补脾益气方药对哮喘大鼠治疗机制的研究
摘要
目的：
支气管哮喘(bronchial asthma)是以嗜酸性粒细胞浸润、T淋巴细胞、肥大细胞、气道上皮细胞等多种炎症细胞和细胞因子参与的气道慢性炎症性疾病,表现以嗜酸性粒细胞为主的炎性细胞浸润、气道高反应性和气道重塑等病理特征。本实验从哮喘病理出发,通过建立大鼠哮喘模型,从多方面、多靶点探讨补脾益气方药治疗哮喘的分子生物学机制,为其临床应用提供实验性理论依据。
材料与方法：
雄性SD大鼠(大连医科大学实验动物中心提供,SPF级)70只,6-8周,体重200±20g,同室,无菌环境下饲养,自由饮水摄食。将大鼠随机分为对照组、模型组、生理盐水组、地塞米松组和低、中、高不同浓度的补脾益气方药组(中药组)。对照组大鼠正常饮食水,生理盐水雾化；哮喘组通过鸡卵白蛋白致敏液(OVA100mg,Al(OH)3200mg,百日咳杆菌5×109,溶于1ml的生理盐水中),分别在实验的1、8天腹腔注射上述致敏液1ml/kg,在第15天起用生理盐水配制的5%OVA30ml雾化进行激发(雾化量1ml/min)连续14天,建立大鼠哮喘模型;生理盐水组和地塞米松组在复制哮喘模型后,在5%OVA雾化前30min,分别腹腔注射1ml/kg生理盐水和0.5mg/kg(?)塞米松做阴性和阳性对照治疗；补脾益气方药组复制哮喘模型后,在每次5%OVA雾化激发前30min,按照1ml/100g给予不同浓度补脾益气中药浓度分别为(0.5g/ml、1g/ml和2g/ml)灌胃,ig1次/d,共14d,统一取材进行指标检测。
第一部分：将大鼠解剖充分暴露气管,夹闭右肺主支气管,左肺注入生理盐水,反复冲洗,收集支气管肺泡灌洗液(BALF),经瑞士姬姆萨染色光镜下计算嗜酸性细胞(eosinophilis, EOS)、巨噬细胞(Macrophage, M)、淋巴细胞(lymphocyte, L)和中性粒细胞(neutrophils, N)比例；取右肺部分中叶组织常规石蜡包埋,HE染色观察各组肺组织病理形态：免疫组化测定肺组织平滑肌肌动蛋白(α-SMA)表达水平,观测哮喘气道炎症特点和病理特征性表现。
第二部分取部分右肺中叶常规石蜡包埋免疫组化观察eotaxin-2在各组表达情况；取右肺上、下叶组织经RT-PCR和Western bloting检测各组肺组织eotaxin-2mRNA和蛋白表达变化,探讨补脾益气方药对eotaxin-2的干预作用。
第三部分：取右肺上叶和下叶组织纯化RNA和蛋白,通过RT-PCR检测IL-4\IFN-γ、以及二者上游的调控因子GATA-3和T-betmRNA的表达水平,探讨工L-4和IFN-γ与GATA-3和T-bet的变化关系；通过RT-PCR、Western bloting检测肺组织GATA-3、T-bet、与细胞因子信号转导子和转录激活子(signal transfector and activator of transcription,STAT)途径的核心转录因子-磷酸化STAT6和磷酸化STAT1表达水平的变化,在蛋白水平上探讨GATA-3和T-bet与磷酸化STAT6和磷酸化STATl变化关系,其目的在于研究哮喘Th1/Th2失衡的机制,探讨补脾益气方药对IL-4/IFN-γ失衡的调节机制。
统计学分析：所有数据均以均数士标准差(x±s)表示,采用SPSS13.0软件处理,组间比较采用单因素方差分析(One-WavANOVA),变量间的关系采用直线相关性分析,P<0.05有统计学意义,P<0.01有显著统计学意义。
结果：
1.通过对补脾益气方药对气道炎症的影响研究,结果显示：模型组与生理盐水组较对照组比较,肺泡灌洗液(BALF)中嗜酸性粒细胞、中性粒细胞、淋巴细胞的比例均明显增高(P<0.01),模型组与生理盐水组比较没有差别。地塞米松治疗组和补脾方药治疗组与模型组组和生理盐水组比较上述细胞的比例下降(P<0.01),尤其在地塞米松和补脾益气方药的中、高剂量治疗组效果显著。
2.病理切片显示,对照组肺组织未见嗜酸性粒细胞,无炎症表现,肺泡扩张良好、支气管粘膜和管壁厚度正常。模型组和生理盐水组肺组织病理可见肺组织内有不规则暗红色充血区,在支气管附近伴有大量红染的嗜酸性粒细胞,EOS.淋巴细胞为主的炎性细胞浸润,管腔狭窄,管壁明显增厚,支气管内有大量粘液栓生成、肺组织发生纤维化,经补脾益气中药及地塞米松治疗后上述现象明显减轻,肺泡扩张得到明显改善。
3.α-SMA又称α平滑肌肌动蛋白,在哮喘发生时由于气道重塑导致位于支气管粘膜内及肺泡间隔内的成纤维细胞转型为成肌纤维细胞合成的蛋白因子,α-SMA只在哮喘肺组织表达,是检测气道重塑的重要指标。实验结果显示：对照组未见α-SMA表达,模型组和生理盐水组与对照组比较α-SMA表达明显增强(P<0.01),模型组与生理盐水组比较没有明显差别(P>0.05)。综合BALF中细胞成分的检测结果,HE的病理特征,还有典型的临床表现,证明哮喘模型复制成功。经地塞米松和补脾方药治疗后α-SMA表达明显减弱(P<0.01)。
4.研究补脾益气方药对eotaxin-2表达的影响,结果显示,模型组和生理盐水组与对照组比较,免疫组化可见支气管附近有大量棕褐色颗粒,eotaxin-2在mRNA和蛋白水平的表达均明显增高；模型组与生理盐水组比较没有明显差别(P>0.05)；地塞米松与补脾益气方药组与模型组和生理盐水组比较,明显下调eotaxin-2的表达水平(P<0.01),且补脾益气方药的中、高剂量组效果显著(P<0.01),但仍比对照组高。
5.通过对哮喘肺组织Th2/Th1免疫失衡及调节的研究,RT-PCR结果显示,模型组和生理盐水组与对照组比较,肺组织工L-4表达水平明显增高,IFN-γ表达水平明显降低(P<0.01),IL-4/IFN-γ(?)匕值明显增加(P<0.01),模型组与生理盐水组比较没有显著差别(P>0.05)。经地塞米松和补脾方药治疗后,能降低工L-4表达水平,上调IFN-γ表达水平,降低IL-4/IFN-γ(?)匕值,在地塞米松组和补脾益气方药的中、高剂量组效果显著(P<0.01)
6.在Th2/Th1失衡机制研究结果表明,模型组和生理盐水组与对照组比较GATA-3mRNA表达水平明显增高,T-bet表达水平明显降低(P<0.01),GATA-3/T-bet的比值增大(P<0.01),模型组与生理盐水组比较没有明显差别(P>0.05)。补脾方药组与模型和生理盐水组比较,能下调GATA-3表达水平,上调T-bet表达水平(P<0.05),降低GATA-3/T-bet的比值(P<0.05),在补脾益气方药的中、高剂量组效果显著(P<0.01)。
7.通过工L-4和IFN-γ,与GATA-3和T-bet在mRNA表达水平上的相关性分析,结果表明,GATA-3与IL-4的表达成正相关(r=0.75,P<0.05),与IFN-γ成负相关(r=-0.73,P<0.05);T-bet与IFN-γ表达成正相关(r=0.78,P<0.05),与Ih-4成负相关(r=-0.71,P<0.05),说明GATA-3具有促进IL-4表达的作用,是Ih-4上游调控因子,T-bet具有促进IFN-γ表达作用,是的IFN-Y上游调控因子；而GATA-3且有抑制IFN-Y的表达,T-bet具有抑制IL-4表达的作用,GATA-3和T-bet存在交互抑制作用,可能是正常生理状态Th2/Th1z衡的基础。
