芪味理肺汤调控转录因子活化蛋白-1（AP-1）治疗支气管哮喘的机制研究
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摘要
芪味理肺汤调控转录因子活化蛋白-1(AP-1)治疗支气管哮喘的机制研究
目的：
1.通过稳定复制的SD大鼠支气管哮喘模型进行给药动物实验,证实AP-1是支气管哮喘发病的关键环节,验证芪味理肺汤治疗支气管哮喘的疗效并获取该方疗效的模式动物学证据。
2.通过分子生物学方法等手段检测AP-1上下游因子的含量,完善芪味理肺汤治疗支气管哮喘的作用机理,探索该方调控AP-1治疗支气管哮喘的具体作用环节。
方法：
70只SPF级SD大鼠适应性喂养7d无异常后,随机分为空白对照组、模型对照组、阻断剂组、中药低、中、高剂量组、西药组7组。阻断剂组先经AP-1的抑制剂姜黄素预处理3d (100mg. kg-1. d-1,50mg/ml×0.2mlBid腹腔注射给药),而后与模型对照组、中药低、中、高剂量组和西药组一起,于d1、d8以五点(腹腔、左右后脚趾、左右腹股沟皮下)注射10%OVA/Al (OH)3+0.0023‰百日咳类毒素混合液共lml(每处0.2m1)致敏；从d15开始用1%OVA生理盐水雾化吸入激发,每天一次,30min/次,共激发7d。空白对照组予等量生理盐水五点注射及雾化激发。
于d9(即末次五点注射完成后)开始进行灌胃,给药组动物每日灌胃一次,共灌胃14d。其中,中药低、中、高剂量组每日分别予以浓度为0.182g/ml、0.363g/ml、0.727g/ml的浸膏水溶液3ml灌胃(即低剂量组每日灌药量为中剂量组的1/2,高剂量组每日灌药量为中剂量组的2倍)；西药组每日予以地塞米松溶液50μg/ml3ml灌胃。空白对照组、模型对照组和阻断剂组每日给予等量生理盐水。
给药结束后各组大鼠行麻醉下肺功能检测,而后采取支气管肺泡灌洗液、动脉血及肺组织标本。分别用于嗜酸性粒细胞计数；血清IgE、IL-4、IL-5、IL-13检测；组织切片HE染色及免疫组化染色法检测AP-1核蛋白；组织匀浆液RT-PCR法及Western blot法分别检测AP-1核蛋白和mRNA.
采用SPSS17.0统计软件：计量资料以均数±标准差(x±s)表示,计数资料如符合正态分布则也以均数±标准差(x±s)表示,如果不符合正态分布,则用中位数±四分位间距(Md±QR)来表示。多组间比较首先采用非参数检验的单样本K-S拟合优度检验(1-Sample K-S Test)进行正态性检验,如符合或近似符合正态分布,使用单因素方差分析(One-way ANOVA),方差齐使用LSD法进行多重比较,方差不齐使用Tamhane'sT2法进行多重比较分析；如果不符合正态分布,则进行数据转化如对数转换等,如数据转换后仍不符合正态分布,采用非参数检验Kruskal-Wallis法进行多重比较,以P<0.05为有统计学差异,P<0.01为有显著统计学差异。
结果：
1.模型判定及疗效指标的观察与检测结果：
一般情况：造模操作后,模型对照组、中药低、中、高剂量组、西药组出现哮喘阳性症状,正常对照组和阻断剂组无阳性表现。
肺功能检测结果：与空白对照组相比,模型对照组气道高反应性(I)、组织迟滞(H)、组织阻尼(G)、动态顺应性(Cr s)较高,但P>0.05,差异无统计学意义；其余各组各组气道高反应性(Ⅰ)、组织迟滞(H)均高,其中阻断剂阻气道高反应性值最低,但P均>0.05,差异无统计学意义,余深吸气量(IC)、中心气道阻力(Rn)、组织阻尼(G)、动态顺应性(Crs)、静态顺应性(Cst)等指标,各组间趋势不一,P均>0.05,差异无统计学意义。
支气管肺泡灌洗液(BALF)涂片镜下可见嗜酸性粒细胞(EOS),初步观察其数量从高到低依次为模型对照组、中药低剂量组、中药中剂量组、中药高剂量组、西药组、阻断剂组、空白对照组。对其进行计数,结果为：与空白对照组相比,模型对照组BALF涂片EOS计数显著升高(P<0.01)；阻断剂组EOS计数稍高,但P>0.05,差异无统计学意义；中药低、中、高剂量组EOS计数升高(P<0.01),且显示出量效关系；西药组EOS计数升高(P<0.01)。与模型对照组相比,阻断剂组EOS计数较低(P<0.01),中药低、中、高剂量组EOS计数也低,但中药低剂量组P>0.05,差异无统计学意义,中药中、高剂量组P<0.01；西药组EOS计数较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组EOS计数较高(P<0.01)；西药组EOS计数也高但P>0.05,差异无统计学意义。与西药组相比,中药低、中、高剂量组EOS计数均高,但中药高剂量组P>0.05,差异无统计学意义,中药低、中剂量组P<0.01。
肺组织切片HE染色镜下观察可见空白对照组未见炎性细胞浸润,且肺泡、支气管黏膜上皮结构完整清晰,支气管无痉挛；阻断剂组可见少量炎性细胞浸润,肺内结构尚完整清晰,未见支气管痉挛；余模型对照组、中药低、中、高剂量组和西药组均可见肺组织炎性细胞浸润,支气管黏膜上皮细胞排列紊乱、成片脱落,肺泡间隔增厚,支气管痉挛等典型支气管哮喘病理表现,但病理损害轻重程度不同。对其进行组织损伤及炎症病理学评分示：在项目炎性细胞浸润方面,得分从高到低依次是模型对照组=中药低剂量组>中药中剂量组=中药高剂量组>西药组>阻断剂组=空白对照组；在项目支气管上皮细胞脱落方面,得分从高到低依次是模型对照组>中药低剂量组=中药中剂量组>中药高剂量组=西药组>阻断剂组=空白对照组；在项目肺泡间隔增厚方面,得分从高到低依次是模型对照组=中药低剂量组>中药中剂量组>中药高剂量组=西药组>阻断剂组=空白对照；在项目支气管痉挛方面,得分从高到低依次是模型对照组=中药低剂量组>中药中剂量组=中药高剂量组>西药组>阻断剂组=空白对照；总分得分从高到低依次是模型对照组>中药低剂量组>中药中剂量组>中药高剂量组>西药组>阻断剂组>空白对照组。以上P值均 血清IgE检测结果：与空白对照组相比,各组血清IgE均升高(P<0.01),中药低、中、高剂量显示出量效关系。与模型对照组相比,阻断剂组血清IgE较低(P<0.01),中药低、中、高剂量组血清IgE也低,但中药低剂量组P>0.05,差异无统计学意义,中药中、高剂量组P<0.05；西药组血清IgE较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组血清IgE较高(其中中药低剂量组P<0.05,中药中、高剂量组P<0.01)；西药组血清IgE也高但P>0.05,差异无统计学意义。与西药组相比,中药低、中、高剂量组血清IgE均高,但中药高剂量组P>0.05,差异无统计学意义,中药低剂量组P<0.05、中药中剂量组P<0.01。
2.AP-1实时荧光定量RT-PCR检测AP-1mRNA表达量结果：
c-jun mRNA表达量：与空白对照组相比,各组c-jun的表达量均较高,其中模型对照组及中药低、中剂量组P<0.05,余组P值均>0.05,差异无统计学意义。与模型对照组相比,中药低剂量组c-jun的表达量略高,其余各组均低,其中,阻断剂组P<0.05,余组P值均>0.05,差异无统计学意义。与阻断剂组相比,中药低、中、高剂量组及西药组c-jun的表达量均较高,其中西药低、中剂量组P<0.05,余组P值均>0.05,差异无统计学意义。与西药组相比,中药低、中、高剂量组c-jun的表达量均高,但P>0.05,差异无统计学意义。
c-fos mRNA表达量：与空白对照组相比,各组c-fos的表达量均较高,但P值均>0.05,差异无统计学意义。与模型对照组相比,各组c-fos的表达量均较低,但P值均>0.05,差异无统计学意义。与阻断剂组相比,中药低、中、高剂量组及西药组c-fos的表达量均低,但P值均>0.05,差异无统计学意义。与西药组相比,中药低、中剂量组c-fos的表达量均高,但P>0.05,差异无统计学意义。
3.AP-1组成成分含量及活性变化的检测结果：
①肺组织病理切片免疫组织化学染色检测AP-1核蛋白c-Jun和c-Fos结果：
镜下初步观察各组间c-Jun蛋白阳性表现强度从高到低依次为：模型对照组、中药低、中、高剂量组、西药组、阻断剂组、空白对照组,c-Fos蛋白阳性表现强度从高到低依次为：模型对照组、阻断剂组、中药低、中、高剂量组、西药组、空白对照组。阳性表现主要分布于支气管上皮细胞,肺泡间隔中也可见。
