当归补血总苷的研制及对实验性肺纤维化的干预作用和分子机制研究
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摘要
特发性肺纤维化(Idiopathic pulmonary fibrosis ,IPF)是一种原因不明、以弥漫性肺泡炎和肺泡结构紊乱最终导致肺间质纤维化为特征的疾病。主要临床表现是逐渐加重的呼吸困难,伴有刺激性干咳,病情一般持续进展,最终因呼吸衰竭而病死,其发病率和死亡率很高,预后极差。IPF的发病机制尚未明了,然而目前肺纤维化治疗尚无特效药物,IPF已成为临床治疗的难点。研究肺纤维化发病机理和寻找行之有效的治疗药物是目前医学界迫切需要解决的课题。
中医经典名方当归补血汤由当归、黄芪组成,两者均有抗肺纤维化作用。前期研究表明,对于博莱霉素诱导的大鼠肺纤维化模型,当归补血总苷能明显减轻肺泡炎症和纤维化程度。本课题拟观察肺纤维化形成过程中胶原基质重塑在IPF发病机制中的动态变化,探索IPF防治新的作用靶点。同时在整体水平上研究当归补血总苷对IPF胶原基质重塑的调控作用从而探讨其对IPF的防治作用及机制,研究当归补血总苷对实验性肺纤维化形成过程中TGF-β1等细胞因子的影响,以及对MMPs和TIMPs局部表达失衡的调节作用,和其对TGF-β1/Smads信号转导通路的影响,为中医药益气活血协同抗肺纤维化提供现代科学依据。
目的制备当归补血总苷并建立其质量控制标准,优选当归补血汤中有效部位的提取工艺,建立当归补血总苷HPLC指纹图谱,并以此考察当归补血总苷的质量。观察当归补血总苷对实验性肺纤维化大鼠肺系数及肺组织病理形态的影响以及血清中HA,LN,PCⅢ、CⅣ,TGF-β1,COLⅠ和IL-13水平的变化;探讨肺纤维化大鼠肺组织中MMP-1、MMP-9与TIMP-1 mRNA的改变,并观察当归补血总苷对肺纤维化大鼠肺组织、MMP-1、MMP-9与TIMP-1表达的影响;探讨PF模型中体内成纤维细胞表型转分化和TGF-β1-Smad3途径在肺纤维化发生发展中的作用,阐明当归补血总苷对肺成纤维细胞表型转分化及TGF-β/Smads信号转导通路的影响及其机制。
方法采用薄层色谱法对当归补血总苷中的黄芪甲苷和阿魏酸进行定性鉴别;采用紫外分光光度法测定当归补血总苷的含量;采用HPLC法测定当归补血总苷中的黄芪甲苷、黄芪苷Ⅱ和阿魏酸的含量。设计L9(34)正交试验,采用HPLC法测定黄芪甲苷、阿魏酸的含量作为质控指标,考察乙醇浓度(A)、乙醇用量(B)、提取时间(C)和提取次数(D)4个因素对当归补血总苷提取的影响,优化提取工艺。采用HPLC-DAD,Diamonsil C18色谱柱(250 mm×4.6 mm, 5μm),乙腈―水二元梯度洗脱,流速1.0 mL·min-1,(λ=203 nm),柱温为30℃,建立了当归补血总苷的HPLC指纹图谱。180只Wistar大鼠随机分为正常对照组、模型对照组、强的松组和当归补血总苷大、中、小剂量组,每组30只。除正常对照组外,其他五组均采用经气管滴注博莱霉素A5建立大鼠肺纤维化模型,造模后第二日起当归补血总苷大、中、小剂量组每天用当归补血总苷(剂量64、32、16 mg·kg-1)灌胃,强的松组用醋酸强的松混悬液(剂量3 mg·kg-1)灌胃,每日一次,每组分别于7、14和28天随机处死10只大鼠采集肺组织,观察各组光镜、电镜(当归补血总苷组中剂量组)下的病理学变化以及比较各组肺系数,以及血清中HA,LN,PCⅢ、CⅣ,TGF-β1,COLⅠ和IL-13水平的变化。RT-PCR法检测当归补血总苷对肺纤维化大鼠肺组织MMP-1、MMP-9与TIMP-1 mRNA表达的影响;Western blotting和RT-PCR法分别检测大鼠正常组、模型组、当归补血总苷组不同组别中a-SMA、TGF-β、COLⅠ、Smad3、P-Smad3等蛋白及mRNA的表达。