8.细胞因子的作用多数都是通过STAT途径起作用,STAT是该途径的核心因子,研究核心因子STAT与转录因子GATA-3和T-bet的关系中发现,经Western bolting检测在蛋白表达水平上表达结果显示,模型组与生理盐水组与对照组比较p-STAT6表达水平明显增高(P<0.01),p-STAT1表达水平明显降低(P<0.01),p-STAT6/p-STAT1的比值增大(P<0.01),模型组与生理盐水组比较没有明显差别(P>0.05)。地塞米松组和补脾方药组与哮喘和生理盐水组比较,均降低p-STAT6表达水平,上调p-STAT1表达水平(P<0.01),降低p-STAT6/p-STAT1的比值(P<0.01)。在地塞米松组和补脾益气方药的中、高剂量组效果显著。通过对p-STAT6与GATA-3以及p-STAT与T-bet表达的相关性分析,结果显示p-STAT6表达水平与GATA-3的表达成正相关(r=0.81,P<0.05), p-STAT1与T-bet的表达成正相关正(r=0.89,P<0.05)
结论：
1.补脾益气方药具有抑制气道炎症,减轻气道重塑的作用。
2.补脾益气方药具有抑制eotaxin表达,减轻气道嗜酸性粒细胞浸润的作用。
3.补脾益气方药能抑制IL-4,上调IFN-γ的表达,对Th2/Th1失衡有调节作用。
4.补脾益气方药的调节机制是通过下调GATA-3和p-STAT6、上调T-bet和p-STAT1的表达,实现对IL-4/IFN-γ的调节。
Purpose:Bronchial asthma is a chronic inflammatory disorder of airway, It involved in inflammatory cells and cytokines, for example, eosinophilia, T lymphocytes, mast cells and epithelial cells of airway. In particular, it has been shown pathological features of eosinophilia infiltration, airway hyperresponsiveness and airway remodel. Base asthma pathology, the experiment explored the effect of "reinforcing the spleen to replenish Qi" on asthma inflammation inhibition in many targets, discuss the role of molecular biology mechanism in order to provide experimental the theory basis for TCM the clinical application
Materials and methods:male SD rats (The Da Lian Medical University experimental animal centre supply, the SPF level,6-8week old) were set in the same room. The free potable water and food. They randomly were divided into7groups:control group, model group,asthmatic control group, Dexamethasone group and group of Reinforcing the Spleen to Replenish QI. group of Reinforcing the Spleen to Replenish QI were divided low dose group, medium dose group and high dose group. control group rats not inflict any processing factors. model group rats Group rats established asthma model by OVA(100mg OVA,200mgAl(OH)3Pertussis Vaccine5×109, All dissolve in100ml normal saline). Model group rats were injected OVA by abdomen at the first and the eighth days. and then they were aroused by5%OVA30ml(OVA dissolved normal saline) aerosol inhalation in lml/min from15to29days. Normal saline group rats and Dexamethasone group rats respectively set up asthma model, and treat with normal saline1ml/kg and dexamethasone0.5mg/kg by intraperitoneal injection before aerosol inhalation30min per day, in all to14days. Group of Reinforcing the Spleen to Replenish QI rats set up asthma model, taked respectively different concentration medicine of reinforcing the spleen to replenish QI (0.5g/ml,1g/ml and2g/ml) by stomach tube before aerosol inhalation30min per day according to1ml/100g,1g1/d, a total of14d.
The first party:Rats were exposed the tracheatied, Closed left lung main bronchial, then put into0.9%normal saline in it, flushed lung and collected bronchoaleolar lavage fluid (BALF). Centrifugal15000rpm for10min. Smeared slide with Cells sediment. Conventional Rachel's-JiM gaza, dyeing, Total and differential cell counts in BALF were measured; by light microscopy in cell classification and counting percentage every200a white blood cell, we calculated eosinophil (EOS), lymphatic cells (L) and neutrophils (N); and lung tissues were embedded in paraffin for histological analysis by HE staining; Lung tissue alpha SMA expression level was determined by immunohistochemical The second part:Taked right middle leaf lung organization, used conventional paraffin embedding, measured eotaxin-2expression in each group by immunohistochemical; Taked the right lung the leaf tissue, extracted total RNA and protein, and detected expression of eotaxin-2by RT-PCR and Western bloting, Which explored effect of medicine of reinforcing the spleen to replenish QI on expression of eotaxin-2in mRNA and protein level.