各组c-Jun积分光密度均较空白对照组高且P<0.01,其中,中药低、中、高剂量组显示出量效关系。与模型对照组相比,阻断剂组c-Jun积分光密度较低(P<0.01)；中药低、中、高剂量组c-Jun积分光密度也低,其中中药低剂量组P<0.05,中药中、高剂量组P<0.01；西药组c-Jun积分光密度较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组及西药组c-Jun积分光密度较高(P<0.01)。与西药组相比,中药低、中、高剂量组c-Jun积分光密度均高,但P>0.05,差异无统计学意义。
各组c-Fos积分光密度均较空白对照组高且P<0.01,其中,中药低、中、高剂量组显示出量效关系。与模型对照组相比,各组积分光密度较低,其中阻断剂组略低但P>0.05,差异无统计学意义,中药低剂量组P<0.05,其余组P<0.01。与阻断剂组相比,中药低、中、高剂量组及西药组c-Fos积分光密度较低,其中西药组P<0.05,其余组P>0.05,差异无统计学意义。与西药组相比,中药低、中、高剂量组c-Fos积分光密度均高,但P>0.05,差异无统计学意义。
②组织匀浆Western blot检测肺组织中AP-1核蛋白c-Jun与c-Fos结果：
初步观察β-actin表达基本一致,c-Jun蛋白各组间表达从高到低依次为模型对照组、中药低、中、高剂量组、西药组、阻断剂组、空白对照组,c-Fos蛋白各组间表达从高到低依次为模型对照组、阻断剂组、中药低、中、高剂量组、西药组、空白对照组。
各组c-Jun平均灰度值均较空白对照组高且P均<0.01,其中,中药低、中、高剂量组显示出量效关系。与模型对照组相比,阻断剂组c-Jun平均灰度较低(P<0.01),中药低、中、高剂量组c-Jun平均灰度也低,但中药中、低剂量组P>0.05,差异无统计学意义,中药高剂量组P<0.01；西药组c-Jun平均灰度较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组及西药组c-Jun平均灰度较高(P<0.01)。与西药组相比,中药低、中、高剂量组c-Jun平均灰度均高,但中药高剂量组P>0.05,差异无统计学意义,中药低、中剂量组P<0.01。
各组c-Fos平均灰度值均较空白对照组高且P均<0.01,其中,中药低、中、高剂量组显示出量效关系。与模型对照组相比,阻断剂组、中药低、中、高剂量组及西药组c-Fos平均灰度均较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组及西药组c-Fos平均灰度较高(P<0.01)。与西药组相比,中药低、中、高剂量组c-Fos平均灰度均高,但中药高剂量组P>0.05,差异无统计学意义,中药低、中剂量组P<0.01。
4.相关细胞因子IL-4、IL-5、IL-13的测定结果：
与空白对照组相比,各组血清IL-4含量均高,中药低、中、高剂量显示出量效关系,其中,阻断剂组P>0.05,差异无统计学意义,其余组P<0.01。与模型对照组相比,各组血清IL-4较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组及西药组血清IL-4较高(P<0.01)。与西药组相比,中药低、中、高剂量组血清IL-4均高,P<0.01。
与空白对照组相比,各组血清IL-5含量均高,中药低、中、高剂量显示出量效关系,但仅有中药低、中剂量组P<0.01,其余各组P>0.05,差异无统计学意义。与模型对照组相比,各组血清IL-5较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组血清IL-5较高,但中药高剂量组P>0.05,差异无统计学意义,中药中、低剂量组P<0.01；西药组血清IL-5也高但P>0.05,差异无统计学意义。与西药组相比,中药低、中、高剂量组血清IL-5均高,但中药高剂量组P>0.05,差异无统计学意义,中药低、中剂量组P<0.01。
与空白对照组相比,各组血清IL-13含量均高,中药低、中、高剂量显示出量效关系,其中阻断剂组和中药高剂量组P<0.05,其余各组P<0.01。与模型对照组相比,各组血清IL-13较低,其中中药低剂量组P<0.05,其余各组P<0.01。与阻断剂组相比,中药低、中、高剂量组血清IL-13较高,但中药高剂量组P>0.05,差异无统计学意义,中药中、低剂量组P<0.01；西药组血清IL-13也高(P<0.05)。与西药组相比,中药低、中剂量组较高(P>0.05),差异无统计学意义,中药高剂量组则略低且P<0.01
结论：
1.本实验中建立的支气管哮喘大鼠模型是成功的,AP-1是支气管哮喘发病的关键环节。
①SD大鼠支气管哮喘模型复制成功。
采用五点(腹腔、左右后脚趾、左右腹股沟皮下)注射10%OVA/A1(OH)3+0.0023‰百日咳类毒素混合液致敏SD大鼠再行1%OVA生理盐水雾化吸入激发的方法可成功复制大鼠支气管哮喘模型。
②姜黄素对支气管哮喘大鼠肺组织AP-1的表达阻断成功。
各种实验手段检测AP-1基因表达产物mRNA和核蛋白含量及Th2细胞下游因子IL-4、 IL-5和IL-13含量：阻断剂组大鼠的c-jun mRNA和核蛋白含量以及IL-4、IL-5和IL-13表达情况接近于空白对照组而远低于模型对照组,c-fos mRNA和核蛋白含量接近于模型对照组而远高于空白对照组,该组大鼠AP-1表达阻断总体而言是成功的。
③AP-1是支气管哮喘发病的关键环节。
阻断剂组大鼠未呈现典型哮喘症状和体征,肺功能、BALF涂片EOS计数、肺组织病理切片HE染色、血清IgE值等实验室检查指标也无典型哮喘模型阳性表现。可以认为该组大鼠未成功复制支气管哮喘模型,提示AP-1是哮喘发病的关键环节,对其进行阻断可在一定程度阻止哮喘发病。
2.芪味理肺汤通过对AP-1的表达及活性的调控,影响其下游细胞因子(IL-4、IL-5、 IL-13)的表达及活性,对支气管哮喘大鼠治疗显著有效。
①芪味理肺汤治疗支气管哮喘大鼠有效。
芪味理肺汤低、中、高剂量组大鼠BALF涂片EOS计数、肺组织病理切片HE染色、血清IgE值等实验室检查指标低于模型对照组而略高于西药组,其中高剂量组接近于西药组、低剂量组则接近于模型对照组。表明芪味理肺汤治疗支气管哮喘大鼠有效,且高剂量灌胃效果接近于西药激素地塞米松灌胃,而低剂量灌胃治疗可认为无效。
OAP-1表达及活性受调情况。
各实验手段检测AP-1基因表达产物mRNA和核蛋白含量,芪味理肺汤低、中、高剂量组三组间呈现量效差异,三组大鼠AP-1mRNA含量与模型对照组、西药组差异均无统计学意义,中药高剂量组大鼠的AP-1核蛋白含量接近于西药组,而中药低剂量组则近似于模型对照组。表明本实验结果尚不能明确芪味理肺汤对支气管大鼠肺组织内AP-1mRNA表达的影响,但可以降低AP-1核蛋白的含量,减少AP-1的合成。
③AP-1下游细胞因子(IL-4、IL-5、IL-13)的表达情况。
芪味理肺汤低、中、高剂量组的Th2细胞下游因子IL-4、IL-5和IL-13含量,三组间呈现量效差异,低剂量组大鼠情况接近于模型对照组而高剂量组则近似于西药组。初步推测芪味理肺汤可能是阻断了AP-1以mRNA为模板合成蛋白这一过程,使大鼠肺组织内的AP-1核蛋白(c-Jun、c-Fos)含量减少,AP-1总量减少,Th2细胞下游因子IL-4、IL-5和IL-13的合成因此受限,表现为IL-4、IL-5和IL-13含量降低。
Objective
1. Using the animal experiments of stable bronchial asthma SD rat model for medicine, to confirm the AP-1is a key link for the pathogenesis of bronchial asthma,and to verify the efficacy of Qiweilifei Decoction and get its evidence.2. Through using the methods of molecular biology to test the upstream and downstream factors of AP-1, to improve the mechanism of bronchial asthma treatment by Qiweilifei Decoction,and to explore the details in regulating AP-1.