结果黄芪甲苷在0.85～6.76μg之间呈良好的线性关系(r=0.9992),平均回收率为97.53%,RSD为0.82%。黄芪苷Ⅱ在1.05～8.4μg之间呈良好的线性关系(r=0.9994),平均回收率为94.82%,RSD为2.16%。阿魏酸在0.07～0.53μg之间呈良好的线性关系(r=0.9998),平均回收率为99.74%,RSD为1.62%。最佳提取条件为采用8倍量70%乙醇提取2次,每次2h。建立的当归补血总苷的HPLC指纹图谱确定了12个共有峰,各峰相对保留时间的RSD在0.2~1.0%之间,相对峰面积的RSD在1.6 ~3.2%之间。结果发现,不同批次当归补血总苷质量基本稳定,通过HPLC指纹图谱能够较好地控制其质量。与模型对照组相比,TGDGBX (16-64 mg·kg-1)及强的松组肺系数明显降低(P<0.01),肺组织病理观察显示肺泡炎及肺纤维化程度均明显减轻;TGDGBX (16-64 mg·kg-1)能显著降低BLM诱导的PF大鼠血清中升高的的HA,LN,PCⅢ和CⅣ水平,显著降低PF大鼠血清中IL-13含量,且造模后7、14和28 d三个时间点均有效。TGDGBX (16-64 mg·kg-1)可抑制BLM诱导的肺纤维化大鼠肺组织在肺纤维化不同阶段MMP-1 mRNA、MMP-9 mRNA的表达,提高TIMP-1mRNA的表达,使MMPs/TIMPs表达趋于平衡。进一步研究发现,TGDGBX (16-64 mg·kg-1)可以显著降低PF大鼠血清中升高的TGF-β1和COLⅠ水平。TGDGBX (16-64 mg·kg-1)可明显抑制肺纤维化不同阶段肺组织α-SMA和TGF-β1的表达,抑制肺组织升高的α-SMA、TGF-β1、COLⅠmRNA和蛋白的表达水平;下调肺纤维化不同阶段肺组织升高的Smad 3、P-Smad 3蛋白和Smad3 mRNA水平。
结论该提取工艺稳定性和重现性良好,简便,快速,可行;本方法制备的TGDGBX质量可控,检测方法精密度、重现性良好,准确,适用于TGDGBX的质量控制。TGDGBX能减轻肺泡炎性水肿、保持肺泡结构及阻抑纤维化形成,能够明显减少博莱霉素诱导肺纤维化大鼠ECM的生成,提示其对肺纤维化有一定防治作用。TGDGBX抗PF可能与其降低血清中升高的TGF-β1水平、促进COLⅠ胶原降解作用有关。提示TGDGBX可能通过对调节MMP1、MMP9/TIMP1表达实现对ECM重塑的调控;TGDGBX可通过TGF-β1环节下调Smad3和P-Smad 3表达,降低COLⅠ表达,从而减少ECM胶原沉积,发挥抗PF作用。但TGF-β1/Smads的改变与MMPs/TIMPs水平的相关性等尚需进一步研究。
Idiopathic pulmonary fibrosis (IPF), caused by unidentified reasons, is a kind of disease featured in diffuse alveolitis and the disordered structure of alveolus that finally lead to the fibrosis of pulmonary interstitial substance. Its major clinical manifestations include increasingly worsening breathing difficulty accompanied by irritable dry cough, progressive development and death due to breathing failure finally.
IPF is marked by higher rates of morbidity and mortality and extremely poor prognosis and its pathogenesis has not been clarified. Even worse, there is no highly effective medicine to treat pulmonary fibrosis and IPF has been a difficult challenge in clinical care. Therefore, it is urgent for medical researchers to study the pathogenesis of pulmonary fibrosis and seek effective medicine to treat the disease.
