松溶液(2.4mg/kg/d)灌胃,每日2次,持续2w。观察各组大鼠反应情况时,病理形态学方法分析气道重塑的相关指标,免疫组化法检测肺组织α-SMA表达,ELISA法测定血清中NGF水平,Western bl ot法检测肺组织TGF-B1及IL-13蛋白相对含量,Real-time PCR法测定肺组织中NGF、TGF-β1及IL-13mRNA的表达。 3.体外实验：分离正常对照组和哮喘模型组大鼠气道上皮细胞,进行原代培养并传至2-3代后,将细胞进行分组：A组为正常气道上皮细胞,不做任何处理；B组为哮喘气道上皮细胞,不做任何处理;C组为哮喘气道上皮细胞+Sema4D:D组为哮喘气道上皮细胞+加入Sema4D、抗Sema4D;E1组、E2组分别为哮喘气道上皮细胞+五虎汤高、中剂量含药血浆;F1组、F2组分别为哮喘气道上皮细胞+Sema4D与五虎汤高、中剂量含药血浆,均处理24h后,应用MTT法和Transwell小室法分别检测气道上皮细胞增殖量及迁移数量,RT-PCR法及Western Blot法分别测定Sema4D受体Plexin-B1和CD72mRNA及蛋白表达水平,ELISA法检测气道上皮细胞中NGF表达。结果：1.建立模型：与正常对照组比较,哮喘模型组大鼠出现明显的喘息、烦躁、竖毛耸肩、口唇发绀、体重增长缓慢及精神差等,HE染色及Masson染色观察发现大量炎性细胞浸润,以嗜酸性粒细胞为主,量化气道学参数明显升高,胶原纤维沉积大量增多等,气道阻力显著增高(P0.01)；地塞米松组较哮喘模型组哮喘症状及相应病理变化均有所减轻。 2.体内实验：(1)与正常组比,模型组大鼠出现明显喘息症状,五虎汤高、中剂量组喘息等症状较模型组明显减轻,地塞米松组大鼠症状亦有所缓解,五虎汤低剂量组大鼠喘息缓解不明显；(2)与正常组比较,模型组病理切片镜下可见大量炎性细胞、管腔狭窄、粘液分泌增多、气道平滑肌显著增厚且排列紊乱、粘膜下可见较多胶原样物质沉积,气道重塑量化指标、肺组织α-SMA表达IOD值均显著增高(P0.01);与模型组比较,五虎汤高、中剂量组及地塞米松组镜下病理改变程度明显减轻,且气道形态学参数各项指标、α-SMA日性表达IOD值明显降低(P0.01),五虎汤低剂量组与其差异不明显(P0.05);(3)与正常组比较,模型组大鼠血清中NGF表达明显增高(P0.01),各治疗组均能降低NGF水平,以五虎汤高、中剂量组较明显(P0.01),且下降程度较低剂量组、地塞米松组差异具有统计学意义(P0.01),五虎汤各剂量组降低NGF表达程度均优于地塞米松组(P0.01,P0.05)；(4)与正常组比较,模型组肺组织TGF-β1、IL-13蛋白的表达均明显升高(P0.01),予药物干预后,五虎汤高、中剂量及地塞米松组TGF-β1及IL-13蛋白表达显著下降(P0.01),且三组间无差异(P0.05),五虎汤低剂量组TGF-β1及IL-13蛋白减弱较模型组差异无统计学意义(P0.05)；(5)与正常组比,其余各组肺组织NGFmRNA相对表达量均升高,与模型组比,各治疗组NGFmRNA的表达均显著下降(P0.01),五虎汤高、中剂量组较低剂量组、地塞米松组下降有明显统计学意义(P0.01),此二组之间无差异(P0.05)；(6)与正常组比较,模型组大鼠TGF-β1mRNA、IL-13mRNA的表达明显升高(P0.01),与模型组比,五虎汤高、中剂量组TGF-β1mRNA表达水平显著降低(P均0.01),且二组之间无明显差异(P0.05),但仍高于正常组(P0.01),低剂量组及地塞米松组与模型组比较无显著差异(P均0.05)；五虎汤高、中剂量组及地塞米松组IL-13mRNA表达水平较模型组均明显降低(P0.01),五虎汤高、中剂量组之间差异无统计学意义(P0.05),较地塞米松组IL-13mRNA表达差异具有显著性(P0.01)；五虎汤低剂量组较模型组差异无统计学意义(P0.05)。 3.体外实验：(1)与A组比较,B组气道上皮细胞增殖量OD值明显升高(P0.01)：与B组比较,C组OD值亦明显升高(P0.01)；D组、F1组、F2组加入OD值有所下降(P0.01);E1、E2组OD值有所降低(P0.01)。以上各组与A组比较,均具有显著性差异(P均0.01)。(2)Transwell小室法测定A组、B组、C组、D组、E1组、E2组、F1组与F2组的细胞迁移数分别为70,102,155,140,102,93,126,137。(3)与A组比较,B组Sema4D及其受体Plexin-B1表达与NGF含量均有升高,受体CD72表达降低；与B组比较,C组在Sema4D作用下,其OD值及迁移细胞数明显增多(P0.01),且Sema4D受体Plexin-B1mRNA及蛋白、气道上皮细胞中NGF表达水平均明显升高(P0.01),CD72受体的表达显著降低(P0.01)与C组比较,E1组、E2组、F1组与F2组Plexin-B1mRNA及蛋白表达水平亦明显下降(P0.01),同时NGF的表达随之显著降低(P0.01)；CD72mRNA及蛋白表达相应地明显升高(P0.01)。