Methods
After7days to feed adaptively,70SPF SD rats were randomly divided into7groups: the blank control group, the model control group,the blocker control group, the Chinese medicine low-dose group,the Chinese medicine middle-dose group, the Chinese medicine high-dose group and the western medicine group. Before the model-making of the blocker control group rats, used curcumin to precondition them for3days (100mg. kg-1. d-1,50mg/ml×0.2mlBid, intraperitoneal injection). Then, ea-ch rat except the blank control group sensitized by injecting the mixture with10%OVA/Al (OH)3+0.0023%o Pertussis toxin from Bordetella pertussis lml into five point(intraperitoneal, subcutaneous of the left/right rear toe and the left/right groin,0.2ml each point)in day1and day8to make model. After sensitization,used1%OVA+normal saline to stimulate immunoreaction by inhaled(30min Qd,7days). The blank control group used normal saline to do all above treatment.
Intragastric administration began in day9(finished the last injection), lasted14days,Qd. The Chinese medicine low-dose, middle-dose and high-dose groups were given by Chinese medical extractum normal saline solution0.182g/ml,0.363g/ml and0.727g/ml (dosage:low-dose group=1/2middle-dose group, middle-dose group=1/2high-dose group)3ml. The western medicine group was given by dexamethasone normal saline solution50μg/ml3ml. And the blank control group, the model control group and the blocker control group were given by normal saline3ml.
After administration,all rats were evaluated pulmonary function under anesthesia. Then taken the samples of bronchoalveolar lavage fluid,arterial blood and the lung tissue to do eosinophil cell count, serum IgE/IL-4/IL-5/IL-13content test,lung tissue sections stained by HE (to observe the morphological alternation) and immumohistochemical (to test the AP-1nucleoprotein),and lung tissue homogenate AP-1mRNA/nucleoprotein content test (by RT-PCR/Western blot).
All statistical analysis was performed using SPSS17.0software package. Calculated data:mean±standard deviation(χ±s).Enumeration data:mean±standard deviation(χ±s)(normal distribution), or median±interquartile range (Md±QR)(abnormal distribution).First, used1-Sample K-S Test of non-parametric test to do normality test.Then, if the data fit normal distribution, used One-Way ANOVA to do the homogeneity test of variances, and used LSD(equal variance) or Tamhane'sT2(unequal variance) to do the multiple comparisons; if the data doesn't fit normal distribution, transformed the data (such as logarithmic transformation) and repeat the above steps,or still doesn't fit normal distribution,used Kruskal-Wallis to do the multiple comparisons. Regarded P<0.05as statistically difference, and P<0.01as statistically significant difference.
Results
1.Observation and test results of the model assessment index and the therapeutic effect index.
General conditions:After the injection for model-making, rats of the model control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group developed asthma symptoms, when rats of the blank control group and the blocker control group did not.
Pulmonary function results:Compared with the blank control group, the results of the model control group's airway hyperresponsiveness (I), tissue sluggish (H), tissue damping (G) and dynamic lung compliance (Crs) were higher (P>0.05, no statistically significant). When the results of the other groups' hyperresponsiveness (I) and tissue sluggish (H) were both higher.Among all the other groups, the airway hyperresponsiveness (I) of the blocker control group was the lowest (P>0.05, no statistically significant). The rest of results such as inspiratory capacity (IC).Central airway resistance (Rn), tissue damping (G), dynamic lung compliance (Crs) and static lung compliance (Cst) had different trends between every group (P>0.05, no statistically significant).
The bronchoalveolar lavage fluid (BALF) smear can recognize some Bosinophil cells (EOS) under the microscope. Preliminary observation the number of them from high to low was the model control group,the Chinese medicine low-dose group, the Chinese medicine middle-dose group, the Chinese medicine high-dose group,the western medicine group,the blocker control group,the blank control group. Count results showed that:Compared with the blank control group,the result of the model control group's BALF smear EOS count was significant elevated(P<0.01); the blocker control group's was a little higher(P>0.05,no statistically significant);the Chinese medicine low-dose, middle-dose and high-dose groups'were higher (P<0.01) and showed the concentration-response relationship; and the western medicine group's was higher too(P<0.01). Compared with the model control group, the blocker control group's was lower (P<0.01); the Chinese medicine low-dose, middle-dose and high-dose groups'were lower too, but the low-dose group's P>0.05(no statistically significant), when the middle-dose and high-dose groups'P<0.01; and the western medicine group's was lower (P<0.01). Compared with the blocker control group,the Chinese medicine low-dose, middle-dose and high-dose groups'were higher (P<0.01); and the western medicine group's was higher too, but P>0.05(no statistically significant). Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups'were higher, but the high-dose group's P>0.05(no statistically significant), and the low-dose, middle-dose groups' P<0.01.
Under the microscope view to observe the morphological alternation from lung tissue sections stained by HE:The blank control group had not showed any change and inflammatory cellular infiltration, when he blocker control group had a little, and the other groups all had inflammatory cellular infiltration in the lung tissue, bronchial epithelial cells irregular arrangement and desquamation, thic-kened alveoli septum and bronchiolar spasm, but the pathological damage severity were different. Results of the score by using tissue injury and inflammation pathological scoring criteria showed that:On the item of the inflammatory cellular infiltration, the score from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group=the Chinese medicine high-dose group> the western medicine group> the blocker control group=the blank control group. On the item of the bronchial epithelial cells desquamation, the score from high to low was the model control group> the Chinese medicine low-dose group=the Chinese medicine middle-dose group>the Chinese medicine high-dose group=the western medicine group> the blocker control group=the blank control group. On the item of the thickened alveoli septum, the score from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group> the Chinese medicine high-dose group=the western medicine group> the blocker control group=the blank control group. On the item of the bronchiolar spasm, the score from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group=the Chinese medicine high-dose group> the western medicine group> the blocker control group=the blank control group. And the total points from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group>the Chinese medicine high-dose group> the western medicine group> the blocker control group> the blank control group, all above P<0.01.
Results of the serum IgE content test showed:Compared with the blank control group, every group's serum IgE content increased (P<0.01), and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, the blocker control group's was lower (P<0.01); the Chinese medicine low-dose, middle-dose and high-dose groups'were lower too,but the low-dose group's P>0.05(no statistically significant), when the middle-dose and high-dose groups'P<0.05;and the western medicine group's was lower (P<0.01). Compared with the blocker control group, the Chinese medicine low-dose,middle-dose and high-dose groups' were higher(the low-dose group's P<0.05, while the middle-dose and high-dose groups' P<0.01); and the western medicine group's was higher too, but P>0.05(no statistically significant). Compared with the western medicine group,the Chinese medicine low-dose, middle-dose and high-dose groups' were higher, but the high-dose group's P>0.05(no statistically significant), and the low-dose group's P<0.01, the middle-dose group's P<0.05.
