摘要
目的：肺间质纤维化是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主要特征的疾病。目前其发病机制尚未完全阐明，又无确切有效的药物治疗。探讨中医药对肺纤维化的防治具有广阔的前景和深远的意义。本课题采用大鼠一次性气管内滴注博莱霉素法造模，通过观察大鼠肺组织的形态学变化、肺泡Ⅱ型上皮细胞凋亡率变化、血清中IL-6和TNF-α的表达水平、肺组织中CTGF的表达、肺组织匀浆中SOD和GSH-PX的活性及MDA的含量，来探讨肺间质纤维化的发病机制，并观察具有“益气通阳、行气活血化痰”功效的化纤汤对这些指标的影响，以探讨化纤汤对大鼠肺纤维化的保护作用及可能机制，为寻求中医中药对肺纤维化的防治进行实验研究。
方法：健康Wistar大鼠240只，体重180-220克，随机分为正常对照组(A组)、模型组(B组)、强的松干预组(C组)、化纤汤大剂量(D组)、中剂量组(E组)、小剂量(F组)干预组共6组。A、B、C、D四组每组各48只(其中24只用于肺泡Ⅱ型上皮细胞的提取)，E、F组各24只。采用一次性气管内滴注博莱霉素(5mg／kg·bw)法造模，各组均于造模后第二天开始灌胃给药。C组每日一次给予醋酸强的松混悬液(0.32mg／ml)灌胃，D、E、F组每日一次给予化纤汤(浓度分别是2g／ml、1g／ml、0.5g／ml)灌胃，A、B组每日给予一次等容积的生理盐水灌胃，其中强的松和化纤汤用量相当于成人常用量，按体表面积折算。分别于第7、14、28天随机处死A、B、C、D四组的大鼠各8只，取材用于肺泡Ⅱ型上皮细胞的分离和纯化；其余各组大鼠亦于第7、14、28天随机处死8只，用于形态学观察及血清、组织匀浆的制备。①在光镜及电镜下观察肺组织病理形态学的变化及超微结构的改变；②用流式细胞仪检测肺泡Ⅱ型上皮细胞凋亡率的变化情况。③用ELISA法检测血清中IL-6、TNF-α表达水平的变化；④用免疫组化SABC法检测肺组织中CTGF的表达；⑤分别用TBA法和比色法检测肺组织匀浆中SOD和GSH-PX的活性及MDA的含量。
结果：①肺大体形态改变：各时间点，A组大鼠双肺呈粉红色，表面光滑，弹性好。B组，第7天，双肺见密集的点状出血灶样瘀斑，肺体积增大；第14天，双肺呈灰白色，局部可见大小不等的结节样改变及陈旧性出血点，肺组织较硬；第28天，双肺苍白，体积缩小，触及较硬，表面结节样改变及条索状凹沟。与B组比较，第7天时，C、D、E、F组两肺外观病理变化明显好于B组，两肺稍暗红，弹性可，其中D、C、E组明显好于F组；第14-28天，D、E组大部分动物两肺颜色稍暗，弹性尚可，偶可见部分肺叶有散在的点状灰色斑块，F、C与B组病变相近。②形态学改变：第7天，B组大鼠肺泡壁增厚，伴有炎性细胞浸润，间质内成纤维细胞增多，病灶内肺泡萎缩，肺泡内大量炎性细胞浸润；第14天，肺泡间隔明显增宽，成纤维化和胶原基质明显增多；第28天，肺泡结构破坏，肺泡壁显著增厚，肺间质纤维化瘢痕形成，毛细血管腔闭塞。超微病理示：B组大鼠第7天，肺泡Ⅱ型上皮细胞增生、肿胀，线粒体肿胀，嵴缩短、断裂或消失，甚至空泡样变性，板层小体数量增多，但是多呈空泡样，细胞微绒毛破坏、稀少或消失；第14-28天，肺泡Ⅱ型细胞数量较第7天减少，核形态不规则，线粒体嵴断裂或消失，大多呈空泡样变性，数量明显减少，板层小体量也减少，大多呈空泡样变。与B组比较，这些病理变化，第7天，D组改善最明显，其次为C、E、F组；第14天，D组改善最明显，其次为E、C、F组；第28天，D组改善最明显，其次为E、F、C组，其中第7天、28天肺泡炎、肺纤维化Szapiel分级比较具有显著性差异(P＜0.01或0.05)。③与A组比较，各时间点，各组大鼠肺泡Ⅱ型上皮细胞凋亡率显著升高(P＜0.05)。与B组比较，第7天时，C、D两组凋亡率均有显著降低(P＜0.05)；第14-28天，C组肺泡Ⅱ型上皮细胞凋亡率有所下降，但统计学无显著差异(P＞0.05)，D组肺泡Ⅱ型上皮细胞凋亡率有显著减少(P＜0.05)。④与A组比较，各时间点各组大鼠血清中TNF-α、IL-6水平明显增高。与B组比较，第7天，C、D、E、F各组大鼠血清中TNF-α、IL-6的水平显著降低(P＜0.01或0.05)；第14-28天，D、E组大鼠血清中TNF-α、IL-6水平明显降低(P＜0.01或0.05)，C、F组虽有降低，但比较差异无统计学意义(P＞0.05)。