与D组比较,E1、E2组PlexinB1mRNA及蛋白与NGF表达水平亦明显下降(P0.01),CD72mRNA及蛋白表达水平亦明显升高(P0.01)。F1、F2组与D组各指标的表达结果一致,差异无统计学意义(P0.05)相对与C组而言,F1、F2组细胞OD值及迁移数量的下降程度,较E1、E2组下降程度不明显(P0.05),其Plexin-BlmRNA及蛋白与NGF表达水平的下降程度和CD72mRNA及蛋白的升高程度均不及E1、E2组对应指标的改变程度(P0.05)。结论：1.应用RSV联合OVA致敏激发方法能够成功制备幼年SD大鼠哮喘气道重塑模型。 2.五虎汤高、中剂量通过改善RSV气道重塑模型大鼠支气管壁、平滑肌增厚及上皮下胶原沉积,并下调a-SMA的表达,从而减慢气道重塑的进程。 3.五虎汤高、中剂量通过降低RSV气道重塑模型大鼠肺组织TGF-β1及IL-13的表达而下调NGF表达实现对哮喘中NEI网络紊乱的调整,从而延缓气道重塑及气道炎症进程。 4.Sema4D参与哮喘气道上皮细胞的增殖与迁移,并通过与NGF相互促进作用,增强其与受体PlexinB1的结合能力,同时抑制与其受体CD72结合力,使气道上皮细胞增殖与迁移过程加速,从而促进气道重塑的发生与发展。 5.五虎汤高、中剂量的含药血浆能够发挥抗Sema4D作用,通过抑制RSV气道重塑模型大鼠Sema4D与NGF结合,降低Sema4D与其受体PlexinB1的结合能力,增强与其受体CD72的结合力,调整哮喘NEI网络紊乱,缓解哮喘气道重塑。
[Abstract]:Objective: To study the effect of five tiger soup on NGF, TGF- beta 1, IL-13 and airway remodeling in the serum of asthmatic rats, and the effect of five tiger soup containing plasma on the expression of NGF and Sema4D receptor Plexin-B1 and CD72 in vitro, and between Sema4D and NGF in vitro. The relationship can help improve airway remodeling by adjusting NEI network disorder, and provide experimental evidence for further elucidate the mechanism of Wuhu Decoction in preventing and treating asthma in children.
Methods: 1. a model was established: 30 young SD rats were randomly divided into normal control group, asthma model group and dexamethasone group, 10 rats in each group. On day 1,2, the asthma model group and dexamethasone group were subcutaneously injected with RSV dripping nose combined with 10%OVA, 1%OVA atomization inhalation, each time 30min each time, continuous 2W, and normal control group with Hep-2. The cells were sensitized with saline and stimulated as control. By measuring airway responsiveness, taking BALF as a cell count and classification, HE staining and Masson staining of lung tissue were used to observe the pathological changes and the deposition of collagen fibers, and the success of the model was verified.