The third part:Taked right lung organization, extracted total RNA and protein and detected mRNA expression level of IL-4, IFN-γ and their regulator-GATA-3and T-bet by RT-PCR, determined expression of of GATA-3, T-bet and phosphorylated STAT6and phosphorylated STAT1in mRNA and protein level by RT-PCR and Western bloting. Correlation analysis of IL-4and IFN-γ with GATA-3and T-bet was made respectivly about expression of mRNA in lung tissue, so did correlation analysis phosphorylated STAT6and phosphorylated STATl with T-bet and GATA-3in expression of protein. In order to study the role of medicine of reinforcing the spleen to replenish QI to intervent in airway inflammation, explore medicine of reinforcing the spleen to replenish Qi to IL-4/IFN-γ unbalanced adjustment and mechanism of asthma, as well as intervene in the above factors of expression. Statistical analysis:all data were mean±standard. and the date was analyzed by a single factor analysis of variance (One-WayANOVA) with SPSS13.0software. the relationship between variables by linear correlation analysis. P<0.05have statistical significance, P<0.01significant statistical significance.
Results:
1. Through studying the the impact of medicine of reinforcing the spleen to replenish Qi on airway inflammation the results showed that The total cellular score of asthma rats in BALF. the cell population of EOS, lymphocytes and macrophage in BALF of the asthma model group and normal saline group increased significantly as compared with that of control group(P<0.01);There were difference between model group and normal saline group(P>0.05); while compared with asthma model group and normal control group, the total cellular score decreased obviously in Dexamethasone group and in complex prescription of medicine of reinforcing the spleen to replenish QI in medium and high dose (P<0.01); the cell population of EOS and macrophage were suppressed remarkably (P<0.01), and have statistical significance in low dose (P<0.05)
2. Pathological results showed there were not eosinophils in the lung tissue of normal group rats. Alveolar expansion was good. Pulmonary histomorphology in the asthma model group and normal saline group, The lung tissue could be seen in irregular dark vision congestion zone, and surrounding tissue of the bronchus was infiltrated by a great quantity of inflammatory cells, which was EOS; Model group and normal saline group compared with controls the airway walls obvious thickening;the air tube narrowed, whose epithelium mucosae also thickened mucous plug was found in the bronchus; the lung tissue happen fibrosis. The pathological appearance above-mentioned demonstrated that the asthma model was successful. Medicine of reinforcing the spleen to replenish QI and dexamethasone after treatment markly reduce the above phenomenon, the thickness of the airway significantly reduced in dexamethasone group and medicine of reinforcing the spleen to replenish QI treatment group. especially Dexamethasone and medicine of reinforcing the spleen to replenish QI, high dose group was even significant. the alveolar expansion have been improved obviously.
