滇龙血竭分期干预对实验性PF炎症暴发级联反应的动态调控研究

Based on the current situation that the mechanism of pulmonary fibrosis (PF), one of refractory disease, is unknown and the case of deficient of treating approaches is there. The present study proposes the hypothetical mechanism, in which inflammatory cascade reaction(ICR) grounded on FZZ1 as core promoter triggers PF, and take it as a research point. To carry out research with Yun Nan ethnic medicine Dian Sanguis Draconis, use it for stage intervention on experimental PF induced by bleomycin. The whole process is divided into two phases: one is the whole and early stage ( alveolitis and pulmonary parenchyma injury), the other is advanced stage ( fibroblast proliferation and fibrosis). Some indicators, such as the level of serum IL- 4/IFN -γ which is detected by ELLSA, the FIZZ1's expressions of protein and mRNA in the lung tissue which are detected by means of immunohistochemistry (IHC) and RT-PCR, the content of Hyp in the lung tissue which is determined by colorimetry, and the Quantitative analysis of pathological morphology which is conducted through HE stain and Masson-trichrome, all those are evaluated dynamically. This research aims to elucidate Dian Sanguis Draconis' dynamic regulation mechanism which take anti-inflammatory as the core, enrich treatment ideas of PF, find the best delivery time, and then supply theoretical and experimental evidences for early intervention of PF. By exploring the effective means of prevention and treatment of PF with the help of ethical medicine resources, and then trying to do some efforts to promote the development of Yunnan ethnic medicine.

鉴于重大疑难疾病- - 肺间质纤维化（PF）机制不明、手段匮乏的现状，提出以FZZ1为核心启动子的"炎症暴发级联反应（ICR）"引发PF的假设机制，并将其作为研究切入点。选择有云南民族医药特色的"滇龙血竭"对博莱霉素诱发的实验性PF进行分期（全程以及早期：肺泡炎及肺实质损伤期；晚期：纤维增殖及纤维化期）干预。通过动态评价相应指标：ELLSA法检测血清IL- 4/IFN -γ水平；免疫组化和RT-PCR技术检测FIZZ1在肺组织中的蛋白及mRNA表达；比色法测定肺组织羟脯氨酸（Hyp）含量；以HE染色、Masson三联染色进行病理形态学定量分析。以期阐明滇龙血竭以抗炎为核心的动态调控作用机制，丰富PF治疗思路；摸索最佳给药时段，可望为早期干预PF提供理论和实验依据；探寻民族医药资源中防治PF行之有效的手段，为促进云南民族医药发展做一点尝试和努力。

背景：鉴于重大疑难疾病——肺间质纤维化（PF）机制不明、手段匮乏的现状，提出以FZZ1为核心启动子的“炎症暴发级联反应”引发PF的假设机制，并将其作为研究切入点，选择有云南民族医药特色的“滇龙血竭”对博莱霉素诱发的实验性PF进行分期干预研究。.主要内容及目的：进行滇龙血竭的制备工艺研究；观察滇龙血竭分期干预对PF大鼠的药效学作用；探讨滇龙血竭抗炎为核心的多靶点作用机制。.方法：分为二个研究进行。.研究一：滇龙血竭制备工艺研究.采用大孔树脂纯化分离提取制备，并建立龙血素A、B的含量测定方法。.研究二：滇龙血竭对PF大鼠的药效学作用昅机制研究.采用经气管注入博莱霉素法诱发SD大鼠PF模型，96只雄性SPF级SD大鼠，随机分为6组，分别于制模后1d灌胃相应药物。以ELISA法检测血清IL– 4/IFN –γ水平；免疫组化和RT-PCR技术检测FIZZ1在肺组织中的蛋白及mRNA表达；比色法测定肺组织羟脯氨酸（Hyp）含量；以HE染色、Masson三联染色进行病理形态学定量分析。.结果：龙血竭总黄酮的最佳制备工艺为龙血竭乙酸乙酯提取物用0.3% NaOH溶解，上样，依次用超纯水洗脱杂质至流出液呈中性为止，再用5BV 80%的乙醇洗脱，洗脱液回收乙醇，浓缩至干；滇龙血竭早期及全程干预可抑制实验性RF大鼠肺组织中FIZZ1的mRNA及蛋白表达水平(P<0.01)；降低血清IL-4水平（P<0.01或P<0.05），提高血清IFN-γ的水平（P<0.01或P<0.05）；降低PF大鼠肺组织中Hyp水平（P<0.01或P<0.05）；组织病理学证实可减轻其肺组织肺泡炎、肺纤维化程度。总体疗效以滇龙血竭全程干预组最佳，与吡非尼酮组无显著差异(P>0.05)。.结论：该工艺在大生产中简便易行，制剂稳定性好，可作为该制剂的制备工艺方案；滇龙血竭早期及全程干预能够有效减缓博莱霉素诱发的肺泡炎及纤维化进程，其机制可能与调节IL–4/IFN–γ失衡，抑制“炎症暴发级联反应”的核心启动子FIZZ1在肺内的表达有关，进而阻止后续胶原蛋白沉积、血管新生等系列变化。说明，滇龙血竭具有以抗炎为核心的多靶位作用机制，在早期防治PF领域有深入研发价值。