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目的观察三步序贯法对哮喘模型大鼠激素撤减过程中TGF-β_1/Smad信号通路上转化生长因子β_1(TGF-β_1)、Smad2、Smad3、Smad6、Smad7蛋白表达的影响。方法将150大鼠随机分为正常对照组、气道重塑组、地塞米松干预组、普米克令舒组及三步序贯组,每组30只。哮喘大鼠激素撤减模型建立方法:以卵蛋白加氢氧化铝致敏,卵蛋白激发,自激发之日开始,于激发前给予地塞米松0.5 mg/kg腹腔注射,连续2周,从第3周开始,按每周0.1 mg/kg速度递减地塞米松用量,至第7周地塞米松全部撤除。三步序贯组在地塞米松干预基础上,于实验第15~28天(撤减前期)给予第一步方颗粒灌胃,第29~63天(撤减中期)给予第二步方颗粒灌胃,第64~77天(撤减后期)给予第三步方颗粒灌胃。气道重塑组仅给予致敏和激发,普米克令舒组在致敏和激发基础上,给予普米克令舒雾化吸入,各组于实验第28、63、77天末次激发2 h后取左肺组织行免疫组化检测,比较各组TGF-β_1、Smad2、Smad3、Smad6、Smad7蛋白表达变化。结果与本组撤减前期比较,地塞米松组撤减中、后期TGF-β_1、Smad2、Smad3蛋白表达升高,Smad6、Smad7蛋白表达降低(P<0.05,P<0.01);普米克令舒组和三步序贯组撤减中、后期TGF-β_1、Smad3、Smad2蛋白表达降低,Smad6蛋白表达升高(P<0.05,P<0.01)。在撤减过程中,与正常对照组同期比较,气道重塑组撤减前、中、后期TGF-β_1、Smad2及Smad3蛋白表达均升高,Smad6、Smad7蛋白表达均降低(P<0.01);与气道重塑组比较,地塞米松组、普米克令舒组、三步序贯组前、中、后期TGF-β_1、Smad2、Smad3蛋白表达均降低,Smad6蛋白表达升高(P<0.01),普米克令舒组、三步序贯组Smad7蛋白表达升高(P<0.01),地塞米松组Smad7蛋白表达撤减前、后期升高(P<0.05);与地塞米松组比较,普米克令舒组、三步序贯组前、中、后期TGF-β_1、Smad2及Smad3均降低,Smad6升高(P<0.01),Smad7中、后期升高(P<0.01)。Smad2与Smad3蛋白表达呈正相关(r=0.909,P<0.01),Smad6与Smad7蛋白表达呈正相关(r=0.873,P<0.01)。结论三步序贯法在哮喘模型大鼠激素撤减过程中能降低TGF-β_1、Smad2、Smad3,升高Smad6、Smad7其阻抑气道重塑的作用可能是通过调节TGF-β_1/Smad信号转导通路来实现的。
Objective To observe the effect of three-step sequential therapy on the expression of TGF-β1, Smad2, Smad3, Smad6 and Smad7 in TGF-β1 / Smad signaling pathway in asthmatic model rats during hormone withdrawal. Methods 150 rats were randomly divided into normal control group, airway remodeling group, dexamethasone intervention group, pulmicort group and three-step sequential group, 30 rats in each group. Establishment of Asthmatic Rat Model of Hypothalamic Hypothesis: Egg protein plus aluminum hydroxide sensitized and ovalbumin-stimulated was administered intraperitoneally before dexamethasone 0.5 mg / kg for 2 weeks starting from the day of challenge. Dexamethasone dosage was decreased by 0.1 mg / kg every week from the third week to the seventh week, dexamethasone was completely removed. On the basis of the intervention of dexamethasone, the three-step sequential group was given intragastric gavage on the 15th to 28th days of the experiment (pre-withdrawal period), and the second-step granulation was given on the 29th to 63rd days (interim withdrawal) Gavage, the first 64 to 77 days (withdrawal period) to give the third step square particles gavage. Airway remodeling group was only sensitized and challenged. Pulmicort was treated with pulmicort respiration inhalation on the basis of sensitization and provocation, each group was left for 2 h after the last challenge on the 28th, 63th and 77th days The lung tissues were examined by immunohistochemistry to compare the expression of TGF-β 1, Smad2, Smad3, Smad6 and Smad7 in each group. Results Compared with the first trimester, the expression of TGF-β_1, Smad2 and Smad3 increased and the expression of Smad6 and Smad7 decreased (P <0.05, P <0.01) The expression of Smad6 and TGF-β1, Smad3 and Smad2 decreased in the middle and late stages of the treatment group and in the three-step sequential group (P <0.05, P <0.01). During the withdrawal process, the expression of TGF-β_1, Smad2 and Smad3 protein in Smad6, Smad6 and Smad7 in the airway remodeling group were significantly lower than those in the normal control group (P <0.01) ; Compared with airway remodeling group, the expression of TGF-β_1, Smad2 and Smad3 in dexamethasone group and pulmicort group were significantly decreased and the expression of Smad6 protein increased in pre-, middle-late and post-treatment groups (P < 0.01). The expression of Smad7 protein in the three sequential groups was increased (P <0.01), while the expression of Smad7 protein in dexamethasone group was increased before and after withdrawal (P <0.05). Compared with dexamethasone group The levels of TGF-β_1, Smad2 and Smad3 were decreased, Smad6 was increased (P <0.01) and Smad7 was increased in the three groups at the early, middle and later stages (P <0.01). There was a positive correlation between the expression of Smad2 and Smad3 protein (r = 0.909, P <0.01), Smad6 and Smad7 protein expression (r = 0.873, P <0.01). Conclusion The three-step sequential therapy can reduce TGF-β_1, Smad2 and Smad3 during the process of hormone withdrawal in asthmatic rats, and increase the expression of Smad6 and Smad7 in airway remodeling may be through regulating TGF-β_1 / Smad signal Transduction pathway to achieve.