3. Alpha SMA also called α-smooth muscle activated protein. It is located in the lungs in bronchial mucosa and alveolar intervals fibroblasts. It was synthesized by myof ibroblasts from fibroblasts transphenotype. It only expressed in asthma lung tissue, Which is an important index of the airway remodeling detection. Normal control group has not seen the alpha SMA expression, asthma group and normal saline group compared with the alpha SMA express enhanced obviously P<0.01), There were difference between asthma model group and normal saline group(P>0.05); dexamethasone therapy and medicine of reinforcing the spleen to replenish QI after treatmen alpha SMA express decreased significantly (P<0.01).
4.Research on influence of medicine of reinforcing the spleen to replenish QI on eotaxin-2expression, Results presented that the expression level of eotaxin increased dramaticly in rats of asthma group and normal saline group compared respectively with normal group. The expression level of eotaxin by reinforcing the spleen to replenish QI treatment was obviously cut, especially in high doses group of medicine of reinforcing the spleen to replenish Qi(P<0.01).
5. Explored Th1/Th2immune imbalances and regulatory mechanism by RT-PCR. The results demonstrated that Asthma group and normal saline group compared with normal lung tissue IL-4significantly higher level and IFN-γ level reduced significantly (P<0.01), IL-4/IFN-γ ratio increased significantly (P<0.01); There were difference between asthma model group and normal saline group(P>0.05);after the dexamethasone and medicine of reinforcing the spleen to replenish Qi treatment, IL-4level could be reduced, IFN-γ level could be increased, the IL-4/IFN-γ ratio could be also reduced. Effect of medicine of reinforcing the spleen to replenish Qi was more significantly in high dose group (P<0.01).
6. Through research of the mechanism of Th1/Th2imbalance in mRNA expression level by RT-PCR, the results of the study confirmed that expression level of GATA-3in asthma group and normal saline groups increased significantly compared with normal group;the expression level of T-bet reduced significantly (P<0.01);the ratio of GATA-3/T-bet increased remarkably (P<0.01). expression level of GATA-3was regulated makbly down and the expression level of T-bet was improved in group of reinforcing the spleen to replenish QI compared with asthma group and normal saline group(P<0.01). GATA-3/T-bet the ratio in high dose group of reinforcing the spleen to replenish QI was reduced significantly (P<0.01).
7. the the results of correlation analysis of IL-4, IFN-γ, GATA-3and T-bet expression showed that the relationship of expression between IL-4and GATA-3was positive correlated (r=0.75, P<0.05), with T-bet was a negative correlation (r=-0.73, P<0.05); The relationship of expression between IFN-γ and T-bet was a positive correlation(r=0.78, P<0.05), and The relationship of expression between IFN-γ and GATA-3was a negative correlation(r=-0.71, P<0.05).
8. Most cytokine play role through the JAK-STAT signal way. STAT(signal transductor and activator transcriptor, STAT)is the core factor in the way. the object of the reseach focused on testing relationship of STAT with GATA-3and T-bet by Western bloting and immunohistochemical in protein expression level. Results indicated that expression level of p-STAT6in asthma group rats and normal saline groups rats increased significantly by compared with normal group rats, however,expression level of p-STATl reduced markly (P<0.01), the ratio of p-STAT6/p-STATl increase (P<0.01). Medicine of reinforcing the spleen to replenish QI could decrease expression level of p-STAT6by compared with asthma and nomal saline group, upgrade the expression level of p-STAT1(P<0.01), and reduce the ratio of p-STAT6/p-STAT1(P<0.01). Th effect was remarkable in high dose group. The relation of expression level of p-STAT6and GATA-3a positive correlation (r=0.89, P<0.05), expression of T-bet and p-STAT1is a positive correlation (r=0.89, P<0.05).
Conclusions:
It is thus concluded that:
1. Medicine of reinforcing the spleen to replenish Qi can inhibit effectly the airway inflammation, relieve airway remodeling.
2. Medicine of reinforcing the spleen to replenish Qi can inhibit eotaxin expression and reduce eosinophils to infiltrate airway around.
3.Medicine of reinforcing the spleen to replenish Qi play its regulatory role in Th1/Th2imbalance through regulating down expression of IL-4and regulating up expression of IFN-γ
4.Medicine of reinforcing the spleen to replenish Qi played role in adjusting IL-4/IFN-γ through regulating down GATA-3and p-STAT6, regulating up T-het and p-STAT1expression to achieve to adjust action.