2. Results of AP-1mRNA expression quantities by Real-time RT-PCR.
c-jun mRNA expression quantities:Compared with the blank control group,every group's c-jun mRNA expression quantities increased, among them, the model control group and the Chinese medicine low-dose, middle-dose groups' P<0.05, the others P>0.05(no statistically significant). Compared with the model control group, the Chinese medicine low-dose group's was a little higher, and the other groups' were lower,but only the blocker control group's P<0.05, all of the rest groups' P>0.05(no statistically significant). Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher, when the the western medicine group and the middle-dose group's P<0.05, the others P>0.05(no statistically significant). Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups'were higher,but P>0.05(no statistically significant).
c-fos mRNA expression quantities:Compared with the blank control group, every group's c-fos mRNA expression quantities increase.Compared with the model control group, every group's was lower. Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was lower.Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups' were higher,but all above P>0.05(no statistically significant).
3. Results of AP-1component content and activity test.
①Result of observing the positive expression of AP-1.
Under the microscope view, the intensity of positive expression from high to low was the model control group, the Chinese medicine low-dose group, the Chinese medicine middle-dose group, the Chinese medicine high-dose group,the western medicine group, the blocker control group, the blank control group. The positive expression mainly located in bronchial epithelial cells, sometimes in alveolar interval.
Compared with the blank control group, every group's c-Jun nucleoprotein integral optical density(IOD) increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, the blocker control group's was lower (P<0.01);the Chinese medicine low-dose, middle-dose and high-dose groups'were lower too, and the low-dose groups' P<0.05, the middle-dose and high-dose groups'P<0.01; the western medicine group's was lower (P<0.01).Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher(P<0.01).Compared with the western medicine group,the Chinese medicine low-dose, middle-dose and high-dose groups'were higher,but P>0.05(no statistically significant).
Compared with the blank control group, every group's c-Fos nucleoprotein integral optical density (IOD) increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, every group's was lower; among them, the blocker control group's P>0.05(no statistically significant), the Chinese medicine low-dose groups'P<0.05, all of the rest P<0.01. Compared with the blocker control group,the Chinese medicine low-dose,middle-dose, high-dose groups and the western medicine group's was lower, the western medicine group's P<0.05, the others P>0.05(no statistically significant). Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups'were higher, but P>0.05(no statistically significant).
②Result of AP-1nucleoprotein (c-Jun and c-Fos) expression quantities by Western blot:
Preliminary observation:All of the β-actin expressed almost the same, while c-Jun nucleoprotein expression quantities from high to low was the model control group,the Chinese medicine low-dose group, middle-dose, high-dose group, the western medicine group, the blocker control group, the blank control group, and c-Fos nucleoprotein expression quantities from high to low was the model control group, the blank control group, the Chinese medicine low-dose group, middle-dose, high-dose group, the western medicine group, the blocker control group.
Compared with the blank control group, every group's c-Jun nucleoprotein mean gray value increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, the blocker control group's was lower(P<0.01); the Chinese medicine low-dose, middle-dose and high-dose groups'were lower too, and the low-dose and middle-dose groups' P>0.05(no statistically significant), the high-dose group's P<0.01; the western medicine group's was lower (P<0.01). Compar-ed with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher (P<0.01). Compared with the western medicine group,the Chinese medicine low-dose, middle-dose and high-dose groups'were higher,but the high-dose group's P>0.05(no statistically significant), whlie the low-dose and middle-dose groups'P<0.01.
Compared with the blank control group, every group's c-Fos nucleoprotein mean gray value increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group,the blocker control group,the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was lower(P<0.01).Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher(P<0.01).Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups'were higher, but the high-dose group's P>0.05(no statistically significant), whlie the low-dose and middle-dose groups' P<0.01.
4.Results of serum IL-4, IL-5, IL-13content test.
Compared with the blank control group,every group's serum IL-4content increased,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship, among them, the blocker control group's P>0.05(no statistically significant) and all of the rest P<0.01. Compared with the model control group, every group's was lower (P<0.01). Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher (P<0.01).Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups' were higher (P<0.01).
Compared with the blank control group, every group's serum IL-5content increased,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship,but only the Chinese medicine low-dose and middle-dose groups'P<0.01, the others P>0.05(no statistically significant). Compared with the model control group, every group's was lower (P <0.01).Compared with the blocker control group,the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher, among them, the high-dose group and the western medicine group's P>0.05(no statistically significant), while the low-dose and middle-dose groups'P <0.01. Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups'were higher,but the high-dose groups' P>0.05(no statistically significant), and the low-dose and middle-dose groups' P<0.01.
Compared with the blank control group,every group's serum IL-13content increased, and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship, among them, the blocker control group and the Chinese medicine high-dose groups'P<0.05, while the others P<0.01. Compared with the model control group,every group's was lower,and the low-dose group's P<0.05, the others P<0.01.Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher,but the high-dose group's P>0.05(no statistically significant), and the low-dose and middle-dose groups'P<0.01,the western medicine group's P<0.05. Compared with the western medicine group,the Chinese medicine low-dose and middle-dose groups'were higher but P>0.05(no statistically significant), while the high-dose group's was a little higher (P<0.01).
Conclusion
1.The bronchial asthma rat model which was established in this experimient had been successful, and AP-1is a key link for the pathogenesis of bronchial asthma.
①The bronchial asthma SD rat model had been successfully duplicated.
The method of sensitized by injecting the mixture with10%OVA/Al (OH)3+0.0023%o Pertussis toxin from Bordetella pertussis into five point (intraperitoneal, subcut-aneous of the left/right rear toe and the left/right groin) to make model,and uses1%OVA+normal saline to stimulate immunoreaction by inhaled can successfully duplicat the bronchial asthma rat model.
②Curcumin had successful blocked the AP-1expression in the lung tissue of bronchial asthma rat.
Through various experimental methods to test the content of AP-1gene expression product (mRNA and nucleoprotein) and downstream factors of Th2cells (IL-4, IL-5and IL-13),we found that the blocker control group's content of c-jun mRNA and nucleoprotein, IL-4,IL-5and IL-13were close to the blank control group but far below the model control group,while the content of c-fos mRNA and nucleoprotein were close to the model control group but far above the blank control group, thus this blocker group had been blocked successful in general.
③AP-1is a key link for the pathogenesis of bronchial asthma.
The blocker control group rats did not present with typical symptoms and physical signs of asthma. Results of pulmonary function,BLAF smear EOS count, lung tissue sections stained by HE and serum IgE did not have positive performance of typical asthma. We can consider that this blocker group failed to copy the bronchus asthma model. All above suggest that AP-1is a key link for the pathogenesis of bronchial asthma, and to block it can prevent asthma pathogenesis to some extent.2. By regulating the expression and activity of AP-1, and affecting its expression and activity of downstream factors (IL-4、IL-5and IL-13), Qiweilifei Decoction is effective in the treatment of asthma.
①Qiweilifei Decoction is effective in the treatment of asthma.
Compare the results of BLAF smear EOS count,lung tissue sections stained by HE and serum IgE had positive performance of typical asthma, the Qiweilifei Decoction low-dose, middle-dose and high-dose groups rats were lower than the model control group but a little higher than the western medicine group. Among them, the high-dose group can be considered equal to the western medicine group,while the low-dose group can be considered close to the model control group. All above indicate that Qiweilifei Decoction is effective in the treatment of asthma, and high-dose gavage can be considered as Dexamethasone, whlie low-dose gavage is ineffective.
②The expression and activity of AP-1were regulated by Qiweilifei Decoction.
Through various experimental methods to test the content of AP-1gene expression product (mRNA and nucleoprotein), the Qiweilifei Decoction low-dose, middle-dose and high-dose groups showed the concentration-response relationship. The content of AP-1mRNA:Compare with the model control group and the western medicine group, three Chinese medicine groups'differences had no statistical significance. The content of AP-1nucleoprotein:The high-dose group can be considered equal to the western medicine group, while the low-dose group can be considered close to the model control group. The results showed that the effect of the AP-1mRNA regulation by Qiweilifei Decoction is not yet clear from this experiment. However, Qiweilifei Decoction can reduce the content of AP-1nucleoprotein and reduce the synthesis of AP-1.
③The expression of downstream factors of AP-1(IL-4, IL-5and IL-13) were affected by Qiweilifei Decoction.