The increasingly rising cases of idiopathic pulmonary fibrosis have caused serious consequences and it is of great significance to seek effective medicines of low toxicity. DangGui BuXue Decoction, the classic recipe of traditional Chinese medicine, contains angelicae,radix and radix astragali, both of which can fight against pulmonary fibrosis. Previous studies indicate that the total glycosides of DangGui BuXue Decoction can obviously relieve the inflammation of pulmonary alveoli and the severity of pulmonary fibrosis according to the pattern of rats’ pulmonary fibrosis induced by the bleomycetin. This paper intends to search the new targets of preventing and curing IPF through observing the dynamic changes of the collagen matrix’remodeling in the pathogenesy of IPF. At the same time, it has also explored the preventive and curing functions and mechanisms on IPF of the total glycosides of DangGui BuXue Decoction by studying its coordinating functions on the collagen matrix’s remodeling of IPF on the incorporated, molecular levels and the effect of the signals’transmitting access, to provide modern scientific evidence of the traditional Chinese medicine’s invigorating and blood-activating functions in concurrently treating IPF.
Objective to prepare the Total Glucosides of DangGui BuXue Decoction and set up the standard of controlling its quality; to optimize the extracting technique of active sites of DangGui BuXue Decoction; to set up the finger print of HPLC in the Total Glucosides of DangGui BuXue Decoction and test the quality of the Total Glucosides of DangGui BuXue Decoction; to observe the effects of the Total Glucosides of DangGui BuXue Decoction on the lung index and pulmonary pathology and histomorphology of experimental rats with pulmonary fibrosis and the changes in serum levels of HA,LN,PCⅢ、CⅣ,TGF-β1,COL and IL-13; to explore the expressions of MMP-1、MMP- 9and TIMP-1 mRNA in the pulmonary tissues of the rats with pulmonary fibrosis and observe how the Total Glucosides of DangGui BuXue Decoction affects the pulmonary tissues and the expressions of MMP-1、MMP-9 and TIMP-1; to probe the fibroblast phenotype transdifferentiation in vivo of PF model and the role played by the access of TGF-β1-Smad3 in the formation and development of pulmonary fibrosis, thus to illustrate the effects and mechanisms of the Total Glucosides of DangGui BuXue Decoction on fibroblast phenotype transdifferentiation in vivo and the signal transduction path of TGF-β/Smads.
Methods Thin-layer chromatography (TLC) was adopted to conduct qualitative identification of the astragalosideⅣand ferulaic acid in the Total Glucosides of DangGui BuXue Decoction; ultraviolet spectrophotometry was applied to measure the content of the total glucosides of DangGui BuXue Decoction; HPLC was used to measure the contents of the astragalosideⅣ, astragalosideⅡand ferulaic acid in TGDGBX to use them as the index of quality control. Orthogonal experiment was designed to explore the effects of four factors-the alcoholic concentration, alcoholic volume, extracting time and times of extracting-on the extraction of TGDGBX by employing HPLC to measure the contents of the astragalosideⅣand ferulaic acid, hence to optimize the extracting technique. The HPLC finger print of TGDGBX was set up by HPLC-DAD: the chromatographic column,Diamonsil C18(250 mm×4.6 mm, 5μm, 30℃)and bidimensional gradient elution using methyl cyanide-water (flowing speed 1.0 mL·min-1,λ=203 nm). 180 Wistar rats were randomly divided into normal control group, model group, cortisone group, and TGDGBX (16,32 and 64 mg·kg-1) groups respectively. With the exception of the normal control group, the other five groups were infused with bleomycin A5 through trachea to build models of the rats with pulmonary fibrosis. And from next day on after the setting-up of the animal models, the three groups treated with different dosages of TGDGBX undertook intragastric administration everyday of TGDGBX (64、32、16 mg·kg-1 for the high, moderate and low dosages respectively) and the cortisone group everyday undertook intragastric administration of suspl. of Prednisoni Acetas. On day 7, 14 and 28, lung tissues were collected by randomly killing 10 rats of each group, and the histopathology changes of the TGDGBX groups were observed under the light microscope and electron microscope and lung index in each group were compared. And the levels of HA,LN,PCⅢ、CⅣ,TGF-β1,COLⅠand IL-13 in serum were also measured. RT-PCR was used to check the effects of TGDGBX on the expressions of TGF-β1、a-SMA、COLⅠ、MMP-1、MMP-9 and TIMP-1 mRNA in the lung tissues of rats with pulmonary fibrosis; Western blotting were employed accordingly to measure the expressions of proteins such as a-SMA, COLⅠ,TGF-β1, Smad3, P-Smad3 of all six groups.