支气管哮喘(bronchial asthma)是以嗜酸性粒细胞浸润、T淋巴细胞、肥大细胞、气道上皮细胞等多种炎症细胞和细胞因子参与的气道慢性炎症性疾病,表现以嗜酸性粒细胞为主的炎性细胞浸润、气道高反应性和气道重塑等病理特征。本实验从哮喘病理出发,通过建立大鼠哮喘模型,从多方面、多靶点探讨补脾益气方药治疗哮喘的分子生物学机制,为其临床应用提供实验性理论依据。
材料与方法：
雄性SD大鼠(大连医科大学实验动物中心提供,SPF级)70只,6-8周,体重200±20g,同室,无菌环境下饲养,自由饮水摄食。将大鼠随机分为对照组、模型组、生理盐水组、地塞米松组和低、中、高不同浓度的补脾益气方药组(中药组)。对照组大鼠正常饮食水,生理盐水雾化；哮喘组通过鸡卵白蛋白致敏液(OVA100mg,Al(OH)3200mg,百日咳杆菌5×109,溶于1ml的生理盐水中),分别在实验的1、8天腹腔注射上述致敏液1ml/kg,在第15天起用生理盐水配制的5%OVA30ml雾化进行激发(雾化量1ml/min)连续14天,建立大鼠哮喘模型;生理盐水组和地塞米松组在复制哮喘模型后,在5%OVA雾化前30min,分别腹腔注射1ml/kg生理盐水和0.5mg/kg(?)塞米松做阴性和阳性对照治疗；补脾益气方药组复制哮喘模型后,在每次5%OVA雾化激发前30min,按照1ml/100g给予不同浓度补脾益气中药浓度分别为(0.5g/ml、1g/ml和2g/ml)灌胃,ig1次/d,共14d,统一取材进行指标检测。
第一部分：将大鼠解剖充分暴露气管,夹闭右肺主支气管,左肺注入生理盐水,反复冲洗,收集支气管肺泡灌洗液(BALF),经瑞士姬姆萨染色光镜下计算嗜酸性细胞(eosinophilis, EOS)、巨噬细胞(Macrophage, M)、淋巴细胞(lymphocyte, L)和中性粒细胞(neutrophils, N)比例；取右肺部分中叶组织常规石蜡包埋,HE染色观察各组肺组织病理形态：免疫组化测定肺组织平滑肌肌动蛋白(α-SMA)表达水平,观测哮喘气道炎症特点和病理特征性表现。
第二部分取部分右肺中叶常规石蜡包埋免疫组化观察eotaxin-2在各组表达情况；取右肺上、下叶组织经RT-PCR和Western bloting检测各组肺组织eotaxin-2mRNA和蛋白表达变化,探讨补脾益气方药对eotaxin-2的干预作用。
第三部分：取右肺上叶和下叶组织纯化RNA和蛋白,通过RT-PCR检测IL-4\IFN-γ、以及二者上游的调控因子GATA-3和T-betmRNA的表达水平,探讨工L-4和IFN-γ与GATA-3和T-bet的变化关系；通过RT-PCR、Western bloting检测肺组织GATA-3、T-bet、与细胞因子信号转导子和转录激活子(signal transfector and activator of transcription,STAT)途径的核心转录因子-磷酸化STAT6和磷酸化STAT1表达水平的变化,在蛋白水平上探讨GATA-3和T-bet与磷酸化STAT6和磷酸化STATl变化关系,其目的在于研究哮喘Th1/Th2失衡的机制,探讨补脾益气方药对IL-4/IFN-γ失衡的调节机制。
统计学分析：所有数据均以均数士标准差(x±s)表示,采用SPSS13.0软件处理,组间比较采用单因素方差分析(One-WavANOVA),变量间的关系采用直线相关性分析,P<0.05有统计学意义,P<0.01有显著统计学意义。
结果：
1.通过对补脾益气方药对气道炎症的影响研究,结果显示：模型组与生理盐水组较对照组比较,肺泡灌洗液(BALF)中嗜酸性粒细胞、中性粒细胞、淋巴细胞的比例均明显增高(P<0.01),模型组与生理盐水组比较没有差别。地塞米松治疗组和补脾方药治疗组与模型组组和生理盐水组比较上述细胞的比例下降(P<0.01),尤其在地塞米松和补脾益气方药的中、高剂量治疗组效果显著。
2.病理切片显示,对照组肺组织未见嗜酸性粒细胞,无炎症表现,肺泡扩张良好、支气管粘膜和管壁厚度正常。模型组和生理盐水组肺组织病理可见肺组织内有不规则暗红色充血区,在支气管附近伴有大量红染的嗜酸性粒细胞,EOS.淋巴细胞为主的炎性细胞浸润,管腔狭窄,管壁明显增厚,支气管内有大量粘液栓生成、肺组织发生纤维化,经补脾益气中药及地塞米松治疗后上述现象明显减轻,肺泡扩张得到明显改善。
3.α-SMA又称α平滑肌肌动蛋白,在哮喘发生时由于气道重塑导致位于支气管粘膜内及肺泡间隔内的成纤维细胞转型为成肌纤维细胞合成的蛋白因子,α-SMA只在哮喘肺组织表达,是检测气道重塑的重要指标。实验结果显示：对照组未见α-SMA表达,模型组和生理盐水组与对照组比较α-SMA表达明显增强(P<0.01),模型组与生理盐水组比较没有明显差别(P>0.05)。综合BALF中细胞成分的检测结果,HE的病理特征,还有典型的临床表现,证明哮喘模型复制成功。经地塞米松和补脾方药治疗后α-SMA表达明显减弱(P<0.01)。
4.研究补脾益气方药对eotaxin-2表达的影响,结果显示,模型组和生理盐水组与对照组比较,免疫组化可见支气管附近有大量棕褐色颗粒,eotaxin-2在mRNA和蛋白水平的表达均明显增高；模型组与生理盐水组比较没有明显差别(P>0.05)；地塞米松与补脾益气方药组与模型组和生理盐水组比较,明显下调eotaxin-2的表达水平(P<0.01),且补脾益气方药的中、高剂量组效果显著(P<0.01),但仍比对照组高。
5.通过对哮喘肺组织Th2/Th1免疫失衡及调节的研究,RT-PCR结果显示,模型组和生理盐水组与对照组比较,肺组织工L-4表达水平明显增高,IFN-γ表达水平明显降低(P<0.01),IL-4/IFN-γ(?)匕值明显增加(P<0.01),模型组与生理盐水组比较没有显著差别(P>0.05)。经地塞米松和补脾方药治疗后,能降低工L-4表达水平,上调IFN-γ表达水平,降低IL-4/IFN-γ(?)匕值,在地塞米松组和补脾益气方药的中、高剂量组效果显著(P<0.01)
6.在Th2/Th1失衡机制研究结果表明,模型组和生理盐水组与对照组比较GATA-3mRNA表达水平明显增高,T-bet表达水平明显降低(P<0.01),GATA-3/T-bet的比值增大(P<0.01),模型组与生理盐水组比较没有明显差别(P>0.05)。补脾方药组与模型和生理盐水组比较,能下调GATA-3表达水平,上调T-bet表达水平(P<0.05),降低GATA-3/T-bet的比值(P<0.05),在补脾益气方药的中、高剂量组效果显著(P<0.01)。
7.通过工L-4和IFN-γ,与GATA-3和T-bet在mRNA表达水平上的相关性分析,结果表明,GATA-3与IL-4的表达成正相关(r=0.75,P<0.05),与IFN-γ成负相关(r=-0.73,P<0.05);T-bet与IFN-γ表达成正相关(r=0.78,P<0.05),与Ih-4成负相关(r=-0.71,P<0.05),说明GATA-3具有促进IL-4表达的作用,是Ih-4上游调控因子,T-bet具有促进IFN-γ表达作用,是的IFN-Y上游调控因子；而GATA-3且有抑制IFN-Y的表达,T-bet具有抑制IL-4表达的作用,GATA-3和T-bet存在交互抑制作用,可能是正常生理状态Th2/Th1z衡的基础。
8.细胞因子的作用多数都是通过STAT途径起作用,STAT是该途径的核心因子,研究核心因子STAT与转录因子GATA-3和T-bet的关系中发现,经Western bolting检测在蛋白表达水平上表达结果显示,模型组与生理盐水组与对照组比较p-STAT6表达水平明显增高(P<0.01),p-STAT1表达水平明显降低(P<0.01),p-STAT6/p-STAT1的比值增大(P<0.01),模型组与生理盐水组比较没有明显差别(P>0.05)。地塞米松组和补脾方药组与哮喘和生理盐水组比较,均降低p-STAT6表达水平,上调p-STAT1表达水平(P<0.01),降低p-STAT6/p-STAT1的比值(P<0.01)。在地塞米松组和补脾益气方药的中、高剂量组效果显著。通过对p-STAT6与GATA-3以及p-STAT与T-bet表达的相关性分析,结果显示p-STAT6表达水平与GATA-3的表达成正相关(r=0.81,P<0.05), p-STAT1与T-bet的表达成正相关正(r=0.89,P<0.05)