2. in vivo experiment: 48 young SD rats were randomly divided into normal control group, asthma model group, dexamethasone group, five tiger soup high, middle, low dose group, except the normal control group, the other groups were established asthma airway remodeling model, from the fifteenth day of the experiment, the five tiger soup high, middle, low dose group were given 4.752g/kg/d, 2.376g/kg/d, 1.188g/kg/d concentration, respectively. The five tiger soup was intragastric 2 times a day and lasted 2W. The normal control group and the asthma model group were given the physiological saline for gastric perfusion. The dexamethasone group was given the dexamethasone solution (2.4mg/kg/d) to gavage, 2 times a day, and 2W. was used to observe the response of each group. The pathological morphological method was used to analyze the correlation index of the airway remodeling and the immunohistochemical method was used to detect the pulmonary tissue alpha -SMA. The expression of NGF in serum was measured by ELISA, and the relative content of TGF-B1 and IL-13 in lung tissue was detected by Western BL ot. Real-time PCR method was used to determine NGF, TGF- beta 1 and IL-13mRNA.
3. in vitro experiment: the airway epithelial cells in the normal control group and the asthmatic model group were isolated and cultured and passed to 2-3 generations. The cells were divided into groups: the A group was normal airway epithelial cells and did not do any treatment; the B group was asthma airway epithelial cells and did not do any treatment; group C was asthma airway epithelial cell +Sema4D:D as asthma. Airway epithelial cells + Sema4D, anti Sema4D, group E1, group E2 were asthma airway epithelial cells + five tiger soup high, medium dose of drug plasma, F1 group, F2 group of asthma airway epithelial cells +Sema4D and five tiger soup high, medium dose of plasma, 24h, MTT method and Transwell cell method were used to detect the proliferation of airway epithelial cell proliferation, respectively. The expression of Sema4D receptor Plexin-B1 and CD72mRNA and protein expression were measured by RT-PCR method and Western Blot method respectively. NGF expression in airway epithelial cells was detected by ELISA method.
Results: 1. the model was established: compared with the normal control group, the rats in the model group of asthma had obvious wheezing, irritability, hair shrug, lip cyanosis, slow weight growth and poor spirit. HE staining and Masson staining found a large number of inflammatory cells infiltrating, mainly eosinophilic cells, quantified airway parameters and collagen fibrous sedimentation Airway resistance increased significantly (P0.01), and asthma symptoms and corresponding pathological changes were relieved in the dexamethasone group compared with asthma model group.
2. in vivo experiment: (1) compared with the normal group, the rats in the model group had obvious wheezing symptoms, the five tiger soup high and the middle dose group were obviously relieved than the model group, and the symptoms of the rats in the dexamethasone group were also relieved. (2) compared with the normal group, the model group showed a large number of inflammation under the pathological section microscope. In sex cells, narrowing of the lumen, mucus secretion increased, the airway smooth muscle was thickened and arranged in disorder, more gel samples were deposited under the mucous membrane, the quantitative index of airway remodeling and the IOD value of the expression of alpha -SMA in the lung tissue were significantly increased (P0.01). Compared with the model group, the five tiger soup was higher, the medium dose group and the dexamethasone group were significantly reduced in the pathological changes. The IOD value of the expression of alpha -SMA was significantly lower (P0.01), and the difference in the low dose group of five tiger soup was not obvious (P0.05). (3) compared with the normal group, the expression of NGF in the serum of the model group was significantly higher (P0.01), and all the treatment groups could reduce the level of NGF, with the five tiger soup high, the middle dose group more obvious (P0.01), and decreased. In the lower dose group, the difference in dexamethasone group was statistically significant (P0.01). The level of NGF expression in each dose group of Wuhu decoction was better than that of dexamethasone group (P0.01, P0.05). (4) compared with the normal group, the expression of TGF- beta 1 and IL-13 protein in the lung tissue of the model group was significantly increased (P0.01), the prognosis of the drug drying, the high level of five tiger soup, the medium dose and dexamethasone. The expression of TGF- beta 1 and