Results AstragalosideⅣshowed good linear correlation between 0.85 and 6.76μg (r=0.9992), with average recovery rate 97.53% and RSD 0.82%. AstragalosideⅡindicated favorable linear correlation between1.05 and 8.4μg ((r=0.9994), with average recovery rate 94.82% and RSD 2.16%. Ferulaic acid demonstrated good linear correlation between 0.07 and 0.53μg (r=0.9998), with average recovery rate 99.74% and RSD 1.62%. The optimal extracting condition could be achieved by using eight times 70% ethanol and extracting twice, and 2 hours every time. The HPLC finger print of TGDGBX had confirmed 12 co-peaks and RSD the of relative retaining time of each peak ranged between 0.2~1.0%, and RSD of relative areas of peaks ranged from 1.6 ~3.2%. The qualities of TGDGBX in different groups were stable and their qualities could be controlled by the HPLC finger print. Compared with the model group, the groups treated with TGDGBX (16-64 mg·kg-1) and cortisone group showed obvious reduction of lung index ((P<0.01). And histopathology observation of lung tissues indicated that there were considerable decreases in the severity of alveolitis and pulmonary fibrosis; TGDGGX(16-64 mg·kg-1) could effectively reduce the rising serum levels of HA,LN,PCⅢand CⅣin rats with pulmonary fibrosis induced by BLM, and evidently decrease the serum content of IL-13 in the PF rats. And this worked in three time points, d7, 14 and 28 after the setting up of the animal model. TGDGBX (16-64 mg·kg-1) could inhibit the expressions of MMP-1 mRNA、MMP-9 mRNA in different stages of pulmonary fibrosis in lung tissues of rats with pulmonary fibrosis induced by BLM, increase the expression of TIMP-1mRNA, and balance the expression of MMPs/TIMPs. Further studies found that TGDGBX (16-64 mg·kg-1) could effectively depress the levels of rising TGF-β1 and COLⅠin the serum of the rats with pulmonary fibrosis; TGDGBX (16-64 mg·kg-1) could evidently suppress the expressions ofα-SMA and TGF-β1 in lung tissues at the various stages of the pulmonary fibrosis and the elevated expressions ofα-SMA, TGF-β1, COLⅠmRNA and proteins; it could remarkably inhibit the elevated level of Smad3 and P-Smad3 proteins and Smad3 mRNA at the different stages of pulmonary fibrosis.
Conclusions This simple, fast and feasible extracting technique shows favorable stability and reproducibility. TGDGBX prepared through this way has controllable quality, precise measuring method, plus good and accurate reproducibility. It can relieve the inflammatory edema of alveolus, retain the structure of alveolus and halt the formation of pulmonary fibrosis. It also can evidently reduce the development of ECM of rats with pulmonary fibrosis induced by bleomycin and demonstrates considerable effect on the treatment of the pulmonary fibrosis. There is great possibility that the resistance of the TGDGBX on PF has something to do its reducing the rising level of TGF-β1 in serum and promoting the decomposition of the COLⅠcollagen. TGDGBX may control the remodeling process of ECM by adjusting the expression of MMP1 and MMP9/TIMP1; it can down regulation the expression of Smad3 and P-Smad3 by the link of TGF-β1, decrease the expression of COLⅠto reduce the sedimentation of the ECM collagen and play a role in fighting against PF. However, the correlation between the change of TGF-β1/Smads and the level of MMPs/TIMPs is to be further explored in the future studies.