结论：
1.补脾益气方药具有抑制气道炎症,减轻气道重塑的作用。
2.补脾益气方药具有抑制eotaxin表达,减轻气道嗜酸性粒细胞浸润的作用。
3.补脾益气方药能抑制IL-4,上调IFN-γ的表达,对Th2/Th1失衡有调节作用。
4.补脾益气方药的调节机制是通过下调GATA-3和p-STAT6、上调T-bet和p-STAT1的表达,实现对IL-4/IFN-γ的调节。
Purpose:Bronchial asthma is a chronic inflammatory disorder of airway, It involved in inflammatory cells and cytokines, for example, eosinophilia, T lymphocytes, mast cells and epithelial cells of airway. In particular, it has been shown pathological features of eosinophilia infiltration, airway hyperresponsiveness and airway remodel. Base asthma pathology, the experiment explored the effect of "reinforcing the spleen to replenish Qi" on asthma inflammation inhibition in many targets, discuss the role of molecular biology mechanism in order to provide experimental the theory basis for TCM the clinical application
Materials and methods:male SD rats (The Da Lian Medical University experimental animal centre supply, the SPF level,6-8week old) were set in the same room. The free potable water and food. They randomly were divided into7groups:control group, model group,asthmatic control group, Dexamethasone group and group of Reinforcing the Spleen to Replenish QI. group of Reinforcing the Spleen to Replenish QI were divided low dose group, medium dose group and high dose group. control group rats not inflict any processing factors. model group rats Group rats established asthma model by OVA(100mg OVA,200mgAl(OH)3Pertussis Vaccine5×109, All dissolve in100ml normal saline). Model group rats were injected OVA by abdomen at the first and the eighth days. and then they were aroused by5%OVA30ml(OVA dissolved normal saline) aerosol inhalation in lml/min from15to29days. Normal saline group rats and Dexamethasone group rats respectively set up asthma model, and treat with normal saline1ml/kg and dexamethasone0.5mg/kg by intraperitoneal injection before aerosol inhalation30min per day, in all to14days. Group of Reinforcing the Spleen to Replenish QI rats set up asthma model, taked respectively different concentration medicine of reinforcing the spleen to replenish QI (0.5g/ml,1g/ml and2g/ml) by stomach tube before aerosol inhalation30min per day according to1ml/100g,1g1/d, a total of14d.
The first party:Rats were exposed the tracheatied, Closed left lung main bronchial, then put into0.9%normal saline in it, flushed lung and collected bronchoaleolar lavage fluid (BALF). Centrifugal15000rpm for10min. Smeared slide with Cells sediment. Conventional Rachel's-JiM gaza, dyeing, Total and differential cell counts in BALF were measured; by light microscopy in cell classification and counting percentage every200a white blood cell, we calculated eosinophil (EOS), lymphatic cells (L) and neutrophils (N); and lung tissues were embedded in paraffin for histological analysis by HE staining; Lung tissue alpha SMA expression level was determined by immunohistochemical The second part:Taked right middle leaf lung organization, used conventional paraffin embedding, measured eotaxin-2expression in each group by immunohistochemical; Taked the right lung the leaf tissue, extracted total RNA and protein, and detected expression of eotaxin-2by RT-PCR and Western bloting, Which explored effect of medicine of reinforcing the spleen to replenish QI on expression of eotaxin-2in mRNA and protein level.