IL-13 protein in the pine group was significantly decreased (P0.01), and there was no difference between the three groups (P0.05). The decrease of TGF- beta 1 and IL-13 protein in the low dose group of the five tiger soup was not statistically significant (P0.05). (5) the relative amount of NGFmRNA in the lung tissues of the other groups increased, compared with the normal group, and the expression of NGFmRNA in each group was significantly higher than that of the model group. Decrease (P0.01), five tiger soup, low dose group, lower dose group, dexamethasone group decreased significantly (P0.01), there was no difference between the two groups (P0.05); (6) compared with the normal group, the expression of TGF- beta 1mRNA, IL-13mRNA increased significantly (P0.01) in the model group, compared with the model group, the five tiger soup was higher, and the medium dose group TGF- beta 1mRNA expression level was significant. There was no significant difference (P 0.01), and there was no significant difference between the two groups (P0.05), but it was still higher than the normal group (P0.01). There was no significant difference between the low dose group and the dexamethasone group (P 0.05). The IL-13mRNA expression level in the medium dose group and the dexamethasone group was significantly lower than that in the model group (P0.01), the five tiger soup and the middle dose group were different. There was no statistical significance (P0.05), and there was significant difference in IL-13mRNA expression compared with dexamethasone group (P0.01), and there was no significant difference between the low dose group and the model group (P0.05).
3. in vitro experiment: (1) compared with group A, the proliferation of airway epithelial cell proliferation in B group increased significantly (P0.01): compared with group B, the OD value of group C was also significantly increased (P0.01); D group, F1 group, F2 group decreased (P0.01) and E1, E2 group O value decreased (0.01). (2) The cell migration number of A group, group B, group C, group D, group E1, E2 group, F1 group and F2 group was 70102155140102,93126137. (3) compared with A group. The expression level of NGF in the airway epithelial cells increased significantly (P0.01), and the expression of CD72 receptor decreased significantly (P0.01), and the expression level of Plexin-B1mRNA and protein in E1, E2, F1 and F2 groups also decreased significantly (P0.01), and the expression of CD72 was significantly decreased (P0.01), and the expression of CD72 was significantly decreased. Compared with group D, the expression level of PlexinB1mRNA and protein and NGF in E1, E2 group also decreased (P0.01), the expression level of CD72mRNA and protein was also significantly increased (P0.01).F1, F2 group was in accordance with the expression results of each index in D group, and the difference was not statistically significant. The decrease degree of E1, E2 group was not obvious (P0.05). The decrease of Plexin-BlmRNA and protein and NGF expression level and the increase of CD72mRNA and protein were not as good as that of E1 and E2 group (P0.05).
Conclusion: 1.. RSV combined with OVA sensitization stimulation method can successfully prepare airway remodeling model of asthmatic SD rats.
2. wutiger soup was high, medium dose was used to improve the bronchial wall of RSV airway remodeling model rats, smooth muscle thickening and collagen deposition, and down regulation of a-SMA expression, thus slowing the process of airway remodeling.
3. five tiger soup was high, and the medium dose reduced the expression of TGF- beta 1 and IL-13 in the lung tissue of the RSV airway remodeling model and downregulated the expression of NGF to adjust the NEI network disorder in asthma, thus postponing the airway remodeling and airway inflammation.
4.Sema4D participates in the proliferation and migration of airway epithelial cells in asthmatic airway, and enhances the binding ability of NGF to receptor PlexinB1, and inhibits the binding force of its receptor with its receptor, accelerating the proliferation and migration of airway epithelial cells, thus promoting the development and development of airway remodeling.
5. five tiger soup, medium dose of drug containing plasma can play the anti Sema4D effect, by inhibiting the RSV airway remodeling model rats Sema4D and NGF binding, reducing the binding capacity of Sema4D and its receptor PlexinB1, enhancing the binding force of the receptor CD72, adjusting the disorder of the asthma NEI network and alleviating the airway remodeling of asthma.
【学位授予单位】：湖南中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R285.5;R-332

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