中医经典名方当归补血汤由当归、黄芪组成,两者均有抗肺纤维化作用。前期研究表明,对于博莱霉素诱导的大鼠肺纤维化模型,当归补血总苷能明显减轻肺泡炎症和纤维化程度。本课题拟观察肺纤维化形成过程中胶原基质重塑在IPF发病机制中的动态变化,探索IPF防治新的作用靶点。同时在整体水平上研究当归补血总苷对IPF胶原基质重塑的调控作用从而探讨其对IPF的防治作用及机制,研究当归补血总苷对实验性肺纤维化形成过程中TGF-β1等细胞因子的影响,以及对MMPs和TIMPs局部表达失衡的调节作用,和其对TGF-β1/Smads信号转导通路的影响,为中医药益气活血协同抗肺纤维化提供现代科学依据。
目的制备当归补血总苷并建立其质量控制标准,优选当归补血汤中有效部位的提取工艺,建立当归补血总苷HPLC指纹图谱,并以此考察当归补血总苷的质量。观察当归补血总苷对实验性肺纤维化大鼠肺系数及肺组织病理形态的影响以及血清中HA,LN,PCⅢ、CⅣ,TGF-β1,COLⅠ和IL-13水平的变化;探讨肺纤维化大鼠肺组织中MMP-1、MMP-9与TIMP-1 mRNA的改变,并观察当归补血总苷对肺纤维化大鼠肺组织、MMP-1、MMP-9与TIMP-1表达的影响;探讨PF模型中体内成纤维细胞表型转分化和TGF-β1-Smad3途径在肺纤维化发生发展中的作用,阐明当归补血总苷对肺成纤维细胞表型转分化及TGF-β/Smads信号转导通路的影响及其机制。
方法采用薄层色谱法对当归补血总苷中的黄芪甲苷和阿魏酸进行定性鉴别;采用紫外分光光度法测定当归补血总苷的含量;采用HPLC法测定当归补血总苷中的黄芪甲苷、黄芪苷Ⅱ和阿魏酸的含量。设计L9(34)正交试验,采用HPLC法测定黄芪甲苷、阿魏酸的含量作为质控指标,考察乙醇浓度(A)、乙醇用量(B)、提取时间(C)和提取次数(D)4个因素对当归补血总苷提取的影响,优化提取工艺。采用HPLC-DAD,Diamonsil C18色谱柱(250 mm×4.6 mm, 5μm),乙腈―水二元梯度洗脱,流速1.0 mL·min-1,(λ=203 nm),柱温为30℃,建立了当归补血总苷的HPLC指纹图谱。180只Wistar大鼠随机分为正常对照组、模型对照组、强的松组和当归补血总苷大、中、小剂量组,每组30只。除正常对照组外,其他五组均采用经气管滴注博莱霉素A5建立大鼠肺纤维化模型,造模后第二日起当归补血总苷大、中、小剂量组每天用当归补血总苷(剂量64、32、16 mg·kg-1)灌胃,强的松组用醋酸强的松混悬液(剂量3 mg·kg-1)灌胃,每日一次,每组分别于7、14和28天随机处死10只大鼠采集肺组织,观察各组光镜、电镜(当归补血总苷组中剂量组)下的病理学变化以及比较各组肺系数,以及血清中HA,LN,PCⅢ、CⅣ,TGF-β1,COLⅠ和IL-13水平的变化。RT-PCR法检测当归补血总苷对肺纤维化大鼠肺组织MMP-1、MMP-9与TIMP-1 mRNA表达的影响;Western blotting和RT-PCR法分别检测大鼠正常组、模型组、当归补血总苷组不同组别中a-SMA、TGF-β、COLⅠ、Smad3、P-Smad3等蛋白及mRNA的表达。
结果黄芪甲苷在0.85～6.76μg之间呈良好的线性关系(r=0.9992),平均回收率为97.53%,RSD为0.82%。黄芪苷Ⅱ在1.05～8.4μg之间呈良好的线性关系(r=0.9994),平均回收率为94.82%,RSD为2.16%。阿魏酸在0.07～0.53μg之间呈良好的线性关系(r=0.9998),平均回收率为99.74%,RSD为1.62%。最佳提取条件为采用8倍量70%乙醇提取2次,每次2h。建立的当归补血总苷的HPLC指纹图谱确定了12个共有峰,各峰相对保留时间的RSD在0.2~1.0%之间,相对峰面积的RSD在1.6 ~3.2%之间。结果发现,不同批次当归补血总苷质量基本稳定,通过HPLC指纹图谱能够较好地控制其质量。与模型对照组相比,TGDGBX (16-64 mg·kg-1)及强的松组肺系数明显降低(P<0.01),肺组织病理观察显示肺泡炎及肺纤维化程度均明显减轻;TGDGBX (16-64 mg·kg-1)能显著降低BLM诱导的PF大鼠血清中升高的的HA,LN,PCⅢ和CⅣ水平,显著降低PF大鼠血清中IL-13含量,且造模后7、14和28 d三个时间点均有效。TGDGBX (16-64 mg·kg-1)可抑制BLM诱导的肺纤维化大鼠肺组织在肺纤维化不同阶段MMP-1 mRNA、MMP-9 mRNA的表达,提高TIMP-1mRNA的表达,使MMPs/TIMPs表达趋于平衡。进一步研究发现,TGDGBX (16-64 mg·kg-1)可以显著降低PF大鼠血清中升高的TGF-β1和COLⅠ水平。TGDGBX (16-64 mg·kg-1)可明显抑制肺纤维化不同阶段肺组织α-SMA和TGF-β1的表达,抑制肺组织升高的α-SMA、TGF-β1、COLⅠmRNA和蛋白的表达水平;下调肺纤维化不同阶段肺组织升高的Smad 3、P-Smad 3蛋白和Smad3 mRNA水平。