The third part:Taked right lung organization, extracted total RNA and protein and detected mRNA expression level of IL-4, IFN-γ and their regulator-GATA-3and T-bet by RT-PCR, determined expression of of GATA-3, T-bet and phosphorylated STAT6and phosphorylated STAT1in mRNA and protein level by RT-PCR and Western bloting. Correlation analysis of IL-4and IFN-γ with GATA-3and T-bet was made respectivly about expression of mRNA in lung tissue, so did correlation analysis phosphorylated STAT6and phosphorylated STATl with T-bet and GATA-3in expression of protein. In order to study the role of medicine of reinforcing the spleen to replenish QI to intervent in airway inflammation, explore medicine of reinforcing the spleen to replenish Qi to IL-4/IFN-γ unbalanced adjustment and mechanism of asthma, as well as intervene in the above factors of expression. Statistical analysis:all data were mean±standard. and the date was analyzed by a single factor analysis of variance (One-WayANOVA) with SPSS13.0software. the relationship between variables by linear correlation analysis. P<0.05have statistical significance, P<0.01significant statistical significance.
Results:
1. Through studying the the impact of medicine of reinforcing the spleen to replenish Qi on airway inflammation the results showed that The total cellular score of asthma rats in BALF. the cell population of EOS, lymphocytes and macrophage in BALF of the asthma model group and normal saline group increased significantly as compared with that of control group(P<0.01);There were difference between model group and normal saline group(P>0.05); while compared with asthma model group and normal control group, the total cellular score decreased obviously in Dexamethasone group and in complex prescription of medicine of reinforcing the spleen to replenish QI in medium and high dose (P<0.01); the cell population of EOS and macrophage were suppressed remarkably (P<0.01), and have statistical significance in low dose (P<0.05)
2. Pathological results showed there were not eosinophils in the lung tissue of normal group rats. Alveolar expansion was good. Pulmonary histomorphology in the asthma model group and normal saline group, The lung tissue could be seen in irregular dark vision congestion zone, and surrounding tissue of the bronchus was infiltrated by a great quantity of inflammatory cells, which was EOS; Model group and normal saline group compared with controls the airway walls obvious thickening;the air tube narrowed, whose epithelium mucosae also thickened mucous plug was found in the bronchus; the lung tissue happen fibrosis. The pathological appearance above-mentioned demonstrated that the asthma model was successful. Medicine of reinforcing the spleen to replenish QI and dexamethasone after treatment markly reduce the above phenomenon, the thickness of the airway significantly reduced in dexamethasone group and medicine of reinforcing the spleen to replenish QI treatment group. especially Dexamethasone and medicine of reinforcing the spleen to replenish QI, high dose group was even significant. the alveolar expansion have been improved obviously.