The Qiweilifei Decoction low-dose, middle-dose and high-dose groups'content of Th2downstream factors(IL-4, IL-5and IL-13)showed the concentration-response relationship. The low-dose group can be considered equal to the model control group while the high-dose group can be considered close to the western medicine group. We preliminary presumption that Qiweilifei Decoction possibly be block the process of AP-1mRNA served as a template to compound a proteins, then reduce content of AP-1nucleoprotein (c-Jun and c-Fos) in the lung tissue of rats. At last, AP-1itself and the Th2downstream factors (IL-4, IL-5and IL-13) also be reduced.
目的：
1.通过稳定复制的SD大鼠支气管哮喘模型进行给药动物实验,证实AP-1是支气管哮喘发病的关键环节,验证芪味理肺汤治疗支气管哮喘的疗效并获取该方疗效的模式动物学证据。
2.通过分子生物学方法等手段检测AP-1上下游因子的含量,完善芪味理肺汤治疗支气管哮喘的作用机理,探索该方调控AP-1治疗支气管哮喘的具体作用环节。
方法：
70只SPF级SD大鼠适应性喂养7d无异常后,随机分为空白对照组、模型对照组、阻断剂组、中药低、中、高剂量组、西药组7组。阻断剂组先经AP-1的抑制剂姜黄素预处理3d (100mg. kg-1. d-1,50mg/ml×0.2mlBid腹腔注射给药),而后与模型对照组、中药低、中、高剂量组和西药组一起,于d1、d8以五点(腹腔、左右后脚趾、左右腹股沟皮下)注射10%OVA/Al (OH)3+0.0023‰百日咳类毒素混合液共lml(每处0.2m1)致敏；从d15开始用1%OVA生理盐水雾化吸入激发,每天一次,30min/次,共激发7d。空白对照组予等量生理盐水五点注射及雾化激发。
于d9(即末次五点注射完成后)开始进行灌胃,给药组动物每日灌胃一次,共灌胃14d。其中,中药低、中、高剂量组每日分别予以浓度为0.182g/ml、0.363g/ml、0.727g/ml的浸膏水溶液3ml灌胃(即低剂量组每日灌药量为中剂量组的1/2,高剂量组每日灌药量为中剂量组的2倍)；西药组每日予以地塞米松溶液50μg/ml3ml灌胃。空白对照组、模型对照组和阻断剂组每日给予等量生理盐水。
给药结束后各组大鼠行麻醉下肺功能检测,而后采取支气管肺泡灌洗液、动脉血及肺组织标本。分别用于嗜酸性粒细胞计数；血清IgE、IL-4、IL-5、IL-13检测；组织切片HE染色及免疫组化染色法检测AP-1核蛋白；组织匀浆液RT-PCR法及Western blot法分别检测AP-1核蛋白和mRNA.
采用SPSS17.0统计软件：计量资料以均数±标准差(x±s)表示,计数资料如符合正态分布则也以均数±标准差(x±s)表示,如果不符合正态分布,则用中位数±四分位间距(Md±QR)来表示。多组间比较首先采用非参数检验的单样本K-S拟合优度检验(1-Sample K-S Test)进行正态性检验,如符合或近似符合正态分布,使用单因素方差分析(One-way ANOVA),方差齐使用LSD法进行多重比较,方差不齐使用Tamhane'sT2法进行多重比较分析；如果不符合正态分布,则进行数据转化如对数转换等,如数据转换后仍不符合正态分布,采用非参数检验Kruskal-Wallis法进行多重比较,以P<0.05为有统计学差异,P<0.01为有显著统计学差异。
结果：
1.模型判定及疗效指标的观察与检测结果：
一般情况：造模操作后,模型对照组、中药低、中、高剂量组、西药组出现哮喘阳性症状,正常对照组和阻断剂组无阳性表现。
肺功能检测结果：与空白对照组相比,模型对照组气道高反应性(I)、组织迟滞(H)、组织阻尼(G)、动态顺应性(Cr s)较高,但P>0.05,差异无统计学意义；其余各组各组气道高反应性(Ⅰ)、组织迟滞(H)均高,其中阻断剂阻气道高反应性值最低,但P均>0.05,差异无统计学意义,余深吸气量(IC)、中心气道阻力(Rn)、组织阻尼(G)、动态顺应性(Crs)、静态顺应性(Cst)等指标,各组间趋势不一,P均>0.05,差异无统计学意义。
支气管肺泡灌洗液(BALF)涂片镜下可见嗜酸性粒细胞(EOS),初步观察其数量从高到低依次为模型对照组、中药低剂量组、中药中剂量组、中药高剂量组、西药组、阻断剂组、空白对照组。对其进行计数,结果为：与空白对照组相比,模型对照组BALF涂片EOS计数显著升高(P<0.01)；阻断剂组EOS计数稍高,但P>0.05,差异无统计学意义；中药低、中、高剂量组EOS计数升高(P<0.01),且显示出量效关系；西药组EOS计数升高(P<0.01)。与模型对照组相比,阻断剂组EOS计数较低(P<0.01),中药低、中、高剂量组EOS计数也低,但中药低剂量组P>0.05,差异无统计学意义,中药中、高剂量组P<0.01；西药组EOS计数较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组EOS计数较高(P<0.01)；西药组EOS计数也高但P>0.05,差异无统计学意义。与西药组相比,中药低、中、高剂量组EOS计数均高,但中药高剂量组P>0.05,差异无统计学意义,中药低、中剂量组P<0.01。
肺组织切片HE染色镜下观察可见空白对照组未见炎性细胞浸润,且肺泡、支气管黏膜上皮结构完整清晰,支气管无痉挛；阻断剂组可见少量炎性细胞浸润,肺内结构尚完整清晰,未见支气管痉挛；余模型对照组、中药低、中、高剂量组和西药组均可见肺组织炎性细胞浸润,支气管黏膜上皮细胞排列紊乱、成片脱落,肺泡间隔增厚,支气管痉挛等典型支气管哮喘病理表现,但病理损害轻重程度不同。对其进行组织损伤及炎症病理学评分示：在项目炎性细胞浸润方面,得分从高到低依次是模型对照组=中药低剂量组>中药中剂量组=中药高剂量组>西药组>阻断剂组=空白对照组；在项目支气管上皮细胞脱落方面,得分从高到低依次是模型对照组>中药低剂量组=中药中剂量组>中药高剂量组=西药组>阻断剂组=空白对照组；在项目肺泡间隔增厚方面,得分从高到低依次是模型对照组=中药低剂量组>中药中剂量组>中药高剂量组=西药组>阻断剂组=空白对照；在项目支气管痉挛方面,得分从高到低依次是模型对照组=中药低剂量组>中药中剂量组=中药高剂量组>西药组>阻断剂组=空白对照；总分得分从高到低依次是模型对照组>中药低剂量组>中药中剂量组>中药高剂量组>西药组>阻断剂组>空白对照组。以上P值均
2.AP-1实时荧光定量RT-PCR检测AP-1mRNA表达量结果：
c-jun mRNA表达量：与空白对照组相比,各组c-jun的表达量均较高,其中模型对照组及中药低、中剂量组P<0.05,余组P值均>0.05,差异无统计学意义。与模型对照组相比,中药低剂量组c-jun的表达量略高,其余各组均低,其中,阻断剂组P<0.05,余组P值均>0.05,差异无统计学意义。与阻断剂组相比,中药低、中、高剂量组及西药组c-jun的表达量均较高,其中西药低、中剂量组P<0.05,余组P值均>0.05,差异无统计学意义。与西药组相比,中药低、中、高剂量组c-jun的表达量均高,但P>0.05,差异无统计学意义。
c-fos mRNA表达量：与空白对照组相比,各组c-fos的表达量均较高,但P值均>0.05,差异无统计学意义。与模型对照组相比,各组c-fos的表达量均较低,但P值均>0.05,差异无统计学意义。与阻断剂组相比,中药低、中、高剂量组及西药组c-fos的表达量均低,但P值均>0.05,差异无统计学意义。与西药组相比,中药低、中剂量组c-fos的表达量均高,但P>0.05,差异无统计学意义。
3.AP-1组成成分含量及活性变化的检测结果：
①肺组织病理切片免疫组织化学染色检测AP-1核蛋白c-Jun和c-Fos结果：
镜下初步观察各组间c-Jun蛋白阳性表现强度从高到低依次为：模型对照组、中药低、中、高剂量组、西药组、阻断剂组、空白对照组,c-Fos蛋白阳性表现强度从高到低依次为：模型对照组、阻断剂组、中药低、中、高剂量组、西药组、空白对照组。阳性表现主要分布于支气管上皮细胞,肺泡间隔中也可见。