结论该提取工艺稳定性和重现性良好,简便,快速,可行;本方法制备的TGDGBX质量可控,检测方法精密度、重现性良好,准确,适用于TGDGBX的质量控制。TGDGBX能减轻肺泡炎性水肿、保持肺泡结构及阻抑纤维化形成,能够明显减少博莱霉素诱导肺纤维化大鼠ECM的生成,提示其对肺纤维化有一定防治作用。TGDGBX抗PF可能与其降低血清中升高的TGF-β1水平、促进COLⅠ胶原降解作用有关。提示TGDGBX可能通过对调节MMP1、MMP9/TIMP1表达实现对ECM重塑的调控;TGDGBX可通过TGF-β1环节下调Smad3和P-Smad 3表达,降低COLⅠ表达,从而减少ECM胶原沉积,发挥抗PF作用。但TGF-β1/Smads的改变与MMPs/TIMPs水平的相关性等尚需进一步研究。
Idiopathic pulmonary fibrosis (IPF), caused by unidentified reasons, is a kind of disease featured in diffuse alveolitis and the disordered structure of alveolus that finally lead to the fibrosis of pulmonary interstitial substance. Its major clinical manifestations include increasingly worsening breathing difficulty accompanied by irritable dry cough, progressive development and death due to breathing failure finally.
IPF is marked by higher rates of morbidity and mortality and extremely poor prognosis and its pathogenesis has not been clarified. Even worse, there is no highly effective medicine to treat pulmonary fibrosis and IPF has been a difficult challenge in clinical care. Therefore, it is urgent for medical researchers to study the pathogenesis of pulmonary fibrosis and seek effective medicine to treat the disease.
The increasingly rising cases of idiopathic pulmonary fibrosis have caused serious consequences and it is of great significance to seek effective medicines of low toxicity. DangGui BuXue Decoction, the classic recipe of traditional Chinese medicine, contains angelicae,radix and radix astragali, both of which can fight against pulmonary fibrosis. Previous studies indicate that the total glycosides of DangGui BuXue Decoction can obviously relieve the inflammation of pulmonary alveoli and the severity of pulmonary fibrosis according to the pattern of rats’ pulmonary fibrosis induced by the bleomycetin. This paper intends to search the new targets of preventing and curing IPF through observing the dynamic changes of the collagen matrix’remodeling in the pathogenesy of IPF. At the same time, it has also explored the preventive and curing functions and mechanisms on IPF of the total glycosides of DangGui BuXue Decoction by studying its coordinating functions on the collagen matrix’s remodeling of IPF on the incorporated, molecular levels and the effect of the signals’transmitting access, to provide modern scientific evidence of the traditional Chinese medicine’s invigorating and blood-activating functions in concurrently treating IPF.