⑤与A组比较，各时间点各组大鼠肺组织中CTGF的表达明显增高(P＜0.01)。与B组比较，各时间点，C、D、E各组CTGF表达均低于B组(P＜0.01或0.05)，F组低于B组，但比较差异无统计学意义(P＞0.05)。⑥与A组比较，各时间点，B、C、D、E、F各组大鼠肺组织中GSH-PX、SOD的活性均降低，MDA的含量均增高，存在显著差异(P＜0.01或0.05)。与B组比较，各时间点，D、E组大鼠GSH-PX与SOD的活性均升高，MDA含量均降低，存在显著差异(P＜0.01或0.05)；第7-14天，C组大鼠GSH-PX与SOD的活性均升高，MDA含量均降低，存在明显差异(P＜0.05)，第28天，C组与B组无明显差异(P＞0.05)。各时间点，F组与B组比较无明显差异(P＞0.05)。
结论：①模型组病理特征变化与文献报道一致，说明造模成功；化纤汤对肺纤维化发展各个阶段的病理变化有明显的改善作用，且大、中剂量组明显优于小剂量组。②ATⅡ细胞过度凋亡可能促进肺泡炎症反应并导致炎症后纤维增生；化纤汤对ATⅡ细胞可能有抗凋亡作用，可能通过抑制ATⅡ细胞的过度凋亡而起到保护和修复肺泡结构及功能的作用，从而减轻BLM对大鼠肺组织的损伤，延缓并减轻肺纤维化的发生发展。③化纤汤能降低肺纤维化大鼠血清中TNF-α、IL-6水平，可能通过下调上述细胞因子水平而对博莱霉素致大鼠的肺损伤起保护作用，减轻肺纤维化形成的程度，化纤汤大、中剂量组疗效优于小剂量组。④CTGF可能在肺泡炎、肺纤维化的发生发展过程中起着重要的作用，其表达程度与肺纤维化的程度密切相关；化纤汤可能通过抑制CTGF的表达对肺组织起着修复及保护作用。⑤化纤汤能调节肺纤维化大鼠体内自由基水平，减轻自由基对肺组织结构的氧化损伤，而且化纤汤大、中剂量组的作用优于小剂量组。
Object: Interstitial pulmonary fibrosis is a kind of disease mainly characterized by inflammation and structural destroy of alveolus, which finally changed into interstitial fibrosis. The therapeutic approach to this not particularly effective because of its unclear mechanism. Tranditional Chinese medicine has reached a several achievements in improving symptoms and reducing death rate, and it's necessary to summarize and improve the methods of TCM for this disease. Huaxian decoction(HD), a prescription of tonifying qi and regulating yang, smoothing qi activing blood and eliminating sputum, has shown a bright future in clinical study of pulmonary fibrosis. In this article, the rat model was induced by blyomicin and we evaluated HD's curative effect objectively and explore its way and mechanism by observing the changing of apoptosis of ATⅡcells, the content of TNF-αand IL-6 in sera, the expression of CTGF, the level of SOD, GSH-PX and MDA in lung, the morphological alternation of the lung tissue.
