【作者】 费雁；

【导师】 孔庆志；

【作者基本信息】 湖北中医学院 ， 中医内科学， 2009， 博士

【摘要】 目的肺纤维化的发病率有逐年增高趋势,然而由于其病因和发病机制尚未阐明,迄今尚无特效疗法,病死率高,成为严重威胁人类健康的难治性疾病。临床表明,中医药治疗不仅能有效改善症状,提高患者的生存质量,而且可以极大降低本病的死亡率,因此,总结和探讨肺间质纤维化的中医药治疗具有很大的必要性和现实意义。芪丹化纤方是根据肺间质纤维化的基本病机特点所拟定的基本方,以益气、行气活血化痰为治法,在临床上以该方为基本方治疗各期肺间质纤维化取得显著疗效。本课题以博莱霉素诱导的大鼠肺纤维化为模型,以影响细胞外基质代谢的细胞因子和基质金属蛋白酶系统为主要切入点,观察芪丹化纤方对肺系数及肺组织病理形态的影响、对基质金属蛋白酶及其组织抑制剂表达的影响、细胞因子PTGFβ1含量的影响以及肺组织SOD的活性及MDA、NO含量的影响来进行研究,目的是通过动物实验,验证和客观评价芪丹化纤方的疗效,并探究其可能的作用途径和机制。方法144只健康Wistar大鼠,体重200±20g,随机分为六组:正常对照组、模型对照组、强的松组、中药大剂量组、中剂量组和小剂量组,每组24只。除正常对照组外,其他五组均采用经气管滴注博莱霉素(BLM)建立大鼠肺纤维化模型,造模后第二日起芪丹化纤方大、中、小剂量组每天用芪丹化纤方(2g/ml、1g/ml、0.5g/ml)灌胃,强的松组用醋酸强的松混悬液(0.32mg/ml)灌胃,正常对照组与模型组生理盐水灌胃,每日一次,剂量为10ml/kg·bw。每组分别于第7、14和28天随机处死8只,HE染色及光镜观察大鼠肺组织显微结构的病理形态学变化;观察大鼠肺系数变化;运用免疫组织化学分析法检测大鼠肺组织TNF-α及CTGF细胞因子含量的变化;运用ELISA法检测大鼠血清中MMP-9及TIMP-1表达的变化;运用样本碱水解法检测肺组织NO、MDA水平及SOD活性的变化;运用RT-PCR法观察大鼠肺组织TGFβ1表达的变化;以及芪丹化纤方对上述各项指标的影响。结果(1)芪丹化纤方可明显降低实验性肺纤维化大鼠肺系数(P＜0.01),芪丹化纤方大剂量组作用优于小剂量组(P＜0.05),强的松组、芪丹化纤方大、中剂量组三组间差异无显著性意义(P＞0.05);肺组织病理形态光镜表明,强的松组、芪丹化纤方大、中、小剂量组与模型组相比病理改变均有不同程度改善,各组间肺泡炎的差异具有显著性(P＜0.01或0.05),由重到轻排序为模型组,芪丹化纤方小剂量组,芪丹化纤方中剂量组,强的松组,芪丹化纤方大剂量组,正常组;各组间肺纤维化的差异具有显著性(P＜0.01或0.05),纤维化程度排序由重到轻依次为模型组,强的松组,芪丹化纤方小剂量组,芪丹化纤方中剂量组,芪丹化纤方大剂量组,正常对照组。(2)与正常对照组比较,各时间点其余各组大鼠肺组织CTGF、TNF-α的表达明显增高(P＜0.01)。与模型对照组比较,各时间点,强的松组、芪丹化纤方大、中剂量组CTGF、TNF-α表达均低于模型对照组(P＜0.01或0.05),芪丹化纤方小剂量组略低于模型对照组,但比较差异无统计学意义(P＞0.05)。(3)与正常对照组比较,模型对照组、强的松组、芪丹化纤方大、中、小剂量组各时间点,SOD的活性均降低,MDA及NO的含量均增高,存在显著差异(P＜0.05);与模型对照组比较,第7、14天时,强的松组、芪丹化纤方大、中、小剂量组SOD的活性均升高,MDA及NO的含量均降低,存在显著差异(P＜0.01或0.05),第28天时,芪丹化纤方大、中组SOD的活性均升高,存在显著差异(P＜0.01或0.05),强的松组、芪丹化纤方小剂量组SOD的活性有所升高,但不存在显著差异(P＞0.05),芪丹化纤方大、中、小剂量组MDA及NO的含量均降低,存在显著差异(P＜0.01或0.05),强的松组MDA及NO的含量有所降低,但不存在显著差异(P＞0.05)。(4)与正常对照组比较,模型对照组、强的松组、芪丹化纤方大、中、小剂量组各时间点,MMP-9及TIMP-1的含量均显著上升(P＜0.01或0.05);与模型对照组比较,各时间点MMP-9及TIMP-1的含量均降低,存在显著差异(P＜0.01或0.05)。(5)与正常对照组比较,模型对照组在三个时间点TGFβ1含量均显著增高(P＜0.01),强的松组、芪丹化纤方大、中、小剂量组在各时间点上TGFβ1的含量比正常对照组明显增加(P＜0.01),但显著低于模型对照组(P＜0.01),芪丹化纤方各组间比较,第7天时,大、小剂量组间的差异具有显著意义(P＜0.05),第14天和第28天时,大剂量组与中、小剂量组的差异均具有显著意义(P＜0.05)。结论芪丹化纤方对博莱霉素诱导的大鼠肺纤维化具有一定的防治作用,这一作用可能是通过以下途径实现的:降低血清MMP-9、TIMP-1的表达,调节MMP-9/TIMP-1系统,使之趋于平衡;降低CTGF、TNF-α含量;减少肺组织中MDA、NO的表达,增强SOD活性;降低肺组织中TGF-β1的表达。芪丹化纤方从多靶点、多环节纠正细胞外基质的过度沉积,有效保护肺组织的正常形态,对肺纤维化的发生和发展起到一定的防治作用。更多还原

【Abstract】 Objective:Pulmonary Fibrosis,with its increasing incidence and unknown cause,is becoming a severe threat to human health with no effective cure and high death rate.The clinical research has already proved that traditional Chinese herbal treatment can effectively ameliorate its symptom,considerably improve the victims’ life quality and substantially reduce its deaths.Therefore,it is of necessity and significance to explore the traditional herbal treatment of pulmonary interstitial fibrosis.Qi Dan Hua Xian Fang（Herbal Prescription to dissolve pulmonary interstitial fibrosis with Qidan,astragalus & moutan bark） takes considerable clinical effects in treating pulmonary interstitial fibrosis at various stages by strengthening and supplementing the