Objective to prepare the Total Glucosides of DangGui BuXue Decoction and set up the standard of controlling its quality; to optimize the extracting technique of active sites of DangGui BuXue Decoction; to set up the finger print of HPLC in the Total Glucosides of DangGui BuXue Decoction and test the quality of the Total Glucosides of DangGui BuXue Decoction; to observe the effects of the Total Glucosides of DangGui BuXue Decoction on the lung index and pulmonary pathology and histomorphology of experimental rats with pulmonary fibrosis and the changes in serum levels of HA,LN,PCⅢ、CⅣ,TGF-β1,COL and IL-13; to explore the expressions of MMP-1、MMP- 9and TIMP-1 mRNA in the pulmonary tissues of the rats with pulmonary fibrosis and observe how the Total Glucosides of DangGui BuXue Decoction affects the pulmonary tissues and the expressions of MMP-1、MMP-9 and TIMP-1; to probe the fibroblast phenotype transdifferentiation in vivo of PF model and the role played by the access of TGF-β1-Smad3 in the formation and development of pulmonary fibrosis, thus to illustrate the effects and mechanisms of the Total Glucosides of DangGui BuXue Decoction on fibroblast phenotype transdifferentiation in vivo and the signal transduction path of TGF-β/Smads.
Methods Thin-layer chromatography (TLC) was adopted to conduct qualitative identification of the astragalosideⅣand ferulaic acid in the Total Glucosides of DangGui BuXue Decoction; ultraviolet spectrophotometry was applied to measure the content of the total glucosides of DangGui BuXue Decoction; HPLC was used to measure the contents of the astragalosideⅣ, astragalosideⅡand ferulaic acid in TGDGBX to use them as the index of quality control. Orthogonal experiment was designed to explore the effects of four factors-the alcoholic concentration, alcoholic volume, extracting time and times of extracting-on the extraction of TGDGBX by employing HPLC to measure the contents of the astragalosideⅣand ferulaic acid, hence to optimize the extracting technique. The HPLC finger print of TGDGBX was set up by HPLC-DAD: the chromatographic column,Diamonsil C18(250 mm×4.6 mm, 5μm, 30℃)and bidimensional gradient elution using methyl cyanide-water (flowing speed 1.0 mL·min-1,λ=203 nm). 180 Wistar rats were randomly divided into normal control group, model group, cortisone group, and TGDGBX (16,32 and 64 mg·kg-1) groups respectively. With the exception of the normal control group, the other five groups were infused with bleomycin A5 through trachea to build models of the rats with pulmonary fibrosis. And from next day on after the setting-up of the animal models, the three groups treated with different dosages of TGDGBX undertook intragastric administration everyday of TGDGBX (64、32、16 mg·kg-1 for the high, moderate and low dosages respectively) and the cortisone group everyday undertook intragastric administration of suspl. of Prednisoni Acetas. On day 7, 14 and 28, lung tissues were collected by randomly killing 10 rats of each group, and the histopathology changes of the TGDGBX groups were observed under the light microscope and electron microscope and lung index in each group were compared. And the levels of HA,LN,PCⅢ、CⅣ,TGF-β1,COLⅠand IL-13 in serum were also measured. RT-PCR was used to check the effects of TGDGBX on the expressions of TGF-β1、a-SMA、COLⅠ、MMP-1、MMP-9 and TIMP-1 mRNA in the lung tissues of rats with pulmonary fibrosis; Western blotting were employed accordingly to measure the expressions of proteins such as a-SMA, COLⅠ,TGF-β1, Smad3, P-Smad3 of all six groups.