各组c-Jun积分光密度均较空白对照组高且P<0.01,其中,中药低、中、高剂量组显示出量效关系。与模型对照组相比,阻断剂组c-Jun积分光密度较低(P<0.01)；中药低、中、高剂量组c-Jun积分光密度也低,其中中药低剂量组P<0.05,中药中、高剂量组P<0.01；西药组c-Jun积分光密度较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组及西药组c-Jun积分光密度较高(P<0.01)。与西药组相比,中药低、中、高剂量组c-Jun积分光密度均高,但P>0.05,差异无统计学意义。
各组c-Fos积分光密度均较空白对照组高且P<0.01,其中,中药低、中、高剂量组显示出量效关系。与模型对照组相比,各组积分光密度较低,其中阻断剂组略低但P>0.05,差异无统计学意义,中药低剂量组P<0.05,其余组P<0.01。与阻断剂组相比,中药低、中、高剂量组及西药组c-Fos积分光密度较低,其中西药组P<0.05,其余组P>0.05,差异无统计学意义。与西药组相比,中药低、中、高剂量组c-Fos积分光密度均高,但P>0.05,差异无统计学意义。
②组织匀浆Western blot检测肺组织中AP-1核蛋白c-Jun与c-Fos结果：
初步观察β-actin表达基本一致,c-Jun蛋白各组间表达从高到低依次为模型对照组、中药低、中、高剂量组、西药组、阻断剂组、空白对照组,c-Fos蛋白各组间表达从高到低依次为模型对照组、阻断剂组、中药低、中、高剂量组、西药组、空白对照组。
各组c-Jun平均灰度值均较空白对照组高且P均<0.01,其中,中药低、中、高剂量组显示出量效关系。与模型对照组相比,阻断剂组c-Jun平均灰度较低(P<0.01),中药低、中、高剂量组c-Jun平均灰度也低,但中药中、低剂量组P>0.05,差异无统计学意义,中药高剂量组P<0.01；西药组c-Jun平均灰度较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组及西药组c-Jun平均灰度较高(P<0.01)。与西药组相比,中药低、中、高剂量组c-Jun平均灰度均高,但中药高剂量组P>0.05,差异无统计学意义,中药低、中剂量组P<0.01。
各组c-Fos平均灰度值均较空白对照组高且P均<0.01,其中,中药低、中、高剂量组显示出量效关系。与模型对照组相比,阻断剂组、中药低、中、高剂量组及西药组c-Fos平均灰度均较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组及西药组c-Fos平均灰度较高(P<0.01)。与西药组相比,中药低、中、高剂量组c-Fos平均灰度均高,但中药高剂量组P>0.05,差异无统计学意义,中药低、中剂量组P<0.01。
4.相关细胞因子IL-4、IL-5、IL-13的测定结果：
与空白对照组相比,各组血清IL-4含量均高,中药低、中、高剂量显示出量效关系,其中,阻断剂组P>0.05,差异无统计学意义,其余组P<0.01。与模型对照组相比,各组血清IL-4较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组及西药组血清IL-4较高(P<0.01)。与西药组相比,中药低、中、高剂量组血清IL-4均高,P<0.01。
与空白对照组相比,各组血清IL-5含量均高,中药低、中、高剂量显示出量效关系,但仅有中药低、中剂量组P<0.01,其余各组P>0.05,差异无统计学意义。与模型对照组相比,各组血清IL-5较低(P<0.01)。与阻断剂组相比,中药低、中、高剂量组血清IL-5较高,但中药高剂量组P>0.05,差异无统计学意义,中药中、低剂量组P<0.01；西药组血清IL-5也高但P>0.05,差异无统计学意义。与西药组相比,中药低、中、高剂量组血清IL-5均高,但中药高剂量组P>0.05,差异无统计学意义,中药低、中剂量组P<0.01。
与空白对照组相比,各组血清IL-13含量均高,中药低、中、高剂量显示出量效关系,其中阻断剂组和中药高剂量组P<0.05,其余各组P<0.01。与模型对照组相比,各组血清IL-13较低,其中中药低剂量组P<0.05,其余各组P<0.01。与阻断剂组相比,中药低、中、高剂量组血清IL-13较高,但中药高剂量组P>0.05,差异无统计学意义,中药中、低剂量组P<0.01；西药组血清IL-13也高(P<0.05)。与西药组相比,中药低、中剂量组较高(P>0.05),差异无统计学意义,中药高剂量组则略低且P<0.01
结论：
1.本实验中建立的支气管哮喘大鼠模型是成功的,AP-1是支气管哮喘发病的关键环节。
①SD大鼠支气管哮喘模型复制成功。
采用五点(腹腔、左右后脚趾、左右腹股沟皮下)注射10%OVA/A1(OH)3+0.0023‰百日咳类毒素混合液致敏SD大鼠再行1%OVA生理盐水雾化吸入激发的方法可成功复制大鼠支气管哮喘模型。
②姜黄素对支气管哮喘大鼠肺组织AP-1的表达阻断成功。
各种实验手段检测AP-1基因表达产物mRNA和核蛋白含量及Th2细胞下游因子IL-4、 IL-5和IL-13含量：阻断剂组大鼠的c-jun mRNA和核蛋白含量以及IL-4、IL-5和IL-13表达情况接近于空白对照组而远低于模型对照组,c-fos mRNA和核蛋白含量接近于模型对照组而远高于空白对照组,该组大鼠AP-1表达阻断总体而言是成功的。
③AP-1是支气管哮喘发病的关键环节。
阻断剂组大鼠未呈现典型哮喘症状和体征,肺功能、BALF涂片EOS计数、肺组织病理切片HE染色、血清IgE值等实验室检查指标也无典型哮喘模型阳性表现。可以认为该组大鼠未成功复制支气管哮喘模型,提示AP-1是哮喘发病的关键环节,对其进行阻断可在一定程度阻止哮喘发病。
2.芪味理肺汤通过对AP-1的表达及活性的调控,影响其下游细胞因子(IL-4、IL-5、 IL-13)的表达及活性,对支气管哮喘大鼠治疗显著有效。
①芪味理肺汤治疗支气管哮喘大鼠有效。
芪味理肺汤低、中、高剂量组大鼠BALF涂片EOS计数、肺组织病理切片HE染色、血清IgE值等实验室检查指标低于模型对照组而略高于西药组,其中高剂量组接近于西药组、低剂量组则接近于模型对照组。表明芪味理肺汤治疗支气管哮喘大鼠有效,且高剂量灌胃效果接近于西药激素地塞米松灌胃,而低剂量灌胃治疗可认为无效。
OAP-1表达及活性受调情况。
各实验手段检测AP-1基因表达产物mRNA和核蛋白含量,芪味理肺汤低、中、高剂量组三组间呈现量效差异,三组大鼠AP-1mRNA含量与模型对照组、西药组差异均无统计学意义,中药高剂量组大鼠的AP-1核蛋白含量接近于西药组,而中药低剂量组则近似于模型对照组。表明本实验结果尚不能明确芪味理肺汤对支气管大鼠肺组织内AP-1mRNA表达的影响,但可以降低AP-1核蛋白的含量,减少AP-1的合成。
③AP-1下游细胞因子(IL-4、IL-5、IL-13)的表达情况。
芪味理肺汤低、中、高剂量组的Th2细胞下游因子IL-4、IL-5和IL-13含量,三组间呈现量效差异,低剂量组大鼠情况接近于模型对照组而高剂量组则近似于西药组。初步推测芪味理肺汤可能是阻断了AP-1以mRNA为模板合成蛋白这一过程,使大鼠肺组织内的AP-1核蛋白(c-Jun、c-Fos)含量减少,AP-1总量减少,Th2细胞下游因子IL-4、IL-5和IL-13的合成因此受限,表现为IL-4、IL-5和IL-13含量降低。
Objective
1. Using the animal experiments of stable bronchial asthma SD rat model for medicine, to confirm the AP-1is a key link for the pathogenesis of bronchial asthma,and to verify the efficacy of Qiweilifei Decoction and get its evidence.2. Through using the methods of molecular biology to test the upstream and downstream factors of AP-1, to improve the mechanism of bronchial asthma treatment by Qiweilifei Decoction,and to explore the details in regulating AP-1.