flow of Qi,promoting blood circulation and dissipating the phlegm. The project is designed to verify and assess the effects of Qi Dan Hua Xian Fang（referred to as QDHXF） via rodent experiment,thus exploring its possible operation ways and mechanism.The project is conducted by observing the effects of QDHXF on the rodents with pulmonary fibrosis induced by bleomycin.Based on QDHXF’s effects on the matrix metalloproteinase system and the cytokine influencing the metabolism of extracellular matrix,the observation is focused on the pathomorphism and ultrastructure of lung tissues,the matrix metalloproteinase and its tissue inhibitors,the content of cytokine PTGFβ1 and the content of lung tissue HYP.The animal experiment is observed to give an objective assessment and evaluation of QDHXF’s effects and provide a possible explanation for its operation ways and mechanism.Methods:144 healthy Wistars,weighing 200±20g,are divided into six groups of 24 members at random,i.e.The normal group,the model control,the group treated with prednisone,the group treated with high-dosed QDHXF,the group treated with medium-dosed QDHXF and the group treated with low-dosed QDHXF.Except the normal group,the other five groups are set with the model of pulmonary fibrosis by intratracheal instillation of bleomycin.From the second day on,the three groups treated with QDHXF are supplemented with the high dose of 2g/ml,medium dose of 1g/ml and small dose of 0.5g/ml.The group treated with prednisone is supplemented with prednisone acetate suspension of 0.32mg/ml.The normal group and the model group are supplemented with 10ml/kg·bw normal saline.8 mice will be executed at random on the 7^th,the 14^th and the 28^th day for observation.The research is focused on（1） the pathomorphism and ultrastructure change of lung tissues via HE dyeing and optical microscope（2） the change of lung coefficients（3） the content change of lung tissue TNF-αand cytokine CTGF via chemical analysis of immune tissues （4） the change MMP-9 and TIMP-1 of blood serum via ELISA testing（5） the change of the NO and MDA level of lung tissues and SOD activity via alkali hydrolysis of samples（6） the change of lung tissue TGFβ1 via RT-PCR testing（7） the effects of QDHXF on the above-mentioned indexes.Results:（1） QDHXF can noticeably reduce the lung coefficients of experimental pulmonary fibrosis（P＜0.01）=.The high-dosed QDHXF group has abetter effect than the low-dosed one（P＜0.05）=.There is no significant differentiation among the group treated with prednisone,the group treated with high-dosed QDHXF and the group treated with medium-dosed QDHXF（P＞0.05）.The microscopic observation of the pathomorphism of lung tissues indicates that in comparison with the model group,there are various improvements among the prednisone group and the three groups treated with QDHXF.Significant differences in the infection of pulmonary alveoli are presented among different groups（P＜0.01 or 0.05）,listed in descending severity as the model group,the low-dosed QDHXF group,the medium-dosed QDHXF group,the prednisone group,the large-dosed QDHXF group and the normal group.There are also substantial discrepancy in the pulmonary fibrosis（P＜0.01 or 0.05）,listed in descending severity as the model group,the prednisone group,the low-dosed QDHXF group,the medium-dosed QDHXF group,the large-dosed QDHXF group and the normal group.