Results AstragalosideⅣshowed good linear correlation between 0.85 and 6.76μg (r=0.9992), with average recovery rate 97.53% and RSD 0.82%. AstragalosideⅡindicated favorable linear correlation between1.05 and 8.4μg ((r=0.9994), with average recovery rate 94.82% and RSD 2.16%. Ferulaic acid demonstrated good linear correlation between 0.07 and 0.53μg (r=0.9998), with average recovery rate 99.74% and RSD 1.62%. The optimal extracting condition could be achieved by using eight times 70% ethanol and extracting twice, and 2 hours every time. The HPLC finger print of TGDGBX had confirmed 12 co-peaks and RSD the of relative retaining time of each peak ranged between 0.2~1.0%, and RSD of relative areas of peaks ranged from 1.6 ~3.2%. The qualities of TGDGBX in different groups were stable and their qualities could be controlled by the HPLC finger print. Compared with the model group, the groups treated with TGDGBX (16-64 mg·kg-1) and cortisone group showed obvious reduction of lung index ((P<0.01). And histopathology observation of lung tissues indicated that there were considerable decreases in the severity of alveolitis and pulmonary fibrosis; TGDGGX(16-64 mg·kg-1) could effectively reduce the rising serum levels of HA,LN,PCⅢand CⅣin rats with pulmonary fibrosis induced by BLM, and evidently decrease the serum content of IL-13 in the PF rats. And this worked in three time points, d7, 14 and 28 after the setting up of the animal model. TGDGBX (16-64 mg·kg-1) could inhibit the expressions of MMP-1 mRNA、MMP-9 mRNA in different stages of pulmonary fibrosis in lung tissues of rats with pulmonary fibrosis induced by BLM, increase the expression of TIMP-1mRNA, and balance the expression of MMPs/TIMPs. Further studies found that TGDGBX (16-64 mg·kg-1) could effectively depress the levels of rising TGF-β1 and COLⅠin the serum of the rats with pulmonary fibrosis; TGDGBX (16-64 mg·kg-1) could evidently suppress the expressions ofα-SMA and TGF-β1 in lung tissues at the various stages of the pulmonary fibrosis and the elevated expressions ofα-SMA, TGF-β1, COLⅠmRNA and proteins; it could remarkably inhibit the elevated level of Smad3 and P-Smad3 proteins and Smad3 mRNA at the different stages of pulmonary fibrosis.
Conclusions This simple, fast and feasible extracting technique shows favorable stability and reproducibility. TGDGBX prepared through this way has controllable quality, precise measuring method, plus good and accurate reproducibility. It can relieve the inflammatory edema of alveolus, retain the structure of alveolus and halt the formation of pulmonary fibrosis. It also can evidently reduce the development of ECM of rats with pulmonary fibrosis induced by bleomycin and demonstrates considerable effect on the treatment of the pulmonary fibrosis. There is great possibility that the resistance of the TGDGBX on PF has something to do its reducing the rising level of TGF-β1 in serum and promoting the decomposition of the COLⅠcollagen. TGDGBX may control the remodeling process of ECM by adjusting the expression of MMP1 and MMP9/TIMP1; it can down regulation the expression of Smad3 and P-Smad3 by the link of TGF-β1, decrease the expression of COLⅠto reduce the sedimentation of the ECM collagen and play a role in fighting against PF. However, the correlation between the change of TGF-β1/Smads and the level of MMPs/TIMPs is to be further explored in the future studies.
引文
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2.魏路清,董彦.肺纤维化发病机制及治疗策略的新观念.国外医学.呼吸系统分册2003,23(I):38-41;
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4.刘巨源,陈永凤,郭萍,高新平,路成吉.黄芪丹参甘草等中药对大鼠肺纤维化的影响.新乡医学院学报, 1999, 16 (4) : 292-95。
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5. Willis BC, Liebler JM, Luby-Phelps K, Nicholson AG, Crandall ED, du Bois RM, Borok Z. Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis. Am J Pathol. 2005; 166(5): 1321 - 32.
6.Sullivan DE,Ferris M,Pociask D,Brody AR. Tumor necrosis factor-alpha induces transforming growth factor-β1 expression in lung fibroblasts through the extracellular signal -regulated kinase pathway. Am J Respir Cell Mol Biol. 2005; 32:342 - 49.
7. Barbarin V, Xing Z, Delos M, Lison D, Huaux F. Pulmonary over expression of IL-10 augments lung fibrosis and Th2 responses induced by silica particles. Am J Physiol Lung Cell Mol Physiol. 2005; 288 (5): L841 - L848.
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