Methods
After7days to feed adaptively,70SPF SD rats were randomly divided into7groups: the blank control group, the model control group,the blocker control group, the Chinese medicine low-dose group,the Chinese medicine middle-dose group, the Chinese medicine high-dose group and the western medicine group. Before the model-making of the blocker control group rats, used curcumin to precondition them for3days (100mg. kg-1. d-1,50mg/ml×0.2mlBid, intraperitoneal injection). Then, ea-ch rat except the blank control group sensitized by injecting the mixture with10%OVA/Al (OH)3+0.0023%o Pertussis toxin from Bordetella pertussis lml into five point(intraperitoneal, subcutaneous of the left/right rear toe and the left/right groin,0.2ml each point)in day1and day8to make model. After sensitization,used1%OVA+normal saline to stimulate immunoreaction by inhaled(30min Qd,7days). The blank control group used normal saline to do all above treatment.
Intragastric administration began in day9(finished the last injection), lasted14days,Qd. The Chinese medicine low-dose, middle-dose and high-dose groups were given by Chinese medical extractum normal saline solution0.182g/ml,0.363g/ml and0.727g/ml (dosage:low-dose group=1/2middle-dose group, middle-dose group=1/2high-dose group)3ml. The western medicine group was given by dexamethasone normal saline solution50μg/ml3ml. And the blank control group, the model control group and the blocker control group were given by normal saline3ml.
After administration,all rats were evaluated pulmonary function under anesthesia. Then taken the samples of bronchoalveolar lavage fluid,arterial blood and the lung tissue to do eosinophil cell count, serum IgE/IL-4/IL-5/IL-13content test,lung tissue sections stained by HE (to observe the morphological alternation) and immumohistochemical (to test the AP-1nucleoprotein),and lung tissue homogenate AP-1mRNA/nucleoprotein content test (by RT-PCR/Western blot).
All statistical analysis was performed using SPSS17.0software package. Calculated data:mean±standard deviation(χ±s).Enumeration data:mean±standard deviation(χ±s)(normal distribution), or median±interquartile range (Md±QR)(abnormal distribution).First, used1-Sample K-S Test of non-parametric test to do normality test.Then, if the data fit normal distribution, used One-Way ANOVA to do the homogeneity test of variances, and used LSD(equal variance) or Tamhane'sT2(unequal variance) to do the multiple comparisons; if the data doesn't fit normal distribution, transformed the data (such as logarithmic transformation) and repeat the above steps,or still doesn't fit normal distribution,used Kruskal-Wallis to do the multiple comparisons. Regarded P<0.05as statistically difference, and P<0.01as statistically significant difference.
Results
1.Observation and test results of the model assessment index and the therapeutic effect index.
General conditions:After the injection for model-making, rats of the model control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group developed asthma symptoms, when rats of the blank control group and the blocker control group did not.
Pulmonary function results:Compared with the blank control group, the results of the model control group's airway hyperresponsiveness (I), tissue sluggish (H), tissue damping (G) and dynamic lung compliance (Crs) were higher (P>0.05, no statistically significant). When the results of the other groups' hyperresponsiveness (I) and tissue sluggish (H) were both higher.Among all the other groups, the airway hyperresponsiveness (I) of the blocker control group was the lowest (P>0.05, no statistically significant). The rest of results such as inspiratory capacity (IC).Central airway resistance (Rn), tissue damping (G), dynamic lung compliance (Crs) and static lung compliance (Cst) had different trends between every group (P>0.05, no statistically significant).
The bronchoalveolar lavage fluid (BALF) smear can recognize some Bosinophil cells (EOS) under the microscope. Preliminary observation the number of them from high to low was the model control group,the Chinese medicine low-dose group, the Chinese medicine middle-dose group, the Chinese medicine high-dose group,the western medicine group,the blocker control group,the blank control group. Count results showed that:Compared with the blank control group,the result of the model control group's BALF smear EOS count was significant elevated(P<0.01); the blocker control group's was a little higher(P>0.05,no statistically significant);the Chinese medicine low-dose, middle-dose and high-dose groups'were higher (P<0.01) and showed the concentration-response relationship; and the western medicine group's was higher too(P<0.01). Compared with the model control group, the blocker control group's was lower (P<0.01); the Chinese medicine low-dose, middle-dose and high-dose groups'were lower too, but the low-dose group's P>0.05(no statistically significant), when the middle-dose and high-dose groups'P<0.01; and the western medicine group's was lower (P<0.01). Compared with the blocker control group,the Chinese medicine low-dose, middle-dose and high-dose groups'were higher (P<0.01); and the western medicine group's was higher too, but P>0.05(no statistically significant). Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups'were higher, but the high-dose group's P>0.05(no statistically significant), and the low-dose, middle-dose groups' P<0.01.
Under the microscope view to observe the morphological alternation from lung tissue sections stained by HE:The blank control group had not showed any change and inflammatory cellular infiltration, when he blocker control group had a little, and the other groups all had inflammatory cellular infiltration in the lung tissue, bronchial epithelial cells irregular arrangement and desquamation, thic-kened alveoli septum and bronchiolar spasm, but the pathological damage severity were different. Results of the score by using tissue injury and inflammation pathological scoring criteria showed that:On the item of the inflammatory cellular infiltration, the score from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group=the Chinese medicine high-dose group> the western medicine group> the blocker control group=the blank control group. On the item of the bronchial epithelial cells desquamation, the score from high to low was the model control group> the Chinese medicine low-dose group=the Chinese medicine middle-dose group>the Chinese medicine high-dose group=the western medicine group> the blocker control group=the blank control group. On the item of the thickened alveoli septum, the score from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group> the Chinese medicine high-dose group=the western medicine group> the blocker control group=the blank control group. On the item of the bronchiolar spasm, the score from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group=the Chinese medicine high-dose group> the western medicine group> the blocker control group=the blank control group. And the total points from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group>the Chinese medicine high-dose group> the western medicine group> the blocker control group> the blank control group, all above P<0.01.
Results of the serum IgE content test showed:Compared with the blank control group, every group's serum IgE content increased (P<0.01), and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, the blocker control group's was lower (P<0.01); the Chinese medicine low-dose, middle-dose and high-dose groups'were lower too,but the low-dose group's P>0.05(no statistically significant), when the middle-dose and high-dose groups'P<0.05;and the western medicine group's was lower (P<0.01). Compared with the blocker control group, the Chinese medicine low-dose,middle-dose and high-dose groups' were higher(the low-dose group's P<0.05, while the middle-dose and high-dose groups' P<0.01); and the western medicine group's was higher too, but P>0.05(no statistically significant). Compared with the western medicine group,the Chinese medicine low-dose, middle-dose and high-dose groups' were higher, but the high-dose group's P>0.05(no statistically significant), and the low-dose group's P<0.01, the middle-dose group's P<0.05.
2. Results of AP-1mRNA expression quantities by Real-time RT-PCR.
c-jun mRNA expression quantities:Compared with the blank control group,every group's c-jun mRNA expression quantities increased, among them, the model control group and the Chinese medicine low-dose, middle-dose groups' P<0.05, the others P>0.05(no statistically significant). Compared with the model control group, the Chinese medicine low-dose group's was a little higher, and the other groups' were lower,but only the blocker control group's P<0.05, all of the rest groups' P>0.05(no statistically significant). Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher, when the the western medicine group and the middle-dose group's P<0.05, the others P>0.05(no statistically significant). Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups'were higher,but P>0.05(no statistically significant).
c-fos mRNA expression quantities:Compared with the blank control group, every group's c-fos mRNA expression quantities increase.Compared with the model control group, every group's was lower. Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was lower.Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups' were higher,but all above P>0.05(no statistically significant).