Methods: Two hundreds and forty healthy Wistar rats, weight from 180 to 220gram were randomly divided into six groups: normal control group(group A, n=48), model group(group B, n=48), prednisone group(group C, n=48), Chinese drugs of high(group D, n=48), moderate (group E, n=24) and low dosage (group F, n=24). The rat model was induced by blyomicin and respective medicine was given to each group. Every group was subdivided into the three groups which was killed in the seventh, fourteenth, and twenty-eighth day respectively.①The rat's lung tissue section was observed under light microscope and electron microscope to study the morphological alternation.②Find the changes of apoptosis of ATⅡcells by flow cytometry.③Assay the sera level of TNF-αand IL-6 by ELISA method.④Detect the expression of CTGF in the lung tissue with immunohistochemical method of analysis.⑤TBA method and colorimetric method were used to measure the content of SOD, GSH-PX and MDA in lung tissue respectively.
Results:①Changes of pathologic morphosis under light microscope in the rat lung fibrosis model: in the 7th day after model was made, interval between alveolus became thicker,, the number of fibroblast increased and alveoli in the focus of infection were atrophied and lots of inflammation cells infiltrated; in the 14th day after model was made, interval between alveolus became thicker and thicker, fibroblast and collagen matrix increased markedly; in the 28th day after model was made, alveoli structure was damaged, the wall of alveoli became thicker remarkably, interstitial fibrotic cicatrix came into being and capillary blocked. Changes under electron microscope in the 7th and 14th day showed that mitochondria of typeⅡalveolar (epithelial) cells became hyperplastic and swollen and its cristae decurtated, collapsed and vanished. Quantities of lamellar body decreased and became vacuole. Microvilli damaged, lessened and disappeared. In the 28th day, quantity of typeⅡalveolar (epithelial) cells decreased more than that in the 7th day or in the 14th day. Cristae in mitochondria and lamellar body damaged and degenerated like vacuole. In the 7th day, amelioration of pathological changes from the most obviously to the most slightly was D, C, E, F, and that in the 14th day was D, E, C, F, that in the 28th day was D, E, F, C. There was significant difference among Szapiel grade of alveolitis and fibrosis in each group (P＜0.01 or 0.05).②the changes of apoptosis of ATⅡcells in group C, D in each phase was lower than that in groupB (P＜0.05).③The sera level of TNF-αand IL-6 in group C, D, E, F in each phase was lower than that in group B respectively(P＜0.01) in the 7th, but only that in group D, E was higher than that in group B in the 14th -28th (P＜0.01 or 0.05).④The expression of CTGF in the lung tissue in the group C, D, E in each phase was lower than that in groupB respectively (P＜0.01 or 0.05).⑤Content of MDA in lung tissue in group C, D, E, F in each phase was lower than that in group B respectively(P＜0.01or0.05), while that of GSH-PX and SOD was higher(P＜0.01, 0.05). In the 7th and 14th day, content of MDA in lung tissue of degressive sequence was F, C, E, D, and in the 28th day was C, F, E, D (P＜0.01or 0.05), while that of GSH-PX was opposite.
Conclusion:①The model was made successfully according to its corresponding manifestation of pathology to human's lung fibrosis and other reports.②HD has therapeutic effect on lung fibrosis in each phase.③HD can decrease the apoptosis of ATⅡcells.④HD can regulate the disbalance of cytokine such as decreacing sera TNF-α, IL-6 level.⑤HD can inhibit the expression of CTGF in lung tissue.⑥HD can decrease harmful free radical and increase beneficial free radical scavenger in lung tissue.
引文
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