3. Alpha SMA also called α-smooth muscle activated protein. It is located in the lungs in bronchial mucosa and alveolar intervals fibroblasts. It was synthesized by myof ibroblasts from fibroblasts transphenotype. It only expressed in asthma lung tissue, Which is an important index of the airway remodeling detection. Normal control group has not seen the alpha SMA expression, asthma group and normal saline group compared with the alpha SMA express enhanced obviously P<0.01), There were difference between asthma model group and normal saline group(P>0.05); dexamethasone therapy and medicine of reinforcing the spleen to replenish QI after treatmen alpha SMA express decreased significantly (P<0.01).
4.Research on influence of medicine of reinforcing the spleen to replenish QI on eotaxin-2expression, Results presented that the expression level of eotaxin increased dramaticly in rats of asthma group and normal saline group compared respectively with normal group. The expression level of eotaxin by reinforcing the spleen to replenish QI treatment was obviously cut, especially in high doses group of medicine of reinforcing the spleen to replenish Qi(P<0.01).
5. Explored Th1/Th2immune imbalances and regulatory mechanism by RT-PCR. The results demonstrated that Asthma group and normal saline group compared with normal lung tissue IL-4significantly higher level and IFN-γ level reduced significantly (P<0.01), IL-4/IFN-γ ratio increased significantly (P<0.01); There were difference between asthma model group and normal saline group(P>0.05);after the dexamethasone and medicine of reinforcing the spleen to replenish Qi treatment, IL-4level could be reduced, IFN-γ level could be increased, the IL-4/IFN-γ ratio could be also reduced. Effect of medicine of reinforcing the spleen to replenish Qi was more significantly in high dose group (P<0.01).
6. Through research of the mechanism of Th1/Th2imbalance in mRNA expression level by RT-PCR, the results of the study confirmed that expression level of GATA-3in asthma group and normal saline groups increased significantly compared with normal group;the expression level of T-bet reduced significantly (P<0.01);the ratio of GATA-3/T-bet increased remarkably (P<0.01). expression level of GATA-3was regulated makbly down and the expression level of T-bet was improved in group of reinforcing the spleen to replenish QI compared with asthma group and normal saline group(P<0.01). GATA-3/T-bet the ratio in high dose group of reinforcing the spleen to replenish QI was reduced significantly (P<0.01).
7. the the results of correlation analysis of IL-4, IFN-γ, GATA-3and T-bet expression showed that the relationship of expression between IL-4and GATA-3was positive correlated (r=0.75, P<0.05), with T-bet was a negative correlation (r=-0.73, P<0.05); The relationship of expression between IFN-γ and T-bet was a positive correlation(r=0.78, P<0.05), and The relationship of expression between IFN-γ and GATA-3was a negative correlation(r=-0.71, P<0.05).
8. Most cytokine play role through the JAK-STAT signal way. STAT(signal transductor and activator transcriptor, STAT)is the core factor in the way. the object of the reseach focused on testing relationship of STAT with GATA-3and T-bet by Western bloting and immunohistochemical in protein expression level. Results indicated that expression level of p-STAT6in asthma group rats and normal saline groups rats increased significantly by compared with normal group rats, however,expression level of p-STATl reduced markly (P<0.01), the ratio of p-STAT6/p-STATl increase (P<0.01). Medicine of reinforcing the spleen to replenish QI could decrease expression level of p-STAT6by compared with asthma and nomal saline group, upgrade the expression level of p-STAT1(P<0.01), and reduce the ratio of p-STAT6/p-STAT1(P<0.01). Th effect was remarkable in high dose group. The relation of expression level of p-STAT6and GATA-3a positive correlation (r=0.89, P<0.05), expression of T-bet and p-STAT1is a positive correlation (r=0.89, P<0.05).
Conclusions:
It is thus concluded that:
1. Medicine of reinforcing the spleen to replenish Qi can inhibit effectly the airway inflammation, relieve airway remodeling.
2. Medicine of reinforcing the spleen to replenish Qi can inhibit eotaxin expression and reduce eosinophils to infiltrate airway around.
3.Medicine of reinforcing the spleen to replenish Qi play its regulatory role in Th1/Th2imbalance through regulating down expression of IL-4and regulating up expression of IFN-γ
4.Medicine of reinforcing the spleen to replenish Qi played role in adjusting IL-4/IFN-γ through regulating down GATA-3and p-STAT6, regulating up T-het and p-STAT1expression to achieve to adjust action.
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