黄芪当归对药不同剂量与配比对IPF小鼠生存状况及TGF-β、IL-6、Foxp3、RORγt基因表达水平影响

Astragalus Angelica Ratio of Drug Doses and on IPF Mice Survival Condition and TGF-beta,IL-6,Foxp3,ROR Gamma t the Influence of the Level of Gene Expression

目的：观察不同剂量与配伍比例的黄芪、当归对药对IPF小鼠生存状况及Th17/Treg细胞分化关键基因TGF-β、IL-6、foxp3、RORγt表达水平的影响,并探讨其相互关系及保护机制。方法：SPF级ICR雄性小鼠80只,体重18~22 g,随机分为正常对照组、模型组、以及6个不同配比与剂量的黄芪当归组成的中药治疗组（1大剂量黄芪当归5∶1组;2大剂量黄芪当归1∶1组;3大剂量黄芪当归1∶5组;4小剂量黄芪当归5∶1组;5小剂量黄芪当归1∶1组;6小剂量黄芪当归1∶5组）,每组10只,除正常对照组外,其余各组采用气管内注射博莱霉素（5 mg/kg）复制小鼠肺纤维化模型。造模第2天起,正常对照组和模型组用生理盐水灌胃,6个中药治疗组给予不同配比与剂量的黄芪当归水煎液灌胃治疗,于第28天处死小鼠,采用Real time-RT-PCR法检测Th17/Treg细胞分化关键基因TGF-β、IL-6、foxp3、RORγt表达水平,HE染色及武兆发简化Mallory氏胶原染色,观察肺组织形态变化。结果：HE染色及胶原染色显示,模型组小鼠可见支气管周围及肺泡间隔有大量炎症细胞浸润,纤维化程度明显。中药第1、6组肺泡炎明显好转,肺泡内未见明显的炎性细胞,肺泡隔、细小支气管周围的纤维化病灶较模型组有明显改善。中药第2、3、4、5组肺泡炎较对照组重,但与模型组比较有很大程度的减轻,肺泡隔纤维增生灶较模型组有所改善,但不如第1、6组明显。第28天时,模型组小鼠体重明显低于对照组,与模型组比较,各组小鼠体重均呈上升的趋势,中药第1组小鼠体重上升趋势较大（P〈0.05）。第28天死亡率,中药治疗组小鼠的死亡率较模型组降低,其中第1组死亡率与模型组的差异有统计学意义（P〈0.05）。模型组TGF-β、RORγt、IL-6 m RNA表达水平均高于对照组,Foxp3 m RNA表达水平均低于对照组（P〈0.05）。结论：从小鼠的生存率与体重变化及病理形态观察来看,大剂量黄芪当归5∶1组能明显改善小鼠的生存质量,其作用机制可能与抑制Th17分化关键基因TGF-β、IL-6、RORγtm RNA的表达水平,促进Treg分化关键基因Foxp3 m RNA的表达水平有关。

Objective：To observe the different doses and compatibility proportion of astragalus,angelica root,weight of medicine of IPF mice survival and differentiation of Th17/Treg cells key genes TGF-beta,IL-6,the influence of foxp3,ROR gamma t expression level,and discuss the relationship and the protection mechanism. Methods：The SPF 80 male ICR mice,18 to 22 g weight,were random Ly divided into normal control group,model group,and six different ratio and dosage of astragalus angelica Chinese medicine treatment group（1a large dose of astragalus angelica 5∶1; 2large dose of astragalus angelica 1∶1 group,3large dose of astragalus angelica 1∶5 group;4small dose of astragalus angelica 5∶1;5small dose of astragalus angelica 1∶1 group;6small dose of astragalus angelica group 1∶5）,10 in each group,in addition to the normal control group,the rest of the group using endotracheal bolai doxycycline injection（5 mg/kg）copy the model of pulmonary fibrosis in mice.Building the second day,normal control group and model group lavage with saline water,six Chinese medicine treatment group give different ratio and dosage of astragalus angelica water decoction lavage treatment,executed 28 days in mice,using Real time rt-pcr method for detection of Th17/Treg cells differentiation key genes TGF- beta,IL- 6,foxp3,ROR γt and c-kit and FGF2 BASCs survive key genes,VEGF,Cyclin D expression level,HE dyeing and reduction WU Zhaofa Mallory＇s,to observe the morphology changes of lung tissue. Results：HE dyeing and reduction WU Zhaofa Mallory＇s,according to the model group mice were visible around the bronchial and alveolar interval has a large number of inflammatory cells infiltration,fibrosis significantly. Alveolar inflammation medicine 1,6 group was obviously better,no obvious inflammatory cells in the alveoli and alveolar septum,the fibrosis around a small bronchial lesions compared with model group obviously improved. Traditional Chinese medicine（traditional Chinese medicine）2,3,4,and 5 groups of alveolar inflammation was heavy,but there are many degree of ease compared with model group,the alveolar septum fiber hyperplasia stove is improved compared with model group,but not equal to 1,6 group obviously.28 d,significantly lower than the control group,model group mice weight compared with model group,each group of mice weight shows ascendant trend,Chinese traditional medicine group 1 larger trend of weight gain in mice（P0.05）.28 d mortality,Chinese medicine treatment group mice reduced mortality compared with model group,the group 1 mortality with model group,the difference was statistically significant（P0.05）.Model group TGF- beta,ROR γt,IL-6 m RNA expression levels were higher than control group,Foxp3 m RNA expression levels were lower than that of control group（P0.05）.Conclusion：From the survival rate of mice with weight and pathological morphology observation,large dose of astragalus angelica 5-1 group can obviously improve the quality of the survival of mice and its mechanism may be related to to silence a key gene Th17 differentiation TGF- beta,IL- 6,ROR γt m RNA expression level,promote the expression of key genes Foxp3 m RNA Treg differentiation level.