3. Results of AP-1component content and activity test.
①Result of observing the positive expression of AP-1.
Under the microscope view, the intensity of positive expression from high to low was the model control group, the Chinese medicine low-dose group, the Chinese medicine middle-dose group, the Chinese medicine high-dose group,the western medicine group, the blocker control group, the blank control group. The positive expression mainly located in bronchial epithelial cells, sometimes in alveolar interval.
Compared with the blank control group, every group's c-Jun nucleoprotein integral optical density(IOD) increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, the blocker control group's was lower (P<0.01);the Chinese medicine low-dose, middle-dose and high-dose groups'were lower too, and the low-dose groups' P<0.05, the middle-dose and high-dose groups'P<0.01; the western medicine group's was lower (P<0.01).Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher(P<0.01).Compared with the western medicine group,the Chinese medicine low-dose, middle-dose and high-dose groups'were higher,but P>0.05(no statistically significant).
Compared with the blank control group, every group's c-Fos nucleoprotein integral optical density (IOD) increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, every group's was lower; among them, the blocker control group's P>0.05(no statistically significant), the Chinese medicine low-dose groups'P<0.05, all of the rest P<0.01. Compared with the blocker control group,the Chinese medicine low-dose,middle-dose, high-dose groups and the western medicine group's was lower, the western medicine group's P<0.05, the others P>0.05(no statistically significant). Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups'were higher, but P>0.05(no statistically significant).
②Result of AP-1nucleoprotein (c-Jun and c-Fos) expression quantities by Western blot:
Preliminary observation:All of the β-actin expressed almost the same, while c-Jun nucleoprotein expression quantities from high to low was the model control group,the Chinese medicine low-dose group, middle-dose, high-dose group, the western medicine group, the blocker control group, the blank control group, and c-Fos nucleoprotein expression quantities from high to low was the model control group, the blank control group, the Chinese medicine low-dose group, middle-dose, high-dose group, the western medicine group, the blocker control group.
Compared with the blank control group, every group's c-Jun nucleoprotein mean gray value increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, the blocker control group's was lower(P<0.01); the Chinese medicine low-dose, middle-dose and high-dose groups'were lower too, and the low-dose and middle-dose groups' P>0.05(no statistically significant), the high-dose group's P<0.01; the western medicine group's was lower (P<0.01). Compar-ed with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher (P<0.01). Compared with the western medicine group,the Chinese medicine low-dose, middle-dose and high-dose groups'were higher,but the high-dose group's P>0.05(no statistically significant), whlie the low-dose and middle-dose groups'P<0.01.
Compared with the blank control group, every group's c-Fos nucleoprotein mean gray value increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group,the blocker control group,the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was lower(P<0.01).Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher(P<0.01).Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups'were higher, but the high-dose group's P>0.05(no statistically significant), whlie the low-dose and middle-dose groups' P<0.01.
4.Results of serum IL-4, IL-5, IL-13content test.
Compared with the blank control group,every group's serum IL-4content increased,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship, among them, the blocker control group's P>0.05(no statistically significant) and all of the rest P<0.01. Compared with the model control group, every group's was lower (P<0.01). Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher (P<0.01).Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups' were higher (P<0.01).
Compared with the blank control group, every group's serum IL-5content increased,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship,but only the Chinese medicine low-dose and middle-dose groups'P<0.01, the others P>0.05(no statistically significant). Compared with the model control group, every group's was lower (P <0.01).Compared with the blocker control group,the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher, among them, the high-dose group and the western medicine group's P>0.05(no statistically significant), while the low-dose and middle-dose groups'P <0.01. Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups'were higher,but the high-dose groups' P>0.05(no statistically significant), and the low-dose and middle-dose groups' P<0.01.
Compared with the blank control group,every group's serum IL-13content increased, and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship, among them, the blocker control group and the Chinese medicine high-dose groups'P<0.05, while the others P<0.01. Compared with the model control group,every group's was lower,and the low-dose group's P<0.05, the others P<0.01.Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group's was higher,but the high-dose group's P>0.05(no statistically significant), and the low-dose and middle-dose groups'P<0.01,the western medicine group's P<0.05. Compared with the western medicine group,the Chinese medicine low-dose and middle-dose groups'were higher but P>0.05(no statistically significant), while the high-dose group's was a little higher (P<0.01).
Conclusion
1.The bronchial asthma rat model which was established in this experimient had been successful, and AP-1is a key link for the pathogenesis of bronchial asthma.
①The bronchial asthma SD rat model had been successfully duplicated.
The method of sensitized by injecting the mixture with10%OVA/Al (OH)3+0.0023%o Pertussis toxin from Bordetella pertussis into five point (intraperitoneal, subcut-aneous of the left/right rear toe and the left/right groin) to make model,and uses1%OVA+normal saline to stimulate immunoreaction by inhaled can successfully duplicat the bronchial asthma rat model.
②Curcumin had successful blocked the AP-1expression in the lung tissue of bronchial asthma rat.
Through various experimental methods to test the content of AP-1gene expression product (mRNA and nucleoprotein) and downstream factors of Th2cells (IL-4, IL-5and IL-13),we found that the blocker control group's content of c-jun mRNA and nucleoprotein, IL-4,IL-5and IL-13were close to the blank control group but far below the model control group,while the content of c-fos mRNA and nucleoprotein were close to the model control group but far above the blank control group, thus this blocker group had been blocked successful in general.
③AP-1is a key link for the pathogenesis of bronchial asthma.
The blocker control group rats did not present with typical symptoms and physical signs of asthma. Results of pulmonary function,BLAF smear EOS count, lung tissue sections stained by HE and serum IgE did not have positive performance of typical asthma. We can consider that this blocker group failed to copy the bronchus asthma model. All above suggest that AP-1is a key link for the pathogenesis of bronchial asthma, and to block it can prevent asthma pathogenesis to some extent.2. By regulating the expression and activity of AP-1, and affecting its expression and activity of downstream factors (IL-4、IL-5and IL-13), Qiweilifei Decoction is effective in the treatment of asthma.
①Qiweilifei Decoction is effective in the treatment of asthma.
Compare the results of BLAF smear EOS count,lung tissue sections stained by HE and serum IgE had positive performance of typical asthma, the Qiweilifei Decoction low-dose, middle-dose and high-dose groups rats were lower than the model control group but a little higher than the western medicine group. Among them, the high-dose group can be considered equal to the western medicine group,while the low-dose group can be considered close to the model control group. All above indicate that Qiweilifei Decoction is effective in the treatment of asthma, and high-dose gavage can be considered as Dexamethasone, whlie low-dose gavage is ineffective.
②The expression and activity of AP-1were regulated by Qiweilifei Decoction.
Through various experimental methods to test the content of AP-1gene expression product (mRNA and nucleoprotein), the Qiweilifei Decoction low-dose, middle-dose and high-dose groups showed the concentration-response relationship. The content of AP-1mRNA:Compare with the model control group and the western medicine group, three Chinese medicine groups'differences had no statistical significance. The content of AP-1nucleoprotein:The high-dose group can be considered equal to the western medicine group, while the low-dose group can be considered close to the model control group. The results showed that the effect of the AP-1mRNA regulation by Qiweilifei Decoction is not yet clear from this experiment. However, Qiweilifei Decoction can reduce the content of AP-1nucleoprotein and reduce the synthesis of AP-1.
③The expression of downstream factors of AP-1(IL-4, IL-5and IL-13) were affected by Qiweilifei Decoction.
The Qiweilifei Decoction low-dose, middle-dose and high-dose groups'content of Th2downstream factors(IL-4, IL-5and IL-13)showed the concentration-response relationship. The low-dose group can be considered equal to the model control group while the high-dose group can be considered close to the western medicine group. We preliminary presumption that Qiweilifei Decoction possibly be block the process of AP-1mRNA served as a template to compound a proteins, then reduce content of AP-1nucleoprotein (c-Jun and c-Fos) in the lung tissue of rats. At last, AP-1itself and the Th2downstream factors (IL-4, IL-5and IL-13) also be reduced.
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