（2） Compares with the normal control group,various time other each group of big mouse lung organizes CTGF,TNF-αexpression obvious markup（P＜0.01）.Compares with the model control group, various time spot,the strong loose group,QDHXF middle monitoring team CTGF,TNF-αthe expression is lower than the model control group（P＜0.01 or 0.05）,QDHXF small monitoring team slightly is lower than the model control group,but comparison difference non-statistics significance（P＞0.05）.（3） Compared with the normal group,the model group has a conspicuous rise in the MDA and NO contents of lung tissues at different times,reaching a summit on the 28^th day.At all times, the prednisone group and the three groups treated with QDHXF has conspicuously lower contents of MDA and NO than those of model group at the corresponding periods（P＜0.01）.Among groups,the three QDHXF groups show negligible differences on the 7^th and 14^th day; however,there is a significant difference between the high-dosed group and the low-dosed group on the 28^th day.Compared with the model group,the high-dosed QDHXF group has substantially higher SOD activity and lower MDA content in their lung tissues.（4） Compared with the normal group,the model group has a notable rise in MMP-9 and TIMP-1 contents in their blood serum on the 7^th,14^th and 28^th day（P＜0.01）.Compared with the model group, the prednisone group and the three groups treated with QDHXF has an obvious decrease in MMP-9 and TIMP-1 contents（P＜0.01 or 0.05）.（5） All experimental groups have TGF-β1 presentation on the 7^th 14^th and 28^th day.Compared with the normal group,the model group has a significant rise on the 7^th day（P＜0.01） and reaches its summit on the 14^th day.With its relative fall on the 28^th day,it is still much higher than the normal group at the corresponding period（P＜0.01）.Compared with the model group,the prednisone group and the three QDHXF groups have conspicuous fall（P＜0.01）.Among the three QDHXF groups,the TGF-β1 of the high-dosed group is considerably lower than that of the low-dosed group（P＜0.05）.Conclusion:QDHXF has significant effects on the prevention and treatment of the pulmonary fibrosis induced by bleomycin,which is likely to be realized by reducing MMP-9 and TIMP-1 in blood serum,regulating MMP-9/TIMP-1 system to its balance,reducing CTGF and TNF-contents, cutting MDA and NO contents in lung tissues, strengthening SOD activity and reducing TGF-β1 in lung tissues. To sum up,modify the over sediment of extracellularmatrix,thus effectively protecting the normal lung tissues,preventing and treating the.更多还原